production and purification of virus like particle (vlp) based vaccine
TRANSCRIPT
Priyabrata Pattnaik, PhDDirector – Worldwide Vaccine Initiative
Bioprocess & Technology Conference, 27-30 October 2015, Singapore
PRODUCTION AND PRODUCTION AND PRODUCTION AND PRODUCTION AND PURIFICATION OF VIRUS PURIFICATION OF VIRUS PURIFICATION OF VIRUS PURIFICATION OF VIRUS LIKE PARTICLE (VLP) LIKE PARTICLE (VLP) LIKE PARTICLE (VLP) LIKE PARTICLE (VLP) BASED VACCINEBASED VACCINEBASED VACCINEBASED VACCINE
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore2
Presentation Outline
VLP as Hepatitis C vaccines
Baculovirus / insect cell expression platform
Challenges in VLP vaccine production and purification
VLP production in insect cell culture
Clarification of VLP
Concentration / Diafiltration of VLP
Chromatographic purification of VLP
Summary
� VLP vaccine candidates have become quite popular of late
� VLP-based processes are, however, currently quite diverse
� We undertook an effort to standardize the process
� We used hepatitis C VLP as a model
� This presentation will explain the approach taken and present the results obtained
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore3
Motivation
4 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
� Contain repetitive high-density displays of viral surface proteins that elicit strong T cell and B cell immune responses
� Non infectious because they do not contain genetic material, thus cannot replicate and are safer
� Their size (40-120 nm diameter) is optimal for uptake by dendritic cells
� Can be produced in a variety of cell culture systems
� Can self assemble in vivo
� Proven technology (Hepatitis B and Human Papilloma Virus vaccines)
Why virus-like particles (VLPs)?
Source: Roldão et al., Expert Reviews in Vaccines 9 (10), 1149-76 (2010)
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore5
Hepatitis C
� 170 million people infected
� Cirrhosis, liver cancer, death
� Current therapies only partially effective, costly and poorly tolerated
� No vaccine currently exists
VLPs for hepatitis C vaccine development
E1 and E2 glycoproteins from Hep C virus
Capsid and structure VLP from retrovirus (murine leukemia
virus)
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore6
Recombinant baculovirus (BV) is used to infect insect cells
Key features
Transient production
High cell densities
Regulatory acceptance
� Cervarix® (GSK)
� Flublok® (Protein Sciences)
� Several late-stage clinicals
Insect cell / baculovirus VLP production platform
~120 nm
VLP
BV
60-80 nm
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore7
� Low production yields
� Stability of enveloped VLPs
� Difficulties in baculovirus (BV) removal lowers recovery
� No established platform processes for purification
Challenges in VLP vaccine production
VLP
BV
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore8
Work carried out in collaboration with iBET
iBET: Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore9
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
UF/DFBaculovirusInactivation
PurificationChromatography
Media and Inoculum Preparation
Cell growth in Bioreactor and
Virus Inoculation
BioburdenReduction
PrimaryClarification
Sterile Filtration
PolishingChromatography
UF/DF
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore10
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
UF/DFBaculovirusInactivation
PurificationChromatography
Media and Inoculum Preparation
Cell growth in Bioreactor and
Virus Inoculation
BioburdenReduction
PrimaryClarification
Sterile Filtration
PolishingChromatography
UF/DF
11 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
� Cell culture was carried out in stirred tank glass bioreactor and disposable bioreactor (Mobius® 3Lbioreactor)
� Sf9 insect cells and Sf900II cell culture media were used in the process
� Mobius® 3L bioreactor was first operated at same conditions previously used for stirred tank glass bioreactors
� Cell aggregation
� Formation of foam
� Longer lag phase
� Lower viable cell concentration
