Viral-vectored vaccines: a new approach in the vaccine manufacturing process

Dr. Priyabrata Pattnaik Director – Asia Vaccine Initiative

Agenda

Vaccine Industry Trends 1

2 Market Trend of Viral based Biologics

Recombinant Virus as Vaccine Vectors 3

4 Growth of R&D on Adenovirus Vaccine

Evaluation and feasibility study for Adenovirus vaccine 5

6 Case study: Ark Therapeutic’s ATOSUS

Process Development Concept and Scale-up of Adenovirus Process 7

8 Conclusion and Acknowledgement

Vaccine Industry Trends

Vaccines segment is growing at 8%.

~ 400 vaccines are in commercial manufacture. 300 are viral based.

~1400 vaccines are in development

~ Half of the vaccines undergoing clinical trials are viral based.

~ 640 viral vaccines

~ 200 viral vectors

~ 60 vaccine like particles

Another ~240 gene therapy products in development that utilize the same technology.

3

Market Trend of Viral based Biologics

0 100 200 300 400 500 600

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 YTD

Vaccine Clinical Trials Viral Vaccine Trials Vaccine Trials

25%

Biologics in Development Vaccines Recombinant Mabs Other

Source: Expression Systems Analysis, G Adams 2011, Clinitrials.gov 4

Recombinant Viruses as Vaccine Vectors

Adenoviruses Non-enveloped virus, 70-100 nm in diameter Genome: linear double-stranded DNA Efficiently transduces non-active and dividing cells Large (>30 kb) transgene capacity Easily produced in high titers

CAR receptor dependent transduction Immunogenic Existing humoral response to certain serotypes

5

Adenovirus Controversy

6

Merck & Co scraps Adenovirus based AIDS vaccine trial Adenovirus vector triggered immune response Body flooded with CD4 cells CD4 is also target for HIV Since then……

Mol. Biol. of adenovirus improved Adenovirus engineered to increase target specificity Adenovirus induced immunology is better understood

Growth of R&D on Adenovirus Vaccine

0

50000

100000

Vaccine Vectored Vaccine

Adenovirus Vaccine

Number of citation in Google Scholar in 2012

0

5000

10000

15000

20000

Citation on Adenovirus vaccine in Google Scholar

7

Oncolytic Vaccine Vector

Source: J.-W. Choi et al. / Advanced Drug Delivery Reviews 64 (2012) 720–729 8

9

Generic Process of Vectored Vaccine Manufacturing

10

Cell Culture for Adenovirus Typical cell culture: HEK293

Cell culture either in continuous or batch mode (w/ media exchange)

Accumulating lactic acid is detrimental to virus production

Virus yield drops significantly when the media pH < 7

Typical cell concentration during virus infection is 2 - 10 x106 cells/ml

Adenovirus titre during harvest generally ranges from 109 to 1011 pfu/ml.

11 Cell culture growth plot, Courtesy: Dr. David Venables, Ark Therapeutics

Fast-Trap™ Lentivirus and Adenovirus Purification & Concentration Kit Kit contains necessary reagent Membrane-based, closed vacuum-driven device High recoveries of purified viable viral particles in under two hours

Source: Merck Millipore Literature No. AN1066EN00 Rev. B

13

Collaboration with GenVec Inc., Gaithersburg, MD USA. Filter sizing for medium exchange, lysate clarification, post- clarification filtration, Ultrafiltration/ diafiltration, and post-hold sterile filtration prior to column chromatography

Adenovirus work before the controversy

Millistak+® Pod filter for lysate clarification

14

Millistak+® filter C0HC and DE30 provided good capacity (>150L/m2) Millistak+® filterC0HC and DE30 provided desired turbidity reduction Millistak+® filter B1HC filter plugged prematurely Millistak+® filter CE30 filter led to turbidity breakthrough

Work in collaboration with GenVec Inc., Gaithersburg, MD, USA. Reference: Bioprocessing Journal, Fall 2006, pp 67-74.

