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• Soyad alfabetik sıralanmıştır.

Terin Adalı 3 Berna Arda 8 Hüseyin Avcı 13 Semra Aydın 17 Turgut Baştuğ 20 N. Ayşe Odman Boztosun 24 Bilge Debeleç Bütüner 31 Gülay Büyükköroğlu 33 Özge Can 35 Eda Çelik Akdur 39 Özlem Çelik Yeşiltaş 41 İsmet Deliloğlu 45 Mert Döşkaya 46 Münis Dündar 51 Berrin Erdağ 55 Arzum Erdem Gürsan 58 Osman Eroğul 59 Hakkı Zafer Güney 60 Nesrin Hasırcı 61 Vasıf Hasırcı 64 Ayşe Göksu Kaya Özsan 65 Kamer Kılınç 69 Tarlan Mamedov 71 Ahmet Mavi 75 Emirhan Nemutlu 79 Sezer Okay 80 Suna Özbaş 85 Sadi Satılmış Özdem 87 Rana Sanyal 88 Dilek Çökeliler Serdaroğlu 91 Emine Şalva 96 Salih Şanlıoğlu 98 Şerif Şentürk 102 Kemal Tekin 104 Dilek Telci 107

Type of the Paper (Extended Abstract, Meeting Report, Preface, Proceeding, etc.)

Curcumin Loaded Pullulan Nano / Micro Particles for

Apoptosis Analysis of MCF-7 Breast Cancer Cells†

Terin Adali1, 2, *, and Pinar Tulay3, 4

1 Department of Biomedical Engineering, Near East University, P.O. Box: 99138, Lefkosa / TRNC, Mersin 10

Turkey. 2 Tissue Engineering and Biomaterials Research Center, Near East University, P.O.

Box: 99138, Lefkosa / TRNC, Mersin 10 Turkey.

3Department of Medical Genetics, Near East University, P.O. Box: 99138, Lefkosa /

TRNC, Mersin 10 Turkey.

4Experimental Health Sciences Institute, Near East University, P. O. Box: 99138,

Lefkosa / TRNC, Mersin 10 Turkey. Emails: terin.adali@neu.edu.tr,

pinar.tulay@neu.edu.tr

* Correspondence: terin.adali@neu.edu.tr; Tel.: +90-542-856-6050

† Presented at the Bio Türkiye / International

Biotechnology Congress, Ottoman Archive Complex,

Istanbul, 5-7 March 2020. Published: date (leave it

empty)

Abstract: Breast cancer is the second most diagnosed cancer in women that over 2.09 million cases exist

worldwide. The aims of this work was to design curcumin (CC) loaded pullulan (PL) nano / micro particles as

drug carrier system and analyze the in vitro MCF-7 cell viability for the therapy of breast cancer. The ionic gelation

method was used in the synthesis of PL / CC nano / micro particles. The apoptotic screening of MCF-7 cells was

carried out with Pl-CC particles, which were cultured for 48 and 72 hours. A cell viability analysis showed a dose

dependent apoptotic effect, indicating significant cell death (P <= 0.05).

Keywords: Breast Cancer, MCF-7; Pullulan; Curcumin; Nanotechnology

1.0 Introduction

Curcumin, the yellow orange compound 1, 7-bis (4-hydroxy-3-methoxyphenyl) – 1, 6-heptadiene – 3, 5-

dione is the main phenolic pigment has been widely employed in Alternative medicine for centuries [1]. Even so,

its chemical instability, poor water solubility reduce its biological effects and bioavailability [2]. Pullulan is a

water soluble, neutral polysaccharide produced from starch by the fungus Aureobasidium pullulans [3].

Encapsulation is an efficient approach to solve this problem. The aim of this work was to design pullulan –

curcumin nano /micro particles by ionic gelation method and evaluate apoptotic effect on MCF-7 breast cancer

cell line.

2.0 Materials and Methods

2.1 Materials

Sayfa 3

Pullulan was purchased from Hayashibara, Japan. Turmeric (Curcuma longa rhizome) was provided from

the market. MCF-7, differentiated mammary epithelium with estrogen receptor (ATCC HTB-22TM, US). 1 %

penicillin / streptomycin (Thermofisher, US), 10 % fetal bovine serum (FBS Thermofisher, US), and insulin in

DMEM / F12 (Thermofisher US). The cell-counting test Kit-8 (CCK-8) was purchased from TEBU-BIO cell

counting. Pentasodium tripolyphosphate (TPP) (Sigma Aldrich, Germany). All other chemical reagents were

analytical grade and purchased from Merck, Germany without any further purification.

2.2 Methods

2.2.1 Isolation of Curcumin from Curcuma Longa rhizome.

The crude extract of Curcuma longa was dissolved in dichloromethane – methanol (1:1) and weighed

amount of silica gel was added. This mixture were dried under vacuum at 50oC. Fraction was performed and those

that were rich in curcumin were evaporated until dryness. Thin layer chromatography was used to separate pure

curcumin.

2.2.2 Preparation of Pullulan-Curcumin Nano / micro particles.

In order to obtain homogenous solution, a given weighed amount of pullulan was dissolved in 20 mL of

ultra-pure water under magnetic stirring. The desired amount of curcumin was blended with pullulan solution. The

PL / CC solution was stirred 30 minutes at 1.5 rpm. The PL/CC solution was added to 0.1 M TPP by dropwise

technique. The preparation conditions are given in Table 1.

Table 1. Pullulan / Curcumin micro / nano particles synthesis conditions for ionic gelation

method.

2.2.3 Characterization of curcumin and PL / CC nano / micro particles

The purity and structure elucidation of curcumin was established by 1H-NMR and 13C-NMR

spectroscopy. The particle size analysis were carried out by using Mastersizer 2000 version from METU Central

Lab Ankara Turkey. Morphological analysis of particles was carried out with a scanning electron microscope (S-

3400 N: Hitachi Japan). FTIR analysis was carried out by FTIR –ATR Spectrophotometer from EMU, department

of chemistry Lab, North Cyprus.

2.2.4 In vitro MCF-7 cells viability Analysis

The MCF-7 cell culture was performed according to the manufacturer’s protocol (ATCC, Canada). The

differentiated mammary epithelium with estrogen receptors, human breast cancer cell lines MCF-7 was cultured

in DMEM/F12 media supplemented with 10 % FBS and 1 5 penicillin / streptomycin mixture 37oC in 5 %

CO2incubator.Approximately 2000 cells were seeded in 96 well plates and after 24 hours after seeding, MCF-7

cells was cultured with three different concentrations of curcumin loaded pullulan m-particles. Ranging from 25

%, 50 % and 100 % for 48 and 72 hours. The cell viability was measured by CCK-8 cell counting kit following

manufacturer protocol.

3.0 Results and Discussions

3.1 The structure and purity elucidation of Curcumin

Sample 2 % Pl C 0.1 M TPP

P 2.5 ml 5.0 ml

PC 2.5 ml 12.5 µm 5.0 ml

P: Pullulan particles PC: Pullulan / curcumin particles

Pl: Pullulan; C: Curcumin

Sayfa 4

The purity and structure elucidation of curcumin was established by 1H and 13C-NMR spectroscopy. The

data obtained for isolated compound were in good agreement with the data reported by Berger & Sicker [4]. The

NMR data indicates the presence of enolic form of curcumin. The singlet at 5.92 pp. integrating for one proton

was assigned to H-4, which confirms the enolic form of curcumin.

3.2 The synthesis and characterization of pullulan encapsulated curcumin particles

The Pl-CC particles size distribution analysis have been carried out by Mastersizer within the size range

of 0.02 – 2000 µm with normal sensitivity and 12.22 % obscuration. The concentration of PL-CC particles is

0.0386 % colume. Surface weighted mean and volume weighted mean values are listed on Table 2 as 22.352 µm

and 164.008 µm , respectively.

Proceedings 2018, 2, x; doi:

Table 2. PL-CC particle size distribution

PL-CC Particle Size Distribution Analysis

Weighted residual : 0.966 %

Concentration : 0.0386 % volume

Specific Surface Area : 0.268 m2 / g

Surface weighted mean D[3, 2] = 22.352 µm

Volume weighted mean D[4, 3] = 73.398 µm

d(0.1) = 8.799 µm

d(0.5) = 73.398 µm

d(0.9) = 431.115 µm

3.2 FTIR Analysis of PL and PL-CC particles

FTIR spectra showed characteristic pullulan and curcumin peaks.

(a) (b)

Figure 1. The FTIR spectrum: (a) Pullulan nanoparticles; (b) Pullulan – curcumin nano- macro

particles.

3.3 Morphological analysis of PL-CC particles

MUSTAFA GAZI 49

Name

Sample 049 By MUSTAFA GAZI Date Friday, February 21 2020

Description

4000 4503500 3000 2500 2000 1500 1000 500

100

61

65

70

75

80

85

90

95

cm-1

%T

486.27cm-1, 62.09%T

1015.35cm-1, 71.21%T

887.73cm-1, 72.21%T

1079.49cm-1, 73.02%T

3234.16cm-1, 84.36%T

1211.33cm-1, 89.01%T

1648.38cm-1, 91.22%T

2931.4

1459.6 1361.3

1099702.4

754.85

MUSTAFA GAZI 52

Name

Sample 052 By MUSTAFA GAZI Date Friday, February 21 2020

Description

4000 4503500 3000 2500 2000 1500 1000 500

100

66

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

cm-1

%T

499.71cm -1, 66 .82% T

886.73cm-1, 73.24%T

1103.07cm-1, 73.51%T

3176.59cm-1, 90.30%T

1652.22cm-1, 94.31%T2924.8

1466.2

1361.3

1220.3

1020.4

971.2

702.4

Sayfa 5

Figure 2. The SEM micrograph of PL-CC nano / microparticles.

Morphological studies were carried out by SEM. All PL-CC particles are spherical in shape as shown in

Figure 2.

3.4 In vitro MCF-7 cells viability Analysis

In vitro MCF-7 cells viability analysis was applied for three different concentrations of curcumin loaded

pullulan micro / nano particles (PL-CC) at 25 %, 50 % and 100 % for 48 and 72 hours, respectively. The results

of this study showed a significant difference between 48 and 72 hours of 100 %, 50 % and 25 % PL-CC for MCF-

7 cells, respectively. Furthermore statistical significance was also observed between 100 % and 75 % PL-CC

concentrations. Table 3 shows the absorbance results of PL and PL-CC particles at 48 and 72 hours. Table 3. MCF-7 Cell viability absorbance results of MCF-7

4.0 Conclusions

The pullulan-curcumin micro / nano particles were successfully prepared by using ionic gelation method for

the first time. The cell viability results showed statistical significance in MCF-7 breast cancer cells compared to

the controls. As a result, PL-CC micro / nano particles synthesized by ionic gelation method is promising in drug

delivery system for breast cancer.

Acknowledgments: The authors thank Prof. Ihsan Calis for his valuable support.

Author Contributions: Terin Adali and Pinar Tulay conceived and designed the experiments; Terin Adali

and Pinar Tulay performed the experiments; Terin Adali and Pinar Tulay analyzed the data; Terin Adali and Pinar Tulay

contributed reagents/materials/analysis tools; Terin Adali wrote the paper.

Conflicts of Interest: The authors declare no conflict of interest.

References

No C RATIO C 48 C 72 P 48 P 72 PC 48 PC 72

1 25:75 3.052 2.931 1.064 1.859 3.184 2.898

2 50:50 1.028 0.585 0.909 1.223 2.599 3.442

3 100:0 0.517 0.554 0.929 1.511 3.074 3.469

C RATIO: Volume of drug : Volume of cell culture medium

C 48: Curcumin at 48 hours; C 72: Curcumin at 72 hours;

P 48: Pullulan at 48 hours; P 72: Pullulan at 72 hours;

PC 48: Pullulan / Curcumin particles at 48 hours;

PC 72: Pullulan / Curcumin particles at 72 hours.

Sayfa 6

1. Wilken, R.; Veena, M. S.; Wang, M. B.; Srivatsan, E. S.; Curcumin: A review of anticancer properties and therapeutic

activity in head and neck squamous cell carcinoma. Mol. Cancer 2011, 10, 1- 9.

2. Strimpakos, A.; Sharma, R. Curcumin: preventive and therapeutic properties in laboratory studies and clinical trails.

Antioxidants & Redox Signaling 2008, 10, 511 – 537.

3. Rekha, M.; Chandra, P.; Pullulan as a promising biomaterial for biomedical applications: a perspective. Trends

Biomater. Artif. Organs 2007, 20(2), 000-000.

4. Berger, S.; Sicker, D. Classic in Spectroscopy. Isolation and Structure Elucidation of Natural Products. Wiley-VCH

Verleg GmbH & Co. KGaA, Leipzing, Germany.

© 2018 by the authors. Submitted for possible open access publication under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

Sayfa 7

Ethics issues in genom editing

Berna ARDA(MD MedSpec PhD)

Ankara University, School of Medicine, History of Medicine and Ethics Department, Professor

World Association for Medical Law, EC Member

Abstract: In this study; emerging relevant technologies in biomedical sciences such as Human

Genom Project and germline manipulations in general , evaluated in the light of bioethics and

medical law.

Keywords: genom editing, bioethics, research ethics, human rights, medical law

Almost everything started with Mendel. Undoubtedly, there may be other predecessors of the

heredity idea, but Mendel should be considered groundbreaking in this context(6). The eugenic

word has the meaning of "congenital well-being" or "being noble on hereditary base". The

history of the views on eliminating diseased and defective genes is quite old. Having “more

qualified”(better, smarter, healtier) people idea extends to Platon. But the modern version of

this approach that came under the eugenic term belongs to Francis Galton(4). The Human

Genome (HUGO) Project was carried out between 1990 and 2006. HUGO is the effort of Homo

Sapiens to reveal “what” is makes us “human” (3). It can even make it possible for people to

re-evaluate "self", "what is happening", "reorganize" with genetic manipulations. At this point,

genetic engineering is faced with an "ethical" questioning, consisting of social norms and a

cluster of values. Genetic manipulations, which have a potential power to be used in a wide

range of diagnosis and treatment of diseases, brought along some concerns relevant to the

applications. At this point, human beings can make their own copies with gene technology and

have the potential to change their environment permanently. In this context, this potential power

of gene technologies has caused a number of ethical questions regarding genetic manipulations.

The deterioration of the natural balance in the human body/ in general, changing the health

quality concept, the risk of using genetic information as a biological weapon, the studies of

creation of superior human as a potential heavy worker or soldier, gender discrimination, being

source of precious information for insurance companies, the employers and the state are all the

main issues(1).

Relevant international legislation: Oviedo Convention

This convention on “Human Rights and Biomedicine” is based on the human rights approach

adopted by the Council of Europe(CoE), to protect the dignity and identity of all people, to

ensure respect for the fundamental rights and freedom of all, without discrimination in the

application of biology and medicine. It is also supported by additional protocols prepared by

the Bioethics Committee. The Convention is also an important legal document for the

establishment of bioethics law in Europe (8). This convention ratified with Law No 5013 by

Turkish Grand National Assembly on December 3rd 2003 as a sort of local legislation(5). The

CoE is concerned with human body interfered technologies, mainly related to advances in

biology and medicine. The main topics are embryo and fetus, cloning, biomedical research,

developing technologies, end of life, genetics, psychiatry, organ transplantation and biobanks.

The convention represents the outcome of an in-depth discussion at the European level

regarding developments in the biomedical field, particularly in the genetic field(7).

Articles 14 and 18 of the Oviedo Convention deal with the protection of the embryo and the

fetus. The creation of human embryos for the purpose of selecting the unborn baby’s sex and

research is prohibited, except in the case of avoiding a serious hereditary disease. In addition,

where existing laws permit research on the embryo, adequate protection of the embryo has to

be ensured. Developments in genetic technologies have created new opportunities for the

Sayfa 8

human genome, on the one hand, leading to ethical problems that have not been encountered

before. While these technologies create an opportunity for the diagnosis, prevention and

treatment of diseases in the future; unwanted harm caused by the use of technology also raises

more complex ethical and human rights issues such as access and consent to these techniques,

eugenics and enhancements(9).

After twin babies were born in China with genome editing in 2018, the Council of Europe

Bioethics Committee repeated its view on genome regulation adopted in 2015(10).

Accordingly, the Bioethics Committee re-emphasized the importance of the Oviedo

Convention, which is the only binding international document addressing human rights in

biomedical studies.

Interventions that cause irreversible changes in the genetic structure of future generations are

prohibited in the Convention, which includes the subject of intervention in the human genome.

However, intervention to change the human genome has been accepted for conservation,

diagnostic and therapeutic purposes. CoE recognizes the principles of the Oviedo Convention

that can be used as reference internationally on key ethical issues posed by the latest

technological developments in this field.

There are three different types of artificial cloning: (1) gene cloning, (2) reproductive cloning

(3) therapeutic cloning. While gene cloning or DNA cloning has different processes, they are

similar in terms of reproductive cloning and therapeutic cloning method. However, the purpose

of both is different from each other. The main method in cloning is based on transferring the

cell nucleus from the somatic cell to an egg cell whose cell nucleus is taken. Gene cloning is a

technique that is widely accepted today and is routinely used in most laboratories. However,

reproductive and therapeutic cloning also raises important ethical issues, particularly with

regard to the use of these methods on humans.

In the 13th article of the Oviedo Convention, the limits of interference with the human genome

are clearly defined. Accordingly, intervention in the human genome can be done only for

preventive, diagnostic and therapeutic purposes. In addition, the intervention should not lead to

a change in subsets. In the 18th article, the production of human embryos is prohibited for

research purposes.

The Council of Europe has different arrangements between the use of embryonic cells in the

cloning method and human cloning. While cell cloning is an ethically acceptable situation; the

use of embryo cells in cloning should be regulated by the relevant articles of the Convention.

Human cloning is also an ethically controversial issue in terms of human identity and dignity.

Human cloning means predetermination of human genetic structure by third parties, which

poses a threat to human identity. Moreover, it is possible to artificially clone person and

instrumentalize human being. This instrumentalization is a situation that clearly undermines

human dignity. It is in human interest to preserve the random nature of the composition of its

genes, since the naturally occurring genetic combination is likely to create greater freedom for

a human than a predetermined genetic construct(11). The additional protocol, which was

opened for signature in Paris on January 12, 1998, concerns the Prohibition of Human Cloning,

is the first and only binding international legal regulation developed in this field. The protocol

entered into force on 1 March 2001. The scope of the protocol concerns only human cloning.

Therefore, there is no comment on the ethical acceptability of cloning of cells and tissues for

research purposes and medicine. This Additional Protocol prohibits any attempt to create a

genetically identical, living or inanimate person to a person. The phrase "genetically identical"

means that a person shares the same nuclear set with another person.

This Protocol includes both Articles 1 (protection of human dignity and identity), 13

(intervention in the human genome), 14 (lack of gender choice) and 18 (research on the embryo

Sayfa 9

in the tube) of the Oviedo Convention. Reveals the ethical principles against the problems that

may arise in this field as a result of the advancement of biology and medicine today and in the

future.

Emerging technologies are technologies that are new, innovative, still developing and expected

to have a great socioeconomic impact. These technologies are not evaluated under the existing

technology classification as they use new concepts, methods and techniques(2). Human rights

problems arising from emerging technologies and convergence technologies (NBIC;

nanotechnology, biotechnology, information technology, cognitive science) ... highlighted

ethical issues related to the use of biomedical technology both within and outside the medical

field. These technologies are well known ethical and social issues such as security, privacy,

autonomy, responsibility, physical and spiritual integrity, informed consent and access to

technology, as well as human enhancement, social enhancement, biological enhancement. it

raises relatively new concepts such as ownership of data, freedom of information, competence

of consumers and medicalization. With the convergence of NBIC, the boundaries of interfering

with the human body will come out of the field of biotechnology and enter nanotechnology and

information technologies. Also, with the development of cognitive technologies, it will be

possible to intervene in the human mind.

The development of new technologies, such as the sequencing of the human genome and DNA

chips, has made human genetics and genomic domains highly dynamic. Under this title,

especially the applications carried out in the prenatal period are focused. In this context, the

Agreement on the Additional Protocol on Genetic Tests for Health Purposes and Statement on

Genome Editing Technologies is important.

The fourth part of the Convention is devoted to the provisions regarding the human genome.

According to the convention, no discrimination can be made to anyone due to their genetic

inheritance. The genetic heritage mentioned here is added to the 14th article of the European

Convention on Human Rights regarding non-discrimination. The Additional Protocol on

Genetic Tests for Health Purposes entered into force on 1 July 2018. Its purpose is to ensure

that the integrity and fundamental freedoms of all are respected, without discriminating the

implementation of genetic testing for health purposes, while maintaining the dignity and

identity of all parties. The genetic tests mentioned in the protocol are the tests performed for

the purpose of identifying the genetic features that are inherited or occur during prenatal

development, which include the analysis of biological materials of human origin(12).

The protocol covers diagnostic, predictive and pharmacogenetic tests and carrier tests

performed on living or dead individuals, or on human-derived biological materials, for health

purposes. However, any test with the human embryo or fetus for research is excluded. For this

reason, preimplantation genetic diagnosis and prenatal genetic diagnosis tests are outside the

scope of the Protocol(13).

The purpose of the Recommendation is to establish the basic rights of individuals whose

personal data are processed for insurance purposes and to secure these rights(15). According to

the decision, the personal data of the insured person related to health should not be processed

for insurance purposes without his written permission. The insured person's consent must be

clear, free and informed. Genetic testing for insurance purposes is also prohibited by the

decision of the Committee of Ministers. It is stated in the 12th Article that tests for the diagnosis

of genetic diseases can only be performed for health purposes or for scientific researches for

health purposes and provided that appropriate genetic counseling services are provided(14).

With this Recommendation of the CoE, strict protection has been introduced for the collection

and processing of personal health data, based on the consent of the insured person. This decision

aims to prevent improper processing and use of health-related data in international legal

Sayfa 10

regulations. The text also emphasizes the need for facilitating access to insurance for eligible

budgets to those with increased health risk, and the importance of promoting fair and objective

resolution of disputes between insured persons and insurance companies(15).

Apart from the CoE, another guiding structure in this regard is the German Ethics

Council's(GEC) view on intervening in the Human Germline. According to this opinion, “the

ethical analysis does not lead to any categorical inviolability of the human germline. The

assesment of the permissibility of germline interventions should not be reduced to mere risk

and opportunity analysis. Rather it should be based on the ethical concepts of human dignity,

protection of life and integrity, freedom, non-maleficence and beneficence, naturalness, justice,

solidarity and responsibility. The prerequisite for permissibility is, in any case, a sufficient

degree of safety and efficacy of such interventions”. GEC also describe decision paths in the

field of preclinical research and also in the transitions to clinical application. Finally they

emphasized the difficulty to differentiate between the conceivable application context.

Furthermore, “the range of complexity” of the respective applications and the related

opportunities and possible risks can vary considerably. A serious ethical evaluation of germline

interventions can only be undertaken on a “case- by- case basis” and with reference to the

respective relevant ethical concepts(16).

As a result;

The ethical issues discussed above, along with all the encouraging positive developments,

should not be ignored. The instrumentalization of man and turning it into a commodity seems

possible with the wrong use of this technology. Genetic manipulations can push the individual

into a normative conflict, such as choosing between fundamental rights and freedoms and

individual dignity. This situation may leave the individual to choose between his / her own

ethical values, individual-society, individual-social norms. In these studies, it is essential to see

people as a purpose and to act accordingly, not as a tool. Ethical principles and behaviors that

comply with these principles will serve a high purpose such as social benefit in the development

of gene technologies.

If the information obtained by deciphering the genetic code becomes a tool that serves political

and ethnic purposes, it will mean that common sense is removed. Gene technology, which will

help develop knowledge about evolution, will lead us to reinterpret "ourselves", "what we are."

It is also possible to determine some privileges with social analysis. If these privileges explain

interpersonal differences such as intelligence, can social responsibility be expected to disappear

altogether? How will the discussion of "determinism's biological origin" explain the concept of

"human responsibility and obligation"? To what extent, for what purposes and by whom the

"information" will be used for the solution of the genetic code.

In ethical inquiry; It would be appropriate to discuss the "framework in which we can fit the

desire to know" that genetic practices will offer us, which motivates people. What will be the

price of the contribution of gene technology to humanity? How clear are the limits of playing

with the human genome? Will a person know where and when to stop? Does man have the right

to implement everything that can be done using the possibilities of technology? Or should there

be a “reasonable” limit of what can be done? Of course, the determination of this "acceptable"

line will be our "own" ethical values, norms, and our ability to resolve ethical problems.

References: 1- Arda B: Ethics and commercial use of genetics. Bulletin of Medical Ethics. 111: 19 - 22.

September 1995.

2- Brey P. Ethics of Emerging Technology. In: Methods for the Ethics of Technology. SO

Hansson ( editor) pp. 175-192. London: Rowman and Littlefield International; 2017.

Sayfa 11

3- Bökesoy I, Arda B: İnsanGenomuProjesi’ nin( HUGO’ nun ) etik ve sosyal yönleri. Türkiye

Klinikleri Tıbbi Etik 1: 22 - 26, 1993 (in Turkish).

4- Güvercin CH, Arda B: Eugenic concept; from Plato to the present. Human Reproduction and

Genetic Ethics, 14(2): 20-26, 2008.

5- Katoğlu T: Türk Hukukunun Bir Parçası Olarak Avrupa Konseyi İnsan Hakları ve Biyotıp

Sözleşmesi. Ankara Üniversitesi Hukuk Fakültesi Derg. 2006;55(1):157–93. (in Turkish)

6- Kurtoğlu A, Arda B: A Biographical Study in the History of Heredity: Gregor Johann Mendel (1822-

1884) Türkiye Klinikleri Tıp Etiği Hukuku Tarihi Dergisi 27(2): 162-177, 2019.

7- Kurtoğlu A, Arda B: Council of Europe and it’s Activities in the Field of Bioethics,

(underreviewed article)

8- Avrupa Konseyi. Biyoloji ve Tıbbın Uygulanması Bakımından İnsan Hakları ve İnsan

Haysiyetinin Korunması Sözleşmesi: İnsan Hakları ve Biyotıp Sözleşmesi . Oviedo; 1997. ETS

No. 164.

9- Parliamentary Assembly of the Council of Europe. Recommendation 2115. The use of new genetic

technologies in human beings. 2017.

10- Council of Europe Committee on Bioethics. Ethics and Human Rights must guide any use of

genome editing technologies in human beings [Internet]. 2018.

https://search.coe.int/directorate_of_communications/Pages/result_details.aspx?ObjectId=0900001

6808fe117

11- Council of Europe. Explanatory Report to the Additional Protocol to the Convention for the

Protection of Human Rights and Dignity of the Human Being with regard to the Application of

Biology and Medicine, on the Prohibition of Cloning Human Beings. 1998.

12- Council of Europe. Additional Protocol to the Convention on Human Rights and Biomedicine

concerning Genetic Testing for Health Purposes. Strazburg; 2008.

13- Council of Europe. Explanatory Report to the Additional Protocol to the Convention on Human

Rights and Biomedicine concerning Genetic Testing for Health Purposes. Strazburg; 2008.

14- Committee of Ministers. Recommendation CM/Rec(2016)8 of the Committee of Ministers to the

Member States on the Processing of Personal Health-Related Data for Insurance Purposes,

including Data Resulting From Genetic Tests. Strazburg; 2016.

15- Committee of Ministers. Recommendation CM/Rec(2016)8 of the Committee of Ministers to the

Member States on the Processing of Personal Health-Related Data for Insurance Purposes,

including Data Resulting From Genetic Tests Explanatory Memorandum. Strazburg; 2016.

16- Deutscher Ethikrat, Intervening in the Human Germline, Opinion, Published by the German Ethics

Council, Berlin, 9 May 2019. pp 36-37, 39-57.