Insect cell culture
12 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
� Increased agitation rate
� Increased cell density of inoculation
� Replaced micro sparger with an open-pipe sparger
Insect cell culture conditions improved based on experience with Mobius®
bioreactor
0
20
40
60
80
100
0.0
0.5
1.0
1.5
2.0
0 24 48 72 96 120 144
Via
bil
ity
(%)
Via
ble
Ce
ll C
on
cen
trat
ion
(10
6ce
lls/m
l)Time (h)
STR2 CR2 CR3 CR4
Viab STR2 Viab CR2 Viab CR3 Viab CR4
13 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
Microscopic evaluation of cells
Stirred glass bioreactor
Mobius® 3L bioreactor
Run CR2
Run CR3
Run CR4
45 hrs 70 hrs 95 hrs
14 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
Western blot analysis of VLPs using three markers
GAG-MLV
STR1
CR2
CR3
CR4
HCV-E2STR1
CR2
CR3
CR4
Sucrose cushion purified VLPs
VLPs pelleted by ultracentrifugation
HCV-E1STR1
CR2
CR3
CR4
GAG-MLV (core protein)
HCV-E1 (envelope protein)
HCV-E2 (envelope protein)
VLP HepC V
15 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
� Successful growth of Sf9 insect cells and infection with baculovirus for production of VLP vaccine using Mobius® 3L disposable bioreactor
� Comparable cell and VLP properties between disposable and glass bioreactors
� Reproducible performance of the disposable bioreactor was seen with identical results for three separate cell culture runs
Successful use of Mobius® bioreactor for VLP production
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore16
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
UF/DFBaculovirusInactivation
PurificationChromatography
Media and Inoculum Preparation
Cell growth in Bioreactor and
Virus Inoculation
BioburdenReduction
PrimaryClarification
Sterile Filtration
PolishingChromatography
UF/DF
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore17
Depth filtration
� Well suited for smaller vaccine batches
� Easier to scale
� Lower cost
� Disposable
� Gentle treatment
� Simpler process development
� Wide choice of depth filters
Centrifugation
� Lab models used early on
� Well suited for large-scale production
� High capital expense
� Shear
Clarification
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore18
Clarification: throughput data
Disposable capsule filters
Polygard® CN, nominal pore sizes of 10, 5, 0.6 and 0.3 µm
Pleated, all-polypropylene depth filters
Filter area: 17 cm2; Inlet flux: 988 LMH
0.0
0.5
1.0
1.5
2.0
0 50 100 150 200 250 300 350
Inle
t pre
ssur
e (b
ar)
Volume filtered (L/m 2)
Polygard CN filter train 5 ���� 0.3 µm
10 µm5 µm
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200 250 300 350
Inle
t pre
ssur
e (b
ar)
Volume filtered (L/m 2)
Polygard CN filter train 10 ���� 5 ���� 0.6 µm
10 µm5 µm0.6 µm
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore19
Clarification: recovery data
Unlike centrifugation, depth filtration resulted in ~70% DNA clearance
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
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore20
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
UF/DFBaculovirusInactivation
PurificationChromatography
Media and Inoculum Preparation
Cell growth in Bioreactor and
Virus Inoculation
BioburdenReduction
PrimaryClarification
Sterile Filtration
PolishingChromatography
UF/DF
21 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
Pellicon® cassettes
Two different ultrafiltration membranes
� 300 kD composite regenerated cellulose (Ultracel® membrane, “CRC”)
� 100 kD polyethersulfone (Biomax® membrane, “PES”)
Similar process conditions employed
� 4-5x concentration factor
� Loading: 72 L/m2; Feed flux: 480 LMH; TMP: 1 bar; Pfeed: 0.6-0.9 bar; Pretent: 1.1-1.4 bar
Concentration of clarified VLP harvest
22 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
Concentration of clarified VLP harvest – results
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
23 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
Clarification
� Filter-only clarification train can be used without compromising recovery yield of VLPs.
