Best Practices in Nucleic Acid Removal from Vaccine Processes 疫苗工艺中去除核酸的最佳方法

Dr. Priyabrata Pattnaik Director – Asia Vaccine Initiative

Agenda 日程

DNA removal needs and regulatory position DNA去除需求和法规立场 1

2 Nucleic acid removal by adsorptive depth filter 使用深层吸附过滤器，去除核酸

Nuclease treatment 核酸酶处理 3

4 Methods for removal and detection of residual nuclease 去除和检测残留核酸酶的方法

Chromatography based removal of nucleic acid 层析法去除核酸 5

6 Tangential flow filtration for DNA removal 切向流过滤去除DNA

Summary 总结 7

Vaccine and DNA 疫苗和DNA

• Viral vaccines and biological products contain contaminating residual DNA from cell substrate •病毒疫苗和生物制品含有来自细胞基质的污染性残留DNA

• WHO Expert Committee on Biological Standardization says................. “DNA considered as cellular contaminant rather than risk factor which requires removal to extremely low levels” • WHO生物标准化专家委员会说................. “DNA视为细胞污染物，而非需要去除至极低水平 的风险因素”

• The amount of residual cell-substrate DNA in a vaccine will depend on the vaccine and the manufacturing process •疫苗中的残留细胞基质DNA的量，将取决于疫苗和生产过程

• DNA makes downstream processing difficult (viscosity, complex formation) • DNA使下游工艺处理困难（粘性，形成复合体） 3

Regulatory requirement on Purity and Safety - Residual DNA content 纯度与安全的法规要求 – 残留DNA含量

10 ng/dose 10 ng/剂

WHO 1998

100 pg/dose 100 pg/剂

WHO 1987 Vero* and MDCK*

Based Viral Vaccine 基于Vero*和MDCK*的病毒疫苗

40 pg/dose 40 pg/剂

Per.C6 Adeno-HIV 腺HIV

10 pg/dose 10 pg/剂

HepB (CHO) EU Pharmaco

欧洲药典

* Non tumerigenic at the passage of production. * 在生产环节无致瘤。 * DNA <10 ng/dose commonly accepted * 通常接受DNA <10 ng/剂

EMEA position on tumerigenic cells of human origin DNA as low as possible with risk assessment study EMEA关于人源性致瘤细胞的立场 风险评估研究，DNA尽可能低

FDA: Case by case Reduce size (<200bp)

and amount (<10ng/dose) FDA：逐个案例 降低大小(<200bp) 和数量(<10ng/剂)

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How much nucleic acid removal is “good enough”? 核酸去除到何种程度，才“足够好”？

Adenovirus-specific regulatory guidance: 10 ng would only be acceptable provided that the DNA was digested to less than 100-200 base pairs in length[1]

腺病毒特异性管理指导：仅当DNA被消化到长度小于100-200碱基对时，才可接受10 ng [1]

Adenoviruses are typically produced at about 104-105 viral particles (vp)/cell[2]

生产出的腺病毒通常约为104-105病毒颗粒(vp)/细胞 [2]

Mammalian cells have a genome of about 10 pg/cell[3]

哺乳动物细胞的基因组约为10 pg/细胞 [3]

Assuming 2x106- 8x108 cell/ml; 20µg - 8 mg DNA /ml at virus harvest

假定：2x106- 8x108细胞/ml； 病毒收获时，20µg - 8 mg DNA /ml

7 logs of DNA clearance would be required in order to attain levels below 100 pg/dose for a high (1012 vp) dose of adenovirus.

对于高剂量(1012 vp)的腺病毒，为了获得低于100 pg/剂的水平，DNA需减少7个数量级。

[1] Bauer et al., Testing of Adenoviral Vector Gene Transfer Products: FDA Expectations. In Adenoviral Vectors for Gene Therapy; Curiel, D. T., Douglas, J. T., Eds.; Academic Press: New York, 2002; pp 615-654. [2] Nadeau and Kamen. Production of adenovirus vector for gene therapy. Biotechnol. Adv. 2003, 20 (7-8), 475-89. [3] Kraiselbuld et al., Presence of aherpes simplex virus DNA fragment in a L cell clone obtained after infection with irradiated herpes simplex virus 1. J. Mol. Biol. 1975, 97, 533-542.0