Filter sizing for manufacturing scale

Work in collaboration with GenVec Inc., Gaithersburg, MD, USA. Reference: Bioprocessing Journal, Fall 2006, pp 67-74.

Process Steps Devices 10 Liters 100 Liters 1,000 Liters

Medium Exchange

ProstakTM 0.33 m2 membrane 1 x 4 Stak Module

3.4 m2 membrane 2 x 20 Stak Module

34.0 m2 membrane 20 x 20 Stak Module

Lysate Clarification

Option 1/Step1: Millistak+® DE30 or Millistak+® C0HC Option 2/Step 1: Clarigard® (3 µm) Option 2/Step 2: Polysep IITM (1/0.5 µm)

0.093 m2 area 0.060 m2 area 1 x 10” device 1 x 2” Opticap® XL

0.7 m2 area 0.47 m2 area 4 x 30” 1 x 10” Opticap® XL

7.0 m2 area 4.7 m2 area 34 x 30” 4 x 30” Opticap® XL

Post-clarification filtration

0.45 µm Durapore® 0.025 m2 membrane 0.25 m2 membrane 2.5 m2 membrane

Concentration/ Diafiltration

Pellicon® 2 Module with Biomax® membrane, 500kD, C screen

0.3 m2 membrane (3X)

2.5 m2 membrane 25 m2 membrane (10X)

Post-Hold/ Pre-Column Filtration

0.22 µm Durapore® 5 Liters Millipak® 20 100 cm2 membrane

50Liters Millipak® 200 1000 cm2 membrane

500 Liters 1 x 20” Capsule 1.38 m2 membrane

Nucleic Acid Removal in Adenovirus Process

General guidelines: 100 pg/dose[1] or <10 ng/dose[2]

Adenovirus-specific regulatory guidance: 10 ng would only be acceptable provided that the DNA was digested to less than 100-200 base pairs in length[3]

Adenoviruses are typically produced at about 104-105 viral particles (vp)/cell[4]

Mammalian cells have a genome of about 10 pg[5]

7 logs of DNA clearance would be required in order to attain levels below 100 pg/dose for a high (1012 vp) dose of adenovirus.

[1] Acceptability of cell substrates for the production of biologicals. Report of a WHO Study Group. In WHO Technical Report Series; World Health Organization: Geneva, 1987. [2] Grachev et al., WHO requirements for the use of animal cells as in vitro substrates for the production of biologicals. Biologicals 1998, 26 (3), 175-193. [3] Bauer et al., Testing of Adenoviral Vector Gene Transfer Products: FDA Expectations. In Adenoviral Vectors for Gene Therapy; Curiel, D. T., Douglas, J. T., Eds.; Academic Press: New York, 2002; pp 615-654. [4] Nadeau and Kamen. Production of adenovirus vector for gene therapy. Biotechnol. Adv. 2003, 20 (7-8), 475-89. [5] Kraiselbuld et al., Presence of aherpes simplex virus DNA fragment in a L cell clone obtained after infection with irradiated herpes simplex virus 1. J. Mol. Biol. 1975, 97, 533-542.0

Benzonase® for Nucleic Acid Removal from Adenovirus Process

rAd5 purification process

Host cell DNA to below a detectable limit

Combination of ultrafiltration and anion exchange chromatography

Source: Konz et al., Biotechnol. Prog. 2005, 21, 466-472 (Merck & Co)

18

Adenovirus Production Process

Kamen and Henry, Development and optimization of an adenovirus production process, J Gene Med 6, S184–S192, 2004

Adenovirus production Harvest

Liquid

Cell lysis

Benzonase® treatment/ Centrifugation

Anion Exchange Chromatography on Fractogel® DEAE media

Solid

Filtration

Ultrafiltration/ Concentration

Ret

enta

te

(ade

novi

rus)

Size Exclusion Chromatography

Purified Adenovirus

19

Fractogel® DEAE-650 M for purification of Adenovirus >92% purity and 70-80% yield

Sample preparation: Viruses propagated in HEK293 cells Lysis by 3 cycles of freeze/thaw of washed cells after centrifugation; Benzonase® (final conc. 100 U/ml) was added for 30 min at RT. Chromatography: -Viral lysate loaded onto a 3 x 6 cm Fractogel® DEAE (M) column -Equilibrated with 50 mM TRIS/HCl, 100 mM NaCl, 2 mM MgCl2, 2% sucrose; pH 8. - Sequential washing with 0.1 & 0.25 M NaCl. Elution: Bound virus was eluted at 0.35 M NaCl.

virus

time

A260

0.1 M

0.25

M 0.

