viral-vectored vaccines: a new approach in the vaccine manufacturing process
TRANSCRIPT
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.
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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
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Adenovirus Controversy
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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
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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
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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
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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
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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)
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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
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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
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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
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Ark’s Ad5 Manufacturing Overview Adherent used for Phase III Cerepro supply
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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
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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
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Acknowledgements Ark Therapeutics Minna Karhinen Robert Shaw Kassim Kolia David Venables Merck Millipore George Adams Nicolas Laroudie Paul Rickets
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