Technical and Regulatory Considerations in Rapid Scale-Up of Vaccine Manufacturing Processes

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na Jones, Ph.D. BioProcess Technology Consultants, Inc.

1 Technical and Regulatory Considerations in Rapid Scale-Up of Vaccine Manufacturing Processes Susan Dana Jones, Ph.D. BioProcess Technology Consultants, Inc. Vaccine Scale up and Manufacturing Conference Brussels, Belgium December 2, 2008

2 When Would Rapid Scale-up Be Needed? Unprecedented spread of an existing strain Vaccine manufacturing process and controls exist Not enough vaccine currently in stock Scale up could be increasing i scale per bth batch or increasing i number of batches processed per unit time Suddenemergence emergence of a new, highly infectious strain No effective vaccine available Manufacturing process and scale up strategy developed simultaneously Vaccine may be manufactured at risk, ie, without sufficient demonstration of efficacy, to meet short timelines Risk based analysis required to choose the vaccine candidate likely l to be most effective

3 WHO Statement on Influenza Pandemic The objective of pandemic planning is to enable countries to be better prepared to recognize and manage an influenza pandemic. Planning may help to reduce transmission of the pandemic virus, to decrease cases, hospitalizations and deaths, to maintain essential services and to reduce the economic and social impact of an influenza pandemic. * *From WHO Checklist for Influenza Preparedness Planning (2005)

4 Considerations in Rapid Vaccine Scale-up Resource availability Multiple organizations must be prepared p to produce the same vaccine with the same quality Virus seed stocks (including egg adapted recombinant seeds) must be produced, qualified, and distributed. Technology Must use scalable technology to develop vaccine Egg based manufacturing for flu vaccine is not rapidly scalable; requires new flock to provide additional eggs Cell based technologies for flu or other vaccines are scalable; well established for biopharmaceuticals Regulatory Harmonized regulatory yprocess required to insure global supply can be achieved in timely manner

5 Influenza Virus Types Influenza Type A Infects humans, birds, pigs, horses Most prevalent, occurs in several Sub Types Most likely to demonstrate Antigenic Drift Shift Major Antigenic Shift may lead to pandemic InfluenzaType B Infects humans, seals Less volatile, no sub types Antigenic drift, slower than Type A Influenza Type C Infects humans Comparatively rare not included in standard vaccines

6 Diagram of Influenza Virus Hemagglutinin HA Neuraminidase NA

7 Influenza Strain Nomenclature A/Brisbane/10/2007(H3N2) Virus Type (A or B) Where Isolated Year Isolated Hemagglutinin Sub-Type Neuraminidase Sub-Type Sequential Number For A Strains

8 Reassortment Avian H3 Human H2 Human H3

9 Identification of New Influenza Virus Strains New A viruses arise by recombination or re assortment (dual infections) in animals, birds Most often in the Far East People living in close proximity to pigs, ducks, etc. Chain of WHO Influenza Diagnostic Laboratories Constantly checking new isolates for antigenic Shift versus Drift Requirement for change in vaccine strains when significant drift or a major shift occurs, MOST OFTEN IN TYPE A. Notified by WHO in January March for Western World. CDC, Atlanta GA issues new strains for manufacturers to use These may need additional adaptation to eggs or cell culture.

10 Traditional Influenza Vaccine Production Recombinant Seed Virus (New Isolate X Well-growing strain) Prepare Substrate Infect & Incubate Remove Cells, Purify Virus Inactivate Virus CURRENT Incubate Embryonated Eggs (1,000s needed) COMING Scale up cell culture from WCB VERO, MDCK, PER-C6, Sf-9 Inoculate Harvest Allantoic Allantoic Sac Fluid, (10-12ml/egg) 12ml/egg) Clarify, Ultra- Centrifuge Infect in Centrifuge, TFF, Bioreactor Chromatography (up to 2,000 L. per batch) Treat with formaldehyde, Sub-unit unit vaccines are then solvent-extracted; extracted;

11 Live, Attenuated Vaccine Isolate Target Strain Attenuate by Laboratory Passage or Recombination Perform Exhaustive Safety Tests REPEAT THE ABOVE WHENEVER A NEW STRAIN ARRIVES Produce Virus on Large Scale Harvest, Clarify, Stabilize Develop Efficient Delivery Method (e.g. FluMist )

12 Current Influenza Vaccines For 2008/9, US product contains three different strains: A/Brisbane/59/2007 / / (H1N1) A/Brisbane/10/2007 (H3N2) B/Florida/4/2006 Three types of products currently licensed in US Egg grown, solvent extracted to release HA and NA antigens Egg grown, inactivated whole virus Live, attenuated virus Additional products licensed in Europe include Optaflu Virus produced in MDCK cell culture Antigens are solvent extracted

13 A Wide Variety of Influenza A Strains Exist H1N1 H1N2 H2N2 H3N1 H3N2 H3N8 H5N1 H5N2 H5N3 H5N8 H5N9 H7N1 H7N2 H7N3 H7N7 H9N2 H9N7 H1N1 was the 1918 Spanish Flu type: 20%world population died H2N2 caused the 1957 Asian Flu H3N2 caused 1968 Hong Kong Flu H5N1 is the type associated with Avian Flu Is this a Potential Pandemic Strain?

