Presentation at IBC's Bioprocess & Technology Conference, 27-30 October 2015, Singapore

1. Priyabrata Pattnaik, PhD
Director – Worldwide Vaccine Initiative
Bioprocess & Technology Conference, 27-30 October 2015, Singapore
PRODUCTION ANDPRODUCTION ANDPRODUCTION ANDPRODUCTION AND
PURIFICATION OF VIRUSPURIFICATION OF VIRUSPURIFICATION OF VIRUSPURIFICATION OF VIRUS
LIKE PARTICLE (VLP)LIKE PARTICLE (VLP)LIKE PARTICLE (VLP)LIKE PARTICLE (VLP)
BASED VACCINEBASED VACCINEBASED VACCINEBASED VACCINE

2. 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

3. 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. 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)

5. 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)

6. 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

7. 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

8. 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

9. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore9
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Primary
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

10. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore10
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Primary
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

11. 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®
3L
bioreactor)
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. 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
Viability(%)
ViableCellConcentration(106
cells/ml)
Time (h)
STR2 CR2 CR3 CR4
Viab STR2 Viab CR2 Viab CR3 Viab CR4

13. 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. 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. 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

16. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore16
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Primary
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

17. 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

18. 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
Inletpressure(bar)
Volume filtered (L/m2)
Polygard CN filter train 5 0.3 µm
10 µm
5 µm
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

19. 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

20. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore20
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Primary
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

21. 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. 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-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

23. 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

24. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore24
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
UF/DF
Baculovirus
Inactivation
Purification
Chromatography
Media and Inoculum
Preparation
Cell growth in
Bioreactor and
Virus Inoculation
Bioburden
Reduction
Primary
Clarification
Sterile
Filtration
Polishing
Chromatography
UF/DF

25. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore25
Purification strategy
Anion exchange chromatography (AEX) resins used
Identify
purification
goal
Ensure
analytics are
available
Batch adsorption
Resin in multiwell plates
Vary pH, conductivity
Measure recovery, purity
Chrom bind/elute
Prepacked columns
Confirm batch
adsorption
Chrom breakthrough
Prepacked columns
Capacity measurements
Scale up
Iterations…
Iterations…

26. 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
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
AEX prototypes – blue
Fractogel® – dotted line
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

27. 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
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
AEX prototypes – blue
Fractogel® – dotted line

28. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore28
Column experiments
Breakthrough 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
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)

29. 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)
NaCl(mM)
Higher recovery
AND
Lower BV LRV

30. 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. 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%

32. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore32
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
UF/DF Baculovirus
Inactivation
Media and Inoculum
Preparation
Bioburden
Reduction
Sterile
Filtration
Polishing
Chromatography
Mobius®
bioreactor
Polygard®-CN
5.0 0.3 µm
filters
Fractogel®
AEX
resins
Pellicon®
Ultrafiltration
cassettes
Ultracel® 300 kD
membrane

33. Production and Purification of Virus like particle (VLP) based Vaccine, Bioprocess & Technology Conference, 27-30 October 2015, Singapore33
Typical VLP-based vaccine process
Insect cell / baculovirus VLP production platform
Mobius®
Bioreactor
Polygard®-CN
5.0 0.3 µm filters
Pellicon®
Ultrafiltration
cassettes
Ultracel® 300 kD
membrane
Fractogel®
AEX resins

34. 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

35. 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

36. THANK YOUTHANK YOUTHANK YOUTHANK YOU
36
Priyabrata Pattnaik, PhD
priyabrata.pattnaik@merckgroup.com
@pattnaik_p
https://sg.linkedin.com/in/priyabratapattnaik
https://plus.google.com/109816383630328905377