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How to remove residual DNA 怎样去除残留DNA Precipitation (Acid/base treatment, organic solvent) 沉淀（酸/碱处理，有机溶剂）

- Ex. Conjugated polysaccharide vaccine - 实例：共轭多糖疫苗 Treatment by β-propiolactone (BPL) β-丙内酯(BPL)处理

- Ex. Killed viral vaccine - 实例：死病毒疫苗 Adsorptive Depth Filters 吸附深度过滤器

- Inactivated Polio - 灭活脊髓灰质炎 Chromatographic methods 层析方法

- Bind and elute (chromatography media) - 结合与洗提（层析介质） - Flow Through (membrane absorber) - 流经（膜吸附器） Nuclease treatment 核酸酶处理

- HepA, LAIV, Rabies, HPV - HepA、LAIV、狂犬病、HPV Tangential Flow Filtration (TFF) 切向流过滤(TFF)

- Several vaccines - 若干种疫苗

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Nucleic Acid Removal by Adsorptive Depth Filtration 采用深层吸附过滤，去除核酸

DNA removal by depth filtration (Millistak+®) mostly by adsorption-based retention mechanism 深层过滤 器(Millistak+®) 去除DNA，主要依靠基于吸附的截留机理

– Attraction forces between particles and filter material

– 颗粒与过滤材料之间的吸引力 – DNA is adsorbed by a combination of

electrostatic and hydrophobic interaction

– 通过静电相互作用和疏水性相互作用，吸收DNA

– Not size-dependent – 与尺寸无关 – Adsorptive capacity is limited and

“breakthrough” eventually occurs – 吸附能力有限，最终发生“穿透” – DNA adsorption depends on solution

composition. pH and conductivity plays a major role

– DNA吸附取决于溶液组成。pH和电导率起重要作用

– DNA reduction: 1 - 2 log – DNA减少：1 – 2个数量级

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Cell based flu clarification by Millistak+®D0HC 使用Millistak+®D0HC，对细胞培养的流感疫苗进行澄清 Performance on HA yield, DNA removal rate and Sterile filter capacity 性能：HA收率、DNA去除率和无菌过滤能力

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9 Millistak+® D0HC can show 50-90% removal of DNA

Millistak+® D0HC可显示50-90%的DNA去除率

Nuclease Treatment 核酸酶处理

FDA briefing document on cell line derived vaccines FDA简报：从细胞系得到的疫苗

http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/VaccinesandRelatedBiologicalProductsAdvisoryCommittee/UCM319573.pdf

……..Benzonase® digestion for live vaccines can reduce the infectivity of DNA by more than 100,000 fold ……. …….. 活疫苗进行Benzonase®消化，可使DNA传染性减少 100,000倍以上…….

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Benzonase® Endonuclease Benzonase®核酸内切酶

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Genetically engineered endonuclease that cleaves all forms of DNA and RNA. 基因工程制造的核酸内切酶，可使所有形式的DNA和RNA断裂。 Origin: Serratia marcescens 来源： 粘质沙雷氏菌 Expression: E.coli K -12 mutant 表达： 大肠杆菌K -12突变株 Molecular mass: ca. 30 kD (subunit, exist as dimer) 分子质量： 大约30 kD（亚单位，作为二聚物存在） Isoelectric point (pI): 6.85 等电点(pl)： 6.85 Functional in pH range: 6–10 具有功能的pH范围： 6–10 Temperature: 0 - 42ºC 温度： 0 - 42ºC Presence of Mg2+ (1-2 mM) is required for enzyme activity. 为维持酶活性，需存在Mg2+ (1-2 mM) 。 One unit of Benzonase® degrades approximately 37mg DNA in 30 min to as low as 3-8 base pairs (<6 kDa). 一个单位的Benzonase®，在30 min内，可将大约37mg DNA降解到低达3-8个碱基对(<6 kDa) 。

Mixture of pLAI DNA and uninfected Jurkat DNA in equal amounts was digested with Benzonase® at 30 °C.等量的pLAI DNA与未受感染的Jurkat DNA的混合物，在30 °C下，用Benzonase®消化。 DNA was purified, analyzed by 1.8% agarose-gel electrophoresis 用1.8%琼脂糖凝胶电泳，纯化、分析DNA 1.5 μg of each time point was transfected into 293T cells followed by co-culture with Jurkat cells. 将每个时间点的1.5 μg DNA转染到293T细胞，然后与Jurkat细胞共培养。 Virus prodn was detected by RT activity and virus prodn from each sample. 用RT活性，检测病毒prodn，检测每个样品的病毒prodn。