35 M

1 M

0.5

M N

aOH

Source: Puresyn, Inc.; Malvern, PA

Presentation title in footer | 00 Month 0000 20 20

Case studies

Case study: ATOSUS Ark Therapeutics Oy Single Use Systems

Thawing and expansion of MCB or WCB

Production of modified adenoviral products in GMP3 facility (ATOSUS)

Cell lysis and clarification of CVL’Clarified Harvest’

Infection of expanded cells with MVSS or WVSS’Bulk Harvest’

Buffer conditioning by crossflow ultrafiltration

’TFF 1 Product’

Capture and polishing with chromatography

’Chromo 1 Product’’Chromo 2 Product’

Concentration and diafiltration by crossflow ultrafiltration

’Purified Bulk’

Fill & Finish’Drug Substance’

’Drug Product’

Final Formulation Buffer preparation

Dow

nstr

eam

pro

cess

ing

Buffer-, storage- & CIP solutions preparations

Ups

trea

m

proc

essi

ng

Medium preparation

High cell densities achieved, up to 30E+6 cells/ml. High upstream infections achieved, cell yields > 60,000 vp/cell. High downstream purified yields achieved, >2x1015vp/10L batch HPLC analysis validated for Crude Viral Lysate, to allow tracking of vps Light scattering analysis to monitor aggregation/precipitation – correlates with HPLC Potency, infectivity, hcDNA, HCP achieved within product specifications Single use processing - plug and play for other AdV projects with tweaks

21

Ark’s Ad5 Manufacturing Overview Adherent used for Phase III Cerepro supply

22

Cell culture ~ 5-6 weeks

Viral Infection – Harvest ~ 2 days

Lysis and Purification ~3 days

Sterile filtration and filling ~1 day

Process Development Concept

10 L 10 L

10 L 10 LProcess Development DSP

10 L 10 L

10 L 10 LDevelop 100L CC Robust 10L GMP Production

100 L 10 L

100 L 100 L Scale Up DSP x10

100 L 100 LRobust 100L GMP Production

Develop 300L CC300 L 100 L

300 L 300 L Scale Up DSP x 3

300 L 300 LRobust 300L GMP Production

Stepwise Progression of Existing Process Each Unit Operation Developed and Scaled Independently Stepwise Scale Up reduces unknowns and risk of failure Time given for Engineering Runs and Requirements for cGMP

Cell Culture Downstream Process

23

Scale-up of developed DSP of Adenoviral ATOSUS Process from bench to pilot scale using commercially available, off-the-shelf systems and single use assemblies

Lysis & Benzonase® Treatment Clarification Intermediate TFF

Tangential Flow Filtration

Last Step Drug

Substance

First step Cell thawing

Cell culture (perfusion) – 5 weeks Virus infection – 2 days

6 weeks

USP week 1 to 5

DSP week 5

DSP week 6

Final Filtration

2 stage Ion Exchange Chromatography

Spanning our clients’ Vaccine Processes

Ingredients/CIP Chromatography

Filtration

Virus Clearance Parenterals

Lipids / PEGs

Enzymes Buffers

EMPROVE® bio

EMPROVE® bio

Clarification

Filtration

EMPROVE® bio

eprova Media Production

Single-use production / sampling

Supplements

Clarification

Services and Solutions Services and Solutions

Services and Solutions

25

Acknowledgements Ark Therapeutics Minna Karhinen Robert Shaw Kassim Kolia David Venables Merck Millipore George Adams Nicolas Laroudie Paul Rickets

26