cell culture influenza vaccines: the current status culture influenza vaccines: the current status...
TRANSCRIPT
Cell Culture Influenza Vaccines:
The current status
Han van den Bosch, Amsterdam, The Netherlands
7th WHO Meeting on Influenza Vaccine Technology Transfer to Developing Country Manufacturers.
Dubai, 25-26 March 2014
Statement
• The presentation contains publicly available information only,
• The presentation gives a limited overview of the subject, and does not intend to be complete in every detail and in all options,
• Examples given about production systems and issues do not provide a guarantee about the performance of a certain system.
Advantages of cell-culture-derived influenza vaccines (1)
• Permits growth of all influenza viruses
• H3N2 strains are difficult to isolate in eggs
• No need for egg adapted High Growth Reassortants
• Available on short notice during any season
• Lead time shorter as compared to egg supply
• No need for embryonated chicken eggs from biosecure flocks
• Not enough chickens may be available in case of avian flu outbreak
• Easier logistics
• Less waste disposal
• Maintained in aseptic closed environment during upstream and downstream
Advantages of cell-culture-derived influenza vaccines (2)
• Reduced risk of contamination during production
• More controlled and consistent production process
• Higher purity of starting material
• Safe whole virus vaccines feasible
• Animal-component-free production feasible
• Reduces vaccine production time
• Might provide broader immunity to influenza variants
• Egg passaging might induce adaptive changes for growth in eggs
• Safe for individuals with allergy to eggs
• Allows for multipurpose facility use (other vaccines, MAbs and other therapeutic proteins)
• W.P. Glezen (2011), The Lancet 377: 698-700
• P.D. Minor et al (2009), Vaccine 27: 2907-2913
Marketing Authorization of cell-culture seasonal IIV
• 2001: Influvac TC, Solvay / Abbott, MDCK-a, EU
– Discontinued after acquisition by Abbott
• 2007: Optaflu, Novartis, MDCK-s, EU
• 2012: Flucelvax, Novartis, MDCK-s, USA
• 2010: Preflucel, Baxter, Vero, EU
• 2013: FluBlok, Protein Sciences, rec.HA in Baculo / SF9 (insect cells), USA
• Multiple (Pre-)Pandemic versions
Ongoing cell culture (P)IIV developments
• GSK (EB66, Valneva / Vivalis)
• Kaketsuken (+GSK)
• Sanofi Pasteur (discontinued PerC6)
• Crucell / J&J (PerC6)
• Takeda (+Baxter, Vero)
• Kitasato Daiichi Sankyo (MDCK)
Cell culture (P)LAIV developments
• MedImmune / AstraZeneca (MDCK)
– Halted after FDA requirements? (Wendy Wolfson, Nature Biotechnology 28, 115 (2010)
• Nobilon / Merck (MDCK, NOBI)
– Discontinued after acquisition by Merck (2010)
• Green Hills Biotech (Vero)
– Ongoing
• Others at earlier pre-clinical stages of development?
WHO Tables on clinical evaluation of influenza vaccines
VACCINE SUBSTRATE EGGS SUBSTRATE CELLS
IIV 178 15 (8%)
LAIV 47 0 (0%)
PIIV 279 38 (12%)
PLAIV 25 3 (11%)
Number (%) of trials mentioned:
http://www.who.int/immunization/diseases/influenza/clinical_evaluation_tables/en/
Barriers / Challenges for cell culture influenza vaccines
• Regulatory
• Technical / Manufacturing
– Cell choice
– Production system
– Purification
– Yields
– Reproducibility & Repeatibility
– Stability of Product
– Timelines
• Financial
– Development costs
– Investments and Cost of Goods (CoG)
Regulatory
WHO Guidelines for National Regulatory Authorities (NRAs)
Regulatory: Guidelines, Directives, Guidance
Regulatory: important cell aspects to consider
• Mammalian or avian
• Suspension or adherent
• Source and record / passage history (TSE)
• Adventitious agents
• Animal Component Free (incl. trypsin and benzonase)
• Stability at passaging (end-of-production passage)
• Suitability for production
• Tumorgenicity (living cells)
• Oncogenicity (host cell DNA remnants)
• Risk assessment
Technical / Manufacturing aspects
• Cell choice
• Production system (“upstream”, USP)
• Purification (“downstream”, DSP)
• Yields
• Reproducibility & Repeatibility (multiple virus strains)
• Stability of Product / Formulation
• Timelines
Cell choice
• MDCK,
• Vero,
• PerC6,
• EB66, or
• Other / New………
• Seed production (MCB, WCB),
• Characterization and Sanitation:
– Tumorgenicity, Oncogenicity, Adventitious Agents, Identity, Stability
• Adherent, or
• Suspension– Suspension cells easier,
higher yields, higher purity, lower CoG
Virus seed preparation, adaptation from egg to cell substrate may be necessaryfor wildtype viruses, HGRs and LAIV reassortants:
Passages
HA
tite
r
eggs TC
140
Production System; Roller bottle
RollerCell40
Bioreactor Steel (Multi-Use, Fixed Piping)
Modes: Suspension cells, Microcarrier, Perfusion
Disposable Bioreactors (Single-Use)
Xcellerex XDR (10-1000L)
WAVE (0.5-500L
CellSTACK® / Cell-Factory™
Disposable(Single-Use)
iCELLis® : fixed-bed, high cell-density, perfusion bioreactor (Single-Use, disposable)
4RB 20RB 100RB 600RB 3000RB
4RB 20RB 40L 200L 1000L
4RB 20RB iCELLis 500
UNIVERCELLS
STAINLESS STEEL VS SINGLE USEINVESTMENT VS OPERATIONAL COSTS
PRODUCTION CAPACITY / YEAR
LVM
INVESTMENTCOGS/DOSE
Single Use facility
HVM
Stainless Steel facility
20
200
300
LVM
SU facility
HVM
Univercells facility
Level of investment iCELLis system similar to single-use approach, BUT
increase of production capacity
Reduction of CoGS enabling affordability of biologics
Typical USP+DSP production process IIV(whole virion, suspension MDCK)
Clarification by low speed centrifugation
Inactivation by BPL
Filtration
Sucrose gradient
Concentration/Dialysis
Adding stabilizer
Blend vaccine
Grow cells in fermentor (2-3 days)
Virus inoculation
Virus harvest (3-5 days)
DNA removal
Sterile filtration
Removal of debris by precipitation
Ultra Filtration
J.G.M. Heldens. Mammalian cells for influenza vaccine production;
comparison of various systems. Visiongain, London UK, May 21. 2010.