SOURCE: Sheng-Fowler et al. (2009) Biologicals, 37(4): 259-269. Division of Viral Products, CBER, FDA

Benzonase® is effective within 4 min Benzonase®在4分钟内生效 Effect of Benzonase® digestion on the infectivity of DNA Benzonase®消化对DNA传染性的影响

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Benzonase® is widely recognized……. Benzonase®受到广泛认可……

...........For Vero cell–produced vaccine, nucleic acid can be reduced in size by treatment with Benzonase® then removed by ultrafiltration using a 50,000 MW membrane or removed by ion-exchange chromatography. It is not necessary to incorporate steps to remove nucleic acid from vaccine produced on diploid cells.......

...........对于Vero细胞生产的疫苗，用Benzonase®处理，可降低核酸的大小，然后使用50,000 MW膜超滤去除，或用离子交换层析去除。不必加入步骤以从二倍体细胞生产的疫苗中去除核酸…… (Page 21)

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Purification of H1N1 (deltaFLU) vaccine H1N1 (deltaFLU) 疫苗的纯化

Adopted from source: Thomas Muster, deltaFLU–A new generation live attenuated vaccine againt influenza, World Vaccine Congress Asia 2010, Singapore. and Elisabeth Röthl, The challenge to purify live influenza virus, Avir Green Hills Biotechnology 15

Benzonase® can prevent Virus-DNA complex formation during purification Benzonase®可防止在纯化时形成病毒－DNA复合体

Fractogel® SO3¯

SOURCE: Chahal et al., Journal of Virological Methods 139 (2007) 61–70.

Adeno-assocuated virus (AAV) and DNA can form aggregates, since there is a net positive charge on AAV at pH 7.5 and negative on DNA 腺病毒相关病毒(AAV) 可与DNA形成聚集体，因为在pH 7.5时，AAV带净正电荷，而DNA带负电荷。 Digesting cellular DNA in by adding Benzonase® in lysis buffer prevents binding of DNA to AAV during and after the cell rupture step 向细胞溶解缓冲液中加入Benzonase® ，以消化细胞DNA，防止DNA与AAV在细胞破裂步骤期间或之后结合 Lysis Buffer: 50mM Tris, 2mM MgCl2 and 5U of Benzonase®/million cells at pH 7.5 细胞溶解缓冲液：50mM Tris，2mM MgCl2 ，5U Benzonase®/百万细胞，pH 7.5

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Benzonase® activity optimization Benzonase®活性优化

Temperature 温度

Magnesium Ions 镁离子

pH

Monovalent Cations 一价阳离子

Detergents 洗涤剂

SDS

Urea 尿素

Phosphate Ions

磷酸盐离子

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Removal and Detection of Residual Benzonase®

去除和检测残留Benzonase®

Benzonase® is not an API or Excipient. Like any other process additives, Benzonase® need to be removed from the vaccine process. Benzonase®并非API或赋形剂。 Benzonase®和其他工艺添加剂一样，需从疫苗工艺中去除。

Removal of Benzonase®

去除Benzonase®

Flow Through TMAE or DMAE Fractogel® pH 7- 8 , 50 – 200 Mm Nacl , 50 mM Tris 流经 TMAE或DMAE Fractogel® pH 7- 8 ，50 – 200 Mm Nacl ，50 mM Tris

Benzonase® is not or only weakly bound to anion exchange resins under a variety of conditions; pH 7.0 – 9.0 at 50 mM NaCl; different Fractogel® anion exchangers 在多种条件下，Benzonase®不与阴离子交换树脂结合，或仅微弱结合；pH 7.0 – 9.0，50 mM NaCl；不同的Fractogel®阴离子交换剂

Benzonase® elutes from cation exchange resins below 200 mM NaCl at pH 6.0 and is not bound to weak cation exchange resin at pH 6.0 pH 6.0，< 200 mM NaCl时，Benzonase®从阳离子交换树脂洗提； pH 6.0时，Benzonase®不与弱阳离子交换树脂结合 Ultrafiltration 500 kDa Biomax® membrane 超滤500 kDa Biomax®膜 Retains Viral Particle 截留病毒颗粒 Diafilter out Benzonase® and small nucleic acid base pairs 渗滤出Benzonase® 和小的核酸碱基对