� 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
� Moderate DNA removal with depth filtration was seen
UF/DF
� Pellicon® cassette with 300 kD regenerated cellulose membrane offered the best combination of recovery and purification
Polygard® CN depth filters and Pellicon® cassettes with Ultracel® membrane offered best results
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore24
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
UF/DFBaculovirusInactivation
PurificationChromatography
Media and Inoculum Preparation
Cell growth in Bioreactor and
Virus Inoculation
BioburdenReduction
PrimaryClarification
Sterile Filtration
PolishingChromatography
UF/DF
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore25
Purification strategyAnion exchange chromatography (AEX) resins used
Identify purification
goal
Ensure analytics are
available
Batch adsorptionResin in multiwell platesVary pH, conductivity
Measure recovery, purity
Chrom bind/elutePrepacked columnsConfirm batch adsorption
Chrom breakthroughPrepacked columns
Capacity measurements
Scale up
Itera
tions…Ite
ratio
ns…
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore26
Batch adsorption experiments (bind-elute)
� Fractogel® and two anion exchnage prototypes approach target of 2 BV LRV
� Yield increases with increasing ligand density for prototypes
DMAETMAE
Q92
Q17
Q54 Q48
0
1
2
3
0
20
40
60
80
BV
LR
V
% y
ield
ligand density ( µmol/g)
Load at 150 mM, elute at 400+700 mM
Fractogel® – redAEX prototypes – blue
Fractogel® – dotted line
DMAE TMAE
Q92
Q17
Q54
Q48
0
1
2
3
0 20 40 60 80
BV
LR
V
% VLP yield
Load at 150 mM, elute at 400+700 mM
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore27
Batch adsorption experiments (flow-through)
� Inadequate performance in pure flow-through mode; Similar trends with ligand density
Adopted strategy: collect the flow-through fraction, then wash/elute the resin to recover more material
DMAE
TMAE
Q92
Q17
Q54 Q48
0
1
2
3
0
20
40
60
80
BV
LR
V
% y
ield
ligand density ( µmol/g)
Flow-through at 300 mM
DMAE
TMAE
Q92Q17
Q54 Q48
0
1
2
3
0 20 40 60 80
BV
LR
V
% VLP yield
Flow-through at 300 mM
Fractogel® – redAEX prototypes – blue
Fractogel® – dotted line
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore28
Column experimentsBreakthrough curves for dynamic binding capacity
� 10% dynamic binding capacity ranges at 900-1300 ng VLP / mL of packed resin
� The prototype resins has about 30% higher DBC compared to Fractogel®
0
500
1000
1500
2000
Q17 Q48 Q54 Q92 TMAE DMAE
DB
C (
ng
VLP
/ m
L o
f re
sin
)
10%
50%
Prototype AEX Fractogel®
Q17
Q48Q54
Q92TMAE
DMAE
0
20
40
60
80
800 1000 1200 1400
% y
ield
10% DBC (ng VLP / mL of resin)
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore29
DOE of flow-through conditions: Fractogel® TMAE
Inputs: load NaCl (100/200/300 mM) and flow rate (100/200/400 cm/hr)Responses: % VLP recovery and BV LRV
Higher flow rate
OR
Higher load conductivity
Recovery LRV
Flow rate (mL/min)
NaC
l(m
M)
Higher recovery
AND
Lower BV LRV
30 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
� Successfully purified VLPs using Fractogel® TMAE commercial resins and AEX prototype resins
� Yield of >60% with ~2 LRV baculovirus can be achieved with a flow-through/wash purification strategy for both resins
� Options to increase recovery or purification depending on product value by varying process conditions
Successful purification of VLPs using Fractogel® and prototype AEX chromatographic resins
31 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
Optimum performance achieved
Traditional lab process
New scalable process
Purity
Baculovirus clearance 94% 97.6%
DNA clearance DNA 99.9%
HCP clearance HCP 82%
Recovery by P30 ELISA
VLP recovery VLP < 10% ~ 65%
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore32
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
UF/DF BaculovirusInactivation
Media and Inoculum Preparation
BioburdenReduction
Sterile Filtration
PolishingChromatography
Mobius®
bioreactor
Polygard®-CN 5.0����0.3 µm
filters
Fractogel®
AEX resins
Pellicon® Ultrafiltration
cassettesUltracel® 300 kD
membrane
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore33
Typical VLP-based vaccine processInsect cell / baculovirus VLP production platform
Mobius®
BioreactorPolygard®-CN
5.0�0.3 µm filtersPellicon®
Ultrafiltration cassettes
Ultracel® 300 kDmembrane
Fractogel®
AEX resins
34 Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore
� Successfully used Mobius® 3L disposable bioreactor for production of VLP-based vaccine in insect cell culture system
� 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
� Purified VLP by using Fractogel® commercial resins and anion exchange prototype resins
� Integrated all the above components to achieve recovery and impurity clearance in line with requirements
Summary
Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore35
EMD/Merck Millipore
Alex Xenopoulos
Elina Gousseinov
Shannon Ryan
Beth Goodridge
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
THANK YOUTHANK YOUTHANK YOUTHANK YOU
36
Priyabrata Pattnaik, [email protected]
@pattnaik_p
https://sg.linkedin.com/in/priyabratapattnaik
https://plus.google.com/109816383630328905377