Sayfa 12

Organ on a Chip: Opportunities and Challenges

Huseyin Avci1-3 1Metallurgical and Materials Engineering Department, Eskisehir Osmangazi University, Eskisehir,

Turkey 2Cellular Therapy and Stem Cell Research Center (ESTEM), Eskisehir Osmangazi University,

Eskisehir, Turkey 3AvciBio Research Group, Eskisehir Osmangazi University, Eskisehir, Turkey www.avcibio.com

Abstract:

Every year studies on new drugs and investments for the treatment of diseases are

increasing rapidly. Nowadays, the total cost of researches to find effective and safe

therapeutic agents exceed $ 2 billion. In addition, a drug already being used in the

market for a long time can be withdrawn after pass all the research and production

stages due to unexpected side effects. Therefore, researchers are working on in vitro

models that can accurately predict the effects of drugs, chemicals and biological

agents in the human body. Here, we briefly investigate a recent effort is being made

to develop highly complex tissue like-cellular structures called organ on a chip to

simulate tissue- and organ-level physiology.

Keywords:

Organ on a chip, drug development, human physiology, opportunities and

challenges.

Introduction:

The change in the concentration and effectiveness of a drug from the first moment

enters the human body, the effect that changes with time; its relationship with blood,

tissues and organs are just a few of the vital interactions that need to be fully

understood. Drugs to be placed on the pharmaceutical market are investigated on in

vivo before proceeding to clinical trials, but they do not give the similar positive

effects as in animal models. For this reason, such systems are needed to simulate the

human body that can show the complex processes of a drug for absorption,

distribution, metabolism and excretion [1]. In this regard, organ on a chip is an in

vitro platform developed recently and has the potential to imitate human tissues or

organs [2]. In fact, multiple tissues or organoids in microfluidics have been brought

together to form human on a chip platform within the chip. In addition, with these

developed models, it is expected to be advantageous in the analysis of new drug

systems by aiming to obtain more accurate results from animal models by allowing

cell and tissue-tissue interfaces, control of transcellular molecules and their

concentration gradients to be performed in relatively small 3D microfluidic chip

setups. On the other hand, thanks to these systems in which the medicines that have

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been withdrawn from the pharmaceutical market have been successfully monitored

the side effects that occur in the human body.

In this study, multisensor-integrated organs on chips platforms are discussed to

analyze physiological functions of target in vivo tissue and organoids after model

drugs administration [3].

Materials and Methods:

Gold-based microelectrode, polydimethylsiloxane (PDMS), chemicals for

immobilization such as 11-mercaptoundodecanoicacid, N-Hydroxysuccinimide,

streptavidine, biot-Ab, antijens (GST-α), primary human hepatocytes, human iPSC-

CMs, HepG2 cells, GelMA and hydrogel were used. Briefly e-beam evaporation for

electrode production, photo and soft lithography for bioreactor and microfluidics, 3D

bioprinting for organoids were utilized [3].

Results and Discussion:

In a study, Zhang et al. have developed a healthy heart integrated liver organoid, and

a healthy heart organoid connected liver cancer on chip platforms to obtain similar

responses like in the human body during drug administration are shown in Figure 1

[3]. Acetaminophen (APAP) and doxorubicin (DOX), which are selected here as

model drugs, were applied to healthy heart - liver organoids and healthy heart - liver

cancer models, respectively. Following APAP application of 72 hours after system

started to work, deterioration in liver organoids and even cell deaths increased,

however no significant changes were observed in heart cells.

On the other hand, in the second platform DOX, a well-known chemotherapy and

anti-cancer drug, has been applied to human healthy heart - liver cancer on chips

platform. As a result of DOX administered for approximately 24 hours from the

beginning of the experiment, liver cancer cells were significantly destroyed. Unlike

the APAP application, DOX has a dramatic side effect on healthy heart cells as

observed in the real clinical cases, resulted decreased beating rate, death of the cells,

and a high amount CK-MB release.

Here, in both in vitro platforms as shown in Figure 1, without conducting any in vivo

animal and clinical human trials, responses and interactions were taken with the

model drugs used, were similar to human physiology.

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Figure 1. Schematic representation of human healthy heart and liver on chips (left), and

human healthy heart and liver cancer on chips (right) with administration of APAP and

DOX, respectively [3].

Conclusions: Benefits and challenges for the future

As a result, with the information and experience to be obtained from these in vitro

microstructures produced, it is expected to accelerate the drug development and

treatment of diseases with reducing the cost significantly. By taking damaged and

diseased cells from the patients can transformed into tissue or organoids inside the

chip, and then the potential for use in personalized medicine or treatment can be

possible. Furthermore, it can help to form different cancer models in in vitro

environments to better understand personalized differences within the same cancer

type and the origin of different diseases can be investigated as well. In addition,

clinicians and researchers can more accurately predict the individual's response to

harmful chemical and biological hazards in food, cosmetics or dietary supplements.

Despite all the advantages of organ on a chip platform, there are still very important

aspects should be developed. More sophisticated microsystems are needed to

combine multiple tissues and vascular channels in a single microsystem to simulate

more accurate and long-term differentiation of target in vivo counterparts. While

studies mainly focused only on a single feature of cells or cell behavior in micro-

organ models, there is not enough number of cells and extracellular factors as the

real organ mimicked. Another important problem is the limited working time of the

systems, i.e. less than a month, therefore chronic diseases, complex reactions at the

endocrine system level, skeletal and nervous systems, and etc. cannot be investigated

in detail. In addition, polydimethylsiloxane (PDMS) is currently widely used in chip

fabrication, but it needs to be improved or replaced with new materials for long-term

stability, reliability, better chemical resistance, biocompatibility, and should

eliminate absorption of small drug hydrophobic molecules to increase drug reliability

with effectiveness.

In the near future, researchers will be put more efforts on standarization and

regulatory endorsement for controllability, long term usage, automation of organ-on-

a-chip system, monitoring, data collection points and times. To obtain robust and

healthy data, a consensus is needed at least formed by engineers, pharmacists,

biologists and medical doctors.

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References

1. Esch, M. B., King, T. L., Shuler, M. L. The role of body-on-a-chip devices in drug and

toxicity studies. Annu Rev Biomed Eng 2011, 13, 55-72.

2. Avci, H., Dogan Guzel, F., Erol, S., Akpek, A. Recent advances in organ-on-a-chip

technologies and future challenges: a review. Turk J Chem 2018, 42(3), 587-610.

3. Zhang, Y. S., Aleman, J., Shin, S. R., Kilic, T., Kim, D., Shaegh, S. A. M., ... & Avci,

H. Multisensor-integrated organs-on-chips platform for automated and continual in situ

monitoring of organoid behaviors. PNAS 2017, 114(12), E2293-E2302.

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Biyoteknolojik Ürünlerde İnvitro Biyotayinler

Invitro Bioactivity Assays of Biopharmaceuticals

Semra AYDIN

Hacettepe University, Institute of Vaccine, Vaccine Technology Department

06100, Ankara

Keywords:Antibody-dependent cellular cytotoxicity (ADCC), complement-

dependent cytotoxicity (CDC), Binding Activity, Enzyme-linked immunosorbent

assay (ELISA), Surface plasmon resonance (SPR),

The biopharmaceutical product, monoclonal antibody (mAb)-based

therapeutic is a large complex biomolecule with a heterogeneous structure, and

prepared by the use of living systems using recombinant DNA technology (1). The

defined functional activity of mAb is based on its specific binding characteristic to a

ligand (commonly known as the antigen). Therefore; the structure of the mAb, which

also determines its immunological properties and capacity to achieve a defined

biological effect, should be clearly assessed in detail by appropriate in vitro assay(s).

Using these assays, not only the mechanism of action for mAb should be justified

with respect to its biological activity, complement binding and activation, cytotoxic

properties, and antibody-dependent cytotoxicity but also the part of the monoclonal

antibody that recognizes and binds to the epitope and specific activity should be

identified (2,3).

To measure binding capacity of mAb such as Fc gamma receptor binding, and

neonatal Fc receptor (FcRn) binding or neutralizing antibodies, an immunoassay

such as ELISA might be deemed appropriate. Many variants of ELISA such as

sandwich, indirect and competitive etc. have been developed and used in different

situations. They all depend on the same basic elements: Coating/Capture (direct or

indirect antigen immobilization to the surface of micro plate wells) Plate Blocking:

covering all unsaturated surface-binding sites of the microplate wells with irrelevant

protein or other molecule. Probing/Detection: incubation with antigen-specific

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antibodies that affinity-bind to the antigens. The detection antibody may be directly

labeled with a signal-generating enzyme or fluorophore or it may be secondarily

probed with an enzyme- or fluor-labeled secondary antibody (or avidin-biotin

chemistry). For enzymatic detection, the appropriate enzyme substrate is added.

Signal Measurement: Signal acquisition generated on the specific antibody (4,5).

Biosensor-based surface plasmon resonance (SPR) technology is an

alternative method which has higher stability, accuracy and precision associated with

enzyme immunoassays. Principles of biosensor; ligand is immobilized/ attached to

the sensor surface, analyte is flown across the surface in a continuous flow of sample

solution. Interface is monitored in real time; kinetics of biomolecular interactions,

the rate of interaction of ligand-antibody and binding level can be determined (6,7).

If effector functions of mAb are relevant then a cell-based bioassay such as

antibody-dependent cellular cytotoxicity (ADCC), complement-dependent

cytotoxicity (CDC), complement dependent cellular cytotoxicity (CDCC) or other

assays such as proliferation/viability can be performed (3) ADCC response is

mediated by binding of NK cells to Fc region of the mAb through receptors (FcɣRIIIa

or CD16), release cytokines/ cytolytic agents which result in the death of the target

cell (8). For these types of in vitro assays, cells are seeded to culture plate.

Appropriate concentration and volumes of drug are added to wells. Respectively,

human serum complement protein, peripheral blood mononuclear cells (PBMC) and

both are added to test wells of CDC, ADCC , CDCC assays and incubated. For

proliferation assay, generally WST-1 reagent is used followed by measurement of

the end point absorbance. For cell viability assay following the exposure of cells to

test compounds for a determined time period, luminescent reagents can be used to

determine metabolically active cells. Stabilized luminescent signal was recorded (9).

In this presentation in vitro assays which can be used to determine biological activity

and effects of mAb-based therapeutic and detailed technical practice will be

reviewed and presented.

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References

[1.] Luz –Rodrigues H, Vaz Carneiro A, Cabrita A, Carrera F, Frazao J.M, Macario F, Neves P, Nolasco

F, Ponce P, Prata M.M, Sa H, Vinhas J., Position statement of the Portuguese Society of Nephrology on

the clinical use of biotechnological drugs in renal patients. Port J Nephrol Hypert 2009, 23(4): 317-321

[2.] EMEA (2007) Guideline on production and quality control of monoclonal antibodies and related

substances. EMEA/CHMP/BWP/157653/2007

[3.] EMEA (2016) Guideline on development, production, characterisation and specification for

monoclonal antibodies and related products. EMA/CHMP/BWP/532517/2008 [4.]Engvall E and Pearlmann P. (1971). Quantitive assay for IgG. Immunochemistry, Vol 8:871.

[5.] http://tools.thermofisher.com/content/sfs/brochures/TR0065-ELISA-guide.pdf [6.] Olaru A, Bala C, Renault N.Z, and Aboul-Eneın H.Y, Surface Plasmon Resonance (SPR) Biosensors

in Pharmaceutical Analysis. Critical Reviews in Analytical Chemistry (2015) 45, 97–105.

[7.] https://www.gelifesciences.co.jp/contact/pdf/BiacoreAssayHandbook.pdf

[8.] Pereira N.A, Chan K.F, Lin P.C, and Song Z., The “less-is-more” in therapeutic antibodies:

Afucosylated anti-cancer antibodieswith enhanced antibody-dependent cellular cytotoxicity. MABS

2018, 10(5), 693–711

[9.] Kizhedath A, Wilkinson S, GlasseyJ., Applicability of Traditional In Vitro Toxicity Tests for

Assessing Adverse Effects of Monoclonal Antibodies: A Case Study of Rituximab and Trastuzumab.

Antibodies 2018, 7, 30; doi:10.3390/antib7030030.

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Molecular Modeling of Sodium Channels

Turgut Baştuğ1, Ali Osman Acar1, Murat Çavuş2, Serdar Kuyucak3

1Department of Biophysics, Faculty of Medicine, Hacettepe University, Ankara, Turkey 2Faculty of Education, Bozok University, Yozgat, Turkey 3School of Physics, University of Sydney, NSW, Australia Abstract:

Action potentials are the electrical signals that regulate physiological processes. Voltage gated ion channels that mediate the electrical signals have different functional roles. Disruption of the voltage gated sodium (NaV) channels may lead to disorders and diseases such as hereditary epilepsy, migraine, periodic paralysis, cardiac arrhythmia and chronic pain syndromes. There are nine NaV1 isoforms (Nav1.1-Nav1.9) with different functions in mammals. Homology modeling studies are widely employed to obtain these structures using prokaryotic channels as templates since a high-resolution eukaryotic channel is not solved yet. Although, several eukaryotic structures have been solved recently using cryo-EM, lower resolution may lead to problems especially at narrow sites such as selectivity filter. In this study, we focus on developing a homology model of NaV1.4 isoform using NaVPaS structure as a template. Considering the results of molecular dynamics simulations and free energy calculations, we present a model that may be useful in understanding the mechanisms of ion conduction, permeation and toxin binding in mammalian channels. Keywords: Voltage Gated Sodium Channels, Molecular Dynamics Simulations, Free Energy Calculations

Introduction

Voltage gated ion channels are a special class of ion channels that open and close in response to changes in the membrane potential, allowing the ions to be conducted in the direction of the electrochemical gradient. Sodium channels have a common structure to include 4 domains along the membrane and a total of 24 α-helices. Each domain contains 6 α-helixes called S1, S2, S3, S4, S5 and S6 helices along the membrane. Among these helices, the S4 helix is highly conserved in all voltage gated ion channels and acts as the voltage sensor of the channel. A pore region and selectivity filter located between the S5 and S6

Sayfa 20

helices, they are mainly responsible for the functions of the channels such as conduction, permeation and selectivity. Although there have been many studies on these functions in prokaryotic channels, our knowledge remains limited on their eukaryotic counterparts. However, homology modeling studies and recently solved cryo-EM structures have made ways for investigations on eukaryotic channels. In this study, we present a homology model of human NaV1.4 channel based on the cryo-EM structure NaVPaS expressed from american cockroach.

Materials and Methods

Sequence alignment processes is performed using the ClustalW [1] to detect the sequence difference between the model and the template. According to this alignment NaV1.4 structure is determined using a model creating software called Modeller [2]. OPM database was used to determine the orientation of the modeled structure across the membrane. Molecular dynamics simulations are employed using the created model. MD parameters used in this study, are optimized parameters for membrane proteins. In all MD simulations performed, the CHARMM36 [3] force field and NAMD [4] v2.11 simulation package were used. A NPT ensemble is used with periodic boundary conditions. Pressure is kept at 1 atm and temperature is kept at 300K using Langevin coupling with damping coefficients of 5 ps-1. Lennard Jones interactions are switched off in a distance of 12 A. Electrostatic interactions are computed with Particle Mesh Ewald algorithm. A time step of 2 fs is used in all simulations. Results and Discussion Before starting MD studies, the features of the model were examined. Eukaryotic channels contain cysteine residues that keep extracellular regions stable. Since both template and model contain these cysteine residues, the disulfide bridges between these cysteines preserved with an average distance of 2 Å. Model is equilibrated with a 125 ns MD simulation and the RMSD profile is calculated using this trajectory data. RMSD values do not exceed 0.85 Å along the whole simulation. In the developed Nav1.4 model, selectivity calculation was made for the filter region. To make this calculation, the process of converting a sodium (Na) ion in the filter region to potassium and a previously added potassium ion into the sodium was performed (forward process). At the same time, a potassium ion in the filter is converted into a sodium ion, while a sodium ion in water is converted into a potassium ion (backward process), to show that the calculation is independent of the hysteresis effect. The calculation was made with the free energy perturbation method, 100 lambda value was used between 0 and 1. The free energy value obtained from the average of forward and reverse processes

Sayfa 21

is 0.8 kcal / mol and is compatible with the experimental data within the error limits. In order to test the Nav1.4 model we created, tetrodotoxin (TTX) binding to the model was investigated. To obtain a binding conformation, docking of TTX was calculated using the Autodock software [5]. Then the TTX – model complex is eqilibrated with a 50 ns MD simulation. To calculate a potential of mean force profile in the protein-TTX complex structure, the TTX molecule along the reaction coordinate is sampled by the US method. Sampling is performed that a window is created every 0.5 Å. A harmonic constraint of 15 kcal/mol is applied in the z direction in the simulations and a cylindrical potential has been applied to keep the TTX molecule on the axis. For unbiasing and combining the results, Weighted Histogram Analysis Method (WHAM) is used [6]. Comparison of the PMF profiles for both template and model is shown in Figure 1. Dissociation constant calculated according to this PMF profile is approximately 62 nM which is in good agreement with the experimental IC50 results. Conclusion In this study, it is aimed to develop a consistent Nav1.4 model. The developed NaV1.4 model was tested using the binding of TTX. The results obtained in the binding modes are in good agreement with the experimental results. The first thing to consider here is that these channels consist of approximately 400

Figure 1: PMF profiles for both template and model.

Sayfa 22

residues, and modeling the entire channel consistently is a long and difficult task. As the developed models are tested through TTX binding, when talking about the consistency of these channels, the active regions of the channels (selective filter region) and the regions that directly interact with these regions (cavity region) should be considered. It can be said that Nav1.4 model developed in this perspective is reliable and can be used as a template to obtain other isoforms of NaV channels. References

1. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice. Nucl Acids Res 22: 4673–4680.

2. B. Webb, A. Sali. Comparative Protein Structure Modeling Using Modeller. Current Protocols in Bioinformatics 54, John Wiley & Sons, Inc., 5.6.1-5.6.37, 2016.

3. Huang, J., & MacKerell, A. D., Jr (2013). CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. Journal of computational chemistry, 34(25), 2135–2145. doi:10.1002/jcc.23354

4. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., … Schulten, K. (2005). Scalable molecular dynamics with NAMD. Journal of computational chemistry, 26(16), 1781–1802. doi:10.1002/jcc.20289

5. Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S. and Olson, A. J. (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J. Computational Chemistry 2009, 16: 2785-91.

6. Kumar, S., Bouzida, D., Swendsen, R. H., Kollman, P. A., and Rosenberg, J. M. (1992) The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comput. Chem. 13, 1011–1021.

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Rekabet Kurulu Kararları Işığında Tıbbi Görüntüleme ve Tanı Aygıtları Pazarı

Bio Türkiye – Uluslararası Biyoteknoloji Kongresi

5-7 Mart 2020, Osmanlı Arşivi Külliyesi Kongre Merkezi, İstanbul

Prof. Dr. N. Ayşe ODMAN BOZTOSUN

Akdeniz Üniversitesi Hukuk Fakültesi

ÖZET

Bu tebliğde, tıbbi görüntüleme ve tanı aygıtları pazarı ile bu aygıtların yedek parça

pazarı, pazardaki firmalar, Türkiye’de bu aygıtların kullanımını etkileyen sosyal güvenlik

düzenlemeleri, rekabeti koruyan düzenlemeler ve fikri mülkiyet düzenlemeleri bağlamında

incelenerek genel durum ortaya konulmuştur.

1. Tıbbi Görüntüleme ve Tanı Aygıtları

Tıbbi görüntüleme ve tanı aygıtları, tıbbi aygıtlar sektörünün bir alt koludur.1 Tıbbi

görüntüleme ve tanı aygıtları aşağıdaki şekilde sayılabilir:

a. Manyetik Rezonans Görüntüleme (MRI)

b. Bilgisayarlı Tomografi (CT)

c. Dijital Radyografi

d. Nükleer Görüntüleme

e. X-ray

f. Ultrason

g. Mamografi

h. Diğerleri

1 Genel olarak tıbbi aygıtlar sektörünün dünyadaki ve Türkiye’deki durumunu ortaya koyan yakın tarihli çalışmalar için bkz.: Türkiye Teknoloji Geliştirme Vakfı, Dünyada ve Türkiye’de Tıbbi Cihaz Sektörü ve Strateji Önerisi, Editör: Kiper, M., Aralık 2018 (2. Baskı), https://ttgv.org.tr/tr/yayinlar/dunyada-ve-turkiye-de-tibbi-cihaz-sektoru-ve-strateji-onerisi; Kılıçarsan, M., Takkasız B., Dünya’da ve Türkiye’de Tıbbi Cihaz Sektöründe Pazarlamanın Önemi, Avrupa Bilim ve Teknoloji Dergisi (EJOSAT) Sayı 17, S. 966-971, Aralık 2019, https://dergipark.org.tr/tr/pub/ejosat/issue/48495/647581

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Bu aygıtlar, farklı çalışma prensiplerine sahip olmakla birlikte, dünyada, uzunca bir

süredir, ağırlıklı olarak belirli firmalar tarafından üretilmektedir.

2. Tıbbi Görüntüleme ve Tanı Aygıtları Pazarı

Siemens Healthcare, GE Healthcare, Philips Healthcare, Canon Medical Systems,

Hitachi Medical, ve Fujifilm Holdings Corp., dünya genelinde tıbbi görüntüleme ve tanı

aygıtları pazarının yaklaşık % 65’ine sahiptir. Bu pazarda faaliyet gösteren diğer firmalar

arasında Carestream Health, Shimadzu Medical, Hologic ve Esaote SpA sayılabilir. Pazarda

rekabet yoğundur, pazarın büyüme potansiyeli yüksektir.2 Teknolojinin gelişme hızı giderek

artmaktadır. Ülkelerin sağlık politikalarındaki ve fiyatlandırmalardaki değişiklikler de pazarı

etkilemektedir.

İlk yatırım maliyetinin yüksek olması ve bakım onarım ağının kurulması gereği, bu

pazara girişlerin önünde iki esaslı engeldir.

Türkiye pazarına bakıldığında, tüm dünyada bu aygıtları üreten yabancı üreticilerin

yavru şirketlerinin veya distribütörlerinin Türkiye’de faaliyet gösterdiği görülmektedir.3 Bu

2 Press Release: The Global Medical Imaging Market is Expected to Grow at a CAGR of 6.8% During the Forecast Period 2018 – 2024, https://www.infoholicresearch.com/press-release/global-medical-imaging-market/ (28 Eylül 2018) Ülkemizde de tıbbi görüntüleme ve tanı aygıtları sayılarının OECD ortalamasının altında kaldığı görülmektedir. Son yıllarda başta Şehir Hastaneleri olmak üzere sağlık kuruluşlarına yapılan yatırımlar dikkate alındığında, mevcut oranlar, ciddi bir büyüme potansiyelinin varlığına işaret etmektedir: 1.000.000 Kişi Başına Düşen MR Cihazı Sayısı: OECD Ort.: 13,2 Türkiye 10,4; 1.000.000 Kişi Başına Düşen Bilgisayarlı Tomografi Cihazı Sayısı: OECD Ort.: 22,5 Türkiye 14,1; 1.000.000 Kişi Başına Düşen PET Cihazı Sayısı: OECD Ort. 1,56 Türkiye 0,94; 1.000.000 Kişi Başına Düşen Mamografi Cihazı Sayısı: OECD Ort. 17,41 Türkiye 12,45; 1.000.000 Kişi Başına Düşen Gamma Kamera Cihazı Sayısı: OECD Ort. 8,74 Türkiye 2,9; 1.000.000 Kişi Başına Düşen DSA Cihazı Sayısı: OECD Ort. 7,23 Türkiye 4,58. Diğer yandan, ülkemizdeki kişi Başı Toplam Sağlık Harcaması da OECD ortalamasının bir hayli altındadır: (Satınalma Gücü Paritesi ABD $) OECD Ort.: 3.243 Türkiye 984 Kaynak: MERTLER,A.A/KARADOĞAN,N/TATARHAN,G, Türkiye’de Tıbbi Cihazların Sayısal Durumu ve OECD Ülkeleri İle Karşılaştırmaları, Uluslararası Sağlık Yönetimi ve Stratejileri Araştırma Dergisi, Cilt 1, Sayı 1, Yıl, 2015, s. 52-70 3 Diğer yandan, tıbbi görüntüleme ve tanı aygıtlarının kullanımına yönelik ülkemize özgü bir sorun da ihale düzenlemelerinden kaynaklanmaktadır. Sorun, Türk Radyoloji Derneği Başkanı Prof. Dr. Tuncay Hazırolan tarafından şu şekilde ifade ediliyor: “Bir hastaneye tomografi veya MR cihazı alınacaksa ihaleye çıkılıyor. Katılan firmalar çekim başına fiyat veriyor. Eğer şartnamede raporları da o firma yaptıracak diye bir madde varsa ya dışarıdan doktor getiriliyor ya da çeşitli yerlere gönderip rapor yazdırılıyor. Rekabet yoğun olduğu için her geçen gün fiyat düşüyor. Bir devlet hastanesinin ihtiyacını görecek ortalama bir MR cihazı bir milyon Euro’dan başlar. İşletim ücretleri, teknisyen, doktor maaşıyla bayağı yüksek bir meblağ. Firma ihaleyi alabilmek için 20-30 liraya çekeceğim dediği zaman cihazın kapasitesi günde 50 hastaysa, çok daha fazla çekmesi lazım ki kârlı çıksın.” (Kaynak: https://www.internethaber.com/cekilen-150-mr-veya-tomografiden-120si-gereksiz-doktorlar-muayene-bilmiyor-2059702h.htm ) Konuyla ilgili istatistikleri derleyen ve bu soruna dair öngörülen iskonto önlemine dair haber için bkz.: “Gereksiz MR'a İskonto Önlemi: Yataklı tedavi kurumlarında MR cihazı başına düşen görüntüleme sayısı 14 bin 992 olarak hesaplanıyor. OECD’de ise bu ortalama sadece 5 bin 125. Yataklı tedavi kurumlarında bin kişiye düşen MR çekim sayısı ortalaması 157 iken, OECD’de bu ortalama 67’de kalıyor. Yataklı tedavi kurumlarında BT cihazı başına düşen görüntüleme sayısı ise 12 bin 993 iken, OECD ortalaması 6 bin 890’u geçmiyor. Yataklı tedavi kurumlarında bin kişiye düşen BT görüntüleme sayısı da 188. OECD’de ise bu uygulamanın ortalaması 147. ..Türkiye’de yılda yaklaşık 12.5 milyon MR (manyetik rezonans görüntüleme), 15

Sayfa 25

şirketler arasında SIEMENS Healthcare Sağlık A.Ş., Türk Philips Ticaret A.Ş. (PHILIPS), GE

Medical Systems Türkiye Limited Şirketi, Medtronic Medikal Teknoloji Ticaret Limited

Şirketi, TMST Tıbbi Sistemler Pazarlama Ticaret ve Servis A.Ş. (TOSHIBA) sayılabilir. Tıbbi

görüntüleme cihazları pazarında dışa bağımlılık oranı halen % 100’dür. Ulusal teknoloji ve ürün

geliştirme çalışmaları devam etmektedir.4 İkincil pazarlarda (yedek parça/yazılım ve sarf

malzemeleri pazarları) üretici firmaların fikri mülkiyet hakları dolayısıyla bağımsız üretim ve

tedarik olanakları, dolayısıyla yerli rekabet olasılığı kısıtlıdır; ancak bu noktada rekabet hukuku

düzenlemeleri ve uygulamaları devreye girmektedir.

3. Rekabet Hukukunun Amacı ve İşleyişi

Piyasalarda rekabeti koruyan düzenlemeler, serbest piyasa ekonomisinin sunduğu özgür

ticaret olanaklarının danışıklılık yoluyla veya pazar gücü baskısıyla kötüye kullanılmasını

önlerler ve ihlal niteliğindeki davranışları cezalandırırlar. Bu düzenlemelerin amacı Pazar

Ekonomisinin Düzgün İşleyişi için Pazarlardaki İşleyebilir Rekabet Ortamının Korunmasıdır.

Son hedef, tüketici yararıdır. Bu bağlamda, teşebbüsler arasında rekabete aykırı anlaşmalar ve

uyumlu eylemler, ilgili pazarda hâkim durumun kötüye kullanılması ve hakim durum yaratacak

firma birleşmeleri yasaktır.

