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DDTEC-212; No of Pages 6

Please cite this article in press as: M.. Matasci, et al., Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects, Drug

Discov Today: Technol (2009), doi:10.1016/j.ddtec.2008.12.003

ECHNOLOGIES

DRUG DISCOVERY

TODAY

Recombinant therapeutic proteinproduction in cultivated mammaliancells: current status and future

prospectsMattia Matasci, David L. Hacker, Lucia Baldi, Florian M. Wurm*Institute of Bioengineering, Laboratory of Cellular Biotechnology, E cole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland

Recombinant therapeutic proteins produced in mam-

malian cells represent a major class of biopharmaceu-

ticals. In recent years, their demand has increased

dramatically and is now driving the development of a

variety of improvements to maximize their expression

in mammalian cells. Advances in media- and process

optimization have already resulted in more than 100-

fold improvement in yield, but further insights and

subsequent targeted modifications with respect to

the general biology of cells (genomics, physiology,

selection and adaptation) in bioreactors are hoped to

further improve protein yields and quality in the near

future.

Section Editors:Marco van de Weert, Eva Horn Moller

Introduction

Over the past two decades recombinant proteins have gained

increasing importance for therapeutic applications, and the

number of proteins either approved or launched into clinical

trials has continually increased over this period. Together

their annual global market is now valued to be more than $50

billion with an annual sales increase of about 20%. Currently

about 60% of all recombinant therapeutic proteins are pro-

duced in mammalian cells, mainly because of the ability of

mammalian hosts to generate high-quality proteins that are

similar in their biochemical properties to the naturally occur-

ring human forms. This growing demand for high-quality

recombinant therapeutics is driving the research and devel-opment of mammalian-cell-based manufacturing systems for

enhanced production yields. In this review we will present

relevant strategies developed to enhance the generation of

high producer cell lines and discuss how newtechnology may

contribute to the further development of cellular processes

for the production of therapeutic proteins.

Current strategy for recombinant cell line

development

The generation of recombinant cell lines follows a well-

defined multistep scheme that begins with the molecularcloning of the gene of interest (GOI) in a mammalian expres-

sion vector (Fig. 1). The GOI is then delivered into cells along

with a selection gene which may be cloned in the same or

different expression vector. Following DNA transfer, cells are

subjected to selective conditions to recover those that have

stably integrated the exogenous genes into a chromosome.

Well-established selection strategies rely upon complementa-

tion of a host auxotrophy. In Chinese hamster ovary (CHO)

cells, the major mammalian host for protein production, the

two most commonly used selection systems are based on the

Drug Discovery Today: Technologies Vol. xxx, No. xx 2009

Editors-in-Chief

Kelvin Lam Pfizer, Inc., USA

Henk Timmerman Vrije Universiteit, The Netherlands

Protein Therapeutics

*Corresponding author: F.M. Wurm ([email protected])

1740-6749/$ 2009 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2008.12.003 e1
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dihydrofolate reductase (dhfr) and the glutamine synthetase

(gs) genes. Selection is achieved by the introduction into cells

of the GOI along with a copy of the gene that complements

the auxotrophy. Cells are then cultivated in medium lacking

the appropriate metabolite(s) (hypoxanthine and thymidine

in the case of DHFR selection and glutamine in the case of GS

selection) so that only transformed clones survive. A com-

mon alternative to the auxotrophic selection method is the

use of genes conferring resistance to antibiotics such as

geneticin (G418), hygromycin B, zeocin, blasticidin or pur-

omycin. With this strategy, transfected cells are selected

using medium containing the appropriate antibiotic. How-

ever, a major advantage of both the DHFR and GS selection

systems is that they allow for amplification of the integrated

recombinant genes. DNA amplification, which normally

results in enhanced productivity, is achieved by exposing

selected cells to increasing concentrations of an inhibitor of

the selection protein. Methotrexate (MTX) and methionine

sulphoximide (MSX) are inhibitors of DHFR and GS, respec-

tively. To survive, cells must produce a high level of the

selection protein. This may be achieved by gene amplifica-

tions of the integrated selection gene. This also results in an

increased copy number of the GOI because it is located at the

same integration site as the selection gene.

Thepool of cells recovered after selectionis highly hetero-

geneous in terms of specific protein productivity and cell

growth. This necessitates the isolation and evaluation of

several hundreds of individual cells to recover a few candi-

date production cell lines that possess the desired character-

istics. This is usually accomplished by one or more rounds of

limitingdilution in which the selected cells aretransferredtomultiwell plates so that on average only one cell is present

per well. Each clonal cell population derived from this pro-

cedure is evaluated for the level of recombinant protein

expression and the highest producers are further studied

for the stability of recombinant protein production because

a decrease in transgene expression over time is commonly

observed in the majority of clonal cell lines. Thus candidate

production lines must be cultivated and monitored for sev-

eral months to ensure a constant level of protein expression

over time [1].

