Process Development for a Peptide Conjugated QbetaVirus Like Particle (VLP) Vaccine

Transcription

1 Engineering Conferences International ECI Digital Archives Vaccine Technology IV Proceedings Spring Process Development for a Peptide Conjugated QbetaVirus Like Particle (VLP) Vaccine Jennifer Thorn Pfizer Follow this and additional works at: Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Jennifer Thorn, "Process Development for a Peptide Conjugated QbetaVirus Like Particle (VLP) Vaccine" in "Vaccine Technology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves, ITQB/IBET; K. Jansen, Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Vaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.

2 NTICE The contents of the following document are or may be subject to copyright restrictions. Do not print, save, copy or forward this document.

3 Process Development for a Peptide Conjugated Qbeta Virus Like Particle (VLP) Vaccine Presented by: Jennifer Thorn Senior Principal Scientist Pfizer, Inc. Vaccine Technology IV Albufeira, Portugal May 20-25, 2012

4 utline Background Confidence in Platform Robustness of process Process consistency Key Quality Attribute Peptide density vs. immunogenicity Manufacturing Drug substance process Process consistency with scaling 3

5 What is Qβ VLP? PNAS November 2, 2004 vol. 101 no Subunit is the capsid protein from Qβ bacteriophage 180 identical subunits selfassemble to form the Virus- Like Particle Proteins and nucleic acids from E. coli host cell are trapped in VLP RNA is necessary for proper VLP assembly 4

6 Qβ Background VLP Capsid 180 monomer subunits 5 external conjugation sites per monomer 2 primary sites identified rganized array of antigens Host Cell RNA E.Coli RNA is packaged during assembly of the VLP Drives assembly and is a potential TLR7 ligand Qbeta Linker HCP & DNA RNA Peptides Technology licensed from Cytos Biotechnology AG in Schlieren Switzerland 5

7 Qbeta Forms Consistently Shaped VLP Particles ¾ Electron microscopy image of purified Qbeta VLP particles ¾ Avg. diameter = 23+/-3 nm Single VLP Particle 6

8 VLP Internal HCP s are Consistent Up to 2% of the total protein present in VLP batches is HCP, of which 98% is contained inside the VLP Capsid structure is disrupted to release internal HCP s Western blot probed with anti-hcp antibodies shows a consistent banding pattern across different lots of VLP Ref. Std. Lot 1 Lot 2 Lot 3 Lot 4 7

9 VLP Internal RNA is Consistent Extracted RNA size Average weighted nt size Ref Std. Lot 1 Lot 2 Lot 3 Lot 4 Nucleotides Lot 1 Lot 2 Lot 3 Lot 4 A B C A B C A B C A B C Extracted RNA samples on Bioanalyzer 25 8

10 Gene expression intensity profile of Pfizer VLP Lots Normalized expression Intensity Pfizer Lot avg. Pfizer Lot 1 Pfizer Lot 2 Pfizer Lot 3 Pfizer Lot Probe set number (sorted by Pfizer lot avg intensity) 9

11 Conjugation Chemistry Activation of Protein Qbeta VLP Bifunctional SMPH (Succinimidyl 6-[(betamaleimidopropionamido)hexanoate]) Linker Activated Qbeta VLP NH 2 + N N N H NHS-ester reacts with free amines to form stable amide bond VLP H N N H N Conjugation of Antigen Activated Qbeta Conjugate VLP H N N H N Maleimide reacts with free sulfhydryl to form thioether bond HS - Peptide HS Peptide VLP H N N H N S Peptide 10

12 Peptide Density by SDS PAGE avlp Increasing SMPH Ratio tetramer Higher MW bands correspond to intra VLP cross-linking trimer dimer monomer Peptide Density is calculated from the monomer bands Peptide Density 11

13 Mechanisms of Cross-linking Cross-linking is mostly intra-vlp SEC data shows < 5% HMWS Mostly likely occurs before peptide addition Extent of cross-linking is reproducible Intra-VLP Crosslink Inter-VLP Crosslink 12

14 Effect of SMPH Ratio on Peptide Density Peptide Density (Peptides/VLP Monomer) ml Reactions PD 1.8 (500 mg) PD 1.7 (2.4g) Peptide For a given peptide density, SMPH 0.5 ratio can be predicted from curve Peptide density is robust with 0.0 increasing scale SMPH Ratio PD (500 mg) PD (1.4g) Peptide 1 13

15 Small-scale Reactions Help Determine Manufacturing Conditions 15ºC Activation 15ºC Activation - P Time and Temp Interaction (10x SMPH) Epitope Density SMPH Molar Ratio 4 Hour Activation 5 Hour Activation 7 Hour Activation 4 Hour Activation at RT Time (hr) Temperature (ºC) Systematic parameter optimization leads to the same conclusion as DoE Increase activation time by 1 hour to adjust for lower temp at pilot plant in order to hit peptide density target

16 ph Effect on Peptide Density Peptide Density x 7.5x 10x ph Peptide density decreases with increasing ph Peptide density increases with increasing molar excess of SMPH 15

17 Immunogenicity is Affected by Peptide Density Peptide Conjugate load adjuvant adjuvant adjuvant adjuvant 2.79 Best responses with peptide density >

18 Drug Substance Manufacturing Process Prefilter And Final Filter Step 1 Activation Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 2 Diafiltration Step 3 Conjugation Step 4 Ultrafiltration/ Diafiltration Description of Steps Step 5 Formulation Step 6 Filtration VLP and bifunctional linker, SMPH, mixed to form activated VLP Excess linker is removed by a DF operation Peptide is added to the activated VLP Excess peptide is removed by a UF/DF operation Addition of excipients Final filter and packaging 17

19 Conjugation Reaction is Consistent Analytical Results for 6 Conjugates (100 mg lab scale) Lot Number ph Peptide Density peptides / monomer Purity % purity RNA Size average NT length RNA Quantity μg/mg Not Tested

20 Process Consistency Immunogenicity Data Mice were vaccinated with samples from the 6 consistency runs Antibody titers to the target antigen were measured Antibody Titers RS=Reference Standard Antibody titers are consistent among runs Pfizer Confidential 19

21 Peptide Density on Scaling Peptide Density is consistent as process is scaled Peptide Density Scale (g) 20

22 SEC Purity on Scaling % SEC Purity Scale (g) 21

23 Conclusions Conjugation to Qbeta VLP is a robust and reproducible platform for vaccine production. The process is robust from lab-scale to clinical manufacturing scale. Key quality attributes of peptide density and SEC purity are tightly controlled and are reproducible in terms of both lot consistency and batch scale. 22

24 Acknowledgements Bioprocess R&D Brian Matthews Renata Crutcher Chandra Gamlath Keshab Bhattacharya Brandi sborne Sydney Hoeltzli Nate Jenkins Andy Espenschied Greg Scheidt Cytos Biotechnology AG Analytical R&D John Amery Aparna Deora Scott Allen Qin Zou Justin Sperry Vaccines Research Brian Champion James Merson Phil White Sandrine Barbanel Maria Hedlund Daisy Hammond Joe Binder 23