development of a production and purification platform for vlp vaccine candidates

Priyabrata Pattnaik, PhDDirector – Worldwide Vaccine InitiativePriyabrata Pattnaik 博士全球疫苗计划-主任

DEVELOPMENT OF A PRODUCTION

AND PURIFICATION PLATFORM FOR

VLP VACCINE CANDIDATES

VLP 疫苗生产和纯化平台的开发

Production & Purification Platform for VLP Vaccine | Priyabrata Pattnaik | 8th China Human Vaccine Industry Summit | 26 June 2016 | Xi'an, China.| Priyabrata Pattnaik | 8 | 2016 26 | VLP疫苗生产和纯化平台博士第届中国人用疫苗行业峰会年6月日中国西安

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Presentation Outline 报告讲述要点

VLP as vaccines 作为疫苗的VLP（病毒样颗粒）

Baculovirus / insect cell expression platform 杆状病毒/昆虫细胞表达平台

Challenges in VLP vaccine production and purification VLP疫苗生产和纯化面临的挑战

VLP production in insect cell culture using single use bioreactor 使用一次性生物反应器采用昆虫细胞培养技术进行VLP生产

Clarification of VLP VLP的澄清

Concentration / Diafiltration of VLP VLP的浓缩/透析

Chromatographic purification of VLP VLP的层析纯化

Summary 总结

VLP vaccine candidates have become quite popular of late VLP疫苗目前十分流行

VLP-based processes are, however, currently quite diverse 但是基于VLP的工艺目前仍千差万别

We undertook an effort to standardize the process 我们努力使该工艺标准化

We used hepatitis C VLP as a model 我们使用C型肝炎（丙肝）VLP作为范例

This presentation will explain the approach taken and present the results obtained 本报告将说明所采取的方法，并阐述所获得的结果

Motivation 目的

4Production & Purification Platform for VLP Vaccine | Priyabrata Pattnaik | 8th China Human Vaccine Industry Summit | 26 June 2016 | Xi'an, China. VLP疫苗生产和纯化平台 | Priyabrata Pattnaik博士 | 第8届中国人用疫苗行业峰会 | 2016年6月26日 | 中国西安

Contain repetitive high-density displays of viral surface proteins that elicit strong T cell and B cell immune responses 含有可重复高密度展现的病毒表面蛋白，可引发强烈的T细胞和B细胞免疫反应（应答）

Non infectious because they do not contain genetic material, thus cannot replicate and are safer 不会传染，因为它们不含有遗传物质，因此不能复制，更加安全

Their size (40-120 nm diameter) is optimal for uptake by dendritic cells 其大小（直径40-120nm）十分有利于通过树突细胞摄取

Can be produced in a variety of cell culture systems 可在各式各样的细胞培养系统中生产

Can self assemble in vivo 可在活体内自组合

Proven technology (Hepatitis B and Human Papilloma Virus vaccines) 成熟技术（B型肝炎（乙肝）疫苗和人乳头瘤病毒疫苗）

Why virus-like particles (VLPs)? 为什么使用病毒样颗粒（VLP）？

Source 来源 : Roldão et al., Expert Reviews in Vaccines 9 (10), 1149-76 (2010)

Production & Purification Platform for VLP Vaccine | Priyabrata Pattnaik | 8th China Human Vaccine Industry Summit | 26 June 2016 | Xi'an, China. | Priyabrata Pattnaik | 8 | 2016 26 | VLP疫苗生产和纯化平台博士第届中国人用疫苗行业峰会年6月日中国西安

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Hepatitis C C型肝炎（丙肝）

170 million people infected 1亿7千万人口受感染

Cirrhosis, liver cancer, death肝硬化、肝癌、死亡

Current therapies only partially effective, costly and poorly tolerated 当前的治疗方法只部分有效，而且治疗费用昂贵，不易耐受

No vaccine currently exists 目前尚未有对应的疫苗

VLPs for hepatitis C vaccine development

E1 and E2 glycoproteins from Hep C virusC型肝炎病毒（丙肝）的E1糖蛋白和E2糖蛋白

Capsid and structure VLP from retrovirus (murine leukemia

virus)逆转录酶病毒（鼠白血病病毒）的衣壳和

结构VLP

Production & Purification Platform for VLP Vaccine | Priyabrata Pattnaik | 8th China Human Vaccine Industry Summit | 26 June 2016 | Xi'an, China. | Priyabrata Pattnaik | 8 | 2016 26 | VLP疫苗生产和纯化平台博士第届中国人用疫苗行业峰会年6月日中国西安6

