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A549: A Novel Cell Substrate Enabling Production of New Vaccine Candidates VRBPAC Silver Spring, MD September 19, 2012.

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Presentation on theme: "A549: A Novel Cell Substrate Enabling Production of New Vaccine Candidates VRBPAC Silver Spring, MD September 19, 2012."— Presentation transcript:

1 A549: A Novel Cell Substrate Enabling Production of New Vaccine Candidates VRBPAC Silver Spring, MD September 19, 2012

2 Presentation Overview
PaxVax Vision Rationale for Selection of the A549 Cell Substrate for adenovirus based vaccines production The origin and regulatory status of the A549 cell line Evaluation of A549 for Vaccine Production Absence of adventitious agents Absence of oncogenic agents Clearance of intact cells and residual DNA Conclusions Proprietary VRBPAC, 19th Sept 2012

3 PaxVax Vision Unique Live Oral
Ad4- and Ad7-Based Replication Competent Vaccines Potential Novel Vaccine Candidates: H5, H7 pandemic flu HIV Anthrax HSV HPV CMV Malaria Tuberculosis Insert gene of relevant antigen(s) Grow in high yield cell substrate A549 Proprietary VRBPAC, 19th Sept 2012

4 A549 is an Ideal Cell Substrate for Ad4-Based Vaccine Production
PaxVax reviewed available cell substrates for suitable Ad4 productivity Ruled out non-human cells due to species specificity Evaluated human diploid cell lines that are used for licensed vaccines: WI-38, which is used for the Ad4/Ad7 vaccine, and MRC-5 Typically, PaxVax vaccines have a lower yield compared to wild type Ad4 Ruled out human diploid cells for our use due to low productivity After screening human continuous cell lines, A549 identified as the optimum candidate for production purposes Proprietary VRBPAC, 19th Sept 2012

5 A549 Cell Substrate Essential for Current Vaccine Development Work
Initial Phase 1 H5 pandemic influenza vaccine candidate produced in MRC-5 cells However, yield in MRC-5 is suboptimal to supply large-scale clinical trials and insufficient to support potential pandemic needs Two Anthrax vaccine candidates in development with NIH/DMID Yield in MRC-5 very low; insufficient for even Phase 1 studies Proprietary VRBPAC, 19th Sept 2012

6 A549 Cell Substrate Essential for Current Vaccine Development Work
HIV vaccine candidates based on gag and truncated env gp145 Phase 1 material produced in MRC-5 but scale insufficient for further vaccine development HIV vaccine candidate based on full length native gp160 Production only possible in A549 cell substrate; gp160 reduces vaccine yield from MRC-5 cells gp160 antigen is of interest to HIV vaccine community Further clinical development of this important antigen in jeopardy if no suitable cell substrate available for production Proprietary VRBPAC, 19th Sept 2012

7 A549 Shows 10-40x Higher Yields for Several PaxVax Ad4 Vaccines
Summary: Anthrax and HIV (gp160) dependent on A549 for development A549 essential for Pan Flu and HIV (gp145) to reach needed production scale Proprietary VRBPAC, 19th Sept 2012

8 Origin and Regulatory Status of the A549 Cell Substrate
Human lung adenocarcinoma-derived (source: 58 yr old Caucasian male, 1972) Deposited at ATCC in 1976 PaxVax prepared Master Cell Bank; undertook comprehensive characterization with support of Wellcome Trust and NIH, with guidance from CBER Biologics Master File submitted to CBER (Oct 2011) Condition of W. Trust funding is that PaxVax A549 cell substrate be made available for licensure to other parties Proprietary VRBPAC, 19th Sept 2012

9 Evaluation of Use of A549 Cell Substrate for Vaccine Production
Extensive Characterization of A549 Cell Substrate Adventitious Agents Testing General screening methods and specific agent PCR tests Chemical induction studies for latent or occult viruses Massively parallel sequencing Oncogenicity testing of cell lysates and DNA Animal studies in mice, rats and hamsters Tumorigenicity profile assessment in mice Review of production process for ability to clear intact viable cells, cellular DNA and proteins Proprietary VRBPAC, 19th Sept 2012