Challenges:
• Gradient from 0 – 55%
• Amount of virus determined per batch
• Separation of
– virus at 42 % sucrose, and
– MDCK host cell protein at 30% sucrose
0.0
10.0
20.0
30.0
40.0
50.0
60.0
0 5 10 15 20 25 30
fraction
HA
0
5
10
15
20
25
30
su
cro
se
sucrose %
HA
> Sterile filtration (220nm)
Particle size: • Virus 150nm
• Others 500 – 1500 nm
> Sucrose gradient
Antigen recovery over the whole process only 2- 6%• 50% antigen loss on sucrose gradient, and • 50% loss on sterile filtration
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
1 6 11 16 21 26
fraction
pro
tein
co
ncen
trati
on
total proteinconcentration(µg/ml)
MDCK proteinconcentration(µg/ml)
Process adaptations IIV (whole virion)
Clarification by low speed centrifugation
Inactivation by BPL
Filtration
Sucrose gradient
Concentration/Dialysis
Adding stabilizer
Blend vaccine
Grow cells in fermentor (2-3 days)
Virus inoculation
Virus harvest (3-5 days)
DNA removal
Sterile filtration
Removal of debris by precipitation
Ultra Filtration
Adapted
Clarification by high speed centrifugation
Inactivation by BPL
Filtration
Sterile filtration
Concentration/Dialysis
Adding stabilizer
Blend vaccine
Grow cells in fermentor (2-3 days)
Virus inoculation
Virus harvest (3-5 days)
DNA removal
Summary adapted production IIV (whole virion, MDCK suspension, NIBRG14/H5N1 example)
Robust scalable process HA yield between 8 and 10
> 95% removal total protein
> 90% removal host cell protein
> 90% removal DNA
NIBRG14 Antigen recovery 50 %4.5 – 5 gram antigen / 2000L
Antigen / 2000L
NIBRG14 Batch 1 4.46 gram
NIBRG14 Batch 2 5.15 gram
NIBRG14 Batch 3 4.64 gram
LAIV upstream production on adherent MDCK cells
Wild type / high growth reassortant vs. cold adapted reassortant
Typical production process LAIV on adherent MDCK cells
Reassortant virus seeds
Grow cells on cell cube (2-3 days)
Virus inoculation
Virus harvest (3-5 days)
Clarification by filtration
Concentration/Dialysis
Adding stabilizer
Blend vaccine
Production wt virus seeds, reassortment
DNA removal
1 d
ay
Human Influenza
A44/Brisbane/59/2007 (H1N1)
Human Influenza
A44/Brisbane/10/2007 (H3N2)
Human Influenza
B56/Brisbane/60/2008
Infectious Titer expressed in log10 TCID50/ml
Infectious titer
Viral Harvest 6.3 6.5 6.2
Infectious titer
Concentrate8.2 9.5 8.5
> Yield critical !
> 98% removal total protein
> 90% removal DNA
Example production LAIV on adherent MDCK cells
MedImmune LAIV-MDCK meeting VRBPAC (2008)(Vaccines and Related Biological Products Advisory Committee, FDA)
MedImmune LAIV-MDCK meeting VRBPAC (2008)
Summary, Cell Culture Influenza Vaccines
• Regulatory requirements and pathway should be clear for cell characterization (EMA, FDA, NRA)
• Use existing approved cell line if feasible (costs, time, IP)
• Suspension cells prefered over adherent cells
– Easier process, higher yield and purity of harvest, lower cost
• Different virus substrates require different DSP procedures
• Different virus strains may require adapted process parameters
• Production system hardware:
– “steel” (higher investment, lower exploitation costs) or
– “disposable” (lower investment, higher exploitation costs; increased flexibility)
• Need for not-egg-passaged vaccine seed viruses
• THANKS