Recombinant baculovirus (BV) is used to infect insect cells

重组杆状病毒（BV）可用于感染昆虫细胞

Key features 主要特性

Transient production 短暂性生产

High cell densities 高细胞密度

Regulatory acceptance 符合法规要求

Cervarix® (GSK) 卉妍康® （GSK）

Flublok® (Protein Sciences) （蛋白质科学）

Several late clinical stage pipeline

几个最近的临床阶段的疫苗

Insect cell / baculovirus VLP production platform昆虫细胞/杆状病毒VLP生产平台

~120 nm

VLPBV

60-80 nm


Low production yields 产品收率低

Stability of enveloped VLPs 包膜VLP的稳定性问题

Difficulties in baculovirus (BV) removal lowers recovery 杆状病毒（BV）清除难度大，从而降低了产品回收率

No established platform processes for purification 尚未建立纯化的平台工艺

Challenges in VLP vaccine production VLP疫苗生产面临的挑战

VLP

BV


Work carried out in collaboration with iBET 与iBET密切合作开展工作

iBET: Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal iBET：葡萄牙奥埃拉斯生物实验技术研究所


Typical VLP-based vaccine process 典型的基于VLP的疫苗工艺Insect cell / baculovirus VLP production platform 昆虫细胞/杆状病毒VLP生产平台

UF/DF BaculovirusInactivation 杆状病毒灭活

PurificationChromatography

纯化层析

Media and Inoculum Preparation

培养基和接种准备

Cell growth in Bioreactor and

Virus Inoculation 生物反应器中的细胞生长和病毒接种

BioburdenReduction 降低生物负载

Clarification 澄清

Sterile Filtration无菌过滤

PolishingChromatography

精制层析

UF/DF



UF/DF BaculovirusInactivation




Virus Inoculation生物反应器中的细胞生长和病毒接种

BioburdenReduction

Clarification

Sterile Filtration


UF/DF


Cell culture was carried out in stirred tank glass bioreactor and disposable bioreactor (Mobius® 3L bioreactor) 细胞培养在搅拌罐玻璃生物反应器和一次性生物反应器（ Mobius® 3L生物反应器）中进行

Sf9 insect cells and Sf-900 II cell culture media were used in the process 本工艺使用Sf9昆虫细胞和Sf-900 II细胞培养基

Mobius® 3L bioreactor was first operated at same conditions previously used for stirred tank glass bioreactors

Mobius® 3L生物反应器首先工作在与先前用于搅拌罐玻璃生物反应器完全相同的条件下

Cell aggregation 细胞积聚

Formation of foam 形成泡沫

Longer lag phase 迟滞期更长

Lower viable cell concentration 活细胞浓度更低

Insect cell culture 昆虫细胞培养


Increased agitation rate 提高搅拌速度

Increased cell density of inoculation 提高细胞接种密度

Replaced micro sparger with an open-pipe sparger 用开管式喷头代替微喷头

Insect cell culture conditions improved based on experience with Mobius®

3L bioreactor 使用Mobius® 3L生物反应器根据经验改进昆虫细胞培养条件

0

20

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100

0.0

0.5

1.0

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0 24 48 72 96 120 144

Viab

ility

(%)

Viab

le Ce

ll Con

cent

ratio

n (10

6ce

lls/m

l)

Time (h)

STR2 CR2 CR3 CR4Viab STR2 Viab CR2 Viab CR3 Viab CR4

GAG-MLV (core protein)（核心蛋白）

HCV-E1 (envelope protein)

（包膜蛋白）

HCV-E2 (envelope protein)

（包膜蛋白）

VLP HepC V


Scale-up to Mobius® 50L Bioreactor 放大至Mobius® 50L生物反应器

Growth kinetics of Sf9 cells in the Mobius® 50 L and 3 L bioreactors. The black arrow indicates the infection of the bioreactors and control shake-flask. Sf9细胞在Mobius® 50 L和 3 L 两种生物反应器中的生长动力学曲线。黑色箭头表示生物反应器和控制摇瓶的感染情形。

GAG-MLV titre in culture bulks of Mobius® 50L and 3L and in glass strirred tank bioreactor (Glass-STR). Mobius® 50L和3L生物反应器的培养液中的GAG-MLV滴定度以及在搅拌罐玻璃生物生物反应器（ Glass-STR ）中的GAG-MLV滴定度


Microscopic evaluation of cells 细胞的微观评价Pictures of the Sf9 cells cultivated in Mobius® 3 L and 50 L at different time points 在Mobius® 3 L and 50 L生物反应器中培养的Sf9细胞在不同时间点的照片