Whereas the described procedure for the generation of

recombinant cell lines is a well-established process, it remainsquite tedious and time-consuming. In an industrial setting,

the whole process usually takes more than six months. This is

particularly inconvenient especially when multiple candi-

date therapeutics need to be produced in high amounts to

be evaluated for efficacy and safety in preclinical studies or

clinical trials.

Drug Discovery Today: Technologies | Protein Therapeutics Vol. xxx, No. xx 2009




Figure 1. Schematic representation of the development of stablecell lines for the production of therapeutic recombinant proteins. Steps

that can be improved by the use of new technologies are shown on the

right. -Omic technologies can contribute to several aspects of the

process including engineering of the parental cell line, identification of

markers for screening, development of the manufacturing process and

media design. Vials represent banks of cells stored in liquid nitrogen.

Spinner flasks indicate scale-down systems for cell line evaluation and

process optimization, and bioreactors represent large-scale production

processes. Abbreviations: methotrexate (MTX), methionine

sulphoximide (MSX).

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Optimization of mammalian expression vectors

The choice of the appropriate expression vector is a crucial

factor to achieve very high and stable expression of recom-

binant proteins in mammalian cells, and nonviral expression

vectors are usually used for the generation of recombinant

cell lines. Strong promoter/enhancer sequences of viral or

cellular origin are typically used to drive expression of the

GOI, which is typically cloned as a cDNA-lacking intron

sequences. However, because the splicing process is known

to promote mRNA nuclear export and stability [2], most

expression plasmids include an intron between the promoter

and the 50-end of the cloned cDNA. Strong transcription

termination sequences added near the 30-end of the cDNA

ensure proper transcript termination. Additional optimiza-

tion of the transgene coding sequence by changing under-

represented codons, removing cryptic splice sites and increas-

ing the overall G + C content may help to improve levels of

recombinant protein expression [3].

The integration site in the host genome has a major effect

on both the level of transgene expression and the progressive

decrease in transcription over time. These two phenomena,

knownas position effect andgene silencing, arethought to be

associated with epigenetic factors resulting in the condensa-

tion and transcriptional inactivation of the chromatin at the

site of integration. Flanking of transgenes with DNA bound-

ary sequences able to block the formation of condensed

chromatin (heterochromatin) may help to obtain stable

transgene expression irrespective of the chromosomal inte-

gration site. These barrier elements include scaffold or matrix

attachment regions (S/MARs), insulators, antirepressor ele-

ments and ubiquitous chromatin opening elements (UCOEs)[4,5].

Cell culture format and media

The pharmaceutical manufacture of recombinant proteins

most frequently employs single-cell suspension cultures in

stirred-tank bioreactors of variable sizes up to 20,000 l [1,6,7].

The cells are typically maintained in media that areoptimized

for suspension growth at high cell density preferentially in

the absence of serum and other animal-derived components.

Cells may be cultivated during the entire protein production

phase without the addition of nutrient additives (batch cul-

ture). Alternatively, nutriments may be periodically added tothe culture to prolong cell viability and protein production

(extended- or fed-batch culture). In some cases, reduction of

the temperature to 30338C, increased osmolarity, or addi-

tion of histone deacetylase inhibitors such as sodium buty-

rate or valproic acid enhance protein productivity in both

batch and fed-batch processes.

Continuous perfusion is another type of process with

suspension cultures, useful for certain products that have a

high sensitivity toward degradative impacts from cell released

proteases or other physico-chemical influences. Perfused cells

are retained in a stirred-tank bioreactor while the culture

medium is exchanged several times each day by the addition

of many bioreactor volumes of fresh medium with the simul-

taneous removal of the same volume of spent medium from

the vessel. The possibility to maintain the perfused cultures

for several weeks or months at high cell densities is a major

advantage of this technology. Additionally the continuous

medium exchange allows the simultaneous recombinant

product harvest and the removal of toxic by-products.

Processes for the production of recombinant proteins from

cells attached to a surface rather than in suspension are also

used in industry [1,7,8]. However, adherent cell culture pro-

cesses are more demanding at large scales owing to the high

surface to volume ratio needed to maximize cell densities.