10 Consistent with human origin
Adventitious Agent Studies: All Tests Negative General Screens for Microbial and Viral Agents Test Type Method Test Article Outcome Identity Karyology MCB Consistent with human origin Isoenzyme Microbial Mycobacteria Negative Sterility test MCB & EOPC No growth Mycoplasmas Viral In vivo screen inapparent viruses None detected In vitro screen viral contaminants Transmission EM for viruses and retroviruses Proprietary VRBPAC, 19th Sept 2012

11 Adventitious Agent Studies: All Tests Negative for Specific Agents
Test Type Method Test Article Outcome Bovine Viruses In Vitro Assay Presence Bovine Viruses 9CFR MCB None detected Qualitative RT-PCR: Bovine polyomavirus Negative PCR – Bovine/Porcine circovirus PCR – Bovine herpesviruses 1 and 4 PCR – Bunyavirus (Cache Valley Virus) Porcine Viruses In Vitro Assay Presence Porcine Viruses Mod 9CFR Infectivity – PRRSV PCR – Porcine cytomegalovirus PCR – Encephalomyocarditis virus (EMCV) PCR – Swine Hepatitis E PCR –Swinepox Virus Rodent Viruses Rat Antibody Production Test (RAP) Hamster Antibody Production Test (HAP) Mouse Antibody Production Test (MAP) Proprietary VRBPAC, 19th Sept 2012

12 Adventitious Agent Studies: All Tests Negative for Specific Agents
Test Type Method Test Article Outcome Simian Virus PCR – Simian virus 40 (SV-40) MCB Negative Human Viruses PCR – HIV1/2 PCR – HTLV I/II PCR – Hepatitis A PCR – Hepatitis B PCR – Hepatitis C PCR – Epstein Barr Virus PCR – Cytomegalovirus PCR – HHV6 (Herpes Virus 6) PCR – HHV7 (Herpes Virus 7) PCR – HHV8 (Herpes Virus 8) PCR – KI polyomavirus PCR – WU polyomavirus PCR – Human polyomaviruses (JCV, BKV) PCR – B19 virus Retrovirus PERT Assay for the Detection of Retrovirus AAV PCR – Adeno-Associated Viruses(AAV) Proprietary VRBPAC, 19th Sept 2012

13 Chemical Induction Studies for Latent or Occult Viruses: None Detected
Viral induction conditions optimized with guidance from Dr. Arifa Khan, CBER* TPA & NaB for DNA viruses IdU or AzaC for RNA viruses No viruses detected by extensive analyses TEM and PERT: Negative for retroviruses Coculture by serial passage with indicator cells: Negative for latent RNA viruses Degenerative PCR: Negative for DNA viruses *Khan et al, Proposed Algorithm to Investigate Latent and Occult Viruses in Vaccine Cell Substrates by Chemical Induction. Biologicals : Proprietary VRBPAC, 19th Sept 2012

14 Massively Parallel Sequencing (MPS) For Detection of Adventitious Agents
Cellular transcriptome analysis (mRNA Shotgun MPS) [Latent/Integrated virus detection] DNA Transcription RNA Extracellular nucleic acids & Culture media components (Amplicon MPS) [Encapsidated/Productive virus detection] A549 DNA RNA Proprietary VRBPAC, 19th Sept 2012

15 Adventitious Agent Studies: Massively Parallel Sequencing (MPS)
A549 MPS Data 2009 Analysis (database of 300,000 entries) 2012 Reanalysis of data (database expanded to >1,000,000); same result No viral sequences detected by analysis of cellular transcriptome Extracellular medium: bovine viral sequences present Bovine viral sequences traced to preparatory serum (MPS and qPCR) A549 MCB negative for the bovine viral sequences (qPCR) Conclusion: No  transcripts associated with replicating, latent or transforming viruses detected in A549 cells (or MRC-5 control) MPS sensitive enough to detect viral sequences Future production serum (γ­irradiated) lots now screened for specific bovine sequences Proprietary VRBPAC, 19th Sept 2012