Western blot analysis of VLPs produced in Mobius® 50L Bioreactor在Mobius® 50L生物反应器中生产的VLP的蛋白质印迹分析

Western-Blot analysis of the kinetics of Gag-MLV and HCV-E1/E2 expression in VLP-HCV.VLP-HCV中Gag-MLV和HCV-E1/E2表达的动力学的蛋白质印迹分析A. Comparison of Gag-MLV and HCV-E1/E2 expression in the pure VLP-HCV samples obtained by

sucrose ultracentrifugation from the culture bulk.对采用蔗糖梯度超速离心方法从培养液中获得的纯VLP-HCV样品中Gag-MLV和HCV-E1/E2表达进行比较

B. Samples of supernatant were collected at 24 hpi, 48 hpi, 72hpi and at the harvesting of the bioreactors (TOH). 上层清夜的样品在24 hpi、48 hpi、72hpi时以生物反应器（TOH）的收率进行收集

VLP-HCV production kinetics in Mobius® 50 L BioreactorMobius® 50 L生物反应器中的VLP-HCV生成动力学曲线


Successful growth of Sf9 insect cells and infection with baculovirus for production of VLP vaccine using Mobius® 3L and 50L disposable bioreactors 使用Mobius® 3L和50L一次性生物反应器生产VLP，成功实现了Sf9昆虫细胞的生长以及与杆状病毒的感染

Comparable cell and VLP properties between disposable and glass bioreactors 比较一次性生物反应器和玻璃生物反应器之间所表达的细胞和VLP的性质

Reproducible performance of the disposable bioreactors was seen with identical results for three separate cell culture runs 从三个独立的细胞培养运行过程来看，它们都获得了相同的结果，表明这些一次性生物反应器具有可复制性能

Linear scale-up of process from 3L to 50L in single use bioreactors with identical performance 使用一次性生物反应器将工艺从3L线性放大到50L，并具有相同的性能

Successful use of Mobius® 3L & 50L bioreactor for VLP production 已成功使用Mobius® 3L和50L生物反应器进行VLP生产







Virus Inoculation

BioburdenReduction

Clarification澄清

Sterile Filtration


UF/DF


Depth filtration 深层过滤

Well suited for smaller vaccine batches 更适合于较小批量的疫苗产品

Easier to scale 更易于放大

Lower cost 成本更低

Disposable 一次性

Gentle treatment 更易于处理

Simpler process development 工艺开发更简单

Wide choice of depth filters 深层过滤器选择范围更广

Centrifugation 离心分离

Lab models used early on 早期主要用于实验室模式

Well suited for large-scale production 更适合于大规模生产

High capital expense 资金费用较高

Shear 有剪切力



Clarification: throughput data 澄清：载量数据

Disposable capsule filters 一次性囊式过滤器

Polygard® CN, nominal pore sizes of 10, 5, 0.6 and 0.3 μm Polygard® CN过滤器，标称孔径10、5、0.6和0.3 μm

Pleated, all-polypropylene depth filters 折叠式全聚丙烯深层过滤器

Filter area: 17 cm2; Inlet flux: 988 LMH 过滤面积：17 cm2；入口通量：988 LMH

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Polygard CN filter train 10 5 0.6 μm

10 μm5 μm0.6 μm

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Polygard CN filter train 5 0.3 μm

10 μm5 μm5 µm0.3 µm


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Clarification: recovery data 澄清：收率数据

Unlike centrifugation, depth filtration resulted in ~70% DNA clearance不像离心分离工艺，深层过滤将导致~70%的DNA清除

0% 20% 40% 60% 80% 100%

10 μm → 5 μm → 0.6 μm

10 μm → 0.6 μm

5 μm → 0.6 μm

5 μm → 0.3 μm

CFG → 0.6 μm

CFG → 0.3 μm

CFG

VLP recovery

HepC VLP clarification







Virus Inoculation

BioburdenReduction

Clarification

Sterile Filtration


UF/DF

22 Production & Purification Platform for VLP Vaccine | Priyabrata Pattnaik | 8th China Human Vaccine Industry Summit | 26 June 2016 | Xi'an, China. VLP疫苗生产和纯化平台 | Priyabrata Pattnaik博士 | 第8届中国人用疫苗行业峰会 | 2016年6月26日 | 中国西安

Pellicon® cassettes Pellicon® 膜包

Two different ultrafiltration membranes 两种不同的超滤膜

300 kD composite regenerated cellulose (Ultracel® membrane, “CRC”)