With roller bottles cells are grown attached to the inner wall

of the vessels filled with medium to 1030% of their nominal

volume while a slow rotation assures the regular wetting and

proper oxygenation of the cells. To increase the cell growth

surface area, adherent cells can also be grown attached to

polymer spheres (microcarriers). Microcarriers seeded with

cells are then maintained in suspension in conventional

stirred-tank bioreactors.

Future prospects for mammalian cell processes

Genomics and proteomics

Genome-scale technologies including genomics, transcrip-

tomics and proteomics are expected to contribute to the

development of mammalian cell-based production systems

[9,10]. Although high-throughput genomic and proteomic

tools are readily available for human- and mouse-derived

production hosts (such as NSO or HEK-293), the lack ofgenome sequence information for the major host (CHO cells)

has hindered the application of these technologies in the

recombinant protein production field. Early transcriptome

studies of CHO cells using DNA microarrays containing

mouse sequences have provided limited results because of

a relevant divergence between the genetic sequences of these

two rodent species. In 2006 the Consortium on CHO cell

genomics was founded with the aim of accelerating the

development of genomic resources for this host (http://

hugroup.cems.umn.edu/CHO/cho_index.html). To date this

cooperation between partners from industry and academia

has made possible the sequencing of more than 80,000expressed sequence tags (ESTs)of CHOorigin. This EST library

that includes more than 27,000 unique sequences have been

used to create tools for the transcriptome analysis of CHO

cells [11].

With further development these tools may aid in under-

standing the overall cellular physiology of this host and its

derived recombinant cell lines, eventually allowing the iden-

tification of features predictive of the level and stability of

transgene expression at production scale [12]. Such predictive

markers will be very useful in the early identification of high

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producing clonal cell lines. For example, several genes related

to cell growth, chromatin modification and protein proces-

sing and secretion have been shown to be differentially

expressed in cell lines producing high levels of recombinant

proteins [13,14]. Other -omic studies have compared host cell

responses associated with changes in culture conditions that

enhance productivity such as hypothermia, hyperosmosis

and exposure to butyric acid. Results from these and similar

studies may help in predicting how a cell line will respond to

changes in environmental conditions relevant to the bior-

eactor and thus be valuable for the optimization of process

conditions and media design [10,15]. In general it is expected

that -omic approaches will provide new insights into the

general physiology of cells that are crucial for protein pro-

duction in mammalian cells thus enhancing cell engineering

efforts to genetically redirect the cells metabolism toward

superior productivity and growth.

Host cell engineering

Many efforts have been made to genetically modify mamma-

lian cells for the purpose of improving recombinant protein

yield and quality. Host cell engineering strategies have

mainly focused on (i) control of cell proliferation; (ii)

enhancement of cellular viability; (iii) reduction of inhibitory

metabolic by-products (i.e. lactate and ammonia); (iv)

increase of protein secretion capacity; and (v) modulation

of post-translational protein modifications (i.e. glycosyla-

tion) [16,17]. Strategies to genetically modify mammalian

cells mainly rely on the overexpression of exogenous effector

genes and the downregulation or elimination of endogenous

genes. Downregulation of host gene expression is usuallyachieved by RNA interference (RNAi) or antisense RNA tech-

nology.

One of the main targets of host engineering has been the

apoptotic pathway. Overexpression of antiapoptotic proteins

such as Bcl-2 or Bcl-xL or the targeted inhibition of caspases, a

family of apoptosis-promoting proteins, has proved to be

effective in maintaining cell viability at high density [18].

Enhancement of the cellular capacity for protein secretion

through engineering of the protein folding machinery in the

endoplasmic reticulum has also been explored. However, the

overexpression of single chaperones or chaperon-like pro-

teins such as binding immunoglobulin protein (BiP) or pro-tein disulfide isomerase (PDI) has yielded mixed results [19].

Recently the overexpression of the X-box binding protein-1

(Xbp-1), a transcription factor which regulates the expression

of multiple genes involved in the unfolded protein response

(UPR), has been shown to enhance recombinant protein

production in CHO cells [20]. Strategies to reduce the amount

of lactate production during cultivation of CHO cells include

the knockdown of lactate dehydrogenase subunit A (LDH-A)

mRNA by antisense and RNAi approaches or the overexpres-

sion of the pyruvate carboxylase enzyme [21,22]. Similarly,

reduced accumulation of ammonium ions has been achieved

by the overexpression of the urea cycle enzymes carbamoyl

phosphate synthase I and ornithine transcarboxylase [23].