14 Reported H5N1 Cases Worldwide WHO reports 387 cases and 245 deaths as of September 2008 Limited human to human transmission Only with close and continuous personal contact Most prevalent in Indonesia Cases also reported in Africa, Asia, Middle East, Turkey Close monitoring should enable rapid response if virus adapts to human propagation Greater human to human transmission will enable this virulent strain to spread rapidly to global lpopulation lti Target age group is unclear from limited epidemiological data One H5N1 vaccine is licensed in Europe for use in pandemic situation Rapid scale up and transfer of H5N1 vaccines to multiple manufacturing sites must occur if pandemic is declared

15 WHO Global Influenza Preparedness Plan (2005) VACCINES 1. Provide tools for countries on methods of assessing the annual burden of influenza as a means to increase vaccine use during interpandemic period. 2. Develop a prioritized global research and development agenda for producing innovative and more efficient vaccines. 3. Explore ways to shorten the time needed for vaccine prototype preparation by working with pharmaceutical companies, national authorities and research institutes. 4. Explore ways to increase availability of pandemic vaccines during pandemic alert and pandemic periods.

16 Baculovirus Expression of Influenza Antigens Influenza antigens can be expressed by cloning into Baculoviruses and growing in insect cell culture Baculovirus has limited host range No replication or infection of mammalian cells Rapid scale up and production of new vaccine strains possible Novavax (Rockville, MD, USA) has produced virus like particles containing HA, NA, and M protein in baculovirus Initiated clinical development in mid 2007 Protein Si Sciences (Meriden, CT, USA) has developed dflublok using their proprietary BEVS baculovirus expression system Contains three recombinantly produced HA antigens Over 50,000 doses delivered with minimal adverse events FiledBLAinApril in April 2008

17 Baculovirus Expression Process Isolate Target Gene Create Recombinant Baculovirus Strain Grow Up a Batch of Insect Cells Infect & Incubate Harvest Infected Culture Clarify Harvest Or Collect Cells Novavax: Purify VLP Protein Sciences: Extract & Purify rha

Other uses Veterinary vaccines Poliovirus vaccines, rabies vaccines Key advantage High viral yield Proven track record, used for other human vaccines Other human vaccines, MAbs, gene therapy,

18 Baculovirus vs. Cell Culture Options Origin MDCK Vero Per.C6 Sf9 Madin Darby African green Human Spodoptera Canine Kidney monkey kidney embryonic retina frugiperda Relative yield High Low Moderate Very high Tumorigenicity High Not Weak None demonstrated Other uses Veterinary vaccines Poliovirus vaccines, rabies vaccines Key advantage High viral yield Proven track record, used for other human vaccines Other human vaccines, MAbs, gene therapy, proteins High susceptibility to influenza virus Established protein expression system High yield, high scalability, low costs Key High Low influenza Newer cell line Immunogenicity disadvantage tumorigenicity virus yield perceived as low Companies involved Novartis Vaccines, Solvay, GSK Baxter Crucell, Sanofi-Pasteur Protein Sciences, Novavax Source: DataMonitor 2007

expressf+ Engineered for Vaccine Manufacturing

pressure in serum free media with added insulin

properties Ideal for Manufacturing serum free

< 1 produces high titer AcNPV cgmp at 500L

19 expressf+ Engineered for Vaccine Manufacturing Cell line developed from Sf9 Cells Selective pressure in serum free media with added insulin (0.4 mm) unique phenotypic and genotypic properties Ideal for Manufacturing serum free stable for > 50 passages infected with low MOI < 1 produces high titer AcNPV cgmp at 500L scale excellent safety record in clinical trials patented used for the commercial production of a USDA licensed veterinary vaccine Uninfected SF+ Infected SF+

20 Protein Sciences FluBlok Expression Baculovirus Expression Vector System (BEVS) Engineer baculovirus with the gene of interest (e.g. Hemagglutinin) Baculoviruses highly specific to insect cells Powerful promoter generates high yield of protein of interest Culture expression of insect cells in a fermenter Infect cells with engineered virus Incubate infection for ~48-72 hours Protein forms rosettes Purify protein to > 90% into final product Formulate with PBS into vaccine