300 kD复合再生纤维素（ Ultracel®滤膜，“CRC”）

100 kD polyethersulfone (Biomax® membrane, “PES”)

100 kD聚醚砜（ Biomax®滤膜，“PES” ）

Similar process conditions employed 使用相同的工艺条件

4-5x concentration factor

4-5x浓度倍数

Loading: 72 L/m2; Feed flux: 480 LMH; TMP: 1 bar; Pfeed: 0.6-0.9 bar; Pretent: 1.1-1.4 bar

载量：72 L/m2；进液通量：480 LMH； TMP：1 bar；Pfeed：0.6-0.9 bar；Pretent：1.1-1.4 bar

Concentration of clarified VLP harvest 澄清后的VLP收获物的浓缩


Concentration of clarified VLP harvest – results 澄清后的VLP收获物浓缩—结果

90%

35%

85%80%

28%

91%

58%

90%96%

38%

0%

25%

50%

75%

100%

HCV-VLPrecovery %

BV removal % Total Proteinremoval %

DNA removal%

HCPremoval%

Pellicon® (PES 100 kD) Pellicon® (CRC 300 kD)Both membranes were fully retentive of the VLP

两种滤膜对VLP 都有充分的截留率



Filter-only clarification train can be used without compromising recovery yield of VLPs.

可使用仅使用过滤器的澄清装置，不会对VLP的收率产生不良影响

Filter cascade composed of a Polygard® CN 5 μm filter followed by a 0.3 μm depth filter showed the highest recovery of HCV-VLP, improving on centrifugation/2° depth filtration

由Polygard® CN 5 μm过滤器和0.3 μm深层过滤器组成的过滤器串联结构显示出其具有最高的HCV-VLP收率，是对离心分离/2级深层过滤的改进

Moderate DNA removal with depth filtration was seen

采用深层过滤器可实现中等（适度）的DNA清除

UF/DF

Pellicon® cassette with 300 kD regenerated cellulose membrane offered the best combination of recovery and purification

采用300 kD再生纤维素膜的Pellicon® 膜包可实现最佳的收率和纯化组合

Polygard® CN depth filters and Pellicon® cassettes with Ultracel® membrane offered best results Polygard® CN深层过滤器和采用Pellicon® Ultracel® 膜包可获得最佳结果





纯化层析



Virus Inoculation

BioburdenReduction

Clarification

Sterile Filtration


UF/DF


Purification strategy 纯化策略Anion exchange chromatography (AEX) resins used 使用阴离子交换层析（AEX）树脂

Identify purification goal 确定纯化目标

Ensure analytics are available 保证分析是有效可行的

Batch adsorption 成批吸附Resin in multiwell plates 多孔板中的树脂Vary pH, conductivity 改变pH、电导率

Measure recovery, purity 测量收率、纯度

Chrom bind/elute 层析结合/洗脱Prepacked columns 预装柱Confirm batch adsorption 确认批次吸附

Chrom breakthrough 层析柱子流穿

Prepacked columns 预装柱Capacity measurements

容量测量

Scale up 放大

Iter

atio

ns…重

复…

Iterations…重

复…


27

Batch adsorption experiments (bind-elute)批次吸附实验（结合-洗脱法）

Fractogel® and two anion exchange prototypes approach target of 2 BV LRV Fractogel®和两个阴离子交换原型接近2 BV LRV的目标

Yield increases with increasing ligand density for prototypes 收率随着原型的配体密度增加而增加

DMAETMAE

Q92

Q17

Q54 Q48

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1

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20

40

60

80

BV

LRV

% y

ield

ligand density (μmol/g)

Load at 150 mM, elute at 400+700 mM

Fractogel® – red 红色AEX prototypes – blue 蓝色

Fractogel® – dotted line 虚线

DMAE TMAEQ92

Q17

Q54

Q48

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LRV

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Load at 150 mM, elute at 400+700 mM


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Batch adsorption experiments (flow-through) 批次吸附实验（流穿法）

Inadequate performance in pure flow-through mode; Similar trends with ligand density 在纯流穿方式下性能不充分；配体密度也具有相同趋势

Adopted strategy: collect the flow-through fraction, then wash/elute the resin to recover more material采用的策略：收集流穿部分，然后冲洗/洗脱该树脂，以回收更多材料

DMAE

TMAE

Q92

Q17

Q54 Q48

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LRV

% y

ield

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Flow-through at 300 mM

DMAE

TMAE

Q92Q17

Q54 Q48

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BV

LRV

% VLP yield

Flow-through at 300 mM

Fractogel® – red 红色AEX prototypes – blue 蓝色

Fractogel® – dotted line 虚线


29

Column experiments 层析实验Breakthrough curves for dynamic binding capacity 动态载量的突破曲线