Ülkelerin rekabet otoriteleri, ihlal teşkil eden davranışları araştırırken, önce ilgili pazarı

saptarlar. Hem coğrafi açıdan hem de ilgili mal veya hizmet açısından ilgili pazarı belirledikten

sonra bu pazarda faaliyet gösteren aktörlerin pazar güçlerini, birbirileriyle ilişkilerini ve

davranışlarını rekabeti koruyan düzenlemelere uygunluk açısından değerlendirirler.

4. Rekabet Hukuku Bağlamında Tıbbi Görüntüleme ve Tanı Aygıtları İle İlgili Pazarlar

Tıbbi görüntüleme ve tanı aygıtları, sağlık sektöründe kullanımı giderek yaygınlaşan

araçlardır. Birçok ülkede satış ve pazarlama faaliyeti yürüten, küresel ölçekte firmaların pazar

milyon BT (bilgisayarlı tomografi), yaklaşık 28 milyon ultrasonografi çekimi ile bu konuda dünya lideri. Sağlık Bakanlığı ise bu duruma karşılık gereksiz MR çekimini engelleyebilmek için ödemeleri iskontolu yapmaya başlıyor. Bunun MR iştahını azaltması bekleniyor.” Kaynak: https://www.hurriyet.com.tr/ekonomi/gereksiz-mra-iskonto-onlemi-41279949 (21/07/2019). 4 T.C. Kalkınma Bakanlığı, Tıbbi Cihaz ve Tıbbi Malzeme Çalışma Grubu Raporu, Ankara 2014, http://www.seis.org.tr/docs/daha-cok-uretmeliyiz/kalkinma-plani/tibbi-cihaz-ve-tibbi-malzeme-calisma-grubu-raporu.pdf T.C. Sağlık Bakanlığı Türkiye İlaç ve Tıbbi Cihaz Kurumu, Türkiye Tıbbi Cihaz Sektörü Strateji Belgesi Ve Eylem Planı (2016-2020), https://titck.gov.tr/Dosyalar/TibbiCihaz/ProjeveStrateji/T%C4%B1bbi%20Cihaz%20Sekt%C3%B6r%20Stratejisi%20Belgesi%20ve%20Eylem%20Plan%C4%B1%2025.11.2015.pdf, yayın tarihi: 25 Kasım 2015, Ayrıca bkz.: “T.C. Sağlık Bakanlığı ve ASELSAN: yerli MR görüntüleme sistemi" (https://www.aa.com.tr/tr/pg/foto-galeri/aselsandan-2-milyar-dolarlik-hamle , haber tarihi: 08.09.2018); “Sağlıklı savunma” (https://www.hurriyet.com.tr/ekonomi/saglikli-savunma-41002282, haber tarihi: 29.10.2018).

Sayfa 26

payının ağırlıkta olduğu tıbbi görüntüleme ve tanı aygıtları pazarında, bu firmalar arasında bir

rekabet söz konusudur.5 Diğer yandan, oldukça pahalı olan ve kapsamlı yatırım gerektiren bu

aygıtların bakım ve tamiri açısından ayrı bir yedek parça pazarının tanımlanması gerekir.

Üretici firma, kendi aygıtlarının yedek parçalarının pazarında tek sağlayıcı konumundadır.

Dolayısıyla, tasarım özgünlüğü ve fikri mülkiyet koruması dikkate alındığında, aygıtın

alıcılarının ve servis firmalarının yedek parçalar açısından aygıt üreticisi firmaya bağımlılığı

doğmaktadır. Bu şekilde tek sağlayıcılık, ilgili pazarda pazar gücünün varlığına, yani piyasa

hâkimiyetine işaret eder.

Bağımsız servis sağlayıcı firmalar, 2005 yılından itibaren üretici firmaların bakım ve

onarımı zorlaştırıcı veya engelleyici olduğunu iddia ettikleri davranışlarına yönelik

şikayetlerini Rekabet Kurulu (“Kurul”)’na iletmeye başlamışlardır. İlk yıllarda bu şikayetlere

yönelik soruşturma başlatmayan Kurul, bu konuda verdiği ve tıbbi görüntüleme ve tanı aygıtları

ikincil pazarı açısından milat olarak kabul edilecek 2009 tarihli kararında ise6, üretici firmaların

kendi ürünlerinin bakım ve onarım pazarlarındaki pazar güçlerinin dizginlenmesine yönelik

tedbirler almıştır. Kurul kararının amacı, tıbbi görüntüleme ve teşhis cihazlarının ardıl pazarı

olarak değerlendirilen bakım-onarım pazarının rekabetçi bir hale getirilmesi, suni engellemeler

ile söz konusu pazarın bağımsız servis sağlayıcılara kapatılmamasıdır. Kararda ilgili pazar, tıbbi

görüntüleme ve tanı aygıtlarının bakım-onarım hizmeti ve yedek parça pazarları olarak

saptanmıştır. Üretici firmaların, bu pazarlarda, kendi ürünlerinin yedek parça ve bakım-onarım

hizmetlerine münhasır olarak, ayrı ayrı hâkim durumda olduklarına hükmedilmiştir. Üretici

firmalara getirilen yükümlülükler, şifre ve yedek parça teminine dairdir. Bu yükümlülükler,

sırasıyla şöyle belirlenmiştir:

i. Tıbbi cihazların garanti sürelerinin bitiminden sonra, cihazları satın alan

müşterilerin yazılı talepte bulunması veya bu yazılı talepleri müşterilerden alan

teknik servislerin yazılı başvuruda bulunması durumunda, cihazlara ilişkin

5 Bununla birlikte, benzer bir pazar olan diagnostik ürünleri pazarında, Rekabet Kurulu, 2010 yılında, “Diagnostik ürünleri ve hizmetleri alanında, hastanelerin en önemli alıcı konumunda olduğu bazı ihalelerde, danışıklı fiyat sunulması ve/veya bölge/hastane paylaşımı yapılması suretiyle rekabeti kısıtlayıcı uygulamalar olup olmadığının tespiti amacıyla Rekabet Kurulu’nun, 12.01.2011 tarih ve 11-03/41-M(a) sayılı kararıyla diagnostik ürünleri ve hizmetleri alanında faaliyet gösteren Beckman Coulter Biomedikal Ürünler San. ve Tic. Ltd. Şti., Siemens Healthcare Diagnostik Tic. Ltd. Şti., Roche Diagnostik Sistemleri Ticaret A.Ş., Abbott Laboratuarları İthalat İhracat ve Tic. Ltd. Şti. ve Mediset Tıbbi Malzemeler İthalat ve Tic. Ltd. Şti. teşebbüslerini kapsayan bir soruşturma yürütmüştür. Soruşturma sonucunda ihlal tespit edilememiştir. (29.05.2012 tarih ve 12-28/832-238 sayılı Rekabet Kurulu kararı) 6 18.02.2009 tarih ve 09-07/128-39 sayılı Rekabet Kurulu kararı.

Sayfa 27

şifrelerin veya bu anlama gelecek her türlü dâhili sistemin firmalar tarafından

mücbir sebepler haricinde, çalışma günlerinde olmak kaydıyla, 24 (yirmidört)

saat içerisinde ücretsiz olarak temin edilmesi,

ii. Cihaz harici takılan ve anılan cihazlara ilişkin teknik servis verilmesine olanak

sağlayan aparatlar/cihazların, müşterilerin yazılı talepte bulunması veya bu

yazılı talepleri müşterilerden alan teknik servislerin yazılı başvuruda bulunması

durumunda, talep anından itibaren en çok 3 (üç) gün içerisinde müşteriye

tesliminin yapılması,

iii. Bu aparatlara ilişkin kiralama ücretlerinin, ayrımcı olmayacak şekilde ve

aparatın maliyetiyle orantılı bir biçimde belirlenmesi,

iv. Cihazların ilk satımı aşamasında yukarıda yer verilen hususlar konusunda

müşterilerin yazılı olarak bilgilendirilmesi,

v. Tıbbi cihazların, son 3 (üç) yıllık satış verilerine dayanarak, en çok kullanılan

100 (yüz) yedek parçanın güncel fiyat listelerinin internet ortamında herkesin

ulaşabileceği şekilde ilan edilmesi,

vi. Müşterilerden ve rakip servis sağlayıcılardan gelen yedek parça fiyat taleplerinin

en geç 3 (üç) işgünü içerisinde cevaplandırılması,

vii. Yedek parça satışında rakip servis sağlayıcılara ve onların müşterilerine yönelik,

objektif kriterlere dayanmayan ayrımcı uygulamalar yapılmaması,

Kararda, üretici firmaların bu yükümlülüklere uymamaları durumunda 4054 sayılı

Rekabetin Korunması Hakkında Kanun çerçevesinde haklarında işlem başlatılacağı uyarısı

yapılmıştır.

Bu tarihten sonra da üretici firmaların Kurul’un öngördüğü yükümlülüklere uymadıkları

iddiasıyla çok sayıda şikayet yapılmış, Kurul, bu şikayetlerin bazılarını soruşturma konusu

yapmıştır. Bugüne kadar sadece Siemens aleyhine, ürünlerinin yedek parça satışını servis satışı

hizmetine bağlayarak hakim durumunu kötüye kullanması gerekçesiyle, yıllık cirosunun

%0,2’si oranında, toplam 2,6 milyon TL idari para cezasına hükmetmiştir. Kurul, bu konuda

2016 yılında da bir karar vermiş, Siemens firmasının yedek parça pazarındaki hakim durumunu

kötüye kullandığına yönelik şikayeti değerlendirmiş ve soruşturma sonucunda firmanın

yükümlülüklere uymadığı yolunda delile ulaşılamadığı gerekçesiyle dosyayı ceza vermeden

Sayfa 28

kapatmıştır.7 En son, Siemens Healthcare Sağlık A.Ş. hakkında verilen Kurul kararının Ankara

7. İdare Mahkemesi tarafından iptali üzerine başlatılan ön araştırma kapsamında, bu firmanın

Kurul uzmanlarının delil tespiti amacıyla yerinde inceleme taleplerini yerine getirmemesi ve

kayıtlarına erişime izin vermemesi üzerine firma aleyhine idari para cezasına hükmedilmiştir.8

Ön araştırmanın firmanın hangi davranışlarına yönelik olduğuna dair bilgi edinilememiştir.

5. Üretici Firmaların Yedek Parça ve Yazılımlar Üzerindeki Fikri Mülkiyet Hakları

Üretici firmaların hem tıbbi görüntüleme ve tanı aygıtlarının hem de bunların yedek

parçalarının üzerinde fikri mülkiyet hakları bulunmaktadır. 6769 sayılı Sınai Mülkiyet Kanunu

kapsamında marka, patent ve tasarım koruması elde etmek mümkündür. Marka koruması

süresizdir. Bununla birlikte, yedek parçalar açısından, malın kullanım amacının belirtilmesi

için, sahibinden izin almadan marka kullanılabilir. [SMK, m. 7(5)(c)] Burada yedek parçanın

menşei konusunda yanlış izlenim yaratılmamasına dikkat edilmesi gerekir. Bu aygıtların

tamamı veya bir kısmı veya üretim yöntemleri üzerinde patent koruması da olabilir. Yerli

teknoloji geliştirmeye girişen firmaların bu sektörde patentle korunan alanları önceden tespit

etmesi ve serbest hareket alanını belirlemesi, patent ihlali iddialarına maruz kalmaması için

elzemdir. Diğer yandan, patent konusu buluşu içeren deneme amaçlı fiiller serbesttir. [SMK,

m. 85(3)(b)] Tıbbi görüntüleme ve tanı aygıtlarının ve yedek parçalarının görünümlerinin

tasarım korumasından yararlanması da mümkündür. Yalnız görünmeyen parçalar tasarım

korumasından yararlanamaz. 9 Görünür parçalar üzerindeki koruma süresi ise 3 yıl ile

sınırlıdır.10 Hatta üretici firmadan başka bir firma tarafından üretilen bir yedek parça Bilim,

Sanayi ve Teknoloji Bakanlığı tarafından eşdeğer parça kabul edilirse bu yedek parçalar,

tasarım hakkı sahibi üretici firmanın iznine tabi olmadan serbestçe üretilebilir.11 Son olarak,

7 Bu konudaki Kurul kararlarının özeti için bkz.: Doğan, İ. Ü., Siemens Soruşturması Hakkında Verilen Karar Akabinde Rekabet Hukuku Kapsamında Sektörel Bir Değerlendirme (http://dogan-law.com/siemens-sorusturmasi-hakkinda-verilen-karar-akabinde-rekabet-hukuku-kapsaminda-sektorel-bir-degerlendirme/, 8 Haziran 2017)

8 07.11.2019 tarih ve 19-38/581-247 sayılı Kurul kararı. 9“(2) Birleşik ürünün parçasının tasarımı, aşağıdaki şartları taşıyorsa yeni ve ayırt edici niteliğe sahip olduğu kabul edilir: a) Parça birleşik ürüne takıldığında, birleşik ürünün normal kullanımında görünür durumda olmalıdır.” [SMK, m. 56(2)(a)] 10 “Birleşik ürünün görünümüne bağımlı olan parçaların, birleşik ürüne orijinal görünümünü yeniden kazandırmak üzere onarım amacıyla ve bu parçaların kaynağı konusunda yanıltıcı olmamak şartıyla tasarımın piyasaya ilk sürüldüğü tarihten üç yıl sonra kullanılması tasarım hakkının ihlali sayılmaz.” [SMK, m. 59(4)] 11 “Bilim, Sanayi ve Teknoloji Bakanlığınca yayımlanan eşdeğer parçaların dördüncü fıkra kapsamında ve tasarımın piyasaya ilk sürüldüğü tarihten itibaren üç yıl içinde kullanımı tasarım hakkının ihlali sayılmaz.” [SMK, m. 59(5)]

Sayfa 29

5846 sayılı Fikir ve Sanat Eserleri Kanunu kapsamında, bu aygıtları çalıştıran yazılımlar

üzerinde eser sahipliği koruması bulunduğu belirtilmelidir.12 Dolayısıyla bu yazılımların hak

sahibi üretici firmanın izni olmadan çoğaltılması ve değiştirilmesi eser sahipliği hakkının

ihlalini teşkil eder.

Sonuç

Tıbbi görüntüleme ve tanı aygıtları pazarı hem ülkelerin sağlık politikalarından ve

mevzuatından hem de genel olarak rekabeti koruyan düzenlemelerden etkilenmektedir.

Ülkemizin rekabet otoritesi Rekabet Kurulu, bu aygıtların bakım-onarım ve yedek parça

pazarını ayrı bir Pazar olarak değerlendirmekte ve üretici firmanın kendi ürününün bakım-

onarım pazarında hakim durumda olduğunu kabul etmektedir. Bu bağlamda üretici firmalara

şifre ve yedek parça temini hususunda bir takım yükümlülükler getirmiştir. Bu yükümlülüklere

uyulmaması Rekabetin Korunması Hakkında Kanun’un ihlali sonucunu doğurmaktadır. Bu

bağlamda, üretici firmaların ürünleri, yedek parçaları ve yazılımları üzerinde sahip oldukları

fikri mülkiyet hakları da koruma amaçlı ileri sürülememektedir. Kaldı ki fikri mülkiyet

düzenlemeleri de, ilgili fikri mülkiyet haklarını, kamu yararını gözeterek çeşitli şekillerde

sınırlandırmışlardır.

12 – İlim ve edebiyat eserleri şunlardır: 1. (Değişik: 7/6/1995 - 4110/1 md.) Herhangi bir şekilde dil ve yazı ile ifade olunan eserler ve her biçim altında ifade edilen bilgisayar programları ve bir sonraki aşamada program sonucu doğurması koşuluyla bunların hazırlık tasarımları [FSEK, m. 2(1)]

• Atıfta bulunulan internet sitelerine en son 25 Haziran 2020 tarihinde erişilmiştir.

Sayfa 30

Dr. Öğr. Ü. Bilge Debeleç Bütüner

CAR-T Cell Therapy Products

Chronic hepatitis B affects 255 million humans worldwide and puts them at high risk to

develop liver cirrhosis or hepatocellular carcinoma (HCC). 880.000 humans die every

year due to the consequences of an HBV infection (WHO 2017). For HCC, HBV

infection is still the leading cause. Nucleos(t)ide analogues are safe and well tolerated

with a high barrier to resistance, and tenofovir (or its derivates) or entecavir have

become the gold standard for the treatment of chronic hepatitis B because of their

ability to efficiently suppress HBV replication.

Since available therapies for hepatitis B do not target the nuclear persistence form of

the hepatitis B virus, the covalently closed circular DNA (cccDNA), they do not result

in virus elimination. Thus, seroconversion from HBsAg to anti-HBs, which is the desired

clinical endpoint and regarded as a “functional cure” remains very rare under this

treatment, committing most patients to long-term therapy with the risk of side effects

and development of antiviral resistance besides being costly.

Capsid assembly modulators (CpAM), an entry inhibitor (Myrcludex B) and siRNAs are

newer antivirals under development. As none of them directly targets the HBV

persistence form, the episomal HBV cccDNA, relapse rates are high after stopping

treatment. So far, the only potentially curative approach remains interferon, which is

already available for more than 20 years but has high side effects and is only effective

in 5-20% of patients. In addition, the cancer risk associated with chronic hepatitis B

remains significant despite the treatment with nucleos(t)ide analogues.

Therefore, hepatitis B still is a disease with high socio-economic importance. There is

a high need for improved diagnosis, prevention and therapy. HBV cure has become of

high interest for academic and pharmaceutical research stimulated by the recent

success in the development of antiviral therapies for hepatitis C. Because a curative

treatment will have to target HBV persistence, we hypothesize that immune stimulation

through an immune therapeutic approach will be needed to achieve HBV cure and

allow terminating antiviral therapy.

Sayfa 31

A complete cure will require (i) the elimination of infected hepatocytes, or alternatively

(ii) non-cytolytic purging of the HBV persistence form by cell-intrinsic defense

mechanisms, or (ii) its loss by cell division if new infection is blocked.

T cell responses seem to be essential to achieve at least a functional cure of hepatitis

B. A functional cure would become obvious by seroconversion from HBs to anti-HBs

and remaining cccDNA will be controlled by the immune response. While HBV-specific

CD4 and CD8 T-cell responses are readily detectable in patients resolving HBV

infection, HBV-specific T cells are scarce and partially impaired in chronic hepatitis B

most likely due to high amount of circulating viral antigens. However, they can be

activated even within the tolerogenic liver microenvironment by appropriate T cell

stimulation. Thus, curative therapeutic interventions will very likely combine antiviral

active drugs with immunotherapeutic approaches enabling the restoration of a

functional adaptive immune response but also minimizing the risk of potential side

effects.

Sayfa 32

Preparation of siRNA drug delivery systems in gene therapy

Gülay Büyükköroğlu

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Anadolu

University, Eskişehir, Turkey

Abstract:

Some diseases are caused by over-expression of a particular gene. Therefore, there

is a need for drugs that can reduce gene expression at the DNA and RNA levels.

Antisense Technology is an approach to change the level of gene expression. The

development of this technology began with the observation that the growth of Rous

sar-coma virus (RSV) is inhibited by introducing oligonucleotides into the cell

infected with this virus . With the use of antisense-based technologies, changes in

the level of gene expression occur at the post-transcription level. The antisense

approach involves the delivery of oligonucleotides which are complementary to the

mRNA or viral RNA to cells. These oligonucleotides have the ability to find and bind

to the target RNA. This binding or hybridization results in suppression of protein

expression, either by blocking the critical steps of the mRNA fragmentation or

translation process. Partial or complete elimination of genetic messages of genes in

these ways is called "knock-down" or "knock-out". Small interfering RNAs

(siRNAs) can be used as tools to study single gene function both in vitro and in vivo

and are an attractive new class of therapeutics, especially against undruggable targets

for the treatment of cancer and other diseases. siRNA based drugs have distinct

advantages over conventional small molecule or protein-based drugs, including high

specificity, higher potency and reduced toxicity. However, siRNA-based drugs have

to be overcome many challenges such as rapid deterioration, poor cellular uptake

and non-target effects before reaching the clinic. Delivery of SiRNA to target tissues

and stability in serum remains a major barrier and is the main focus of current

research and development efforts. To overcome these challenges, different

approaches suhc as rational design strategies, selection algorithms, chemical

modifications and nanocarriers are using.

Clinical trials with siRNA-based therapeutics were introduced after validation in

in vivo models and reached to more than 50 involving 26 different siRNAs. Initial

studies were conducted in diseases requiring localized delivery mostly mostly such

as eye. Since advances in nanocarrier technology, the number of systemically

administered siRNA-based therapeutics has increased considerably compared to

delivered locally. These These new therapeutics approch holds great promise for the

treatment of various cancers by targeting signaling pathways and oncogenes that

Sayfa 33

promote cell proliferation, cell cycle progression, invasion/metastasis and resistance

mechanisms in tumors.

Keywords:

Antisense Technologies, siRNA, gene therapy, oligonucleotides, gene silencing.

Refernces :

Zamecnik, P. C., & Stephenson, M. L. (1978). Inhibition of Rous sarcoma virus replication

and cell transformation by a specific oligodeoxynucleotide. Proceedings of the Nat

ional Academy of Sciences, 75(1), 280-284.

Dallas, A., & Vlassov, A. V. (2006). RNAi: A novel antisense technology and its therapeutic

potential. Medical science monitor, 12(4), RA67-RA74.

IDTutorial (2005). Antisense Technologies, Integrated DNA Technologies, 1-12.

Özcan, G., Özpolat,B., Coleman, R.L., Sood, A.K., Lopez-Berestein, G. (2016). Preclinical

and clinical development of siRNA-based therapeutics, Advanced drug delivery

reviews, 29(87), 108–119.

Bora, R.S., Gupta, D., Mukkur, T.K.S. Kulvinder Singh Saini, K.S. (2012). RNA interference

therapeutics for cancer: Challenges and opportunities, Molecular Medicine Reports, 6,

9-15.

Sayfa 34

Title:

Downstream Process for Monoclonal Antibodies from Small Scale to GMP

Production

Authors:

Özge Can1, Emre Burak Erkal2, Dilara Baş2, Melisa Köprülü2, Deniz Bayçın2, R.

Serdar Alpan2

Affiliations: 1 Acıbadem Mehmet Ali Aydınlar University, Ataşehir / İstanbul 2 Turgut İlaçları A.Ş., Levent / İstanbul

Abstract:

Monoclonal antibodies (mAbs) have gained considerable importance and this

molecule family has become increasingly important for the biopharmaceutical

industry market. The industrial manufacturing of mAbs is complex that needs

significant effort in both upstream and downstream processes. Mammalian cell

culture for the production of mAbs is increased in recent years because of high

specific productivity and right characteristics. Downstream processing focuses on

the purification and recovery of protein of interest.1 The early stages of development

of mAb purification used plasma fractionation because of simplicity.2 The modern

processes used for the purification of recombinant mAbs mainly use

chromatographic steps. Modern downstream processing has high level yields ranging

from 60 to 80%, depending on the number of steps.2 Filtration steps are considered

for virus-reducing step and a final diafiltration has to be included to get drug based

mAb. Each step of downstream processing should be handled with care to avoid the

risk of contamination and to ensure that material loss is kept at a minimum. High

resolution, high recovery, high speed and high capacity are indicators of the success

of the downstream processing. Every mAb for downstream processing represents

specific purification or recovery process. The first step in downstream processing is

the removal of cells and cell debris with filtration or centrifugation. Purification for

mAbs usually begins with Protein A chromatography, where they can provide a high

degree of purity in a single step. Following Protein A chromatography, ion exhcange

chromatography (IEX) or hydrophobic interaction chromatography (HIC) are

generally used.3-4 In this review, each type of unit operation information that forms

a downstream processing for mAb production is provided. The basic recovery and

purification processes for mAbs such as cell harvesting, chromatographic steps and

filtration steps are summarized. In addition, some important considerations required

for the scale-up and GMP production are also discussed.

Sayfa 35

Keywords:

Monoclonal antibodies; Downstream mAb processing; Antibody purification; GMP

antibody production; mAb purification scale-up

Main Text:

Hundreds of mAbs are currently available on the market or during development.

Many companies are investing in this issue, as mAbs have become prominent in the

biotechnological pharmaceutical industry. Efficient recovery and purification of

mAbs from cell culture at the end of the biopharmaceutical product manufacturing

processes is the most critical part of the production, and the purification steps are the

most important part of the total cost of the process. At the same time, maximum

attention should be paid to maintaining product quality, purity and integrity, while

dealing with numerous challenges. mAbs need to be separated from contaminants

(e.g. host cell proteins (HCP), DNA, endotoxin, residual Protein A and some cell

culture media additives) and product related impurities (e.g. high molecular weight

aggregate and low molecular weight species). In addition, viruses must be removed

from the system to ensure complete safety of the product. First step in the separation

of the molecule at the end of the monoclonal antibody production process is cell

harvesting which is called as clarification process. The aim of this process is to

separate cells and cell debris from supernatant. Protein A chromatography is most

commonly used as a capture step in mAb purification processes. Although, the

specific regions and sequences for mAbs have some differences, the purification

steps are essentially similar since Fc region is common. This region shows high

specificity for Protein A and constitutes the first step of purification as an affinity

chromatography in all mAb production processes. However, it is not possible to

collect protein of interest as 100% pure because it is eluted with HCP and other

contaminations (residual Protein A, DNA, high molecular weight aggregate and low

molecular weight species) from the chromatography column.5 According to the FDA

Q5A guideline, there are specific procedures that should be performed for viral

reduction as well as chromatographic steps to ensure safety of products produced by

mammalian cell culture.6 Protein A column eluate is at low pH and most mAbs are

stable under low pH conditions, it is relatively easy to include the low pH incubation

step to inactivate viruses. Retroviruses can be successfully inactivated at low pH. pH

of the protein A elution pool can be adjusted by adding weak acid/base at high

concentrations (after acid inactivation, neutralization is applied to more stable pH

range).7-8 Polishing steps following downstream processes aim to reduce impurities.

The choice of which chromatography methods to use for the polishing steps depends

on the nature of the impurities. Anion Exchange (AEX) or Cation Exchange (CEX)

Sayfa 36

chromatography techniques are useful for the polishing step. Anion exchange

chromatography operates with a positively charged group (weakly basic such as

diethylamino ethyl, DEAE or dimethylamino ethyl, DMAE; or strongly basic such

as quaternary amino ethyl, Q or trimethylammonium ethyl, TMAE or quaternary

aminoethyl, QAE) of the resin. This technique is used to remove process-related

impurities such as HCP, DNA, endotoxin and residual Protein A, and product-related

impurities such as aggregates. Also, AEX provides more than 4 log reduction for

viral clearance in general.9-10 Hydrophobic interaction chromatography (HIC) is a

useful tool for separating proteins according to their hydrophobicity and is

complementary to other techniques that separate proteins by charge, size or affinity.

Salt buffer interacts with water molecules to reduce the dissolution of protein

molecules, resulting in hydrophobic regions in protein molecules that bind to the HIC

resin. The basic principle of this chromatography technique is based on the salting-

out / salting-in mechanism.11 Ultrafiltration is a pressure-operated membrane process

commonly used for protein concentration and buffer exchange.12 Ultrafiltration is a

separation in which molecules larger than membrane pores are retained and smaller

ones pass freely. Separation is achieved by differences in membrane filtration rates

of different components under a certain pressure. Buffer exchange is carried out

using a diafiltration mode in which the buffer of the final desired composition is

added to the retentate system at the same rate at which the filtrate is removed, thereby

maintaining a constant retentate volume.13 The scale-up for manufacturing processes

is divided into a number of stages. Firstly, calculations are performed to determine

what small scale process be like if scaled-up linearly. Then, experimental stages are

performed in a laboratory scale to optimize the process. For chromatographic

operations, linear flow rate (cm/hr), resin bed height is the key for the scale-up

studies. Other studies should be performed for buffers, volume and membrane area

for filtration. Considering these process parameters optimized at laboratory scale, the

most suitable large-scale strategy is determined in pilot scale studies. After the pilot

scale studies, Good Manufacturing Practices (GMP) can be realized by making

improvements in full compliance with regulatory authorities.