Reference: Yi Lu et al, Development of Economic Production Platform for Live Attenuated Influenza Vaccine. IMVAC Aug 2009. 19

Process step

工序

Total Benzonase®

input (ng) 总

Benzonase®

输入(ng)

Total Benzonase® output (ng)

总

Benzonase®

输出(ng)

Benzonase® Clearance

Benzonase®

清除

Anion Exchange阴离子交

换

3 0.006 98%

Gel Filtration 凝胶过滤

3.9 0.12 97%

Samples were assayed using the Benzonase® ELISA Kit II 使用Benzonase® ELISA试剂盒II，测定样品

• Solid line shows UV at 280 nm • 实线表示280 nm的紫外线 • Solid columns represent the Benzonase® concentration 实心柱代表Benzonase®浓度 • Arrows indicate the target Ad5-GFP peaks • 箭头指出目标Ad5-GFP峰

SOURCE: Eglon et al., Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography. J Gene Med 2009; 11: 978–989. 20

Benzonase® clearance by AIEX and GF 使用AIEX和GF，清除Benzonase®

Detection systems for Benzonase® Benzonase®的检测系统

Semi-quantitative assay for the detection of residual activity of nucleases (digestion of salmon-sperm, photometric measure of cleavage products, LLOQ ~ 1 U/mL, Merck Millipore monograph) 探测核酸酶残留活性的半定量测定（消化鲑鱼精子，分裂产物的光度测量，LLOQ ~ 1 U/mL，默

克密理博专题论文） Electrophoretic test (based on a publication by Nycomed) 电泳试验（基于Nycomed的一份出版物） Fluorescence test (see publication by MedImmune, R. Strouse et. al., BioPharm April 2000;

LLOQ ~ 0.001 U/mL) 荧光试验（参见MedImmune的出版物，R. Strouse et. al., BioPharm April 2000; LLOQ ~ 0.001

U/mL ） Benzonase® standard activity assay (volume activity measuring Benzonase® activity of the

active Benzonase®) Benzonase®标准活性测定（活性Benzonase® 的体积活性测量） ELISA II by Merck Millipore (is measuring Benzonase® protein concentration, this does not

correspond to activity) ELISA II，由默克密理博执行（测量Benzonase®蛋白质浓度，该浓度与活性并不对应） As the EU patent of HAV vaccine (VAQTA®) of Merck & Co. indicated, the residual Benzonase® is lower than 0.0001ng per dose. 正如Merck & Co. 的HAV疫苗(VAQTA®) 的欧洲专利所指出，残留Benzonase®低于每剂0.0001ng。

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Benzonase® ELISA Kit II Benzonase® ELISA试剂盒II

Description: Immunological detection of Benzonase®

说明：免疫学检测Benzonase® Sensitivity: ca. 0.2 ng/ml Benzonase® (0.2

ng/ml (correspond to < 1ppm in the presence of other proteins at conc. > 0.5mg/ml.)

灵敏度：约0.2 ng/ml Benzonase® (0.2 ng/ml （相当于：存在浓度 > 0.5 mg/ml的其他蛋白质时，< 1ppm）

Validation: Test method is validated 验证：试验方法经过验证

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Nucleic Acid Removal by Chromatography 层析法去除核酸

1. Bind and elute based removal (packed bed chromatography) 基于结合与洗提的去除（填充床层析） 2. Flow-through chromatography (membrane absorber) 膜层析法（膜吸附器）

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Bind & elute based process 基于结合与洗提的工艺 Example: Adenovirus purification 实例：腺病毒纯化

Kamen and Henry, Development and optimization of an adenovirus production process, J Gene Med 6, S184–S192, 2004

Adenovirus production 腺病毒生产

Harvest 收获

Liquid 液体

Cell lysis细胞溶解

Benzonase® treatment/ Centrifugation Benzonase®处理/离心

Anion Exchange Chromatography on Fractogel® DEAE media 阴离子交换层析，Fractogel® DEAE介质

Solid 固体

Filtration 过滤

Ultrafiltration/ Concentration 超滤/浓缩

Ret

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滞留物（腺病毒）

Size Exclusion Chromatography 尺寸排除层析

Purified Adenovirus 纯化的腺病毒

Performance level of different Fractogel® media for DNA removal from rabies vaccine 不同的Fractogel®介质，从狂犬病疫苗中去除DNA的性能水平