10% dynamic binding capacity ranges at 900-1300 ng VLP / mL of packed resin 900-1300 ng VLP / mL的预装树脂时具有10%的动态载量

The prototype resins has about 30% higher DBC compared to Fractogel®

原型树脂拥有比Fractogel®高约30%的DBC

0

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Q92TMAE

DMAE

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DOE of flow-through conditions: Fractogel® TMAE流穿条件的DOE：Fractogel® TMAEInputs: load NaCl (100/200/300 mM) and flow rate (100/200/400 cm/hr)输入：负载NaCl（ 100/200/300 mM ）和流速（ 100/200/400 cm/hr ）Responses: % VLP recovery and BV LRV响应：% VLP收率和BV LRV

Higher flow rate高流速

OR或Higher load conductivity高负载

电导率

Recovery 收率 LRV

Flow rate (mL/min) 流速（ mL/min ）

NaC

l(m

M)

Higher recovery 高收率

AND 和Lower BV LRV 低BV LRV


Successfully purified VLPs using Fractogel® TMAE commercial resins and AEX prototype resins

使用Fractogel® TMAE商用树脂和AEX原型树脂，成功纯化了VLP

Yield of >60% with ~2 LRV baculovirus can be achieved with a flow-through/wash purification strategy for both resins

对于两种树脂，采用流穿/清洗纯化策略，使用~2 LRV杆状病毒可实现收率>60%

Options to increase recovery or purification depending on product value by varying process conditions

可通过改变工艺条件，提高收率或纯化程度，这由产品价值确定

Successful purification of VLPs using Fractogel® and prototype AEX chromatographic resins 使用Fractogel®和AEX层析树脂原型样品对VLP成功进行了纯化


Optimum performance achieved 实现了最佳性能

Traditional lab process 传统的实

验室工艺

New scalable process 新型可

放大工艺

Purity 纯度

Baculovirus clearance 杆状病毒清除率

94% 97.6%

DNA clearance DNA清除率 99.9%

HCP clearance HCP清除率 82%

Recovery by P30 ELISA 通过P30 ELISA回收

VLP recovery VLP收率 < 10% ~ 65%



UF/DF BaculovirusInactivation杆状病毒灭活


培养基和接种准备

BioburdenReduction 生物负载减少

Sterile Filtration除菌过滤


精制层析

Mobius®

bioreactor 生物反应器

Polygard®-CN 5.00.3 μm filters 过滤器

Fractogel®AEX

resins 树脂

Pellicon® Ultrafiltration

cassettes 超滤膜包Ultracel® 300 kD

membrane 膜

35Production & Purification Platform for VLP Vaccine | Priyabrata Pattnaik | 8th China Human Vaccine Industry Summit | 26 June 2016 | Xi'an, China. | Priyabrata Pattnaik | 8 | 2016 26 | VLP疫苗生产和纯化平台博士第届中国人用疫苗行业峰会年6月日中国西安

Summary 总结

Successfully used Mobius® 3L & 50L disposable bioreactor for production of VLP-based vaccine in insect cell culture system

使用Mobius® 3L和50L一次性生物反应器，在昆虫细胞培养系统中成功生产出了VLP疫苗

Optimized downstream processing using Polygard® CΝ 5.00.3 μm depth filters followed by UF/DF using Pellicon® cassette with Ultracel® 300 kD membrane

使用Polygard® CΝ 5.00.3 μm深层过滤器及Pellicon® Ultracel® 300 kD膜包的UF/DF，是优化的下游工艺方案。Purified VLP by using Fractogel® commercial resins and anion exchange prototype resins

使用Fractogel®商用树脂和阴离子交换原型树脂，纯化了VLP

Integrated all the above components to achieve recovery and impurity clearance in line with requirements

整合了上述所有部件，实现了收率提高和杂质清除，符合相关要求

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Alex XenopoulosElina GousseinovShannon RyanBeth GoodrichAchim SchwaemmleAndreas SteinAnnika AldingerSylvain RibaudLenaig Savary

Cristina PeixotoRicardo SilvaRute CastroAna Sofia CoroadinhaPaula AlvesManuel Carrondo

Team and acknowledgments 团队和致谢

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Thank You 谢谢大家

Priyabrata Pattnaik博士[email protected]

@pattnaik_p

https://sg.linkedin.com/in/priyabratapattnaik

https://plus.google.com/109816383630328905377