Sayfa 37

References

1. Marichal-Gallardo, P. A., & Álvarez, M. M. (2012). State-of-the-art in

downstream processing of monoclonal antibodies: Process trends in design and

validation. Biotechnology Progress, 28(4), 899–916. doi: 10.1002/btpr.1567

2. J.V. Fiore, W.P. Olson, S.L. Holst, in: J. Curling (Ed.), Methods of Plasma

Protein Fractionation, Academic Press, New York, 1980, p. 239.

3. L.S. Hanna, P. Pine, G. Reuzinsky, S. Nigam, D.R. Omstead, Biopharm. Int.

(October) (1991) 33. R.L.

4. G. Blank, Recovery of Biological Products X Conference, Cancun, Mexico,

June, 2001. Keller, T. Friedmann, A. Boxman, Trends Biotechnol. 19 (11) (2001)

438.

5. Shukla, Abhinav A., et al. "Downstream processing of monoclonal antibodies—

application of platform approaches." Journal of Chromatography B 848.1 (2007):

28-39.

6. FDA Q5A Guidance Document: Viral Safety Evaluation of Biotechnology

Products Derived from Cell Lines of Human or Animal Origin, Federal Register,

vol. 63(185), 1998, p. 51074. Available at

http://www.fda.gov/cder/guidance/Q5A-fnl.PDF.

7. G. Sofer, Biopharm. Int. 16 (1) (2003) 50.

8. K. Brorson, S. Krejci, K. Lee, E. Hamilton, K. Stein, Y. Xu, Biotechnol. Bioeng.

82 (2003) 321

9. Curtis S, Lee K, Blank GS, Brorson K, Xu Y. Generic/ matrix evaluation of SV40

clearance by anion exchange chromatography in flow-through mode. Biotechnol

Bioeng 2003; 84:179-86.

10. Zhou JX. Development of future downstream process for commercial

monoclonal antibody production. Presentation at Asia bioLogic 2007; Beijing,

China

11. Porath J. Salt-promoted adsorption: recent developments. J Chromatog 1986;

376:331-41

12. R. van Reis, E. Goodrich, C. Yson, L. Frautschy, S. Dzengeleski, H. Lutz,

Biotechnol. Bioeng. 55 (1997) 737

13. vanReis R, Zydney AL. Protein ultrafiltration. in Encyclopedia of Bioprocess

Technology-Fermentation, Biocatalysis and Bioseparation. Flickinger MC,

Drew SW, Editors. John Wiley & Sons 1999; 2197-214.

Sayfa 38

Title:

Production of ScFv Antibody Fragments and Their Applications

Authors:

İlkay Koçer1, Dilek Şahinbaş2, Selen Cilasun2, Eda Çelik1,2

Affiliations:

1Department of Chemical Engineering, Hacettepe University, Beytepe, 06800

Ankara, Turkey

2Institute of Science, Bioengineering Division, Hacettepe University, Beytepe,

06800 Ankara, Turkey

Abstract:

Intact antibodies, soluble serum glycoproteins are highly specific targeting reagents

and are involved in the defence against pathogenic organisms and toxins. IgG is the

main serum antibody with a Y-shaped structure, divided into two separate domains:

antigen-binding region (Fab) and crystallizable (Fc) region. In addition to whole

structure antibodies, the individual protein fragments of antibodies can be isolated or

novel classes of antibody fragments can be generated to be used in biomedical

research and applications with the help of protein engineering tools. Single chain

variable fragments (scFvs), first engineered in 1988 (Bird et al.), are comprised of

the heavy (VH) and light (VL) chains of antibody’s variable domains that are joined

by a flexible peptide linker, which varies from 10 to 30 amino acids in length.

The scFv is the smallest active component of an antibody that shows specificity to a

target antigen and has a wide range of uses in medical, diagnostic and

biotechnological applications as a substitute for full-length antibodies (Holliger &

Hudson, 2005), which have an expected market value of about 250 billion USD by

2024 (Fu et al., 2018). Compared with the antibodies produced by hybridoma

technology, the scFv antibody fragments can be easily modified to improve

selectivity and affinity, and the production cost can be reduced (Wang et al., 2013).

Moreover, they penetrate tissues easier and have lower immunogenicity compared to

whole antibodies.

In literature, there are number of studies on the development and production of scFv

molecules using various bacteria and yeast systems (Frenzel et al., 2013). E. coli is

the most popular host for the production of scFvs due to its numerous advantages

including low cost, fast growth, easy genetic manipulation, high biomass with

scalable cultivation in bioreactors and scFv production levels upto 3.5 g/L (Joosten

et al., 2003; Gupta et al., 2017).

Sayfa 39

This review will provide an overall evaluation of the tremendous potential of

antibody fragments as robust diagnostic reagents or as nonimmunogenic

biopharmaceuticals, in addition to an overview of recombinant production schemes

especially towards their bioconjugation. Finally, our efforts towards bioprocess

development for highly soluble and functional recombinant anti-HER2-scFv

production in E. coli will be summarized.

Keywords:

Single chain variable fragment, Recombinant Escherichia coli, Theranostics

References:

Bird, R., Hardman, K., Jacobson, J., Johnson, S., Kaufman, B., Lee, S., Lee, T., Pope, S.,

Riordan, G., Whitlow, M. 1988. Single-chain antigen-binding proteins. Science,

242(4877), 423-426.

Frenzel, A., Hust, M., Schirrmann, T. 2013. Expression of recombinant antibodies. Front

Immunol, 4, 217.

Fu, R., L. Carroll, G. Yahioglu, E. O. Aboagye, and P. W. Miller. 2018. Antibody Fragment

and Affibody ImmunoPET Imaging Agents: Radiolabelling Strategies and Applications.

ChemMedChem 13 (23):2466-2478.

Gupta, S.K., Shukla, P. 2017. Microbial platform technology for recombinant antibody

fragment production: A review. Critical Reviews in Microbiology, 43(1), 31-42.

Holliger, P., Hudson, P.J. 2005. Engineered antibody fragments and the rise of single domains.

Nat Biotechnol, 23(9), 1126-36.

Joosten, V., Lokman, C., van den Hondel, C.A., Punt, P.J. 2003. The production of antibody

fragments and antibody fusion proteins by yeasts and filamentous fungi. Microbial Cell

Factories, 2, 1-1.

Wang, R., Xiang, S., Feng, Y., Srinivas, S., Zhang, Y., Lin, M., Wang, S. 2013. Engineering

production of functional scFv antibody in E. coli by co-expressing the molecule chaperone

Skp. Front Cell Infect Microbiol, 3, 72.

Sayfa 40

Microphysiological Systems in Theraphy

Ozlem Yesil-Celiktas

Department of Bioengineering, Faculty of Engineering, Ege University

35100, Bornova – Izmir / Turkey

One of the hurdles that has slowed down the development and approval of therapies

for neural disorders such as hereditary, neurodegenerative and neurodevelopmental

is the lack of preclinical models that can be utilized to identify molecular, cellular

and biophysical features of these diseases. This is due to the fact that the majority of

the in vitro neural models fail to fully recapitulate the local tissue and

microenvironment, thereby delimitating the understanding of complex

pathophysiology. On the other hand, current animal models also have limited

predictivity for drug efficacy in humans as the large majority of drugs fails in clinical

trials. Moreover, the difficulties in investigation of interactions between human

genetics and environmental factors lead to lack of knowledge about the mechanisms

that induce neural diseases. Microphysiological systems provide complex in vitro

human models that better emulate the physiology and function of the organ by

integrating various cell types and extracellular matrix components in a specific three-

dimensional (3D) configuration. Particularly, organoids derived from induced

pluripotent stem cells (iPSCs) can be differentiated into multiple neural cell types

and present a range of possibilities allowing cellular studies of individuals with

different genetic backgrounds. The aim of this paper is to highlight the recent

developments in microphysiological systems utilized as neural models to better

understand mechanisms of the disease as these systems can be novel tools in drug

development, toxicology and medicine.

Keywords: microphysiological systems; organ-on-chips; organoids, 3D cell culture;

disease modelling; theraphy

Introduction

Microphysiological systems that mimic in vivo physiology and tissue

microenvironment for pre-clinical evaluation of the efficacy and safety of potential

new drugs have become imperative for modelling various diseases, mainly

hereditary, infectious, neurodegenerative, neurodevelopmental and cancer. Three-

dimensional cell culture techniques that overcome the inadequacy of traditional two-

dimensional models are not only new tools in drug discovery, but also potential

models for the treatment of diseases. The discovery of multipotent stem and

progenitor cells provided the possibility to natively differentiate and self-organize

into defined structures under in vitro conditions, which has revolutionized the disease

modeling field (Tropepe et al., 2001). These defined structures resembling human

Sayfa 41

organs at varying levels of accuracy, have been referred as organoids, where

generation of complex cell types has become possible as well. Current omics

approaches have contributed to the increased characterization and understanding of

the complexity of human tissue types, which has led to more sophisticated culture

methods for generating organoids of varying tissue types. As these approaches are

refined and adapted, more organoid types are continually added, such as neural

organoids. Human induced pluripotent stem cells are important sources to form

organoid based models in vitro (Yan et al., 2018). The aim of this paper is to highlight

the recent developments in microphysiological systems utilized as neural models.

Fabrication and maintenance of neural organoids

Organoids are 3D multicellular tissue structures that can be widely self-organized by

pluripotent stem cell-derived cells and mimic the in vivo conditions in a physiological

and functional manner. As for neural organoids, the multicellular aggregates from

iPSCs, so-called embryoid bodies are encapsulated in a biomaterial mimicking

extracellular matrix for neuroepithelial expansion in a 3D architecture (Lancaster et

al., 2013). This extracelular matrix like biomaterial, which is in most of the cases is

Matrigel, not only sustains homeostasis but also enhances the diffusion of growth

factors and metabolites. Subsequently, the embryoid bodies are transferred into

spinning bioreactors or shakers for suspension and generation of neural organoids

(Vogel, 2013). While these conditions promote self-neuronal organization, they also

support the passage of oxygen and nutrient into the tissue. It has been noted that this

promotes the formation of larger neural organoids with fluid-filled cavities

resembling ventricles (Liu et al., 2019; Di Lullo and Kriegstein, 2017). Neural

organoid protocols are formulated differently to produce specific brain regions such

as the medial ganglionic region, cerebral cortex, cerebellum, midbrain, forebrain,

hypothalamus, hippocampus, and even cortical internerone) by adding various

factors (Wang, 2018). Cerebral organoids contain forebrain regulation centers that

identify hidden growth factors that may be located in the dorso-ventral pattern (Luo

et al., 2016). In addition to neuron cells with mature morphologies in its structure,

there are astrocytes and oligodendrocytes. It also enables modeling of later events

such as neuronal survival, maturation, and degeneration beyond the stage of brain

development, as they can be maintained for a long time (Eisenstein, 2018). As for in

vivo conditions, neural development is characterized by a dynamic process beginning

with early gestation and tissue morphogenesis and organogenesis are tightly

regulated by the stem cell microenvironment in the developing embryo, which

contains factors such as mechanical fluid flow and intrinsic or extrinsic biochemical

signals (Wang et al., 2018). Although stem cell-derived organoids recapitulate the

early developmental process of organs, differences from the native organs still exist,

which suggest that the microenvironment factors, such as biochemical, physical

signals and multicellular interactions are either lacking or presented incorrectly.

Organ-on-a-chip technology can fill this gap as these platforms recapitulate the

Sayfa 42

mechano-transduction effect on the cells, provide control over physiological stress,

chemical signaling, and cell-cell interactions, while reducing the consumption of

nutrients and ensuring cells are studied under physiological fluid flow conditions.

Neural organoids-on-a-chip

Microfluidic technology, among many popular tissue engineering approaches, brings

new insights in the study of both physiology and pathophysiology. Microfluidic

systems mimic dynamic physiological microenvironment as cells maintain their

original functions and respond to pathological changes as well as external stimuli,

thereby serving as versatile platforms for neuronal models (Yesil-Celiktas et al.,

2018). In a recent study, iPSCs-derived 3D brain organoids were generated using an

organ-on-a-chip system by incorporating Matrigel, fluid flow and multicellular

architectures of tissues that allowed in situ neural differentiation, and organization

of brain organoids on a single device. The generated brain organoids displayed well-

defined neural differentiation, regionalization and cortical organization under

perfused culture conditions. Moreover, the brain organoids exhibited an enhanced

expression of cortical layer markers under perfused cultures as compared to that

under static cultures, indicating the role of mechanical fluid flow in promoting brain

organogenesis (Wang et al., 2018). Similarly, spheroids also demonstrate some

characteristics such as expression of tight junctions, adherent junctions and cell

specific markers (Nzou et al., 2018). Another study was conducted using

neurospheroids and a brain-on-a-chip was fabricated to investigate the effect of

interstitial flow on the size of the neurospheroids, neural network, and cell

differentiation. Larger and more robust neurospheroids as well as complex neural

network were obtained when cultured under flow in comparison to the static

conditions, which was concluded to possess a positive effect of diffusion based flow

enabling continuous transport of oxygen, nutrients, and cytokines as well as

discharge of metabolic wastes (Park et al., 2014). In one of our on-going projects,

we are generating cerebral organoids, where maturation is planned in both spinning

bioreactors and a newly designed organ-on-chip platform. Organoids and organ-on-

chips are rapidly evolving in vitro platforms which hold great promise for the

modeling of neuronal development and disease.

Conclusions

When comparing techniques used to mimic neuronal tissue, microfluidic platforms

have more advantages. Organoid models combined with microfluidic platforms

provide more precise control of the microenvironment. Thus, they are relatively

useful to understand the physiology of human brain and mechanisms of

neurodegenerative diseases. Microfluidic platforms combined with organoids will be

effective models after specific design and optimization requirements are fulfilled. A

Sayfa 43

rapid growth is envisioned in this field providing new opportunities for personalized

medicine.

Acknowledgement

The financial support provided by TUBITAK through grant no 119M578 is highly

appreciated.

References

Akhtar, A.A., Sances, S., Barrett, R., Breunig, J.J. (2017). Organoid and organ-on-a-chip

systems : new paradigms for modeling neurological and gastrointestinal disease, Current

Stem Cell Reports, 3, 98-111.

Di Lullo, E., Kriegstein, A.R. (2017). The use of brain organoids to investigate neural

development and disease, Natural Review of Neuroscience, 18 (10): 573–584.

Eisenstein, M. (2018). Organoids: the body builders, Nature Methods, 15,19–22

Jorfi, M., D'Avanzo, C., Kim, D. Y., Irimia, D. (2018). Three-dimensional models of the human

brain development and diseases, Advanced Healthcare Materials, 7(1).

Lancaster, M.A., Renner, M., Martin, C.A., Wenzel, D., Bicknell, L.S., Hurles, M.E., Homfray,

T., Penninger, J.M., Jackson, A.P., Knoblich, J.A. (2013). Cerebral organoids model human

brain development and microcephaly, Nature, 501 (7467): 373-9.

Liu, F., Huang, J., Zhang, L., Chen, J., Zeng, Y., Tangaand, Y., Li, Z. (2019). Advances in

cerebral organoid systems and their application in disease modeling, Neuroscience, 399, 28-

38.

Luo, C., Lancaster, M. A., Castanon, R., Nery, J. R., Knoblich, J. A., & Ecker, J. R. (2016).

Cerebral organoids recapitulate epigenomic signatures of the human fetal brain, Cell Reports,

17(12), 3369-3384.

Nzou, G., Wicks, R. T., Wicks, E. E., Seale, S. A., Sane, C. H., Chen, A., . . . Atala, A. J. (2018).

Human cortex spheroid with a functional blood brain barrier for high-throughput

neurotoxicity screening and disease modeling, Scientific Reports, 8(1), 7413.

Park, J., Kim, S., Park, S.I., Choe, Y., Li, J., Han, A. (2014) A microchip for quantitative

analysis of CNS axon growth under localized biomolecular treatments, Journal of

Neuroscience Methods, 221,166-74.

Tropepe, V., Hitoshi, S., Sirard, C., Mak, T. W., Rossant, J., van der Kooy, D. (2001). Direct

neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell

stage acquired through a default mechanism, Neuron, 30(1), 65-78.

Vogel, G. (2013). Lab dishes up mini-brains, Science, 341(6149), 946-947.

Wang, H. (2018) Modeling neurological diseases with human brain organoids, Frontiers in

Synaptic Neuroscience, 10, 15.

Wang, Y., Wang, L., Guo, Y., Zhu, Y., Qin, J. (2018) Engineering stem cell-derived 3D brain

organoids in a perfusable organ-on-a-chip system, RSC Advances, 8(3), 1677-1685.

Yan, Y., Song, L., Madinya, J., Ma, T., & Li, Y. (2018). Derivation of cortical spheroids from

human ınduced pluripotent stem cells in a suspension bioreactor, Tissue Eng Part A, 24(5-6),

418-431.

Yesil-Celiktas, O., Hassan, S., Miri, A.K., Maharjan, S., Al-kharboosh, R., Quiñones-Hinojosa,

A., Zhang, Y.S. (2018) Mimicking human pathophysiology in organ-on-chip devices.

Advanced Biosystems, 2 (10) 1800109.

Sayfa 44

Monoklonal Antikorlar, Üretim Prosesleri ve İmmunoterapi Yaklaşımları

Monoclonal Antibodies, Production Processes and Immunotherapy

Applications

Prof Dr. S. İsmet DELILOĞLU-GÜRHAN*1

Prof. S. Ismet DELILOGLU-GÜRHAN, D.V.M.,Ph.D.*1

Doktor Öğretim Üyesi Sultan GÜLÇE İZ1,2

Assist Prof. Sultan GÜLÇE-IZ, Ph.D.1,2

1Ege Ünivesitesi, Mühendislik Fakültesi, Biyomühendislik Bölümü, İzmir, Türkiye 1Ege University, Engineering Faculty, Department of Bioengineering, Izmir, Turkey 2Eindhoven Teknoloji Üniversitesi, Biyomedikal Mühendisliği, BioInterface Science Lab, Eindhoven, Hollanda 2Technology University, Biomedical Engineering, Biointerface Science in Reg. Med. Eindhoven, NL

After the great invention of hybridoma technology by Köhler and Milstein at 1975, there have been breakthrough

innovations on the monoclonal antibody (Mabs) development and production up to date. Monoclonal antibodies

are declared to be the pioneers for the immunotherapeutic applications as they are highly specific compared to

unspecific surgery, radiotherapy and chemotherapy treatments. Thus, there is an increasing number of Mabs

marketed each year.

There are several methods of Mabs production. Classical hybridoma method which is known as Mabs from rodent

hybridomas combines the characteristics of both cell types, namely, the immortality of the myeloma cells and the

antibody secretion capability of the antibody secreting B cell. However, it was not successful for several reasons

such as the high immunogenicity of these murine proteins that leads to the induction of a human antimouse

antibody (HAMA). The elimination of mouse sequences in Mabs seems the only way for generation of therapeutic

Mabs. Alternative techniques were developed to find out the best therapeutic Mabs for humans. Chimeric

antibodies, humanized antibodies, fully human antibodies by using transgenic animals, phage display, yeast cell

display, ribosome display, mRNA display, human B cell hybridoma technologies.

Only a few micro- or milligrams of Mabs are required for analytical purposes, but rather gram or even sometimes

kilogram amounts are required for human and veterinary medical diagnostics, in agriculture and also in

therapeutic procedures in humans. Nowadays, large amounts of Mabs are produced essentially entirely in vitro.

Bioprocess development to operations with bioreactors following by downstream processing is one of the most

popular approaches in mass production of these valuable bio pharmaceutics.

Here, we will try to review the production processes of diagnostic and immunotherapeutic Mabs as well as future

expectations.

Sayfa 45

Importance of Recombinant Protein Antigen Production Bioprocess

Quality and its Implication on the Immune Response Elicited by

Vaccination

Mert Döşkaya1 1Ege University Faculty of Medicine, Vaccine Research and Development

Laboratory

Abstract:

The biotechnological vaccine antigen production process is a unique process that

uses the components of living cells to obtain the desired antigen in its pure form.

Each step of this process has utmost importance to obtain the desired immune

response. This process is divided into upstream and downstream processes. The

upstream part of the biotechnological vaccine production bioprocess is the

expression of the protein to be used as antigen in bacteria / yeast / insect / mammalian

cells by recombinant technology. The downstream part of the biotechnological

vaccine production bioprocess is the purification process of the expressed protein

either kept inside or secreted outside the host cell. The quality and amount of the

recombinant protein is assessed by various techniques such as ELISA, Western blot,

and Mass Spectroscopy. The recombinant protein which can be purified to

homogeneity or do not precipitate can have excellent immunostimulant properties.

On the other side, the epitopes of the protein are important and just before initiation

of the biotechnological vaccine antigen production process the epitopes of target

antigen should be analyzed using in silico and in vitro screening methods. We should

also keep in mind that 3D properties of these antigens and docking with Immune

globulins/MHC molecules should be analyzed in order to prevent immunogenic

inefficiency of recombinant protein. In this study, we analyzed the quality and

quantity of several antigenic recombinant proteins of Toxoplasma gondii, Breast

cancer, Foot-and-mouth disease virus and Crimean-Congo haemorrhagic fever virus

and their efficiency in stimulating immune response.

Keywords:

Recombinant protein; Vaccine; Bioprocess; Upstream; Downstream; Immune

response; Toxoplasma gondii; Foot-and-mouth disease virus; Crimean-Congo

haemorrhagic fever virus; Her2/Neu

Main Text:

Introduction

The biotechnological vaccine antigen production process is a unique process that

uses the components of living cells to obtain the desired antigen in its pure form.

Sayfa 46

Each step of this process has utmost importance to obtain the desired immune

response. This process is divided into upstream and downstream processes. The

upstream part of the biotechnological vaccine production bioprocess is the

expression of the protein to be used as antigen in bacteria / yeast / insect / mammalian

cells by recombinant technology. The upstream process covers all the stages used in

the development of the cell that will produce the antigen with recombinant

technology and the optimization of the production parameters. Upstream ends as the

cells reach sufficient density and collection of the host cells expressing the

recombinant protein (1).

The downstream part of the biotechnological vaccine production bioprocess is the

purification process of the expressed protein kept inside or secreted outside by the

bacterial / yeast / insect / mammalian cell. There are roughly three main processes in

this section; cell destruction / separation, purification and polishing. If we explain

these steps in more detail, first, after the release of the recombinant vaccine antigen

formed in the bacterial / yeast / mammalian cell by cell destruction, the

macromolecule residues and the medium are separated by centrifugation and

filtration processes; Separation of the antigen secreted in the yeast / mammalian cell

from the medium / producing pathogen from which it is produced by centrifuge-

ultracentrifuge can also be performed. If the vaccine antigen is secreted out of the

cell, then the medium is concentrated. As a result of these processes, a clear

supernatant is obtained. In the next step, the process of purification and polishing of

the target antigen to be used in the vaccine from the clear supernatant described

above initiates. During this process, various dialysis, concentration and column

chromatography approaches can be used according to the physico-chemical

properties of the antigen. The process ends with the 98-100% purification of the final

product (1).

The quality and amount of the recombinant protein is assessed by various techniques

such as ELISA, Western blot, and Mass Spectroscopy. The recombinant protein

which can be purified to homogeneity or do not precipitate may have excellent

immunostimulant properties. On the other side, the epitopes of the protein are

important and just before initiation of the biotechnological vaccine antigen

production process the epitopes of target antigen should be analyzed using in silico

and in vitro analyses methods. We should also keep in mind that 3D properties of

these antigens and docking with Immune globulins/MHC molecules should be

analyzed in order to prevent immunogenic inefficiency of recombinant protein

antigens. In this study, we aimed to analyze the quality of several antigenic

recombinant proteins of Toxoplasma gondii, Breast cancer (Her2/Neu), Foot-and-

mouth disease (FMD) virus and Crimean-Congo haemorrhagic fever (CCHF) virus

and their efficiency in stimulating immune response.

Materials and Methods

Sayfa 47

Antigenic recombinant proteins of T. gondii (rGRA1, rBAG1, rSporoSAG,

TGME49_047370_4, TGME49_026020_8, TGME49_053730_9,

TGME49_061020_40, TGME49_047390_4, TGME49_024710_6,

TGME49_104950_2, TGME49_086450_1, TGME49_021710_4,

TGME49_121520_3, TGME49_037880_1, TGME49_093730_6,

TGME49_015980_1, TGME49_001840_1, TGME49_039440_1,

TGME49_019310_8, TGME49_027280_1, TGME49_003310_1,

TGME49_048540_4, TGME49_100120_9, TGME49_095650_3,

TGME49_051630_2, TGME49_054720_1, TGME49_005360_12,

TGME49_058660_1, TGME49_034360_1, TGME49_113440_1,

TGME49_013390_6, TGME49_073130_2, TGME49_075490_12,

TGME49_114850_1, TGME49_047540_5, TGME49_110780_1,

TGME49_026110_8, TGME49_093000_2, TGME49_078100_1,

TGME49_092280_1, TGME49_026380_1, TGME49_090680_8,

TGME49_005360_17, TGME49_109590_1, TGME49_114500_1,

TGME49_055180_14, TGME49_055180_7, TGME49_025340_13,

TGME49_022370_1, TGME49_026510_1, TGME49_013390_5,

TGME49_095700_1) Breast cancer (Her2/Neu), FMD virus (synthetic protein

containing T and B cell epitopes), and CCHF virus (S, M and L segment) previously

analyzed by in silico and in vitro screening approaches and expressed by our study

group have been included to this study (2-12). We retrospectively analyzed the

quality and immunogenicity of these recombinant proteins as determined by the

Western blot and ELISA results and correlate them with immune responses elicited

by vaccinations or by natural infection in humans or animals models.

Results

The results showed that the in silico and in vitro analyzed as well as abundantly

expressed and successfully purified recombinant proteins of Toxoplasma gondii

(rGRA1, rBAG1, rROP6, DnaK family protein, a hypothetical protein, SRS30A,

ubiquitin carboxyl-terminal hydrolase, and plectin) and CCHF virus (S and M

segment) have high antigenic properties according to ELISA and Western blot results

conducted by serum samples obtained from vaccinated animals or naturally infected

humans or animals. These immunogenic properties are further supported by induced

protective T cell responses as determined by flow cytometry or extracellular cytokine

levels obtained from vaccinated animals.

Discussion

As shown by immunogenicity results, the quality and quantity of the recombinant

protein are very important to stimulate potent B cell and T cell responses. The quality

and in silico properties of the recombinant protein directly influences the potency of

the immune response. The quality of this response lies within the bioprocess

protocols applied during the upstream and downstream processes (1). During the

Sayfa 48

bioprocess steps, if the recombinant protein is not expressed abundantly or purified

to homogeneity, it may not be preferred for vaccine development.

Conclusions

Overall, the biotechnological vaccine antigen production process has utmost

importance to obtain the desired immune response. The in silico and in vitro

screenings as well as the steps taken under upstream and downstream processes have

direct influence on the quality and quantity of the recombinant protein to be used as

antigen in vaccine development.

References

1. Döşkaya M., Konvansiyonel ve Biyoteknolojik Aşı Antijen Üretimi

Biyoproseslerine Genel Bakış, AŞI Akademik, Endüstriyel ve Resmi Otorite

Yönüyle, Hipokrat Yayıncılık, Ankara, pp.61-72, 2019.

2. Döşkaya, M., Kalantari-Dehaghi, M., Walsh, C.M., Hiszczynska-Sawicka, E.,

Davies, D.H., Felgner, P.L., Larsen, L.S., Lathrop, R.H., Hatfield, G.W., Schulz,

J.R., Gürüz, Y., Jurnak, F. (2007). GRA1 protein vaccine confers better immune

response compared to codon-optimized GRA1 DNA vaccine. Vaccine,

25(10):1824-37.

3. Döşkaya, M., Caner, A., Değirmenci, A., Jurnak, F., Gürüz, Y. (2009).

Investigation of folding of purified recombinant GRA1 protein using web based

protein disorder servers and trypsin digestion. Protein and Peptide Letters.

16(7):834-41.

4. Liang L, Döskaya M, Juarez S, Caner A, Jasinskas A, Tan X, Hajagos BE,

Bradley PJ, Korkmaz M, Güruz Y, Felgner P, Davies DH. (2011). Identification

of potential serodiagnostic and subunit vaccine antigens by antibody profiling of

toxoplasmosis cases in Turkey. Mol Cell Proteomics, 10(7):M110.006916.