16 Conclusion: Adventitious Agents Not Detected in A549
None detected in general screens (broad detection) No viruses detected by extensive type-specific assays 30 standard plus 11 human specific viruses per FDA suggestion Rare bovine, porcine, rodent, simian Human retroviruses, adeno-associated viruses General antibody production: hamster, mouse & rat Latent or occult DNA and RNA viruses not detected by chemical induction tests Massively Parallel Sequencing did not detect viral signatures in A549 cells Also, PRNP gene sequencing found no mutations that predispose to transmissible spongiform encephalopathies (TSEs) Proprietary VRBPAC, 19th Sept 2012

17 A549 DNA or Cell Lysate is Not Oncogenic
Oncogenicity: Ability of DNA or acellular materials to induce tumor formation in animals PaxVax studies showed A549 cell lysates and A549 DNA did not induce tumors in nude newborn mice, rats or hamsters The lack of tumor formation by high molecular weight A549 cellular DNA result is consistent with publicly available data, including that in Newborn CD3 Epsilon mice (Dr Keith Peden, CBER) Proprietary VRBPAC, 19th Sept 2012

18 A549 Cell Substrate Tumorigenicity Profile
A549 cells, known to be tumorigenic, had a TPD50 value of log in a study in athymic nude mice conducted by PaxVax Group N Tumors at Injection Site Spontaneous Tumors at Other Locations Negative Control 10 Positive Control 1 (ear sarcoma) 101 A549 Cells 103 A549 Cells 105 A549 Cells 8 107 A549 Cells Proprietary VRBPAC, 19th Sept 2012

19 Clearance of A549 Cells and DNA
Concern is about potential presence of transforming genes from residual cell substrate DNA and presence of intact viable cells in final vaccine product The clearance of intact viable A549 cells, DNA and cellular protein is achieved by multiple purification steps in the PaxVax manufacturing process Proprietary VRBPAC, 19th Sept 2012

20 PaxVax Manufacturing Process Removes A549 Components at Multiple Steps
A549 MCB/WCB Clarified Virus Benzonase A549 DNA Digestion Virus Infected A549 Intermediate #1 Detergent Lysis Intact Cell Destruction Anion Exchange Chromatography A549 DNA & Protein Removal Virus Lysate #1 Intermediate #2 Freeze-Thaw Intact Cell Destruction Diafiltration A549 DNA & Protein Removal Virus Lysate #2 Intermediate #3 Filtration #1 Depth 0.8/0.22 µm Intact Cell Removal Filtration #2 0.22 µm Intact Cell Removal Clarified Virus Bulk Drug Substance Proprietary VRBPAC, 19th Sept 2012 Oral Dosage Form

21 Residual Cellular DNA Below Limit of Detection
Several process steps remove or degrade DNA No residual cellular DNA detected in 5 small-scale Ad4 wild type BDS lots made using the PaxVax process High sensitivity qPCR (non-validated) All results below the level of detection (LOD <0.13 ng/mL) Equivalent to <0.013 ng/dose (1010 vp vaccine dose) Absence of residual DNA makes DNA fragment size determination impractical Any DNA fragments are likely to be <200 bp due to Benzonase treatment in the PaxVax process Benzonase able to reduce DNA to 2-5 nucleotides in length PaxVax process uses 25,000 U/L for 3 hours (exhaustive digestion) Proprietary VRBPAC, 19th Sept 2012

22 The A549 Cell Substrate is Suitable for Production of Human Vaccines
A549 cell substrate is uniquely suited for Ad4-based vaccine production at a scale to meet potential global medical needs 10 to 40 fold higher yield than MRC-5 Extensive characterization failed to identify any adventitious agents in the A549 MCB General microbial and viral screening tests and specific virus PCR tests Chemical induction for latent/occult viruses Massively parallel sequencing Lack of oncogenicity of A549 DNA or cell lysates in standard animal models Robust manufacturing process includes orthogonal clearance steps which remove A549 DNA and intact viable cells PaxVax recommends that the A549 cell substrate be accepted for use for the production of human vaccines Proprietary VRBPAC, 19th Sept 2012


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