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 Number (%) of trials mentioned: VACCINE
SUBSTRATE EGGS
SUBSTRATE CELLS
IIV
178
15 (8%)
LAIV
47
0 (0%)
PIIV
279
38 (12%)
PLAIV
25
3 (11%)
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………
• Adherent, or • Suspension – Suspension cells easier, higher yields, higher purity, lower CoG
• Seed production (MCB, WCB), • Characterization and Sanitation: – Tumorgenicity, Oncogenicity, Adventitious Agents, Identity, Stability
Virus seed preparation,
HA titer
adaptation from egg to cell substrate may be necessary for wildtype viruses, HGRs and LAIV reassortants:
0
14
eggs
TC
Passages
Production System; Roller bottle
RollerCell40
Bioreactor Steel (Multi-Use, Fixed Piping) Modes: Suspension cells, Microcarrier, Perfusion
Disposable Bioreactors (Single-Use)
Xcellerex XDR (101000L)
WAVE (0.5-500L
CellSTACK® / Cell-Factory™
Disposable (Single-Use)
iCELLis® : fixed-bed, high cell-density, perfusion bioreactor (Single-Use, disposable)
4RB
20RB
100RB
4RB
20RB
40L
4RB
20RB
600RB
3000RB
200L
1000L
iCELLis 500
STAINLESS STEEL VS SINGLE USE INVESTMENT VS OPERATIONAL COSTS INVESTMENT
COGS/DOSE
300
Stainless Steel facility
200
20 Single Use facility
HVM
LVM
PRODUCTION CAPACITY / YEAR
Level of investment iCELLis system similar to single-use approach, BUT increase of production capacity Reduction of CoGS enabling affordability of biologics
SU facility Univercells facility
LVM
HVM
UNIVERCELLS
Typical USP+DSP production process IIV (whole virion, suspension MDCK) Grow cells in fermentor (2-3 days) Virus inoculation Virus harvest (3-5 days) Clarification by low speed centrifugation Filtration Inactivation by BPL DNA removal Ultra Filtration Removal of debris by precipitation Sucrose gradient Sterile filtration Concentration/Dialysis Adding stabilizer Blend vaccine
J.G.M. Heldens. Mammalian cells for influenza vaccine production; comparison of various systems. Visiongain, London UK, May 21. 2010.
• • •
Gradient from 0 – 55% Amount of virus determined per batch Separation of
HA
> Sucrose gradient
– virus at 42 % sucrose, and – MDCK host cell protein at 30% sucrose
60.0
30
50.0
25
40.0
20
30.0
15
20.0
10
10.0
5
0.0
sucrose
Challenges:
sucrose % HA
0 0
5
10
15
20
25
30
fraction 18000
Particle size:
• Virus 150nm • Others 500 – 1500 nm
16000 protein concentration
> Sterile filtration (220nm)
14000 12000
total protein concentration (µg/ml)
10000 8000
MDCK protein concentration (µg/ml)
6000 4000 2000 0 1
6
11
16
21
26
fraction
Antigen recovery over the whole process only 2- 6% • 50% antigen loss on sucrose gradient, and • 50% loss on sterile filtration
Process adaptations IIV (whole virion) Grow cells in fermentor (2-3 days) Virus inoculation
Adapted Grow cells in fermentor (2-3 days) Virus inoculation
Virus harvest (3-5 days) Clarification by low speed centrifugation
Virus harvest (3-5 days)
Filtration
Clarification by high speed centrifugation
Inactivation by BPL
Inactivation by BPL
DNA removal
DNA removal
Ultra Filtration Removal of debris by precipitation
Filtration
Sucrose gradient
Sterile filtration
Sterile filtration
Concentration/Dialysis
Concentration/Dialysis
Adding stabilizer
Adding stabilizer
Blend vaccine
Blend vaccine
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 Antigen / 2000L NIBRG14 Batch 1
4.46 gram
NIBRG14 Batch 2
5.15 gram
NIBRG14 Batch 3
4.64 gram
NIBRG14 Antigen recovery 50 % 4.5 – 5 gram antigen / 2000L
LAIV upstream production on adherent MDCK cells Wild type / high growth reassortant vs. cold adapted reassortant
Typical production process LAIV on adherent MDCK cells Production wt virus seeds, reassortment Reassortant virus seeds Grow cells on cell cube (2-3 days) Virus inoculation Virus harvest (3-5 days)
DNA removal Concentration/Dialysis Adding stabilizer Blend vaccine
1 day
Clarification by filtration
Example production LAIV on adherent MDCK cells
Human Influenza
Human Influenza
Human Influenza
A44/Brisbane/59/2007 (H1N1)
A44/Brisbane/10/2007 (H3N2)
B56/Brisbane/60/2008
Infectious Titer expressed in log10 TCID50/ml
Infectious titer Viral Harvest
6.3
6.5
6.2
8.2
9.5
8.5
Infectious titer Concentrate
> Yield critical ! > 98% removal total protein > 90% removal DNA
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