5. Polat C., İz S.G., Döşkaya M., Can H., Caner A., Değirmenci A., Balcan E.,

Gürüz Y. (2013). Comparison of Immune Responses Elicited by Adjuvanted

Tachyzoite Lysate Vaccines Developed from Two Different Toxoplasma gondii

Strains Isolated in Turkey. Mikrobiyol Bul, 47(1): 122-134.

6. Gülçe İz S, Döşkaya M., Borrego B, Rodriguez F, Gürüz Y, Gürhan ID. (2013).

Co-expression of the Bcl-xL antiapoptotic protein enhances the induction of Th1-

like immune responses in mice immunized with DNA vaccines encoding FMDV

B and T cell epitopes. Vet Res Commun, 37(3):187-96.

7. Felgner J., Juarez S., Hung C., Liang L., Jain A., Döskaya M., Felgner P.L., Caner

A., Gürüz Y., Davies D.H. (2015). Identification of Toxoplasma gondii antigens

associated with different types of infection by serum antibody profiling.

Parasitology, 142(6):827-38.

Sayfa 49

8. Döşkaya M., Caner A., Can H., Gülçe İz S., Gedik Y., Değirmenci Döşkaya A.,

Kalantari-Dehaghi M., Gürüz Y. (2014). Diagnostic value of a Rec-ELISA using

Toxoplasma gondii recombinant SporoSAG, BAG1, and GRA1 proteins in

murine models infected orally with tissue cysts and oocysts. PLoS One,

9(9):e108329.

9. Anıl M., Gülçe İz S., Metıner P.S., Sahar E.A., Can H., Zekioğlu O., et al.,

"Tumour forming efficiencies of TUBO cell line expressing Her2 in two different

mice strains with Matrigel matrix to develop animal models for novel

immunotherapies", EUROPEAN JOURNAL OF CANCER, vol.55, pp.S12-S12,

2016.

10. Döşkaya M., Liang L., Jain A., Can H., Gülçe İz S., Felgner P.L., Değirmenci

Döşkaya A., Davies D.H., Gürüz A.Y. (2018). Discovery of new Toxoplasma

gondii antigenic proteins using a high throughput protein microarray approach

screening sera of murine model infected orally with oocysts and tissue cysts.

Parasit Vectors. 11(1):393.

11. Can H, Erkunt Alak S, Köseoğlu AE, Döşkaya M, Ün C. (2019). Do Toxoplasma

gondii apicoplast proteins have antigenic potential? An in silico study. Comput

Biol Chem. 107158.

12. Atalay Şahar E., Can H., Gülçe İz S., Değirmenci Döşkaya A., Kalantarı-Dehaghi

M., Deveci R., Gürüz A.Y., Döşkaya M. (2020). Development of a hexavalent

recombinant protein vaccine adjuvanted with Montanide ISA 50V and

determination of its protective efficacy against acute toxoplasmosis. BMC Infect

Dis. Accepted for publication.

Sayfa 50

Recent Developments in Biotechnology and European Biotechnology

Network Association

Munis DUNDAR1*, Nilgun KARASU1 1Erciyes University, Faculty of Medicine, Department of Medical Genetics, Kayseri, Turkey

Country: Turkey

Emails: dundar@erciyes.edu.tr, nilgunkarasu@erciyes.edu.tr

* Correspondence: dundar@erciyes.edu.tr; Tel.: +905326486116

† Presented at the Bio Turkey 2020 International Biotechnology Congress, İstanbul/Turkey, 5-7 March 2020

Published: date

Abstract

Biotechnology enables the production of biosynthetic substances, processes or even organisms

with the DNA technology by combining the knowledge of natural sciences and other disciplines

such as engineering sciences, veterinary sciences and medicine. The progress of biotechnology

has accelerated rapidly in the last two decades with advancements in other scientific fields such

as fastly developing computer sciences which revolutionized the way of producing or analyzing

the human genome project data. Although media attention focuses on the impacts of

biotechnology in medicine and pharmacy, biotechnology had an impact in many other fields

such as agriculture or food industry. The EBTNA (European Biotechnology Thematic Network

Association) has long been recognized as a professional institution leading to innovation,

formation, knowledge, and education. The EBTNA is an extensive network of relationships

with representatives in 50 countries. The task of spreading, nurturing, and announcing all these

developments has fallen to EBTNA. From now on to the next decades, EBTNA’s

interdisciplinary and educational network will be much more needed for the development of

science without borders.

Keywords: Biotechnology, EBTNA, education, laboratory, network

Main Text

Biotechnology is, as its name implies, the common name given to technologies involving a

biological substance or process to produce an outcome or product that may or may not exist in

the nature by combining different fields of study such as biology, medicine, veterinary medicine

or engineering. If we consider biology as a bright white sunlight, each of the subfields of the

biotechnology can be a color of the rainbow spectrum, forming a harmonious mixture, a

methodology referred to as the 'Rainbow Methodology'. The Rainbow Methodology allows us

to reduce the complexity of all sub-areas correlated with biotechnology [1].

Completion of the Human Genome Project in 2003 lead to rapid improvements in many areas.

The most significant developments were in the field of biotechnology, which is defined as the

science of the 2000s. For example, the development of personalized medicine in the

pharmacogenetic field can be held responsible from the emergence of various new cancer and

Sayfa 51

biotechnological treatments. Even more, the idea of curing genetic diseases became a reality in

the societies’ minds [2]. Biotechnological drugs are also known as biopharmaceuticals and the

number of biotechnological molecules produced per year has increased significantly in the last

few years thanks to biotechnological advances [3]. Another important application of

biotechnology to medicine is the CRISPR/CAS9 genome editing method which was basically

stemmed from pure genetic engineering studies with lower organisms. In addition, gene therapy

in many diseases, for example, metabolic diseases, cardiovascular diseases, and monogenic

diseases have been recruited more than ever before in the recent years [4].

The gene therapy is treating a disease by correcting the aberrant gene’s function. This is

generally obtained by replacing the disease-causing incorrect gene with a "normal" copy. Put

another way, it is an empirical technique for repairing a defective gene that is responsible for

disease growth [5]. Improvements in genetic engineering methods (such as CRISPR/CAS9

systems for gene deletion and gene insertion) have provided ease in building biological circuits

to combine DNA fragments and control cellular behavior. On the other hand, synthetic

biologists can reunite the modular DNA parts by using engineering approaches and

computational models. Bioinformatics is a relatively new, indispensable tool in every area of

biotechnology when looked in another perspective [6]. Researchers specialized in

bioinformatics are needed for the processing, integration, and organization of big data obtained

from the human genome project [7]. Besides, with the rapid advances in computer science,

brain-controlled machines or human-like thinking software can be developed using computer-

based software. In this sense, artificial intelligence and machine learning have recently become

popular topics [8]. Also, subjects such as 3-D printers, NGS (Next-generation sequencing),

minimal genome, and liquid biopsy can be counted as other popular topics arising with

advances in biotechnology. As a powerful tool, NGS is revolutionizing the areas such as

personalized medicine, genetic diseases, and clinical diagnosis by offering a high throughput

option with the ability to line multiple people at the same time [9]. Liquid biopsy is another

non-invasive method that can provide many advantages. For instance, the phenotypic and

genotypic analyses can be now made with high specificity, low cost and real-time in a portable

system [10].

As the biotechnology is developing rapidly in many fields all over the world, the task of

spreading and announcing all these developments has become a major issue and this task was

undertaken by the EBTNA (European Biotechnology Thematic Network Association). The

EBTNA is an international institution established in 1996, operates in 50 countries throughout

the world. Since its establishment, it has been contributed to the both developments in

biotechnology and education of researchers as well as connecting scientists via academic,

industrial meetings or projects. The aims of this non-profit association are to unite together the

world of biotechnology as a ‘sunshine’ and increase the knowledge and skill capacity of

scientists (and more importantly students). Meanwhile, the EBTNA also to carries out

consultation, supervision, and implementation processes. For this purpose, it organizes

meetings that have become a tradition each year. The ODL (open distance education) modules,

the first one of which was created within the framework of the BIOTECHN8ET project, is one

the useful products of this association. In addition, the European Biotechnology School is

Sayfa 52

organized in collaboration with EBTNA (European Biotechnology Thematic Network

Association). Moreover, the EuroBiotech Project aims at implementing the development of

Biotechnological Industries by creating synergies between Institutions and Enterprises. Last but

not least, in order to form a universal language and to prevent misdiagnosis and

miscommunication among medical genetics laboratories, the quality assurance and EBTNA

application certification systems were implemented with collaboration of the EBTNA

members. Because the accreditation of the genetic laboratories against national and

international standards is important [11].

In conclusion, EBTNA is more crucial than ever in carrying out the task of educating, spreading

knowledge and maintaining a universal language of quality.

References

[1] De la Vega I, Requena J, Fernández-Gómez R. The colors of biotechnology in

Venezuela: A bibliometric analysis. Technol Soc 2015;42:123–34.

https://doi.org/10.1016/j.techsoc.2015.03.007.

[2] Wang F. The Once and Future of Medicine. Sci Insights 2018;2018:1–7.

https://doi.org/10.15354/si.18.re016.

[3] Luz H, Carneiro AV, Cabrita A, Carrera F, Frazão JM, Macário F. Position statement of

the Portuguese Society of Nephrology on the clinical use of biotechnological drugs in

renal patients 2009;23:317–21.

[4] Lino CA, Harper JC, Carney JP, Timlin JA. Delivering crispr: A review of the challenges

and approaches. Drug Deliv 2018;25:1234–57.

https://doi.org/10.1080/10717544.2018.1474964.

[5] Gonçalves GAR, Paiva R de MA. Gene therapy: advances, challenges and perspectives.

Einstein (Sao Paulo) 2017;15:369–75. https://doi.org/10.1590/S1679-

45082017RB4024.

[6] McCarty NS, Ledesma-Amaro R. Synthetic Biology Tools to Engineer Microbial

Communities for Biotechnology. Trends Biotechnol 2019;37:181–97.

https://doi.org/10.1016/j.tibtech.2018.11.002.

[7] Oliveira AL. Biotechnology, Big Data and Artificial Intelligence. Biotechnol J

2019;14:1–6. https://doi.org/10.1002/biot.201800613.

[8] Miller DD, Brown EW. Artificial Intelligence in Medical Practice: The Question to the

Answer? Am J Med 2018;131:129–33. https://doi.org/10.1016/j.amjmed.2017.10.035.

[9] Smith HS, Swint JM, Lalani SR, Yamal JM, de Oliveira Otto MC, Castellanos S, et al.

Clinical Application of Genome and Exome Sequencing as a Diagnostic Tool for

Pediatric Patients: a Scoping Review of the Literature. Genet Med 2019;21:3–16.

https://doi.org/10.1038/s41436-018-0024-6.

[10] Li X, Ye M, Zhang W, Tan D, Jaffrezic-Renault N, Yang X, et al. Liquid biopsy of

Sayfa 53

circulating tumor DNA and biosensor applications. Biosens Bioelectron 2019;126:596–

607. https://doi.org/10.1016/j.bios.2018.11.037.

[11] Precone V, Dundar M, Beccari T, Turanli ET, Cecchin S, Marceddu G, et al. Quality

assurance of genetic laboratories and the EBTNA practice certification, a simple

standardization assurance system for a laboratory network. EuroBiotech J 2018;2:215–

22. https://doi.org/10.2478/ebtj-2018-0052.

Sayfa 54

Title:

Antibody Engineering for the Development of Therapeutic Antibodies

Authors:

Berrin ERDAG,Assoc,Prof.

Affiliations:

TÜBITAK Marmara Research Centre Genetic Engineering and Biotechnology

Institute

Baris Mah. Dr. Zeki Acar Cad. No:1 P.K. 21 41470 Gebze Kocaeli ; Turkiye

T +90 262 677 2000 – 3345 F +90 262 641 2309

www.mam.gov.tr berrin.erdag@tubitak.gov.tr

Abstract:

Over the last four decades, monoclonal antibodies have made a dramatic

transformation from scientific tools to powerful human therapeutics. At present,

approximately 90 therapeutic monoclonal antibodies are marketed in the United

States and Europe. In this presentation, I draw attention to how antibody engineering

has revolutionized drug discovery and what are considered the key areas for future

development in the monoclonal antibody therapy field.

Keywords: Therapeutic antibodies, Monoclonal antibody, Humanized and human

antibodies Antibody engineering, , recombinant antibody fragments , Phage display,

, scFv

Main Text:

Therapeutic antibodies represent one of the fastest growing areas of the

pharmaceutical industry. Antibodies can swiftly provide therapeutics to target the

disease-related molecules that have been discovered in genomic research because 1)

the high level of specificity and affinity to the target molecule or antigen achieves a

high level of efficacy and fewer adverse events, 2) their ability to target diverse

molecules and the modes of action of the antibodies allow them to be applied to a

wide range of therapeutic targets, and 3) modification and refinement by genetic

engineering technology and the establishment of recombinant manufacturing

technology has made industrial manufacturing possible. The efficacy of therapeutic

antibodies stems from various natural functions of antibodies — neutralization,

antibody-dependent cell-mediated cytotoxic (ADCC) activity, or complement-

dependent cytotoxic (CDC) activity —, or the antibody can be utilized as a drug

Sayfa 55

delivery carrier. Development of therapeutic antibodies boomed in the 1980’s, and

the first therapeutic antibody, a mouse antibody, was launched in 1986 as an

immunosuppressive agent used during organ transplantation Georges Köhler and

César Milstein invented a means of cloning individual antibodies, thus opening up

the way for tremendous advances in the fields of cell biology and clinical diagnostics

. However, in spite of their early promise, monoclonal antibodies (MAbs) were

largely unsuccessful as therapeutic reagents resulting from insufficient activation of

human effector functions and immune reactions against proteins of murine origin.

These problems have recently been overcome to a large extent using antibody

engineering techniques to produce chimeric mouse/human and completely human

antibodies. In chimeric antibodies, 33% of the structure originates from mouse, with

variable regions from mouse and constant regions from human, and in humanized

antibodies, up to 90% of the structure originates from human, with only the antigen

binding site in the variable region (complementarity-determining region) originating

from mouse. Such an approach is particularly suitable because of the domain

structure of the antibody molecule where functional domains carrying antigen-

binding activities (Fabs or Fvs) or effector functions (Fc) can be exchanged between

antibodies . On the basis of sequence variation, the residues in the variable domains

(V-region) are assigned either to the hypervariable complementarity-determining

regions (CDR) or to framework regions (FR). It is possible to replace much of the

rodent-derived sequence of an antibody with sequences derived from human

immunoglobulins without loss of function. Furthermore, genetically truncated

versions of the antibody may be produced ranging in size from the smallest

antigenbinding unit or Fv through Fab' to F(ab')2 fragments. More recently it has

become possible to obtain totally human antibodies from human antibody phage

libraries and transgenic mice bearing human immunoglobulin loci.

During tle last 25 years in vitro technologies opened powerful routes to combine the

generation of large libraries together with fast selection procedures to identify lead

candidates. One of the them is based on the use filamentous phages. Antibodies

fragments can be displayed successfully on the surface of phage by fusing the coding

sequence of the antibody variable (V) regions to the phage minor coat protein pIII.

By creating large libraries, antibodies with affinities comparable to those obtained

using traditional hybridomas technology can be selected by a series of cycles of

selection on antigen. As in this system (Phage display technology) antibody genes

are cloned simultaneously with selection they can be easily further engineered for

example by increasing their affinity, modulating their effector function or their

specificity.

Characterization of immunoglobulins and technological improvements have resulted

in the engineering of antibodies to produce protein therapeutics with

unique properties, both biological and biophysical, that are leading to novel

Sayfa 56

therapeutic approaches. Antibody engineering includes the introduction of the

antibody combining site (variable regions) into a host of architectures including bi

and multi-specific formats that further impact the therapeutic properties leading to

further advantages and successes in patient treatment. This presentation will be

focused on the structural and functional characteristics of antibody and the antibody

engineering for the generation and optimization of therapeutic antibodies.

Sayfa 57

ELECTROCHEMICAL NUCLEIC ACID BIOSENSORS WITH

RECENT APPLICATIONS

Arzum Erdem Gürsan *

Ege University, Faculty of Pharmacy, Analytical Chemistry Department

35100 Bornova, Izmir, TURKEY

*arzum.erdem@ege.edu.tr and arzume@hotmail.com

After the discovery of electroactivity in nucleic acids in 1960, many electrochemical

methods were developed for the quantification of nucleic acids. There has been a growing

interest for the development of electrochemical nucleic acid biosensors since 1995.

Herein, an overview to electrochemical nucleic acid biosensors has been presented with

their recent applications on detection of spesific biomolecular recognitions such as, sequence-

selective nucleic acid hybridization and DNA interactions etc.

Keywords: Electrochemical nucleic acid biosensors; Electrochemical genosensors;

Aptasensors

Acknowledgements. A.E acknowledges the financial support from Turkish Scientific and

Technological Council (TÜBİTAK Project no.111T073, 114Z400 and 115Z099), and she also

would like to express her gratitude to the Turkish Academy of Sciences (TÜBA) as the principal

member for its partial support.

Sayfa 58

Ongoing Biotechnology Applications in CERN Osman EROGUL

Department of Biomedical Engineering, TOBB University of Economics and

Technology,

Ankara, Turkey,

erogul@etu.edu.tr

Abstract

European Organization for Nuclear Research (CERN) is a scientific organization based in Geneva,

Switzerland that studies mainly on particle physics. CERN transfers know-how and technology to other

fields and thus maximizes the societal impact of the Laboratory’s research. One of the integral part of

CERN is medical applications focused on R&D projects, using technologies and infrastructures that are

uniquely available at CERN. The CERN-Member States KT (knowledge-transfer) Forum is arranged

every year in order to discuss medical applications-related activities and facilitate planned initiatives for

the transfer of CERN technologies for the purpose of medical applications. The content of high-impact

medical projects includes radioactive isotopes for medical diagnostics, radiobiology, nuclear physics,

instrumentation for dosimetry, accelerator design for future hadron therapy facilities, simulation for

health applications (modeling the effects of radiation on biological tissues, big-data management,

analysis for personalized medicine), medical accelerators, medical imaging and applications of high-

field superconducting magnets. The aim of CERN-MEDICIS project is to develop non-conventional

radioisotopes for medical research. More than 1000 isotopes of 70 chemical elements have been

produced in the scope of this project. New radiopharmaceuticals have also been developed for therapy

such as Xofigo which has been approved by the FDA (Food Drug Administration) and in Europe for

castration resistant prostate cancer with metastasis. In this presentation, biotechnologies developed in

CERN are explained briefly and collaboration opportunities are discussed in details.

Keywords: CERN, medical technologies, healthcare, collaboration, medical physics

Sayfa 59

The Clinical Trial Methods Used in the Development of

Medical Devices

Hakkı Zafer Güney

Gazi University Medical School Department of Pharmacology

The lecture will be about the clinical trial methods used in the development of

medical devices. Beyond the methods, the approaches of the national and the

international authorities will also be discussed. The development of medical devices

is a very important process which takes a long time and intense efforts. Beyond

regulatory process, the quality assurance is also a very important process.

Keyword 1; Medical device keyword 2; Clinical Trial keyword 3: Methods

Today, biomedical professionals, field service and other medical personnel must

meet increasing regulatory guidelines, higher quality standards, and rapid

technological growth while performing their work faster and more efficiently than

ever.

There are several international fairs which take place in different parts of the world

every year. In Turkey, the research and development of medical devices is not so

common however in order to be more active in the field the research and

development of medical devices is a very important issue which has to be taken into

consideration seriously.

Medical devices are classified based on their intended use, invasiveness, duration

of use, and the risks and potential harms associated with their use. Adhesive

bandages, crutches and tongue depressors are class 1 agents with a minimal risk.

They do not need clinical trials. Hypodermic needles, Gauze dressings and TENS

devices are class IIa agents and the risk is low to moderate. Lung ventilators,

Insulin pens and diagnostic X-rays are class IIb agents with a moderate to high

risk. Heart valves, spinal & vascular stents are class III agents with a high risk.

Pacemakers, spinal cord stimulators and defibrillators are examples of Active

Implantable Medical Devices (AIMD) with a high risk.

The medical devices which are class I do not need clinical trials. Class Iı a and IIb

agents may need clinical trials while class III agents and AIMD devices do need

clinical trials.

Sayfa 60

Biotechnology in Health: From Molecules to Engineered Systems

Nesrin Hasırcı

Near East University (NEU), Lefkosa, TRNC, Mersin-10, Turkey

Middle East Technical University (METU), Chemistry Department,

METU BIOMATEN - Center of Excellence in Biomaterials and Tissue

Engineering, Ankara, Turkey

Abstract:

Biomaterials are made of atoms and molecules as every material. The important point

is the biomaterial created by the arrangement of the atoms and molecules should have

high biocompatibility with the living system without causing any adverse effect. We

work with biocompatible polymers and prepare their composites to enhance their

bioactivity in the desired direction. For tissue engineering applications, we prepared

fibrous and porous scaffolds by using electrospinning, wet-spinning or 3D printing

techniques. 3D printing technology gained high importance in medical device

production since it becomes possible to create ‘patient specific’ systems with a

predetermined shape and property. In this talk, different systems prepared for soft

and hard tissue engineering purposes will be summarized.

Keywords:

Biomaterial; tissue engineering; artificial organs; tissue models; organ mimics

Introduction:

Life is based on carbon containing molecules. Although there are many other

elements in our body, the essential life molecules as DNA, RNA, enzymes or

hormones are mostly made of carbon together with hydrogen, oxygen, nitrogen,

phosphorus and others. Scientists are trying to develop novel molecules and materials

in order to treat the damaged or non-functioning parts of the patients, and

developments in science, biology, nanotechnology and material engineering are

creating promising solutions to health problems. One of the well-organized hard

tissues is bone which contains 32% organic material mostly collagen and cells.

Nanocrystals of hydroxyapatite (a calcium phosphate composition) are placed among

the collagen fibrils. Although we cannot make an exactly similar and functional bone

tissue in the lab, we can prepare some mimics and some supporting devices to be

used in orthopedic treatments. In the cases when the bone defect is large and has

irregular shape, the treatment approaches, are not very successful due to the type and

the shape of the implant material. Scientists trying are to find the best materials and

bioactive agents which would have high biocompatibility and will enhance the tissue

regeneration. In the recent years, ‘patient specific treatments’ became important in

Sayfa 61

medicine and 3D printing technology helped to the production of devices which can

be prepared according to the need of a patient.

Materials and Methods:

In tissue engineering systems, polymers and polymeric composites are taking the

place of the metals. In our studies, the materials used are either synthetic

(polycaprolactone (PCL), polyurethane (PU), etc.) or natural (collagen, chitosan,

etc.) polymers. As bioactive agents, antibiotics (for antibacterial surfaces), heparin

(for anticoagulant surfaces), growth factors (for tissue regeneration) and

hydroxyapatite (HAp) (for enhancing bone bioactivity) were used. In some cases,

cells were added on the scaffolds to examine their cell-compatibility. Materials were

prepared by solvent casting (for films), freeze drying (for porous sponges),

electrospun or wet spun techniques (for fibrous scaffolds) or 3D printing technic (for

scaffolds with predetermined design). Characterization studies were achieved via

chemical (NMR, FTIR), physical (porosity, density, topography), mechanical

(tension, compression, bending), biological (in vitro cell culture) and preclinical (in

vivo animal experiments with rats or rabbits).

Results and Discussion:

The materials used in our studies were mostly synthetic or natural polymers and they

were biocompatible. Many of them were already approved by authorities. The

analyses carried out according to the standards approved that the materials had no

toxic, allergic or immunogenic reactions. Especially, the 3D printed bone supporting

scaffolds prepared from PCL, modified with polypropylene fumarate (PPF) and

HAp, and also having bone marrow stem cells (BMSC) demonstrated excellent bone

fusion (observed by micro-computed tomography (μ-CT) analysis) and even better

mechanical strength compared to the natural bone (determined by 4-point bending

analysis) after 8 weeks of implantation on rabbit femurs. In these studies, cylindrical

PCL scaffolds (10 mm diameter 1.5 mm height for characterization and in vitro

studies; and 5 mm diameter 2.5 mm height for in vivo studies) were produced by 3D

printing, coated with HAp and a second coating with PPF was applied. Biological

studies after seeding with BMSCs showed enhanced cell adhesion for PCL/HAp

scaffolds, and the cell proliferation rate was higher on PCL/HAp/PPF scaffolds

compared to that in the others. For all samples in vitro and in vivo biocompatibility

tests were carried out and none of the scaffolds had any cytotoxicity, irritation or

inflammation.

Conclusions:

It can be concluded that the scaffolds prepared by 3D printing technique using PCL

and modified with HAp and PPF have great potential as regenerative materials for

bone tissue engineering applications. Developments in science and technology will

lead to well-designed medical implants and devices which would perfectly fit the

Sayfa 62

host area, will have high biocompatibility, and prepared in a patient specific way.

The most important point is to establish the path needed to carry the materials and

devices from lab to bed side. Universities, hospitals, industry and governmental

authorizes should build this path.

Sayfa 63

New Scaffold Production Techniques and Tissue Models in Tissue Engineering

Vasif Hasirci, Acibadem University, Department of Medical Engineering, Atasehir 34752, Istanbul TURKEY, and BIOMATEN, METU Center of Excellence in Biomaterials and Tissue Engineering, Ankara TURKEY Abstract: Tissue engineering one of the main components is the scaffold. Developments in this field lead to new, significant opportunities in obtaining products with exceptional properties and also to new testing environments. In this presentation, some of the newer scaffold production and testing methods will be discussed. Keywords: Scaffold, in vitro testing, tissue models Main Text: In the production of a tissue engineered product scaffolds (or cell carriers), stem and mature cells and bioactive agents such as growth factors are needed. With the developments in these fields production of new tissues become possible. Among them the most interesting is the additive manufacturing. With this approach products with carefully designed inner and exterior architecture can be printed especially for patient specific applications which eliminates the need for fitting of the implants to defects. The products in this field moved gradually from hard and stiff materials to gel-like soft products. Another exciting development in the field is the attempts made to replace in vivo (animal) experiments with tissue mimics or models. One of the most important developments in this line are the cancer models where instead of establishing the disease in a rodent followed by treatment protocols this can now be mimicked on material and cell based models which are becoming alternatives to animal use. The major development in this direction happened with realization of 3D culture media represent the biological environment much better than 2D culture plates.

Sayfa 64

Title:

Intellectual Property of Biotechnological Drugs

Authors:

Ayşe Göksu KAYA ÖZSAN1

Affiliations: 1 Turkish Patent and Trademark Office, Ankara, TURKEY

Abstract:

Patents, the main intellectual property rights for biopharmaceuticals, are crucial for

the safeguard of the biopharmaceutical industry’s development and innovation in the

future. According to the Turkish Law No. 6769 on Industrial Property, products, in

particular substances or compositions, for use in the treatment of the human body

can be protected by patents. Pharmaceuticals and biotechnology are important

industrial sectors and they progress leadingly in the world. The statistics also show

the relative specialization of Turkey in biopharmaceuticals and the road ahead in

Turkey, indicating the importance of the field.

Keywords:

Intellectual property (IP); patent; patentability; biopharmaceuticals; biotechnology;

pharmaceuticals

Main Text:

Creations of the mind are protected by intellectual property (IP) rights. Inventions,

literary and artistic works, designs, symbols, names and images used in commerce

are protected by patents, copyright and trademarks, in order to enable people to earn

recognition or financial benefit from what they invent or create. During the

development of a drug, the inventor can request a patent for his/her invention to

exclude others from making, using, distributing, selling and importing the invention

for a limited period of years (generally 20 years from the filing date of the

application), in exchange for revealing the invention by public disclosure.

The Turkish Law No. 6769 on Industrial Property covers several industrial property

matters including patents. Article 82 of the Law is related to the patentable inventions

and exceptions to patentability. According to the article, discoveries shall not be

considered as inventions, consequently mere discoveries stay out of patentability.

