Case study of formulating two Subtype C gp120 proteins

Transcription

1 Case study of formulating two Subtype C gp120 proteins Antu Dey, DPhil. Senior Director, IAVI (formerly at GSK Vaccines) Associate Project Leader HIV Research Head Formulation Sciences & Process Development (Development) 2 nd Env Vaccine Manufacturing Workshop September 15, 2016

2 Outline of Presentation Introduction Selection of 2 Subtype C protein; Research & early Development results Strategy of formulating gp120 proteins Targeted formulation studies Instability of 1086.C gp120 & ph selection Conclusions 2 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

3 Introduction To support P5 (Pox-Protein Public Private Partnership) s plan to evaluate safety and efficacy of a prime-boost strategy with a canary-pox vector as the prime (Sanofi-pasteur) followed by a boost with a bivalent gp120 protein vaccine (GSK Vaccines) administered with MF59 TM* adjuvant. A commitment was made to produce and deliver ~50,000 doses of two selected subtype C gp120 antigens for use as boosts in proof-of-concept clinical trials to be conducted by the HVTN. Two gp120 antigens were selected - TV1.C gp120 and 1086.C gp120. Both gp120s were recombinantly expressed in stable CHO-cell lines as secreted proteins and purified from culture media. The strategy was to manufacture frozen monovalent drug product for each of the gp120 antigens and perform bed-side mixing with MF59 TM adjuvant prior to intramuscular administration. *MF59 is a trade mark of Novartis, used under license by the GSK group of companies 3 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

4 Introduction Research study conclusion (mid-2012) Based on pre-set criteria and rigorous selection, TV1.C and 1086.C gp120s were selected for bivalent subtype C boost (with MF59 TM ) for RSA trials. Stable CHO-cell lines selected for both gp120s; two-step ion-exchange based purification process developed that generates >95% pure, homogenous gp120s. Both gp120 proteins were stable as DS and can be readily formulated in MF59 TM as stable DP at ph 6.5; supported by in vitro and in vivo studies at <0.8mg/mL. 4 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

5 Formulation of gp120 proteins Formulation development was geared towards the strategy of gp120 antigens being bed-side mixed with MF59 TM adjuvant prior to IM administration. The components of the frozen formulation for the 2 subtype C gp120 proteins were selected based on prior development studies for a HIV gp140 protein (Chiron), consisting of citrate buffer (10mM) and sodium chloride (300mM) at ph 6.5. MF59 TM adjuvant Oil-in-water emulsion; aqueous phase is Sodium citrate buffer ph 6.5 All Research and early Development studies performed in Drug Product (DP) formulation buffer: 10mM citrate ph 6.5, 300mM sodium chloride. 5 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

6 Scale-up and late Development Process scale-up and testing (mid-2013) During drug substance and drug product scale-up studies, residual protease activity was found to be high at low phs ( ), resulting in high clipping of 1086.C drug product upon storage. The high clipping and the cleavage sites on 1086.C gp120 protein were investigated through N-terminal sequencing studies. Clipping in V1V2 and V3 loops Studies were subsequently performed to optimize the ph and to control protease-mediated clipping and an optimized formulation condition was proposed. 6 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

7 ph Range Studies TV1.C and 1086.C Selection of ph range for both gp120 DPs TV1.C gp120 ph range study Results: DP concentration 800±120µg/mL, ph 6.5 ± 0.3; Minimal degradation observed at C gp120 ph range study Results: DP concentration 800±120µg/mL, ph 7.0 ± 0.3; degradation observed at ph <6.5 7 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

8 SEC and SDS-PAGE to estimate 1086.C gp120 clipping MW = Molecular weight marker; NR = non-reduced; R= Reduced; C19 = Batch # 1086.C gp120 ph 6.5 (37 C), 1week data 8 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

9 monomer % monomer % ph Range studies: 1086.C gp120 - I SEC and SDS-PAGE (reduced condition) Results C (reduced samples) ph 6.5 ph 7.0 ph 7.5 ph 6.5 ph 7.0 ph C 1d 1 week 2 week 4 week At 4 C (accelerated temperature), 1086.C gp120 DP is stable across ph range ( ) for 1, 2 and 4 weeks via both SEC and SDS-PAGE No change at -80 C as well. 1-day -80 C sample used as reference standard in both assays SEC SDS-PAGE C (reduced samples) ph 6.5 ph 7.0 ph 7.5 ph 6.5 ph 7.0 ph C 1d 1 week 2 week 4 week At 25 C (stressed temperature), 1086.C gp120 DP is not stable at ph 6.5; evident after 2 and 4 weeks via both SEC and SDS-PAGE 1-day -80 C sample used as reference standard in both assays SEC SDS-PAGE

10 monomer % monomer % ph Range studies: 1086.C gp120 - II SEC and SDS-PAGE (reduced condition), 37 C, Results 37 C, SEC (reduced samples) 37 C (reduced samples) time (weeks) C, ph C, ph C, ph C1d, ph C1d, ph C1d, ph ph 6.5 ph 7.0 ph 7.5 ph 6.5 ph 7.0 ph 7.5 SEC SDS-PAGE -80C 1d 1 week 2 week 4 week At 37 C (stressed temperature), 1086.C gp120 DP is not stable at ph 6.5; evident after 1, 2 and 4 weeks via both SEC and SDS-PAGE 1-day -80 C sample used as reference standard in both assays Based on these results, unlike TV1.C gp120, 1086.C gp120 cannot be formulated as DP at ph with set-point Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

11 Selection of ph for 1086.C gp120 DP Therefore, 1086.C gp120 DP concentration 800±120µg/mL had to be formulated in 10mM Citrate, 300mM NaCl ph 7.0 ± Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

12 ATM selection key to stability studies Protein quantitation methods, A280 and RP-HPLC, were largely non-discriminatory Gp120 Concentration using A280 RP-HPLC - main peak % DLS and MFI results showed no significant difference across ph ranges ATM=Analytical Test Method; DLS=Dynamic Light Scattering; MFI=Micro-Flow Imaging 12 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

13 Conclusions Small-scale (Research & early development) studies were not indicative of final (scaled-up process-produced) gp120 protein stability. Scaled-up process generated Drug Substance and Drug Product, at their intend-use concentration, should be assessed for stability Upon scale-up, impurities (Process-related and product-related) may vary Impurities, and levels therewith, do have an impact on DS/DP stability Despite previous experience of formulating gp120 proteins in 10mM Citrate, 300mM NaCl ph 6.5, stability studies of 1086.C gp120 indicated use of ph 7.0 ± 0.3 (to reduce risk of V1V2 & V3 loop clipping ). Choice of appropriate stability-indicating Analytical Test Methods are key to understanding DS/DP stability under various conditions. 13 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop

14 Acknowledgements Bill and Melinda Gates Foundation National Institute of Health GSK Vaccine Project Team Sanofi Pasteur Project Team Duke University/CHAVI Project Team All collaborators of P5 14 Case study of formulating 2 Subtype-C gp120 proteins Antu Dey 15 Sept., nd Env Vaccine Manufacturing Workshop