Variations in the glycan content of glycoproteins can signif-

icantly affect the stability, activity, immunogenicity and

pharmacokinetics of recombinant therapeutic proteins. In

this regard, engineering of the glycosylation pathways in

mammalian cells with the final aim to generate proteins with

a more humanized glycosylation profile and/or to enhance

the biological activity of therapeutic proteins has met with

some success [24].

High-throughput recombinant cell line selection methods

The traditional gene transfer, selection and amplification

methods used for the generation of stable cell lines are not

readily scalable to allow screening of more than a few

hundred cell lines. Therefore, current research focuses on

the development of high-throughput technologies with

increased screening capacities [25,26]. Particularly promising

are methods based on flow cytometry and cell sorting tech-

nologies. One elegant screening strategy employs the

cotransfection of cells with the GOI and a gene coding for

the green fluorescent protein (GFP). If the two genes are

genetically linked, a linear correlation between fluorescence

intensity and expression of the GOI is usually seen. Single

clones with high level of GFP expression are then recovered

by fluorescence-activated cell sorting (FACS) [27]. Coexpres-

sion of a nonfluorescent cell surface protein that can be

labeled with a fluorescent antibody before screening by FACS

is an alternative strategy [28]. Strategies to directly measure

the level of the recombinant protein rather than a reporterprotein are expected to be even more effective in the screen-

ing of high producing cell lines. However, because most of

the recombinant proteins produced in mammalian cells are

secreted, techniques to keep the recombinant protein in the

vicinity of the producing cell are required. This can be

accomplished by embedding individual recombinant cells

in a gel or semisolid matrix to limit the diffusion of the

secreted product and allow its detection using standard

immunohistochemical methods [29]. Automation of these

new screening methods will definitely reduce the time

needed to generate recombinant cell lines and increase the

probability of recovering high producing clones.

Transient gene expression

Transient gene expression (TGE) is a relatively new technol-

ogy that is being considered for the production of recombi-

nant proteins at large scale [30]. A key advantage of this

technology is thespeed by which significant amounts (grams)

of recombinant protein can be produced. Thus large-scale

TGE has attracted interest for the rapid production of recom-

binant proteins for basic research and preclinical studies.

Usually, the protein production phase for TGE lasts up to







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14 days following transfection, avoiding the time-consuming

process of generating stable clonal cell lines. The recombi-

nant gene is usually cloned in a nonviral expression vector

and transfected into single-cell suspension cultures by means

of a chemical delivery agent such as polyethylenimine (PEI).

To date, the largest volumetric scale for TGE has been about

100 l [31]. TGE has been performed in stirred-tank bioreac-

tors, orbitally shaken containers and wave-type bioreactors.

Whereas several host cell lines have been used for TGE, CHO

and HEK-293 cells are preferred owing to their high transfect-

ability and the ability of these cells to support high-density

growth in suspension [32,33]. Recently, volumetric yields of

recombinant protein comparable to those obtained for opti-

mized processes with stable lines have been achieved, under-

lining the potential of TGE as an economic alternative for the

manufacture of low-dose therapeutic proteins at moderate

volumetric scales [34].

Conclusions

Despite recent progress, recombinant protein expression

technology using mammalian cells is still in its infancy.

Much remains to be done to enhance the productivity of

production processes. In highly optimized fed-batch manu-

facturing processes yields of several grams of per liter of

secreted protein are frequently attained today for antibody

products and some other well-behaved proteins, a 10100-

fold increase compared to the yields that were obtained two

decades ago. This increase in volumetric productivity has

been primarily achieved through optimization of the manu-

facturing process by improvements in media composition,nutrient feeding strategies, cell line development and cell

screening strategies. However, even with these impressive

improvements in yield, opportunities still exist to further

enhance production levels. Yields of 1020 g/l in fed-batch

processes are expected to be reached in the near future. This

may be primarily achieved by an increase in viable cell

densities and duration of the culture process through opti-

mization of the culture conditions. The cell concentrations

for batch and extended-batch processes typically peak at

about 10 million cells/ml, meaning the cell biomass only

occupies about 34% of the volume of the culture. By contrast

microbial cultures may reach a biomass of 2030% of theculture volume.

The current strategies for the development of cell lines for

the recombinant expression of therapeutic proteins remain

empirical to a large extent. This is mainly due the lack of

consistent methods to predict production capacity and

growth characteristics of clonal cell lines at production scale.

In this regard the knowledge gained from -omic studies is

expected to provide a much better understanding of the

biochemistry and physiology of mammalian cells so that

higher product yields and quality may be achieved through

genetic engineering of the host or by a more rationale opti-

mization of the culture conditions.

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