Also, a patent cannot be granted to the inventions of;

- “plant or animal varieties or biological processes for the production of plants

or animals with the exception of microbiological processes or products

obtained in the result of such processes; where “microbiological process”

Sayfa 65

means any process involving or performed upon or resulting in

microbiological material and “biological process” means any completely

natural plant or animal production procedures such as hybridization or

selection”;

- “methods for treatment of the human or animal body by surgery or therapy

and diagnostic methods practiced on the human or animal body with the

exception of products, in particular substances or compositions, for use in any

of these methods”;

- “discovering one of the elements of human body including the human body, a

gene sequence or a partial gene sequence at the various stages of its formation

and development”;

- “the human cloning processes, the changing processes of genetic identity of

human sextinked inheritance, using human embryos for industrial or

commercial purposes, changing processes of genetic identity in a way that

may agonize the animals without providing any significant medical avails for

human or animals and animals that are obtained in the result of such

operations”.

Turkey is a member of European Patent Convention (EPC) and more than half of the

applications made to Turkish Patent and Trademark Office are coming through the

EPC route. According to EPC, any substance or composition comprised in the state

of the art shall not exclude the patentability, provided that its use for any treatment

method is not comprised in the state of the art. As a result, specific molecules

(product), manufacturing processes, effects of the molecules on body (medical

indication), combination of products, dosage forms, dosage regimens, patient groups,

routes of administration, modified compounds (e.g. polymorphs) etc. can all be

protected by a patent. Accordingly, a single drug can be protected by a large number

of separate patents, which are related to a different invention.

Theoretically, discoveries are not patentable under the EPC. “However, if a

substance found in nature can be shown to produce a technical effect, it may be

patentable.” A substance occurring in nature which is found to have an antibiotic

effect; a microorganism discovered to exist in nature and to produce an antibiotic; a

gene which is discovered to exist in nature to use in making a certain polypeptide or

in gene therapy are examples of patentable inventions1. Thus, a biological material

which is isolated from its natural environment or produced by means of a technical

process, susceptible of industrial application, including the sequence or partial

sequence of a gene, may constitute a patentable invention, even if the structure of

that element is identical to that of a natural element. The industrial application of a

1 European Patent Office; “Guidelines for Examination in the European Patent Office”,

Part G, Chapter II, 3.1, 2019

Sayfa 66

sequence or partial sequence must be disclosed in the patent application as filed2.

Without indication of a function, a mere nucleic acid sequence cannot be patented3.

The sequence shall specify which protein or part of a protein is produced and what

function this protein or part of a protein performs. Otherwise, when a protein or part

of a protein is not produced, the function shall be indicated4.

Patents for pharmaceutical products constitute 55% of the patents in biotechnology

at the EPO. Annual Report 2018 of the EPO reveals that, pharmaceuticals was the

top 7th and biotechnology was the 8th technological field in 2018, by nearly 8% of all

patent applications filed. With chemical engineering and control instruments, they

constitute the top four technological fields having the highest increase in application

numbers5. Although biopharmaceuticals have a part in our life more than 30 years,

the increase of their dignity continues.

The leading country in biopharmaceutical patent applications published up to 2017

is the United States of America (USA). One third of the applications is granted,

which indicates the highest number of granted patents having country in this

technical area6. In a study in 2015 on FDA-approved biologics new medical entities,

first-identifiable patents were collected with a total number of 1374. Approximately

one quarter of all biologics-based medicines were invented by academic institutions

(including government laboratories) in the 80s, where almost three quarters were

from pharmaceutical companies. Although, biotechnology and pharmaceutical

companies share roughly the same number of patents in 2015, biotechnology

companies have increasingly displaced academic institutions and pharmaceutical

companies over time7.

For an indication of the level of invention in biopharmaceuticals, the Relative

Specialization Index (RSI) of 30 biggest applicant countries and other G20 countries

were calculated within the published biopharmaceutical patent application dataset in

2007-2016. The USA, Japan and China are the top three applicant countries and

appear relatively specialized in the field of biopharmaceuticals, but this is now

reversed when the RSI is applied as these countries rank below several others

including Denmark, Argentina and Australia. These three high-ranking countries,

show much greater levels of patenting in biopharmaceuticals than expected. The

2 European Patent Office; “Guidelines for Examination in the European Patent Office”,

Part G, Chapter II, 5.2, 2019 3 EU Dir. 98/44/EC, rec. 23

4 European Patent Office; “Guidelines for Examination in the European Patent Office”,

Part G, Chapter III, 4, 2019

5 European Patent Office; “Annual Report 2018”, 2020

6 Kaya Özsan; “Biopharmaceuticals and Patent”, 2017

7 Kinch and Raffo; “Sources of Biopharmaceutical Innovation: An Assessment of

Intellectual Property”, 2015

Sayfa 67

leading country in biopharmaceuticals patent applications, the USA, ranked 13th.

Turkey ranked 30th with a relative specialization on biopharmaceuticals8.

The complexity of biopharmaceutical molecules causes a change in the pattern of

innovation. In chemical pharmaceutical industry, after regulatory approval, patents

are filed very rare. The innovation increases after years by the generic competition

loom. Because of the intensive effort required in biopharmaceutical industry for

scaling up from laboratory volumes to largescale production, the pattern is different.

The scaling up process for the phase III clinical trial stage and the market demand

after the launch create a high rate of innovation. The innovation has two peaks at that

terms, before and after drug approval, without any stopover9.

In conclusion, having a portion of 17.2% in prescribed medicines in Turkey,

biopharmaceuticals, is gaining a more important place in both global and national

industry10. Global biopharma industry is driven by scientific knowledge rather than

manufacturing know-how. In order to prevent the products to be reproduced, industry

needs to develop IP policies, management and strategies. Patents are the main IP

right for the biopharmaceutical industry and should be earned and managed properly.

8 Kaya Özsan; “Biopharmaceuticals and Patent”, 2017 9 Lim and Suh; “Product and Process Innovation in the Development Cycle of

Biopharmaceuticals”, 2015

10 İEİS; “Türkiye İlaç Pazarı”, 2019

Sayfa 68

Title:

Stability of Biotechnological Medicinal Products

Authors:

Kamer Kılınç

Affiliations:

TOBB ETU Faculty of Medicine, Depatment of Biochemistry

Abstract:

Biopharmaceuticals, also called ‘biological medicinal products’ or ‘biological

medicines’, are medicines produced by a living organism by means of recombinant DNA

technology. Unlike traditional chemical drugs, biopharmaceuticals with their protein or

glycoprotein structure are complex molecules. The structure of the final biopharmaceutical

and eventually its efficacy can be influenced at every stage of production, formulation,

transport and storage. Any chemical and physical changes in the structure of the protein form

the basis of the instability problem of biopharmaceuticals.

Since biopharmaceuticals are produced via a host cell (e.g., bacteria, yeast, or

mammalian, insect, or plant cell lines), some amount of non-product, host cell-derived

material will inevitably be introduced into the process stream. This process results in a

mixture of the desired product and host cell derived impurities, including host cell proteins

(HCPs), and other process-related impurities. The primary concern with HCPs in

biopharmaceutical products is their potential to induce anti-HCP antibodies that could induce

a clinical effect in patients. HCPs may possibly act as adjuvants, which can induce anti-drug

antibodies that can affect the safety or efficacy of the drug. HCPs with hydrolytic activity,

even in minute quantities, can cleave the desired protein product over time, reducing or

eliminating biological potency or altering stability.

The purity of biopharmaceuticals does not guarantee their stability. Purified proteins

may lose their structure and efficacy by undergoing chemical and physical changes over time.

Causes of protein instabilities can be divided into two general classes: chemical instability and

physical instability. Chemical instabilities involve processes that produce a new chemical

group or break covalent bonds in proteins, generating new chemical entities (e.g.,

rasemization, deamidation, isomerization, N-terminal pyroglutamat formation, oxidation,

glycation). Conversely, there are physical instabilities for proteins in which the chemical

composition is unaltered, but the physical state of the protein does change. This includes

denaturation, aggregation, precipitation, and adsorption. Physical instabilities decrease the

solubility and availibility of active protein concentration. Since proteins can be modified

easily during manufacturing and storage, some protective strategies are necessary to improve

stability of biopharmaceuticals.

Keywords:

1. Stability of biotechnological medicinal products 2. Chemical and physical basis

Sayfa 69

Biyoteknoloji Ürünü Protein İlaçların Stabilitesi

Kamer Kılınç

TOBB ETÜ Tıp Fakültesi, Biyokimya Anabilim Dalı

Özet

'Biyolojik tıbbi ürünler' veya 'biyolojik ilaçlar' olarak da adlandırılan biyoteknoloji

ürünü protein ilaçlar, rekombinant DNA teknolojisi ile canlı bir organizmada üretilen

ilaçlardır. Protein veya glikoprotein yapısına sahip olan biyofarmasötikler, geleneksel

kimyasal ilaçların aksine, karmaşık yapıda ve stabil olmayan moleküllerdir. Ürün haline gelmiş

biyofarmasötiklerin yapısı ve nihayetinde etkinliği, üretim, formülasyon, taşıma ve

depolamanın her aşamasında etkilenebilir. Proteinin yapısındaki herhangi bir kimyasal

ve/veya fiziksel değişiklik, biyofarmasötiklerin instabilite probleminin temelini oluşturur.

Biyofarmasötikler bir konakçı hücre (örn., bakteri, maya veya memeli) aracılığı ile

üretildiğinden, bir miktar ürün dışı, konakçı hücre kaynaklı proteinler de kaçınılmaz olarak

ortama dahil olacaktır. Saflaştırma sürecinde bazı konakçı hücre proteinleri (HCP'ler)

tamamen uzaklaştırılamayabilir ve hedef ürünle birlikte bulunabilir. Biyofarmasötik

ürünlerdeki HCP'ler, hastalarda klinik bir etki oluşturabilecek anti-HCP antikorlarını

indükleme potansiyeline sahiptir. HCP'ler adjuvan gibi davranarak hastada anti-ilaç

antikorlarının sentezini indükleyebilirler ve bu nedenle ilacın etkinliğini baskılayabilirler.

Hidrolitik aktiviteye sahip HCP'ler, çok düşük miktarlarında bile, protein ürünü zaman içinde

parçalayabilir, biyolojik etkinliğini azaltabilir veya ortadan kaldırabilir ya da stabilitesini

etkileyebilirler.

Biyofarmasötiklerin tamamıyla saf olmaları da stabilitelerini garanti etmez.

Saflaştırılmış proteinler, zaman içinde kimyasal ve fiziksel değişikliklere uğrayarak yapılarını

ve neticede etkinliklerini kaybedebilirler. Protein instabilitelerinin nedenleri iki genel sınıfa

ayrılabilir: kimyasal instabilite ve fiziksel instabilite. Kimyasal instabilite, protein üzerinde

yeni bir kimyasal grup oluşturan veya proteinlerde bulunan kovalent bağları kırarak yapı

değişimine neden olan tepkimelerden (örn. Rasemizasyon, deamidasyon, izomerizasyon, N-

terminal piroglutamat oluşumu, oksidasyon, glikasyon) kaynaklanır. Fiziksel instabilitede ise

proteinin kimyasal bileşiminde değişiklik olmaz. Ortamın fiziksel kompozisyonu ve/veya

kimyasal içeriğinin değişmesi sonucu proteinin fiziksel durumu değişir. Fiziksel değişim

denatürasyon, agregasyon, presipitasyon ve adsorpsiyon şeklinde gözlemlenir. Fiziksel

instabilite, aktif protein konsantrasyonunu, çözünürlüğünü ve elde edilebilirliğini azaltır.

Proteinler üretim, transport ve depolama süreçlerinde kolayca bozunabildiğinden,

biyofarmasötiklerin stabilitesini temin etmek üzere koruyucu stratejiler gereklidir.

Anahtar kelimeler

1. Stability of biotechnological medicinal products 2. Chemical and physical basis

Sayfa 70

Transmission-Blocking Vaccine Candidate Against Malaria

Tarlan Mamedov1, Kader Cicek1, Kazutoyo Miura 2, Burcu Gulec1 & Gulnara

Hasanova1

1Akdeniz University, Department of Agricultural Biotechnology, Dumlupınar

Boulevard 07058 Campus, Antalya, Turkey

2Laboratory of Malaria and Vector Research, National Institute of Allergy and

Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway,

Rockville, MD, USA

This work describes the development of transmission blocking (TB) vaccine against

malaria using plant transient expression system. Pfs48/45 is one of the leading

candidates for TB vaccine development and has been shown to play an essential role

in parasite fertilization. Over the past 20 years, attempts have been made to express

the full-length Pfs48/45 antigen using several systems. However, efficient and

conformationally-correct expression of full-length Pfs48/45 was problematic (no or

low expression, poor solubility, did not elicit TB antibodies in mice, etc.). In this

study, we demonstrate for the first time that the full length, Endo H in vivo enzymatic

deglycosylated Pfs48/45 antigen is produced at a high level in plants and is

structurally stable at elevated temperatures. Moreover, sera from mice immunized

with this antigen showed strong inhibition in standard membrane-feeding assay

(SMFA). Thus, Endo H in vivo enzymatic deglycosylated, full-length Pfs48/45

antigen is a promising candidate for the development of an affordable TB vaccine,

which may have the potential to save millions life.

Keywords:

malaria, TB vaccine, Pfs48/45 antigen, plant expression system

Introduction

Malaria is one of the most common infectious diseases caused by protozoan

parasites of the genus Plasmodium and transmitted by the female Anopheles

mosquito. According to the latest World Malaria Report, there were 238 million

cases of malaria in 2018 with 405 000 deaths and nearly half of the world’s

population was at risk of malaria. Although there have been many decades of

effort, malaria remains the leading cause of morbidity and mortality among the

human population globally and no vaccine is currently available that provides a

satisfactory level of protection against malaria. Pfs48/45 is one of the leading

candidates for TB vaccine development and has been shown to play an essential

Sayfa 71

role in parasite fertilization. Since Pfs48/45 is a complex protein and has seven

potential N-linked glycosylation sites and contains 16 cysteines, which are involved

in disulfide bond formation, therefore, the production of correctly folded

recombinant Pfs48/45 has been the main obstacle to the development of a

Pfs48/45-based vaccine. In fact, over the past 20 years the production of full-length

Pfs48/45 antigen has been attempted using a number of available expression

systems. However, those efforts were unsuccessful and efficient and

conformationally-correct expression of full-length Pfs48/45 was problematic (no or

low expression, poor solubility, did not elicit TB antibodies in mice, etc.) in used

expression platforms.

In recent years, numerous studies have demonstrated the plant transient

expression systems as a promising expression platform with high expression

capacity, which provide safe, cost-effective production of a variety of biologically

active proteins in a relatively short period of time. Plant transient expression system

has a number of advantages including safety, rapid, high scalability and high

production capacity and have the ability to accumulate hundreds of milligrams of

target protein per kilogram of biomass in less than a week. However, the ability of

the plant expression systems (all eukaryotic expression systems) to glycosylate

proteins limits this system for the production of some proteins, for example, a wide

range of bacterial proteins, malaria antigens, and also some human proteins, which

are particularly important for pharmaceuticals. At this point, in previous efforts,

there were attempts to produce a full-length Pfs48/45 protein in N. benthamiana

plant, but the resulting plant produced antigen did not show TB activity. We

hypothesized that it could be due to aberrant N-glycosylation of Pfs48/45 protein

when expressed in eukaryotic expression systems including plants. Native Pfs48/45

proteins of P. falciparum do not carry N-linked glycans, but contain potential N-

linked glycosylation sites, therefore, are aberrantly glycosylated during expression

in most of eukaryotic systems, including plants. To address this need, we developed

a robust strategy for the production of proteins in a non-glycosylated form, by in

vivo co-expression of target proteins with the bacterial deglycosylation enzyme,

Endo H (Mamedov et al., PLoS One, 2017). Using this strategy, a full-length

Pfs48/45 antigen was produced in N. benthamiana plant, and purified protein was

tested for functional activity. We demonstrate that the full-length, Endo H in vivo

enzymatic deglycosylated Pfs48/45 antigen is produced at a high level in plants and

is structurally stable at elevated temperatures. Importantly, sera from mice

immunized with this full-length Pfs48/45 antigen showed strong inhibition in

SMFA.

Materials and Methods

Sayfa 72

Cloning of Endo H, Pfs48/45 genes, and co-expression of Endo H with Pfs48/45

antigen, purification of deglycosylated form of full-length Pfs48/45 from N.

benthamiana plant leaves and stability analysis of Pfs48/45 were performed as

described previously (Mamedov et al., PLoS One, 2017; Mamedov et al., Scientific

Reports, 2019). The standardized methodology for performing the SMFA was

performed as described elsewhere (Mamedov et al., Scientific Reports, 2019).

Results

Recombinant full-length Pfs48/45protein was produced in N. benthamiana plant

and purified as decried previously ((Mamedov et al., PLoS One, 2017; Mamedov et

al., Scientific Reports, 2019). The purification yield of Endo H deglycosylated,

full-length Pfs48/45 protein, was about ~52 mg/kg of leaf biomass. The stability of

plant produced in vivo deglycosylated form of Pfs48/45 was examined after

incubation at 37°C for a longer period of time: 48, 96, and 144 hours. Based on

these results it was confirmed that plant produced, deglycosylated full-length

Pfs48/45 protein was structurally stable and recognized by the conformational

specific Pfs48/45 antibody, when incubated at elevated temperature. For functional

analysis, serum samples from mice immunized with the full-length Pfs48/45

antigen, were collected and used for SMFA. Deglycosylated Pfs48/45 IgG showed

the strongest activity in SMFA. The results demonstrate that deglycosylation of the

Pfs48/45 protein was required to induce functional antibodies.

Discussion

Plasmodium falciparum is one of the most deadly parasites in human history and

therefore, the development of a safe, low-cost and highly efficient malaria vaccine

with long-term stability is urgently needed. It has been demonstrated that the

Pfs48/45 antigen is one of the leading candidates for TB. However, since the full-

length Pfs48/45 protein is complex cysteine rich protein, and, therefore, its proper

folding depends on the correct formation of disulfide bridges. At this point, it has

been challenging to achieve high yield production, stable, and functionally active

full-length Pfs48/45 using different expression systems. Plant transient expression

system becomes one of the promising expression platforms for a variety of

recombinant proteins and has a number of advantages including safety, rapid, high

production capacity and high scalability. However, the ability of the plant

expression systems to glycosylate proteins limits this system for the production of

some proteins, for example, a wide range of bacterial proteins, malaria antigens,

and also some human proteins, which are particularly important for

pharmaceuticals. For example, a plant produced full-length glycosylated Pfs48/45

protein that was expressed in N. benthamiana had no transmission blocking (TB)

activity. In the current study, a non-glycosylated full-length Pfs48/45 of P.

Sayfa 73

falciparum was produced in N. benthamiana plant by in vivo co-expression with

bacterial Endo H enzyme, and the purified proteins were injected into mice to

generate antisera for immunogenicity and SMFA analysis. We demonstrate for the

first time that the full-length, Endo H in vivo enzymatic deglycosylated Pfs48/45

antigen is produced at a high level in N. benthamiana plants and is structurally

stable at elevated temperatures for a prolonged period and sera from mice

immunized with this plant produced malaria antigen showed strong inhibition

activity in SMFA. It should be noted that up to date, the full-length recombinant

Pfs48/45, which was produced using different expression systems did not show

strong inhibition in SMFA. Thus, this is the first report demonstrating the

production of a functionally active recombinant full-length Pfs48/45 antigen.

Conclusion

Overall all the above findings demonstrate that the plant produced, Endo H in vivo

deglycosylated, full-length Pfs48/45 malaria antigen is the most promising

candidate for the development of an affordable, safe, stable, and full-length

Pfs48/45-based TB malaria vaccine, and therefore, may have the potential to save

millions. All of these features support further characterization, formulation, pre-

clinical testing and finally early stage clinical development of plant produced Endo

H deglycosylated full-length deglycosylated Pfs48/45.

Sayfa 74

Magnetic Nanoparticles for More Effective Cancer Treatment

Ahmet Mavi

Department of Nanoscience and Nanoengineering, Atatürk University, Erzurum,

Turkey

Abstract: Magnetic nanoparticles (MNPs) have been used for the dozens of applications such as

labeling of cells, enzyme immobilization, hyperthermia, targeted delivery of molecules such

as drugs, genes and antibodies. Surface modifications are made to increase the stability of the

particles and make them biocompatible. Silica coating to MNPs, which is more resistant to

adverse conditions such as mechanical stress, high temperature and hydrolysis induced

degradation compared to polymers, is the most widely used agent in this field. Mesoporous

silica nanoparticles (MSPs) are nanotechnology-based drug delivery systems that are used in

several application areas such as labeling, separation of molecules and tissue-specific release

of therapeutic agents such as drugs, DNA, RNA, oligonucleotides, proteins or enzymes.

Biocompatible MSPs exhibit excellent colloidal stability, minimize undesired protein

adsorption on the surface and have high drug loading capacity with high surface area (>700

m2/g) and pore volume (> 1 cm3/g).

Keywords:

Magnetic nanoparticle, Magnetic Hyperthermia, Cancer

Main Text

Nanomaterials often have atomic dimensions (from 1 to 100 nanometers in at least one

dimension) and therefore exhibit physical, chemical, optical or electronic properties that

differ from the bulk form of the same material. Nanomaterials are classified as their

dimensions like spherical (nanoparticles, NPs), tubes or films. They have been produced by

physical, chemical or biological methods. In biology and medicine, a variety of nanomaterials

such as gold NPs, magnetic NPs, carbon and boron nitride nanotubes have been used for

therapy of any disease, labelling and tracking of cells/molecules. Small size of nanomaterials

give opportunity to reach the cells even subcellular locations. Nanomaterials can be used for

delivery of targeted drug or gene after appropriate surface modifications. NPs can also be

modified for multifunctional purposes.

Multifunctional NPs allow multiple treatments at the same time, such as chemotherapy,

photothermal therapy and hyperthermia, while facilitating the addition of fluorescent labels to

monitor nanomaterials in the body.

Iron oxides are the most widely used magnetic nanoparticles (MNPs) because of their

cheapness, high magnetization properties (Figure 1) and low toxicity. MNPs have been used

in various applications such as drug and gene delivery, magnetic resonance imaging and

magnetic hyperthermia. Different shape and size of MNPs have been produced in small sizes

up to 4 nm by diverse methods such as co-precipitation, chemical vapor deposition,

hydrothermal or thermal decomposition.

Sayfa 75

Figure 1 Direction of MNPs with a magnetic field

To functionalize the MNPs, organic or inorganic coating processes are applied. Gold, silica,

polymers such as polyethyleneimine and nucleic acids are preferred as material for coating or

surface modification. The coated MNPs do not lose their magnetic properties. Surface of

MNPs can also be modified by ligand molecules which are specific to targeted cells or tissue.

Surface modifications are also made to increase the stability of the particles and make them

biocompatible. Changing surface chemistry adds extra chemical and physical properties to

NPs. For example, silica, which is more resistant to adverse conditions such as mechanical

stress, high temperature, hydrolysis induced degradation compared to polymers, is the most

widely used agent in this field [1].

Mesoporous silica has advantages such as narrow pore size distribution, adjustable pore

diameter, very large surface area and pore volume [2], [3]. Since it has both internal and

external surface areas, it allows the addition of different functional groups to the surface,

which increases the use of mesoporous silica coated materials [3].

Recently, mesoporous silica coated MNPs (MNP@mSi) (Figure 2) have been used for

multifunctional nanomaterials. These particles can carry drug (Figure 3), gene in the pores

and also generate heat by magnetic hyperthermia (MHT). MHT has been paid attention

because of the opportunity of destroying localized or deep-seated tumors with reduced side

effects [4].

Sayfa 76

Figure 2 Image of MNP@mSi

Figure 3 Rout from synthesis to application

MHT is based on the magnetic particles releasing heat due to magnetic hysteresis under the

high frequency AC (Alternative Current) external magnetic field (Figure 4). The advantage of

this method is that the magnetic field has high penetration in living tissues. MHT application

heats tissues to temperatures above 42 °C where the cancer growth stops while healthy cells

can be preserved [4]. Cancer cells are more sensitive to heat more than normal cells. For this

reason, MHT is a method that has been intensively researched in cancer treatment in recent

years.

Figure 4 Magnetic hyperthermia

MHT can also be used to activate heat inducible promoter regions of genes. Recombinant

DNAs with heat shock protein (HSP) promotors can be transfected by MNPs to targeted cells

and thus gene expression can be achieved by MHT. This strategy can provide controlled

production of therapeutic proteins with reduced clinical side effect in cancerous tissues.

References

AMF Generator

Magnetic

Hyperthermia

Heat

Release

Sayfa 77

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Chuan, “Magnetic iron oxide nanoparticles : Synthesis and surface coating techniques for

biomedical applications,” Chin. Phys. B Vol., vol. 23, no. 3, pp. 037503-1-037503–19, 2014.

[2] Z. A. ALOthman, “A Review : Fundamental Aspects of Silicate,” Materials (Basel)., vol.

5, no. 2874–2902, 2012.

[3] A. Liberman, N. Mendez, W. C. Trogler, and A. C. Kummel, “Synthesis and surface

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pp. 132–158, 2014.

[4] C . E. Demirci Donmez, P. K. Manna, R. Nickel, S.Aktürk and Johan van Lierop,

"Comparative Heating Efficiency of Cobalt-, Manganese-, and Nickel-Ferrite Nanoparticles

for a Hyperthermia Agent in Biomedicines” ACS Appl. Mater. Interfaces, vol. 11, pp.

6858−6866, 2019,

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Biyolojik /Biyoteknolojik İlacların Analizi: Analitik Validasyon

Emirhan Nemutlu

Hacettepe Üniversitesi Eczacılık Fakültesi Analitik Kimya Bölümü

Sıhhiye Ankara, 06100 enemutlu@hacettepe.edu.tr

Biyofarmasötik ürünler, küresel ilaç pazarında hızlı bir büyüme oranını sahiptirler.

Pazardaki biyofarmasötik ürünlerin sayısı her yıl, özellikle yenilikçi ürünlerin patent

korumasının sona ermesi nedeniyle artan biyobenzerler sayısına bağlı olarak

artmaktadır. Düşük araştırma ve geliştirme maliyetleri nedeniyle biyobenzerler

yenilikçi ürüne uygun maliyetli bir alternatif sunmak ve bu avantajlarından dolayı

ilaç pazardaki en çekici ürün haline getirmektedir. Öte yandan, biyobenzerlerin

güvenlik, saflık, aktivite ve etki gücü açısından çok kapsamlı bir karakterizasyonu

yapılmalıdır. Bu nedenle, biyobenzerler de dahil olmak üzere biyofarmasötiklerin

fizikokimyasal karakterizasyonu, çoklu, tamamlayıcı ve aynı zamanda ortogonal ve

en gelişmiş analitik yöntemler kullanılarak yapılmalıdır. Seçilen karakterizasyon

yöntemleri, birincil amino asit sekansını (örn., Kesilme, deamidasyon, oksidasyon),

çevrim sonrası modifikasyonları (örn., Glikosilasyon) daha yüksek dereceli yapısal

değişiklikleri (örn., İkincil katlama, agregasyon) ve safsızlıkları kapsamalıdır.

Karakterizasyon çalışmaları dışında bitmiş ürün ve hammaddelerin kontrollerinin

yapılası da gerekmektedir. Tüm bu analizlerde kullanılan çok farklı analitik

tekniklerin yöntem validasyonu, doğrulama ve yöntem transferi çalışmaları da

yapılaması gerekenler ve dikkat edilmesi gereken kısımlar tartışılacaktır. Ayrıca ICH

ile uyum çalışmaları içinde getirilecek yeni düzenlemeler ile ilgili gelişmeler de

aktarılacak ve CTD uygulamasındaki yaygın hatalar ve yanlış validasyon çalışmları

da incelenecektir. Son olarak, analitik yöntem transferi çalışmaları da tartışılacaktır.