SOURCE: Method for Purifying the rabies virus, Patent-US2010/0260798A1, Date: Oct 14. 2010 (Sanofi Pasteur)

®

®

®

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Flow-through based chromatography for DNA removal using ChromaSorbTM membrane adsorber 膜层析法去除DNA，使用ChromaSorbTM膜吸附器

0.08 mL

50 mL

Polyethelyene (0.65µm) 聚乙烯(0.65mm) Positively Charged gel (PAA) 带正电荷凝胶(PAA)

500 mL

8 layers of membrane 8层膜 Membrane bed volume = 0.0798mL 膜床层体积 = 0.0798mL

Virus-DNA separation using ChromaSorbTM membrane adsorber in flow-through mode 流经模式，使用ChromaSorbTM膜吸附器，分离病毒－DNA

Feed 进料

MDCK cell culture Influenza A/WS (10KHAU/ml), DNA (1-2µg/ml) in Buffer 在缓冲液中，MDCK细胞培养流感 A/WS

(10KHAU/ml)，DNA (1-2µg/ml)

Pure Virus 纯病毒

Most DNA bound 结合大部分DNA

100% DNA removal corresponds to <10ng of whole DNA 100% DNA去除，相当于<10ng 完整DNA

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• Complete flow-through of virus in presence of multivalent ions • 存在多价离子时，病毒全部流经 • High capacity for DNA and high throughput (~187CV) • 高DNA容量和高处理量(~187CV) 27

Nucleic Acid Removal by Tangential Flow Filtration (TFF) 切向流过滤（TFF）去除核酸

Clearance of Benzonase® digested DNA across TFF (Pellicon® 2, Biomax® 500kDa) 通过TFF (Pellicon® 2, Biomax® 500kDa)去除用于消化DNA的Benzonase®

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1 2 3 4 5 6 7 8 9 Various UF samples 不同的UF样品

Lane 1 – Marker (100 BP) 道1 – 标志物(100 BP) Lane 2 – Undigested DNA in

Feed 道2 – 进料中的未消化DNA Lane 3 – After Benzonase®

digestion 道3 – Benzonase®消化后 Lane 4 – Post Recirc

retentate 道4 – 再循环后的滞留物 Lanes 5, 6, 7, 8 – Retentate

samples after 1, 3, 5, 8 DV 道5、6、7、8 – 在1、3、5、

8 DV后的滞留物样品 Lane 9 – Permeate at 5DV 道9 – 在5DV时的渗透物

Diafiltration of Residual Benzonase®

残留Benzonase®的透析

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99.5% clearance at 5 diavolumes and > 99.9% (3 log) clearance after 8 diavolumes across the UF/DF step 通过UF/DF步骤，在5倍透析体积时，达到99.5%清除；在8倍透析体积时，达到> 99.9% (3 log) 清除

Summary 总结

There are multiple methods for DNA removal from vaccine processes 从疫苗工艺去除DNA的方法有多种 Adsorptive depth filter (Millistak+ ®) can also remove nucleic acid from vaccine

process 深层吸附过滤器(Millistak+ ®) 也可从疫苗工艺去除核酸 Benzonase® is the proven endonuclease for digestion of nucleic acid in vaccine

processes Benzonase®是可靠的核酸内切酶，用于疫苗工艺中的核酸的消化 Optimization of reaction conditions using Benzonase® is critical for success of

DNA digestion 使用Benzonase® 来优化反应条件，关键在于成功地消化DNA Combination of Chromatography (Fractogel®) and TFF (Pellicon® 2) is good

enough for removal of residual DNA and residual Benzonase®

层析(Fractogel®)和TFF (Pellicon® 2)的组合，足以去除残留DNA和残留Benzonase® Multiple analytical methods (Benzonase® ELISA Kit II) are available for

quantization of residual Benzonase® in final product 有多种分析方法(Benzonase® ELISA试剂盒II)，可对最终产品中的残留Benzonase®

进行量化 31

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Thank you 谢谢各位