Cell Culture Influenza Vaccines: The current status

Transcription

1 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, March 2014

2 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.

3 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

4 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: P.D. Minor et al (2009), Vaccine 27:

5 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

6 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)

7 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?

8 WHO Tables on clinical evaluation of influenza vaccines Number (%) of trials mentioned: VACCINE SUBSTRATE EGGS SUBSTRATE CELLS IIV (8%) LAIV 47 0 (0%) PIIV (12%) PLAIV 25 3 (11%)

9 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)

10 Regulatory WHO Guidelines for National Regulatory Authorities (NRAs)

11 Regulatory: Guidelines, Directives, Guidance

12 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

13 Technical / Manufacturing aspects Cell choice Production system ( upstream, USP) Purification ( downstream, DSP) Yields Reproducibility & Repeatibility (multiple virus strains) Stability of Product / Formulation Timelines

14 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

15 Virus seed preparation, adaptation from egg to cell substrate may be necessary for wildtype viruses, HGRs and LAIV reassortants: HA titer 0 14 eggs TC Passages

16 Production System; Roller bottle RollerCell40

17 Bioreactor Steel (Multi-Use, Fixed Piping) Modes: Suspension cells, Microcarrier, Perfusion

18 Disposable Bioreactors (Single-Use) Xcellerex XDR ( L) WAVE ( L

19 CellSTACK / Cell-Factory Disposable (Single-Use)

20 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

21 STAINLESS STEEL VS SINGLE USE INVESTMENT VS OPERATIONAL COSTS COGS/DOSE INVESTMENT 300 Stainless Steel facility 200 Level of investment icellis system similar to single-use approach, BUT Single Use facility 20 increase of production capacity LVM HVM PRODUCTION CAPACITY / YEAR Reduction of CoGS enabling affordability of biologics SU facility Univercells facility LVM HVM UNIVERCELLS

22 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

23 Challenges: > Sucrose gradient Gradient from 0 55% Amount of virus determined per batch Separation of virus at 42 % sucrose, and HA sucrose sucrose % HA MDCK host cell protein at 30% sucrose fraction > Sterile filtration (220nm) Particle size: Virus 150nm Others nm protein concentration total protein concentration (µg/ml) MDCK protein concentration (µg/ml) fraction Antigen recovery over the whole process only 2-6% 50% antigen loss on sucrose gradient, and 50% loss on sterile filtration

24 Process adaptations IIV (whole virion) 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 Adapted Grow cells in fermentor (2-3 days) Virus inoculation Virus harvest (3-5 days) Clarification by high speed centrifugation Inactivation by BPL DNA removal Filtration Sterile filtration Concentration/Dialysis Adding stabilizer Blend vaccine

25 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 NIBRG14 Batch 2 NIBRG14 Batch gram 5.15 gram 4.64 gram NIBRG14 Antigen recovery 50 % gram antigen / 2000L

26 LAIV upstream production on adherent MDCK cells Wild type / high growth reassortant vs. cold adapted reassortant

27 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) Clarification by filtration DNA removal Concentration/Dialysis Adding stabilizer Blend vaccine 1 day

28 Example production LAIV on adherent MDCK cells Human Influenza A44/Brisbane/59/2007 (H1N1) Human Influenza A44/Brisbane/10/2007 (H3N2) Human Influenza B56/Brisbane/60/2008 Infectious titer Infectious Titer expressed in log10 TCID50/ml Viral Harvest Infectious titer Concentrate > Yield critical! > 98% removal total protein > 90% removal DNA

29 MedImmune LAIV-MDCK meeting VRBPAC (2008) (Vaccines and Related Biological Products Advisory Committee, FDA)

30 MedImmune LAIV-MDCK meeting VRBPAC (2008)

31 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