Anahtar Kelimeler: Analitik yöntem validasyonu, Farmakope, ICH,

Biyofarmasötik

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Title:

Recombinant Vaccines against Infectious Diseases

Author:

Sezer Okay

Affiliation:

Department of Vaccine Technology, Vaccine Institute, Hacettepe University, Ankara

Abstract:

Vaccines are biological preparations that contain the whole or a part of the pathogen

to make the immune system recognize the disease agent, so easily fight with it when

infected. Attenuated live or inactivated cells of the pathogen, or its subunits like

toxin, DNA, proteins etc. can be used for vaccine development. Recombinant

vaccines may include the live organisms/viruses, the pathogenic ones attenuated via

mutation of virulence gene(s), and their subunits such as DNA or proteins obtained

using recombinant DNA techniques. Recombinant antigens can be obtained from

bacteria, plants, mammalian or insect cells. Next-generation vaccine platforms take

the advantage of nanotechnology and reverse vaccinology and utilize nanoparticles

and short peptides targeting immune cells to obtain nanovaccines. These

technologies aim to increase the vaccine efficacy and safety.

Keywords: DNA vaccine; edible vaccine; nanovaccines; recombinant protein;

reverse vaccinology

1. Importance of vaccination

Vaccines are biological preparations saving lives against deadly diseases. High

vaccination rates protect individuals as well as the community preventing the spread

of the disease to non-vaccinated or vaccinated but vulnerable individuals, thus the

outbreaks of the infectious diseases are prevented as well. World Health

Organization (WHO) reported 67% and 76% decreases in the mortality rates of

measles and pertussis, respectively in between 1980-1990 due to increase in

vaccination against these diseases from 20% to 75% [1]. Unfortunately, near-

eradication of a disease may lower the vaccination rates, and WHO reported a 300%

global increase in measles in the first three months of 2019, compared to the same

time last year. Vaccination also reduces the costs of health services by preventing

diseases.

2. Strategies for vaccine development

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At the end of the 18th century, the term “vaccine” was first introduced by Edward

Jenner for the cowpox-infected materials to immunize against smallpox. A century

later, Louis Pasteur invoked the original scientific strategy behind vaccinology as

isolate, inactivate, and inject. Conventional attenuated or inactivated vaccines are

obtained using this strategy. However, 150 years after Pasteur’s invention, today

next-generation vaccines are obtained utilizing novel technologies such as reverse

vaccinology, bioinformatics, nanotechnology, genetic engineering, and synthetic

biology.

3. Recombinant vaccines

There are different types of recombinant vaccines involving attenuated live cells as

well as their subunits such as toxin, DNA or proteins. Recombinant attenuated viral

vectors are used to carry the pieces of a viral or bacterial pathogen to host cell. The

viral vectors can be replication-competent which have high risk of being revertant

and recombination with wild type viruses or replication–incompetent (partially

replicating) with low risk of being revertant and recombination [2]. Virus-like

particles (VLPs), self-assembling viral proteins lacking nucleic acids or lipids, can

also be used in vaccine development.

Other than viruses, live recombinant bacteria are also used as vectors in vaccine

development. The pathogenic ones among these bacteria possess several mutations

and lack virulence. Salmonella enterica serovars typhi and typhimurium, Listeria

monocytogenes, lactic acid bacteria (LAB) such as Lactococcus lactis, Pseudomonas

aeruginosa, Bacillus subtilis, and Mycobacterium smegmatis are among the bacteria

used as vectors [3].

3.1. DNA vaccines

Some types of vaccines involve DNA sequences from pathogens, especially the

genes encoding for immunogenic proteins. In this type of vaccines, the gene of

interest is cloned into an expression vector such as pCMV or pVAX1 and introduced

into Escherichia coli. The recombinant bacteria carrying desired vector are selected

according to the antibiotic resistance provided by the plasmid. Recombinant

plasmids are recovered from bacteria using endotoxin-free plasmid isolation kits, and

the expression of gene of interest is verified on mammalian cells. Later, the plasmids

are used for vaccination of mice to show immunogenicity pre-clinically [4].

3.2. Protein-based recombinant vaccines

There are high number of studies on the development of recombinant vaccines using

antigenic proteins and peptides belonging to viral or bacterial pathogens. The best

known example is the recombinant vaccine against hepatitis B virus (Figure 1). In

this vaccine, the gene encoding HBsAg protein is cloned to a plasmid vector, and

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introduced into a yeast cell. The recombinant yeast is cultivated in a bioreactor while

producing HBsAg inside. Then, the yeast cells are disrupted and the antigen is

purified. The purified HBsAg protein is formulated with adjuvant to obtain

commercial vaccine.

Figure 1 Representation of the production of recombinant hepatitis B vaccine

schematically [modified from 5].

The basic principles in the development of recombinant protein-based vaccines are

(I) cloning of the gene(s) encoding antigen(s) to a vector, plasmid or viral, (II)

introduction of the recombinant vector into a host cell, (III) overproduction of the

protein, (IV) purification of the protein from the host, and (V) preparation of the

vaccine formulation using recombinant protein and generally an adjuvant [6].

The host organisms for the production of recombinant proteins are diverse. A wide

range of microorganism including bacteria and yeast cells are used for protein

overproduction. In some strategies, recombinant proteins are obtained via

mammalian, insect or plant cell cultures. Edible vaccines are also generated by the

expression of the genes of pathogens in plants or insect larvae. The recombinant

plasmids are introduced into plants via Agrobacterium tumefaciens transformation

or gene gun techniques, and into insect larvae via baculovirus expression system.

The edible vaccines are directly used for immunization without a need for protein

purification.

3.3. Nanovaccines

A recent technology in vaccine development is utilization of nanoparticles (NPs) for

delivery of the antigens to immune cells. Sizes of the NPs are similar to those of

pathogens; therefore, immune system recognizes them resulting in stimulation of

cellular and humoral immune responses. Additionally, stability of the nanovaccines

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in blood flow is well enough to remain in blood, as well as booster doses and cold

chain requirements are not needed [7]. Various polymer-based NPs have been used

for nanovaccine development, and the most widely used one is polylactide-co-

glycolide (PLGA) copolymer [8]. These NPs carry proteins or short peptides, and

according to the epitopes they carry, humoral and/or cellular immune responses are

induced.

3.4. Reverse vaccinology

The omics technologies and bioinformatics are powerful tools for the development

of vaccines. Omics studies provide us a whole picture of a cell and we can analyze

the each component using bioinformatic tools. Reverse vaccinology is a

methodology to screen the components of the cell to capture potential antigens and

search for epitopes. Thus, the number of proteins to investigate their potency is

reduced [9]. For instance, 2158 candidates were predicted in the whole genome of

Neisseria meningitidis serogroup B (MenB), 600 of them were predicted as surface

exposed by in silico analyses. Then, 350 proteins were produced and injected to mice,

and 91 of them were found to be surface exposed. Next, 28 proteins were identified

as inducing immune responses, and 3 antigens were selected for vaccine

development [10].

References

[1] who.int/news-room/campaigns/world-immunization-week/world-immunization-

week-2019/vaccines-and-the-power-to-protect

[2] Sakurai A, Ogawa T, Matsumoto J, et al. Regulatory aspects of quality and safety

for live recombinant viral vaccines against infectious diseases in Japan. Vaccine.

2019;37(43):6573-6579.

[3] da Silva AJ, Zangirolami TC, Novo-Mansur MT, Giordano Rde C, Martins EA.

Live bacterial vaccine vectors: an overview. Braz J Microbiol. 2015;45(4):1117-29.

[4] Okay S, Ozcengiz E, Ozcengiz G. Immune responses against chimeric DNA and

protein vaccines composed of plpEN-OmpH and PlpEC-OmpH from Pasteurella

multocida A:3 in mice. Acta Microbiol Immunol Hung. 2012; 59(4):485-98.

[5] https://www.eduhk.hk/biotech/eng/classrm/class_health5.html

[6] Okay S, Çetin R, Karabulut F, Doğan C, Sürücüoğlu S, Kızıldoğan AK. Immune

responses elicited by the recombinant Erp, HspR, LppX, MmaA4, and OmpA

proteins from Mycobacterium tuberculosis in mice. Acta Microbiol Immunol Hung.

2019;66(2):219-234.

[7] Gheibi Hayat SM, Darroudi M. Nanovaccine: A novel approach in immunization.

J Cell Physiol. 2019;234(8):12530-12536.

[8] Luo M, Samandi LZ, Wang Z, Chen ZJ, Gao J. Synthetic nanovaccines for

immunotherapy. J Control Release. 2017;263:200-210.

[9] Sette A, Rappuoli R. Reverse vaccinology: developing vaccines in the era of

genomics. Immunity. 2010;33(4):530-41.

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[10] Masignani V, Pizza M, Moxon ER. The development of a vaccine against

meningococcus B using reverse vaccinology. Front Immunol. 2019;10:751.

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Preparation of Gene Therapy Drugs withPlasmid DNA Delivery Systems

Asst.Prof. Suna ÖZBAŞ

Marmara University, Faculty of Pharmacy, Department of Pharmaceutical

Biotechnology, Istanbul/Turkey

Keywords: Gene Therapy, Plasmid DNA, Non-viral Gene Delivery Systems,

Gene Drugs

In the recent years, medicine verge to eliminate the cause of disease rather than treat

the symptoms and so genetic material (DNA and RNA molecules) gain importance.

Gene therapy, modification of genetic material in viable cells or transfer the genetic

material into the cells, is an alternative approach to treat different types of diseases

as well as genetic disorders. First studies to administrate the genetic material to the

patient for therapeutic protein production in the cells started with development of

molecular biology at 1960s and carried into practice with first clinical gene therapy

at 1990s. Nowadays, gene therapy is a fast growing field and a promising therapy

method for most diseases. Even though cancer types are the most common diseases

for the gene therapy strategies, there are also many studies to treat monogenic genetic

disorders, infectious diseases and vascular diseases.

Nucleic-acid based pharmaceutical products can control progression of disease with

induction or inhibition of genes. Nucleic acid-based therapeutics include i) transgene

plasmids, ii) antisense and antigen oligonucleotides (gene silencers), iii) ribozyme,

iv) DNazyme v) aptamers, vi) small interfering RNA and micro RNA (siRNA and

miRNA), vii) genome editing molecules (i.e., ZFNs, TALENs, CRISPRs). Even

most of these molecules are still at clinical trial phase, especially antisense

oligonucleotides, aptamers, siRNAs and gene modified cellular therapy products

have a great potential to treat serious diseases like AIDS, cardiovascular and

neurological diseases. After Vitravene®, the first drug of gene therapy, was

approved in 1998 by FDA, number of approved nucleic acid-based pharmaceutical

products/gene drugs are increased until today. Gendicine®, Macugen®, Oncorine®,

Rexin-G®, Neovasculgen®, Glybera®, Kynamro®, Imlygic®, Zalmoxis®,

Spinraza®, Strimvelis®, Exondys51®, Invossa®, Defitelio®, Yescarta®,

Kymriah®, Luxturna®, Tegsedi®, Onpattro®, Collategene®, Waylivra®,

Zynteigo®, Zolgensma® are approved gene drugs since 1998 to 2020.

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Major advantage of nucleic-acid based drugs to conventional drugs is to be specific

to targets and pathways but it is hard to transfer genes to the cells directly. Thus, gene

delivery systems are important to develop a successful gene drug. Gene delivery

systems are classified as; viral and non-viral delivery systems. Viral vectors can

obtain long term, stable and high level gene expression but they have crucial

disadvantages like low transgene capacity, high immunogenicity, risk of

pathogenicity, mutagenicity and carcinogenicity and difficulties of production and

purification processes.

Their high transgene capacity, low immunogenicity, ease of production and

purification processes, low cost and scalability of production make non-viral delivery

systems good candidates for DNA and gene delivery studies even though they have

lower transfection efficiency than viral vectors.

Plasmids, a non-viral delivery system, are high molecular weighted, double stranded,

poly-anionic extrachromosomal DNAs that contain gene, coding specific protein. In

molecular perspective, plasmids are thought as pro-drugs that provide transfer of

DNA to the cell for biosynthesis of therapeutic molecule (protein) after transcription

and translation. A plasmid gene delivery vector consist of protein coded therapeutic

gene and regulator components that control gene expression. Gene delivery system

design and regulatory component choice are very important for successful gene

expression. Especially, promoters and enhancers as regulatory sites have curial effect

on gene expression levels, therefore tissue- or tumor-specific promoters can be used

for higher efficiency.

Beside the studies to improve properties of existing delivery vector there are many

new approaches like viral/non-viral hybrid vectors, targeting, using of tissue-specific

promoters and modification of cell receptors. It is important to design a suitable

vector for the success of gene therapy.

In the future, scientist in pharmaceutical field will play a key role to determine

efficiency of gene transfer with design of gene delivery systems. First generation

gene drugs, as a new class of therapeutics, are promising for treatment and/or

prevention of genetic disorders. Despite the challenges and mysteries, over the first

approved gene drug, past 22 years indicate that gene therapy will probably be the

rising star of next century.

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Clinical studies for the biosimilars Dr. Sadi S. ÖZDEM

Akdeniz University, Medical Faculty, Department of Medical Pharmacology, Antalya, Turkey Analytical comparability forms the basis of biosimilarity. However, further non-clinical and clinical studies are needed to prove the comparability of biosimilar test drug with the reference product, of which the extent usually depends on the degree of analytical comparability together with the level of residual uncertainty. In terms of clinical studies, at least a comparative pharmacokinetic/pharmacodynamic study is required to support demonstration of biosimilarity of test product with the reference product in addition to well-performed comprehensive non-clinical comparative physicochemical and biological activity studies. The decision to go further with clinical comparative efficacy and safety studies will be dependent on level of residual uncertainty regarding the similarity of test agent with the reference on the basis of the structural and functional characterization, in vitro and in vivo (if needed) preclinical studies, clinical pharmacokinetic and pharmacodynamic data and immunogenicity assessment. Provided that there are surrogate pharmacodynamic markers for the disease the biosimilar is developed for and that both the test product and the reference product produce comparative alterations in these surrogate markers in a clinical pharmacodynamic study, the need for comparative clinical efficacy and safety studies are reduced substantially. Unfortunately, currently the number of surrogate pharmacodynamic markers are quite few and further comparative clinical efficacy and safety studies are required within the totality of evidence approach to support the biosimilarity of test product with the reference product for several large, structurally complex biological medicinal products including fusion proteins. Besides that, given that most biological medicinal products including biosimilars are therapeutic proteins, following administration to patients they are recognized by the immune system with a resultant immune response ranging from a transient and clinically non-significant antidrug antibody formation to more detrimental clinical results such as loss of efficacy due to binding and neutralization of the therapeutic product, serious immune effects including anaphylaxis and cross-reactivity, particularly with the endogenous substances for which the therapeutic agents are used as substitutes. Factors related both to the product administered and the disease treated as well as the patients are important in immunogenicity of biological medicinal products. Genetic background, pre-existing immunity and immune status of the patients, administration route and the dosing schedule of the biotechnological medicinal products as well as the stability characteristics, formulation and the manufacturing process may all be predisposing factors/determinants of immune responses to therapeutic proteins including biosimilars. Therefore, in order to provide further support for the biosimilarity, comparison of immunogenicity of the test and the reference products using appropriate methods and measures should be part of clinical comparative pharmacokinetic/pharmacodynamic and efficacity/safety studies.

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New Approaches to Ecosystems: Start-up Model to Realize a Science Dream

Authors:

Rana Sanyal

Affiliations: Department(s) and institution(s) of all contributors (use superscript numbers (i.e. 1) to link department affiliations)

1 RS Research, Teknopark Istanbul, Pendik, Istanbul, TURKEY 2 Bogazici University, Department of Chemistry, Bebek, 34342, Istanbul, TURKEY 3 Bogazici University, Center for Life Sciences & Technologies, 34342, Istanbul, TURKEY

Abstract:

A single paragraph to give a brief introduction to your work

RS Research is a VC-backed academic spin-off in phase-I clinical development of the lead candidate developed based on a highly flexible nanomedicine platform with reduced side effects and enhanced efficacy for cancer chemotherapy. The start-up has developed a targeted smart chemotherapy technology - a novel drug delivery platform that packages the cytotoxic agent until it reaches the tumor. This helps reducing the side effects of chemotherapy, enabling a higher amount of drug delivery per dose, and increasing the half-life of the drug. RS Research’s success is based on a comprehensive business model that brings together various skill sets to create an even bigger pool of capabilities to lead into a multi-beneficial collaboration ecosystem. With the increasing role of start-ups in R&D studies globally, the ecosystems and how they connect to each other have become a larger focus for sustainability and improvement of bringing scientific research to human aid.

Keywords: 1: New approaches to designing the ecosystem 2: Targeted Chemotherapy 3: Drug Discovery

Main Text: -For presentations that report original research, you should use the titles:“Introduction”, “Materials and Methods”, “Results”, “Discussion” and“Conclusions”

Introduction According to the Tufts Center for the Study of Drug Development (CSDD) Report, average cost to develop a new drug is $2.6 billion. Details of the Tufts report show that the total cost of discovery and development, preclinical studies and phase-I clinical trials is around 500 million USD. On the other hand, according to data from FDA, in the last 10 years, 67% of the newly approved drugs have been developed up to the end of phase-I by small sized enterprises (SME’s). There lies a Sayfa 88

discrepancy: Small sized companies do not have the means and resources to cover such an expense reported by Tufts CSDD. But, the escalating “67%” shows that

SMEs’ role and contribution to drug discovery studies all around the world is gaining more importance every day. This is only possible through changing one’s

perspective about the business model.

Discussion

The idea that resulted in the establishment of RS Research, is ignited by this paradigm change. Innovative treatments cannot be realized by repeating what has

already failed in the laboratory or brought to the bedside unless conventional business models are replaced by inventive models. Our start-up, RS Research, has been established in Istanbul in March 2015 with the passion to demonstrate that

new drugs can be developed in Turkey; utilizing a new collaboration model between relevant parties in a bigger ecosystem who merge their power together.

While extreme costs on spreadsheets intimidate everyone to take the first step, RS Research has proven that reaching the end of pre-clinical research wasn’t impossible, owing largely to a well-established university-industry collaboration.

The win-win business model developed by Boğaziçi Univesity’s Center for Life Sciences and Technologies (LifeSci) and RS Research maintains a favorable

relation for both sides. LifeSci is an international center of excellence, home to indispensable labs and researchers, a heaven for a start-up who is in dire need of scientific quality as well as exceptional man-power. The start-up takes advantage

of the fully equipped infrastructure while the center collects additional funds for the sustainability of this platform. A similar game plan can be followed by many

entrepreneurs or academicians, who intend to develop new drugs; as the Center is has the desire to become a hub, supporting this process.

As mentioned, start-ups bake a very big share of the drug discovery pie but don’t necessarily have the rumored financial strength necessary for drug development.

These companies are usually backed by modest venture capital funding. Our start-up RS Research develops its globally patented, novel nanomedicine platform by using polymer technology. This proprietary drug delivery platform consists of a

tunable polymer and biodegradable drug linkers, enabling tailor made designs with targeting units. The drug candidates in the pipeline target a broad range of cancer

types. This approach combines the effectiveness of chemotherapy with the precision of tumor targeting in order to maintain a “targeted smart chemotherapy”. In 2017, a seed venture capital investment, one of the top three highest biotech

investments of the last decade, helped the team to work on its pipeline and successfully complete the pre-clinical studies of the first drug candidate. Following

promising results from its initial pre-clinical studies, the start-up received Investigational New Drug (IND) approval from the Turkish Ministry of Health for

the leading candidate of the pipeline, RS-0139. This approval is not only a milestone for the company, but also for the history of Turkey, as it is the first ever nationally developed drug starting from bench top moving into clinic.

Being an academician and an entrepreneur at the same time brings the risk of

shifting one’s focus in an unfavorable way. Instead of carrying all the burden of finances and logistics of research, relying on a strong business model that’s based on a strong collaboration network helps gathering an ideal set of skills and

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capabilities for one’s ultimate goal. This is way beyond contracting and delegating duties that one cannot do or don’t have time to do. While scientists are putting all

they have forth; accomplishing the undone; creating what doesn’t exist yet, they are also in a real world and the real world comes with legal requirements,

approvals, tax responsibilities, and all those “other” important procedures. The start-up approach to science helps scientists continue uninterruptedly, with a longer breath to run the marathon. Although the science is at the core of it, start-

up functions create the essential conditions.

The ecosystem steps in here, strengthening the unseen construction that makes science stand straight up. Although we’re observing that the field of R&D is improving in terms of structure and infrastructures, the current research requires

this ecosystem to build new muscles to survive.

With a desire to help fellow start-ups gain speed for commercialization of their ideas, creating a tangible evidence of success of the first steps ever taken in Turkey for an original drug going through clinical trials was a must. With this motivation,

RS Research has taken the lead of “Network for Antineoplastic Nanomedicine: GMP Production and Phase I/II Clinical Studies.” project in response to TUBITAK Call on

“Industry Innovation Network Mechanism to Support Product Development with High Added-Value”. Stakeholders in this project range from universities to industry, consultants to hospitals including strong members like Boğaziçi

University and Koç University. With the productization roadmap as the outcome of this project, RS Research hopes to transfer the knowledge and experience to other

researchers in the field, eventually creating an ecosystem where many novel medicines are discovered and brought to patients. And it is this ecosystem that the researchers yearn for is what will make future of scientific research brighter in

Turkey.

Conclusion Start-ups act as the core strength to build up the ecosystem for research to

flourish. This will be evident in the nearer future if not so already. Success stories through this model will pave the way for more scientists to bring their inventions

to human benefit.

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Biotechnology Assisted Biosensor Technology Dilek Çökeliler Serdaroğlu1 1Biomedical Engineering Department, Başkent University, Bağlıca Campus, Ankara, cokeliler@baskent.edu.tr Abstract: Sensor is defined as a measuring device that exhibits a characteristic of a measurable signal when it is subjected to a phenomenon. We can divide sensors into two types, namely (i) physical sensors for measuring physical indicators such as distance, mass, temperature, pressure, (ii) chemical sensors which measure chemical substances by chemical or physical responses. Biosensors are subgroup of chemical sensors, which measure chemical substances by using a biological sensing layer. The one objective of this speech is to understand the possible impact of selection of suitable transducer for particular target. Than, selection of recognition mechanism could be critical and changed depend on the type of transducer. Our studies and methods that include target detections that have been summarized with its selection criteria and performance parameters are summarized. Particularly, as a field of biotechnology, biological elements are discussed for improvement sensitivity and selectivity in immunosensor or biosensor applications. Keywords: Biosensor; Quartz Tuning Fork; Electrochemical Sensor; Antibody Introduction:

A chemical sensor is defined as a device, which responds to a particular analyte in a selective way through a chemical reaction and can be used for the qualitative or quantitative determination of the analyte. Such a sensor is concerned with detecting and measuring a specijfic chemical substance or set of chemicals. Biosensors are really a subset of chemical sensors, but are often treated as a topic in their own right. One might consider the ears, eyes and fingers to be physical sensors as they detect physical sensations of sound, light and heat, etc., respectively. What we detect with the nose - smells - are in fact small quantities of chemicals. The nose is an extremely sensitive and selective instrument, which is very difficult to emulate artificially. It can distinguish between many different chemical substances qualitatively and can give a general idea of ‘quantity’ down to very low detection limits. The chemicals to be detected pass through the olfactory membrane to the olfactory bulbs, which contain biological receptors that sense the substrate. The response is an electrical signal, which is transmitted to the brain via the olfactory nerves. The brain then transduces this response into the sensation we know as smell. The tongue operates in a similar way. The nostrils collect the ‘smell sample’, which is then sensed by the

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olfactory membrane, i.e. the sensing element. The responses of the olfactory receptors are then converted by the olfactory nerve cell, which is the equivalent of the transducer, into electrical [1].

During analysis, when the sample (analyte) are added to a biosensor, its bio-recognition elements (enzymes, aptamers, antibodies and ligands) selectively react with the analyte by means of biochemical reaction (electrochemical, colour, light, etc.) and its signals are further processed through a transducer device (e.g., reading the colour intensity, light emission, current or voltage produced), to provide a quantitative or semi quantitative analyte level in the sample.

The major tasks in developing a biosensor for a target analyte and the necessary skills involved are: Bioreceptors are typically enzymes or binding proteins, such as antibodies, immobilized onto the surface of a physico-chemical transducer. Specific interactions between the target analyte and recognition sites within the bioreceptor produces a physico-chemical change which is detected and may be measured by the transducer. In principle, any biomolecule or molecular assembly that has the capability of recognizing the analyte can be used as a bioreceptor. The immobilization is done either by physical entrapment or chemical attachment. Note that only minute quantities of bioreceptor molecules are needed, and they are used repeatedly for measurements The transducer can take many forms depending upon the parameters being measured - electrochemical, optical, mass and thermal changes are the most common..

A mass-sensitive biosensor is defined as any device that measures the property that scales proportionally to mass associated with or bound to its sensitive surface assembled with capture probes. The most commonly known mass sensitive sensors applied for immunoassay approaches are surface acoustic wave (SAW) bulk acoustic wave (BAW), and Quartz Crystal Microbalance (QCM) device. In this speech new type of mass sensitive transducer, quartz tuning fork is presented. Moreover, design of electrochemical sensor is showed for detection pollutant as bisphenol-A.

Materials and Methods: For systematic design of biosensor, selection of transducer could be the keypoint. There are options to use different transducers for same target. One example from our recent study showing that for beta transferrin. An increased plasma transferrin level is often seen in patients suffering from iron deficiency anemia, during pregnancy, and with the use of oral contraceptives, reflecting an increase in transferrin protein expression. When plasma transferrin levels rise, there is a reciprocal decrease in percent transferrin iron saturation, and a corresponding increase in total iron binding capacity in iron deficient states [2]. A decreased plasma transferrin can occur in iron overload diseases and protein malnutrition. In addition this, it can be marker for head

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injury. For this reason, preparation of quartz tuning forks is offered for detection beta transferrin as a preliminary test for nazal fluids in our study. In that design, our patented mass sensitive system (TR 201605031B) is modified for anti-transferrin immobilization [3]. In this study, to supply antibody for beta transferrin was also challenging. Quartz tuning fork is modified with antibody oriented design for anti-transferrin molecules (Figure 1). This orientation is very important for obtaining higher sensitivity. Since mass load is critical, the modification must have low mass loading too.

Figure 1. Quartz tuning fork and idea for antibody immobilisation

On the other hand, electrochemical sensors are also very common systems. Since, specified electron transfer is critical point, the idea for electrochemical transducers modification could be totally different. One idea could be the improvement of electron transfers by conductive layers and nanomaterial can assist structures.

Figure 2. Preparation of electrochemical sensor surface [4].

In our electrochemical system, carbon nanotube / graphene oxide / gold nanoparticle nanocomposite structure is prepared for detection of bisphenol which is one of the important pollutant (Figure 2). It is a chemical which could potentially interfere with the endocrine system of humans and animal. This study is one example shows that also nanomaterials could be way for supplying specifity tinstead of biological elements. Results and Discussion:

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Since quartz tuning fork is used as biosensor very newly, immobilization of antibody is first and important stage (Figure 1). Generally, increasing of antibody concentration will support higher frequency shift. However we must be carefull that it didnt mean we prefer higher antibody loading.

Figure 1. Optimisation of antibody immobilization on quartz tuning fork system

However, higher amount of electroactive components for electrochemical sensor could be the general approach to get higher sensivity (Figure 2).

Figure 2. Performance of nanomaterial assisted electrochemical sensor

Conclusions: In biosensor design, selection of transducer is also critical for depending on targets. Each tranducer type has it own concerns. In addition this, selection of biorecognition layer due to transducer is other challenge. At that stage, biotechnology has important role to supply varying biological component with both higher affinity and selectivity. References: [1] Eggins BR. Chemical sensors and Biosensors, John Wiley & Sons, 2008.

