Development of a production and purification platform for VLP vaccine candidates

Priyabrata Pattnaik, PhD
Director – Worldwide Vaccine Initiative
Priyabrata 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

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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
 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)

. | 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 virus
C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

. | 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
VLP
BV
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
Baculovirus
Inactivation
杆状病毒灭活
Purification
Chromatography
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
Bioburden
Reduction 降低生物负载
Clarification 澄清
Sterile
Filtration
无菌过滤
Polishing
Chromatography
精制层析
UF/DF

UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
生物反应器中的细胞
生长和病毒接种
Bioburden
Reduction
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

11
 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

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 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
40
60
80
100
0.0
0.5
1.0
1.5
2.0
0 24 48 72 96 120 144
Viability(%)
ViableCellConcentration(106cells/ml)
Time (h)
STR2 CR2 CR3 CR4
Viab 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

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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

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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

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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 Bioreactor
Mobius® 50 L生物反应器中的VLP-HCV生成动力学曲线

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 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

UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Clarification
澄清
Sterile
Filtration
Polishing
Chromatography
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
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200 250 300 350
Inletpressure(bar)
Volume filtered (L/m2)
Polygard CN filter train 10  5  0.6 μm
10 μm
5 μm
0.6 μm
0.0
0.5
1.0
1.5
2.0
0 50 100 150 200 250 300 350
Inletpressure(bar)
Volume filtered (L/m2)
Polygard CN filter train 5  0.3 μm
10 μm
5 μm
5 µm
0.3 µm

20
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

UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

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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

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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-VLP
recovery %
BV removal % Total Protein
removal %
DNA removal
%
HCP
removal%
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

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 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

UF/DF
Baculovirus
Inactivation
Purification
Chromatography
纯化层析
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Clarification
Sterile
Filtration
Polishing
Chromatography
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
Iterations…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
DMAE
TMAE
Q92
Q17
Q54 Q48
0
1
2
3
0
20
40
60
80
BVLRV
%yield
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 TMAE
Q92
Q17
Q54
Q48
0
1
2
3
0 20 40 60 80
BVLRV
% VLP yield
Load at 150 mM, elute at 400+700 mM

28
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
0
1
2
3
0
20
40
60
80
BVLRV
%yield
ligand density (μmol/g)
Flow-through at 300 mM
DMAE
TMAE
Q92
Q17
Q54 Q48
0
1
2
3
0 20 40 60 80
BVLRV
% 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
500
1000
1500
2000
Q17 Q48 Q54 Q92 TMAE DMAE
DBC(ngVLP/mLofresin)
10%
50%
Prototype AEX Fractogel®
Q17
Q48
Q54
Q92
TMAE
DMAE
0
20
40
60
80
800 1000 1200 1400
%yield
10% DBC (ng VLP / mL of resin)

30
DOE of flow-through conditions: Fractogel® TMAE
流穿条件的DOE：Fractogel® TMAE
Inputs: 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 ）
NaCl(mM)
Higher recovery 高收率
AND 和
Lower BV LRV 低BV LRV

31
 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

32
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 Baculovirus
Inactivation
杆状病毒灭活
Media and Inoculum
Preparation
培养基和接种准备
Bioburden
Reduction
生物负载减少
Sterile
Filtration
除菌过滤
Polishing
Chromatography
精制层析
Mobius®
bioreactor 生物反
应器
Polygard®-CN
5.00.3 μm
filters 过滤器
Fractogel®
AEX
resins 树脂
Pellicon®
Ultrafiltration
cassettes 超滤膜包
Ultracel® 300 kD
membrane 膜

34
Mobius®
Bioreactor 生物反
应器
Polygard®-CN
5.00.3 μm filters
过滤器
Pellicon®
Ultrafiltration
cassettes 超滤膜包
Ultracel® 300 kD
membrane 膜
Fractogel®
AEX resins
树脂

35
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 5.00.3 μm depth filters followed by UF/DF
using Pellicon® cassette with Ultracel® 300 kD membrane
使用Polygard® CΝ 5.00.3 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
1
2
3
4

Alex Xenopoulos
Elina Gousseinov
Shannon Ryan
Beth Goodrich
Achim Schwaemmle
Andreas Stein
Annika Aldinger
Sylvain Ribaud
Lenaig Savary
Cristina Peixoto
Ricardo Silva
Rute Castro
Ana Sofia Coroadinha
Paula Alves
Manuel Carrondo
Team and acknowledgments 团队和致谢

37
Thank You
谢谢大家
Priyabrata Pattnaik博士
priyabrata.pattnaik@merckgroup.com
@pattnaik_p
https://sg.linkedin.com/in/priyabratapattnaik
https://plus.google.com/109816383630328905377

Development of a production and purification platform for VLP vaccine candidates

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