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