00,20,40,60,8

11,21,41,61,8

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Antibody Concentration, TRC2 (µg/µL)

R2 = 0,940

Sayfa 94

[2] Geoffrey FH, Sergio AM, Stuart LW, Toru N, Wendy S, Orthop J. 2004; 24: 115–118. [3] Dedeoğlu A. Validation of the Quartz Tuning Fork System by Electrochemical Method and Fabrication of Transferrin Detecting Biosensor, undergraduate thesis, Başkent University, 2018 (adviser: Prof. Çökeliler). [4] Wang YC, Çökeliler D, Gunasekaran S, Electroanalysis, 2015; 27: 2527–2536

Sayfa 95

miRNA Delivery Systems to Prepare Drugs in Gene Therapy

Emine Şalva

Inonu University Faculty of Pharmacy Department of Pharmaceutical

Biotechnology, Malatya

Non-coding RNAs in the genome are important gene expression regulators.

MicroRNAs (miRNAs), which make up about 3% of the human genome, regulate

the expression of 60-70% of 25000 genes. miRNAs are singe-stranded, small and

about 22 nucleotides in length endogenous RNAs that posttranscriptionally repress

the expression of genes by binding to mRNAs with base pairing. miRNAs play a role

in cell growth, differentiation, proliferation, apoptosis, and many cellular processes.

Impaired expression of miRNA causes many diseases, including cancer. miRNAs in

cancer act as oncogenes (oncomirs) or tumor suppressors. miRNA replacements

(miRNA mimics) or inhibitors (antimiRs) are given to restore the physiological

functions of the regulator miRNAs in cells. miRNAs associated with cancer

metastasis are an important part of the cancer genome. Cancer-related miRNAs can

be used both as a therapeutic target and as a biomarker.

While small RNA drugs enter the clinic, the preclinical and clinical

applications of miRNAs continue. Candidate miRNAs reached phase 1 and phase 2

stages. Effective and safe delivery of miRNAs is important in the clinic applications.

The most important obstacle for miRNA-based therapeutics is the correct selection

of target miRNA candidates. In addition, miRNAs should be given in appropriate

doses to avoid side and off-target effects. Therefore dose studies are required. Kaban

et al (2017) showed that dose studies for an effective therapy by miRNA are

important, because high dose of miRNAs is lead to severe side effects. In particular,

some miRNAs play both tumor suppressor and oncogenic roles in cancer. Another

major problem is the design of the appropriate miRNA delivery system. The

effectiveness of naked miRNAs is limited due to the short half-life, off-target effects

and poor targeting ability. To overcome these problems, miRNAs must be

formulated with delivery systems. Delivery systems extend the duration of miRNAs

in the blood and increase their accumulation in the blood. Development of miRNA-

based therapeutics can be accomplished with effective cellular uptake and tissue-

specific transport. Viral and non-viral delivery systems have been developed for

miRNA delivery. The delivery systems should have long circulatory half-lives, allow

controlled release, release in the target region, and should not cause off-target effects.

Commercially available transfection reagents or electroporation methods are

used to delivery miRNAs to cells in vitro. In addition, in vivo stability of miRNAs

can be increased by chemical modifications. For example, chemically modified

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miRNAs can be developed with locked nucleic acids (LNA) or peptide nucleic acids

(PNA). Nanoparticular delivery systems are preferred to increase the in vivo delivery

and stability of miRNA. So far, synthetic (PEI, PLGA, PCL, PU) and natural

(chitosan, HA, alginate, protamine) polymeric nanoparticles, lipid-based

nanoparticles and inorganic (silica, iron-oxide, calcium phosphate, glod)

nanoparticles for miRNA delivery has been extensively investigated. The advantages

of nanoparticular delivery systems include increasing loading efficacy, providing

target-specific therapy, facilitating transport into the cell, providing controlled

release, increasing endosomal escape, reducing toxicity by reducing the required

dose and consequently increasing therapeutic efficacy.

References:

1. Lee SVW, Paolettia C. et al. MicroRNA delivery through nanoparticles.

Journal of Controlled Release, 313 (2019) 80-95.

2. Kaban K., Şalva E., Akbuğa J. In vitro dose studies on chitosan nanoplexes

for microRNA delivery in breast cancer cells. Nucleic Acid Therapeutics, 27(1):45-

55, 2017.

3. Kaban K., Şalva E., Akbuğa J. Modulation of the dual faced effects of miR-

141 with chitosan/miR-141 nanoplexes in breast cancer cells. The Journal of Gene

Medicine, 21(9), 1-9., 2019.

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Current progress in clinical and experimental gene therapy approaches for

genetic diseases

Salih Sanlioglu 1

1 The Department of Gene and Cell Therapy, Akdeniz University, Antalya, 07058, Turkiye

Abstract:

Gene and cell therapy are the overlapping fields of biomedicine with similar therapeutic

goals purposefully designed for the treatment of inherited and/or acquired diseases. Not to

mention a single shot of gene therapy has the potential to tackle currently incurable complex

genetic diseases like diabetes and cancer etc. It has been more than 30 years since Rosenberg

and his team performed a retroviral-mediated gene transduction to introduce neomycin

resistance gene into human tumor-infiltrating lymphocytes (TIL) before their infusion into a

52-year-old truck driver from Indiana [1]. The end result of the first in human clinical trial was

the prolonged survival of the transferred cells in the patient demonstrating that the engineered

virus can be used safely in humans. However in 1999, unintended loss of Jesse Gelsinger, 18-

year-old teenager from Tucson, Arizona, (the 18th person to receive the modified virus) in a

clinical trial involving adenovirus mediated gene therapy against ornithine transcarbamoylase

(OTC) deficiency conducted by Dr. James M. Wilson, director of the Institute for Human Gene

Therapy at the University of Pennsylvania caused a substantial setback in the gene therapy field

due to unjustified negative publicity [2, 3]. Despite facing numerous obstacles and a great deal

of failures over the last three decades, fruitful results ultimately gathered from the experimental

and clinical trials of gene and cell therapy led to the approval of numerous gene and cell therapy

products (Glybera, T-VEC, Strimvelis, Kymriah, Yescarta, Luxturna, Zolgensma, Zynteglo)

for marketing [4]. Furthermore, the advent of the genome editing tool CRISPR-Cas9 is expected

to revolutionize the medicine due to its potential to mediate targeted gene editing despite

existing technical pitfalls and ethical considerations.

Keywords: Gene and cell therapy; genetic diseases; diabetes; islet cell transplantation

Preclinical studies involving animal models of human diseases are the key to clinical

success and considered to be essential to assess therapeutic efficacy of novel gene and cell

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therapy products under development. Since diabetes is one of the most prevalent disease with

a high mortality rate like cancer, novel gene and cell therapy approaches against diabetes

developed at the Human Gene and Cell Therapy Center of Akdeniz University are summarized

below.

Type 1 diabetes (T1DM), characterized by permanent destruction of insulin-producing

beta cells, is lethal unless conventional exogenous insulin therapy or whole-organ

transplantation is employed [5]. Although pancreatic islet transplantation is a safer and less

invasive method compared with whole-organ transplant surgery, its treatment efficacy has been

limited by islet graft malfunction and graft failure. Thus, ex vivo genetic engineering of beta

cells is necessary to prolong islet graft survival [6]. For this reason, a novel gene therapy

approach involving adenovirus-mediated TRAIL gene delivery into pancreatic islets was tested

to determine whether this approach would defy autoreactive T cell assault in streptozotocin

(STZ)-induced diabetic rats. To test this, genetically modified rat pancreatic islets were

transplanted under the kidney capsule of STZ-induced diabetic rats, and diabetic status (blood

sugar and body weight) was monitored after islet transplantation. STZ-induced diabetic rats

carrying Ad5hTRAIL-infected islets experienced prolonged normoglycemia compared with

animals grafted with mock-infected or AdLacZ-infected islets. In addition, severe insulitis was

detected in animals transplanted with mock-infected or AdLacZ-infected islets, whereas the

severity of insulitis was reduced in animals engrafted with Ad5hTRAIL-infected islets. Thus,

TRAIL overexpression in pancreatic islets extends allograft survival and function, leading to a

therapeutic benefit in STZ-induced diabetic rats [7].

Type 2 Diabetes (T2DM) is characterized by insulin resistance, glucose intolerance and

beta cell loss leading to hyperglycemia. Because vasoactive intestinal peptide (VIP) displayed

insulinotropic and anti-inflammatory properties, it has been regarded as a novel therapeutic

agent for the treatment of diabetes [8]. Despite all these beneficial properties, VIP is extremely

sensitive to peptidases (DPP-4) requiring constant infusions or multiple injections to observe

any therapeutic effect. Thus, an HIV-based lentiviral vector encoding human VIP (LentihVIP)

was constructed to test the therapeutic efficacy of hVIP in diet induced obesity (DIO) animal

model of T2DM. Intraperitoneal (IP) delivery of LentihVIP vectors into C57BL/6J mice

significantly increased serum VIP concentrations compared to control animals. Consequently,

LentihVIP delivery in DIO animal model of T2DM resulted in improved insulin sensitivity,

glucose tolerance, and protection against STZ-induced diabetes in addition to reduction in

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serum triglyceride/cholesterol levels. All these beneficial results suggested that LentiVIP

delivery should be evaluated as a novel therapeutic approach for the treatment of patients with

T2DM [9].

Agents that increase beta cell mass are considered useful in managing T2DM, where

the ideal beta cell preserving agent should reduce insulin resistance, increase glucose-induced

insulin secretion, promote beta cell replication and/or islet neogenesis, and protect islet cells

from apoptosis [10]. Postprandial glucose-induced insulin secretion from the islets of

Langerhans is facilitated by glucagonlike peptide-1 (GLP-1)—a metabolic hormone with

insulinotropic properties. Among the variety of effects it mediates, GLP-1 induces delta cell

secretion of somatostatin, inhibits alpha cell release of glucagon, reduces gastric emptying, and

slows food intake [11]. These events collectively contribute to weight loss over time. During

T2DM, however, the incretin response to glucose is reduced and accompanied by a moderate

reduction in GLP-1 secretion. To provide constant GLP-1 synthesis and secretion in vivo, a

lentiviral vector carrying the GLP-1 gene (LentiGLP-1) was constructed, and its therapeutic

efficacy was tested in obese diabetic rats [12]. LentiGLP-1 administration significantly reduced

blood glucose and concurrently improved insulin sensitivity and glucose tolerance. Importantly,

normoglycemia correlated with an increase in blood GLP-1 and pancreatic beta cell

regeneration following LentiGLP-1 delivery. The data obtained suggest the clinical potential of

GLP-1 gene transfer therapy for the treatment of T2DM.

References:

1. Rosenberg, S.A., et al., Gene transfer into humans--immunotherapy of patients with

advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene

transduction. N Engl J Med, 1990. 323(9): p. 570-8.

2. Somanathan, S., R. Calcedo, and J.M. Wilson, Adenovirus-Antibody Complexes

Contributed to Lethal Systemic Inflammation in a Gene Therapy Trial. Mol Ther, 2020.

3. Raper, S.E., et al., Fatal systemic inflammatory response syndrome in a ornithine

transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet

Metab, 2003. 80(1-2): p. 148-58.

4. Shahryari, A., et al., Development and Clinical Translation of Approved Gene Therapy

Products for Genetic Disorders. Frontiers in Genetics, 2019. 10.

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5. Sanlioglu, A.D., et al., Clinical utility of insulin and insulin analogs. Islets, 2013. 5(2): p.

67-78.

6. Sanlioglu, A.D., et al., Molecular mechanisms of death ligand-mediated immune

modulation: a gene therapy model to prolong islet survival in type 1 diabetes. J Cell

Biochem, 2008. 104(3): p. 710-20.

7. Dirice, E., et al., Adenovirus-mediated TRAIL gene (Ad5hTRAIL) delivery into

pancreatic islets prolongs normoglycemia in streptozotocin-induced diabetic rats. Hum

Gene Ther, 2009. 20(10): p. 1177-89.

8. Sanlioglu, A.D., et al., Therapeutic potential of VIP vs PACAP in diabetes. J Mol

Endocrinol, 2012. 49(3): p. R157-67.

9. Tasyurek, H.M., et al., HIV-based lentivirus-mediated vasoactive intestinal peptide

gene delivery protects against DIO animal model of Type 2 diabetes. Gene Ther, 2018.

25(4): p. 269-283.

10. Tasyurek, M.H., et al., GLP-1-mediated gene therapy approaches for diabetes

treatment. Expert Rev Mol Med, 2014. 16: p. e7.

11. Tasyurek, H.M., et al., Incretins: their physiology and application in the treatment of

diabetes mellitus. Diabetes Metab Res Rev, 2014. 30(5): p. 354-71.

12. Tasyurek, H.M., et al., Therapeutic Potential of Lentivirus-Mediated Glucagon-Like

Peptide-1 Gene Therapy for Diabetes. Hum Gene Ther, 2018. 29(7): p. 802-815.

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Opportunities for Young Researchers in Genome Editing Market

Serif Senturk 1, 2

1 Izmir Biomedicine and Genome Center, Balcova, Izmir 2 Dokuz Eylul University, Izmir International Biomedicine and Genome Institute, Balcova,

Izmir

The ability to modify genetic information is important for the examination of gene function and

the discovery and modulation of biological mechanisms. Genome editing is a group of

technologies that allow scientists to achieve meticulous modification of the characteristics of

an organism by shaping its genetic material. These technologies allow any genetic material to

be added, removed or modified at specific locations of the target genome. Various tools and

approaches have been developed for use in genome engineering since the 1970s. These genome

editing tools include recombinases, meganucleases, zinc finger nucleases, transcription

activator-like effector nucleases, and various forms of programmable nucleases of the CRISPR

systems.

The adaptation of CRISPR-Cas9-based genome editing systems from prokaryotes to eukaryotic

cells has improved our ability to specifically manipulate, detect and visualize DNA sequences,

plus add or remove genetic as well as epigenetic information in the genomes of various species.

The superior application ease, robustness and low cost of this latest genome editing technology,

when compared to other tools, have transformed and revolutionized the face of genome

engineering and the pace of basic biological research and biotechnology. In addition to those

modalities of genome editing based on non-homologous end joining and homology-directed

repair following an efficient double-strand break by wild-type Cas9 enzyme, other approaches

based on base editing, reverse transcriptase, transposase and DNA polymerase enzyme fusions

have recently been developed. More importantly, in addition to targeting the DNA molecule,

CRISPR-Cas-based RNA targeting tools have also been developed.

The scientific developments in the past few years have made genome editing more efficient,

precise and robust than ever. As a result, the idea that CRISPR-Cas systems can diagnose and

treat many inherited and acquired diseases that pose a threat to human health has become of

great interest around the globe. Current pre-clinical applications on genome editing focus

particularly on the development of treatment strategies for viral infections, cardiovascular

diseases, metabolic disorders, blindness and deafness, primary defects of the immune system,

hemophilia, Alzheimer's disease, muscular dystrophy, and T cell-based anti-cancer

immunotherapies. A large body of evidence suggests that CRISPR-Cas technologies are

expected to be successfully utilized for producing fuel and manufacturing chemicals and for

generating disease-resistant and environment-adaptive livestock and agricultural products to

meet the nutritional needs of the growing global population.

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Finally, CRISPR-Cas-based genome editing technologies support sustainable development and

growth in biomedicine, agriculture, industrial biotechnology and other sectors related to

bioeconomics. Countless research centers, start-ups and established biotechnology companies

are now working hard to bring to life the benefits of genome engineering products. The

development and implementation of these revolutionary technologies that shape the future of

biotechnology require equal progress. To achieve this, there is a growing need for competent

and innovative biomedical researchers to perform studies on genome engineering and develop

the cutting-edge CRISPR-based technology platforms for research and therapeutic applications

as well as skilled professionals who can be employed in the development, marketing and

evaluation of economic and environmental benefits and ethical issues of the biotechnological

innovations and products. Accordingly, various career advancement opportunities are regularly

becoming available in public and private scientific institutions and biotechnology companies.

Keywords:

Genome editing; CRISPR-Cas; biotechnology

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Title:

Medical Device Manufacturers' Roadmap and Legal Requirements in the “CE”

Certification Process Under the New Legal Regulations in Turkey and in the World

Authors:

Dr. Kemal TEKİN / Presenter: Dr. Kemal TEKİN

Affiliations:

Q Men Consultancy

Abstract:

The medical device industry is one of the health technology assessment areas

where innovation, competition and costs are very high. Medical devices are among

the high-risk health technology types. First-class medical devices in EU member

states must obtain Conformité Européenne (CE) certification. Second and third class

medical devices are evaluated by 80 different evaluation institutions worldwide to be

approved by each country's local authority.

Keywords:

Medical Device; Manufacturer; MDR; CE Certification; Notified Body;

Certification;

Main Text:

Within the framework of the “new approach” related to technical standardization, the

EU has been subject to common harmonized rules at the level of unity since 1990,

which medical devices must meet. Since the 1990s, the regulation of the medical

device industry in Europe has changed relatively.

Before a medical device was introduced to the European market, manufacturers had

to produce technical documentation providing evidence of compliance with the

relevant legislation. Technical documentation should comply with the Medical

Devices Directive (MDD) 93/42 / EEC or Active Implantable Medical Devices

Directive (AIMDD) 90/385 / EEC (hereinafter referred to as "MDD / AIMDD").

However, the incident that jeopardized two large patient safety events within the

European Union triggered this process (Hip prostheses and breast implant crisis).

These events emphasized the need for urgent improvement in standards, processes

and procedures and served as catalysts for reform.

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On September 26, 2012, the European Commission agreed to address the European

Parliament and Council Regulation on medical devices (MDR) and in vitro

diagnostics (IVDR). These regulations are intended to replace MDR and IVDR's

three existing medical device directives after implementation. The aim of these new

regulations is to ensure that products are effective and safe and can be bought and

sold freely and fairly across the EU.

MDR (2017/745) entered into force on May 26, 2017. A 3-year transition period is

envisaged for compliance. Until May 26, 2020, manufacturers' or Medical Devices

Regulation (MDR) of their technical documentation to the European Union (EU)

2017/745 (hereinafter referred to as "MDR") to renew a CE certificate or issue a

Declaration of Conformity (DoC) must be appropriate. However, as stated in Article

120 of MDR, after 26 May 2020, medical devices may be placed on the market under

the provisions of the MDD / AIMDD, provided that the certificate continues to

comply with the condition that the certificate is issued before this date.

Certificates issued in accordance with MDD / AIMDD after 25 May 2017 will remain

valid until their expiration date, but in any case the certificates will expire on 27 May

2024 at the latest.

This will require changes in the processes and existing documents of manufacturers,

Competent Authorities (CAs) and Notified Bodies (NBs). In the MDR process, it

will bring additional responsibilities to “Notified Bodies” (independent third parties

conducting conformity assessments for medium and high risk devices). Notified

Bodies will be subject to further scrutiny from the competent authorities and the

appointment process will be coordinated at European level.

It is understood that new requirements for the technical documentation generated by

the medical device manufacturer are emerging and will also be subject to further

review by the Competent Authority (CA) / Notified Body (NB) as appropriate. It will

be the manufacturer's responsibility to prepare the technical documentation required

for all medical device classes, to provide access to these documents at the request of

the CA or NB.

As stated in article 61 of MDR. Manufacturers are required to follow all applicable

MDR PMS-PMCF requirements from 26 May 2020, even if the related devices are

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still released under MDD / AIMDD. Therefore, MDR now requires a subdivision

within the technical documentation that specifically addresses the PMS activities

established by the manufacturer. Details on what information should be provided in

this section of the technical documentation are available under MDR Annex III. More

precisely, the technical documentation should now include a PMS plan, a Periodic

Security Update Report (PSUR) for devices larger than class I, in line with the

manufacturer's obligations set out in Article 84 of MDR (Article 86 of MDR) or it

should contain a PMS report (article 85 of MDR) for class I devices.

As a result, the change from MDD / AIMDD to MDR requires manufacturers to

make some important adjustments to the technical documentation of a device.

Although the total number of documents to be included in the technical documents

is generally the same;

Unique Device Identification-Reference to Device Identifier (UDI-DI),

Necessity of post-market surveillance plan

Necessity of post-market clinical follow-up plan and implementation,

General safety and performance requirements (PSUR),

Necessity of the person responsible for compliance with the legislation,

The conditions related to the authorized representatives have been defined, etc.

However, the expected quality of technical documents has increased, which should

be robust enough to duly prove any claim, especially when it comes to clinical data.

Manufacturers will need to improve the scientific quality and clarity of their technical

documentation.

Manufacturers should also raise awareness among their employees on how to

properly implement MDR and train their employees to achieve the qualifications

required by this new Regulation.

Sayfa 106

Cell banks in the production of biological / biotechnological drugs

and new cell lines

Title:

Cell banks in the production of biological / biotechnological drugs and new cell lines.

Authors:

Dilek Telci

Affiliations:

Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe

University, Istanbul, Turkey.

Since the approval of biotherapeutic proteins in the early 1980s, they have become

the mainstream group of products in pharmaceutical industry and used in the

treatment of numerous conditions such as genetic, metabolic and hormonals

disorders, cancer, autoimmune and hematologic diseases (6). Protein therapeutics are

mostly produced in microbial systems, rodent and human cell lines and albeit

seldomly in insect and plant cells. With the advanced technology, more cost effective

approaches including new host cell engineering, clone screening and disposable cell

culture scale-up strategies have been developed to decrease the manufacturing cost

of such recombinant biotherapeutic proteins (8). Here presented a birds-eye view of

in-use and in-development cell culture processes and expression platform

technologies used for the manufacturing of protein therapeutic.

Selection of a production cell line for the development of pharmaceutical depends

on the nature and complexity of recombinant protein including the aspect of post-

translational modifications (PTM) and folding structure. Microbial system offers the

benefit of low cost, rapid production timeline, and high yields (2). Insulin, the first

recombinant bio-drug authorized was manufactured in Escherichia coli, while

Saccharomyces cerevisiae was used in the production of vaccines, enzymes, peptides

and clotting factors. As the demand was arisen for the production of more complex,

multi-subunit proteins with high glycosylation patterns, shortcomings of microbial

systems on protein misfolding, aggregation and limited capacity of PTM forced the

industry to use specialized mammalian cells (2, 8). Chinese hamster ovary cells

(CHO) isolated from ovary biopsy sample was genetically engineered to adapt

growth in suspension and produce products with desired PTM and increased yield.

Auxotrophic metabolic mutants such as dihydrofolate reductase (DHFR) deficient

CHO DG44 or glutamine synthase (GS) lacking murine myeloma cells NS0 were

used for their ability to amplify the biotherapeutic transgene (BT) under the selection

pressure of DHFR inhibitor methotrexate (MTX) and GS inhibitor methionine

Sayfa 107

sulfoximine (MSX), respectively (1, 3, 10). Enhanced expression of BT without the

need of gene amplification was achieved with the development of novel expression

systems. Inclusion of epigenetic chromatin modeling elements such as matrix

attachment regions (MARs) and ubiquitious chromatin opening elements (UCOE)

into the expression vectors as well as targeted delivery of BT to transcriptionally

active euchromatic sites using site specific recombinases (Cre-loxP and FLP/FRT

systems), transposons and nucleases (CRISPR/Cas) are some of the recent cell line

development processes used to improve BT expression levels (8, 9).

Deviations in glycosylation patterns such as galactose-a1,3-galactose (α-gal) and N-

glycolylneuraminic acid (NGNA), and different degree of galactosylation,

sialylation, and fucosylation in biopharmaceuticals produced by non-human cell

lines brought about the development of human cell lines and genetically enhanced

mammalian cell lines (4). Although the theoretical contamination of human

adventitious agent due to lack of cross-species barrier pose a risk for potential safety,

advances in down-stream clearance steps have provided means for the use of human

cell lines in the production of biotherapeutics (8). Derived from human embryonic

kidney cells by adenovirus type 5 DNA transfection, HEK293 and fibrosarcoma HT-

1080 are used in vaccine production, enzymes and blood factors. Engineering of new

human cell lines such as human embryonic retinal cells PEC.C6, hybrid HKB-11,

and human amniocyte CAP for use in industrial process is currently underway and

some therapeutic proteins utilizing these cell lines in early phase trials (5, 7).

However, due to the experience in process parameters and in-process controls, CHO

and NS0 still remains as the main choice of biotherapeutic protein production with

engineering efforts now focusing on generation of cell lines that can provide human-

like glycosylation patterns (13).

Strategies for screening clones with high specific productivity, product quality and

desired cell density vary in cost, time and throughput stage. Traditional method of

limiting dilution, whereby a single cell/well transferred a multiwell plate forming a

colony is scaled up for the evaluation of stability, production efficiency and product

quality, requires time-consuming rounds of repeat screening to accomplish

monoclonality (8). To increase the throughput, fluorescent activated cell sorting and

automated systems such as ClonePixTM, Cell XpressTM, GenePixTM, which utilize

selection of clones in liquid or semisolid media by linking protein expression level

with fluorescent intensity, are used. The viability and growth of the clone and the

expression levels and quality of biotherapeutic protein can be different at small scale

than at large scale. As the use of shake flasks or Tube-Spin tubes are not ideal to

mimic the parameters in scale-up bioreactors, single use microbioreactors operating

with volumes as small as a few hundred microliter liter are developed for the

screening of many clones concurrently (11).

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Leading candidate clones that meet up with the standards of specific productivity,

volumetic productivity, and product quality are cryopreserved as pre-master cell

bank and evaluated for their phenotypic and genetic stability, in addition to their

product yield and quality with increasing passaging. Having acquired the desired cell

clone, master cell bank is generated under cGMP and tested for purity, identity, viral

contamination and genetic characterization. Master cell bank is subcultured to be

expanded to generate working cell bank in which BT genetic stability along with

product quality is investigated until the harvest from the end of production cell bank

(12).

The demand in pharmaceutical industry for the production of protein therapeutics is

growing and hence new host cell manufacturing platforms are needed to provide

products with enhanced pharmacological properties and lower costs. As advances in

technology such as genetic editing, -omics, automated screening and single-use

apparatuses continue, the generation of new biopharmaceutical production cell line

will evolve to achieve the production of better superior bio-drugs.

References

1. Assaraf, Y.G. and Schimke, R.T., Identification of methotrexate transport

deficiency in mammalian-cells using fluoresceinated methotrexate and flow-

cytometry, Proc. Natl. Acad. Sci. U.S.A, 1987; 84 (20): 7154–7158.

2. Castan A., Schulz P., Wenger T., Fischer S., Chapter 7—Cell line development,

Biopharmaceutical Processing, 2018; pp. 131–146.

3. Cockett, M.I., Bebbington, C.R., and Yarranton, G.T., High-level expression of

tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using

glutamine-synthetase gene amplification, Nat. Biotechnol., 1990; 8 (7): 662–667.

4. Dumont J, Euwart D, Mei B, Estes S, Kshirsagar R., Human cell lines for

biopharmaceutical manufacturing: history, status, and future perspectives, Crit.

Rev Biotechnol. 2016; 36(6):1110-1122.

5. Fliedl L, Grillari J, Grillari-Voglauer R., Human cell lines for the production of

recombinant proteins: on the horizon, N Biotechnol. 2015; 32(6):673-9.

6. Gadgil, M., Cell culture processes for biopharmaceutical manufacturing, Current

Science, 2017; 112(7): 1478-88.

7. Hu J, Han J, Li H, Zhang X, Liu LL, Chen F, Zeng B. Human Embryonic Kidney

293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology,

and Electrophysiology, Cells Tissues Organs, 2018; 205(1):1-8

8. Hunter M, Yuan P, Vavilala D, Fox M., Optimization of Protein Expression in

Mammalian Cells, Curr Protoc Protein Sci., 2019; 95(1):e77.

9. Inao, T., Kawabe, Y., Yamashiro, T. et al.. Improved transgene integration into

the Chinese hamster ovary cell genome using the Cre-loxP system., J. Biosci.

Bioeng., 2015;120 (1): 99–106.

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10. Kuo, C. C. et al., The emerging role of systems biology for engineering protein

production in CHO cells, Curr. Opin. Biotechnol. 2018; 51, 64–69.

11. Priola JJ, Calzadilla N, Baumann M, Borth N, Tate CG, Betenbaugh MJ. High-

throughput screening and selection of mammalian cells for enhanced protein

production, Biotechnol J, 2016, 11(7):853-65.

12. Shukla AA, Rameez S, Wolfe LS, Oien N., High-Throughput Process

Development for Biopharmaceuticals., Adv Biochem Eng Biotechnol, 2018;

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