21 FluBlok: Potential Benefits Influenza rha antigens are produced in insect cells protein based vaccine with low endotoxin content rha protein is highly purified and does not contain egg protein or other contaminants from eggs Selection or adaptation of influenza virus strains that produce at high levels in eggs is not required the best genetic match Cloning, expression and manufacture of FluBlokwithin 2 months FluBlok does not require large amounts of embryonated chicken eggs Manufacturing of FluBlok does not require biocontainment facilities Manufacture of rha does not include formalin inactivation or organic extraction procedures

22 Rapid Scale up Options for Vaccine Mfg Universal disposable bioreactor and purification equipment will enable use of multiple facilities with minimal impact on product quality Disposable systems reduce overall manufacturing costs Utilities Cleaning Maintenance and validation Disposable bioreactors are available from companies including Thermo fisher h GE Healthcare Xcellerex Downstream processing disposable modules are in development

XDR Single Use GMP Bioreactor Exhaust filter heater Bag

feed View Ports Probe Ports Tank weight control - Δ P or load

23 XDR Single Use GMP Bioreactor Exhaust filter heater Bag pressure protection and ΔP weight control Disposable Bag Assembly (incl. Filters & Tubing) Jacketed Stainless Steel Support Tank PLC or Delta V Controller: ph, DO, agit., temp, gas sparge, CO2 strip, headspace gas, weight, media feed View Ports Probe Ports Tank weight control - Δ P or load cells Shaftless, seal-less Bottom Mag. Drive Agitator Integrated Acid/Base, Media Feed Pumps Temperature Control Unit -cooling and heating no utilities needed

24 XDR GMP Single-Use Bioreactor Family Fully integrated systems with modular process control 2000 liter reactor (working volume) available now 5000 liter working volume system pending Characterized and modeled optimized performance on first run XDR-200 XDR-500 XDR-2000

25 Xcellerex: XDR Bioreactor Performance Cell type Product Mode Scale (w/v L) Cell Density CHO fusion proteins batch, fed batch 200L 2000L 5 13M cells/ml NSO mabs fed batch 200L 20M cells/ml Insect S2 subunit vaccine fed batch 200L 44M cells/ml Human HEK biotherapeutic fed batch 500L 19M cells/ml Human HEK therap. Enzyme perfusion 200L, 1,000L 10M cells/ml

26 Insect Cells - West Nile Vaccine Manufacturing XDR-200 Growth Curves Vials # E E E+07 Added Gl XDR-200 reaches 25% higher cell density than conventional bioreactor Cell Densi ty 3.50E E E E+07 Added Antifoam and Glucose Added Antifoam #21-1A Viable cells/ml #21-1A Viability (%) Added Glucose 1.50E E+07 Induce with 0.2 M CuSO E Added Antifoam 0.00E Time in XDR-200 (h) Highest cell density achieved in stainless steel bioreactors was 32 E6 cell/ml

27 Many Vaccine Types Need Universal Mfg. Facility Toxoid (Bacterial) Live, attenuated Virus- like particle Inactivated Subunit DNA Empirical Killed, metabolically active Purified Recombinant Recombinant Vector Prime boost combinations Conjugate (protein -polysaccharide)

28 Cell Culture and Disposable Platforms are Key to Rapid Scale up Egg based vaccine timeline (US) Seasonal strains available from CDC in February March First product available in November Elapsed time 8 9 months Unsuitable for pandemic response Cell culture timeline (recombinant antigen) Identification of seasonal antigens in October November Cloning, expression, and characterization of production cells Product available in 6 8 months Baculovirus timeline (recombinant or VLP) Cloning and generation of virus stock 2 months Scale up and production 3 months Elapsed time 5 6 months

29 Quality Control of Influenza Vaccines Safety Absence of live virus (inactivated/sub unit vaccines) Lack of pathogenicity (attenuated vaccines) Absence of microbial contaminants Lack of toxicity (whole virus vaccines may be pyrogenic) Purity Absence of contaminating ti (host cell) protein ti Potency Hemagglutinin Content Ability to produce HI antibodies in test animals Specificity Test for strain specific antigens (H and N)

30 Acknowledgements BioProcess Technology Consultants Alex Kanarek, Ph.D. Howard Levine, Ph.D. Protein Sciences Corporation Manon Cox, Ph.D. Xcellerex Parrish Galliher, Ph.D.

31 THANK YOU! BioProcess Technology Consultants, Inc. 289 Great Road, Suite 303 Acton, MA USA (phone) (fax) sjones@bioprocessconsultants.com

Challenges in Vaccine Production and Rapid Scale up to Meet Emerging Pandemic Threats

Challenges in Vaccine Production and Rapid Scale up to Meet Emerging Pandemic Threats Susan Dana Jones, Ph.D. BioProcess Technology Consultants, Inc. BIO 2009 Process Zone Theater Atlanta, GE May 20, 2009