Verification of the removal of thrombogenic activity in Australian manufactured immunoglobulins

Transcription

1 Verification of the removal of thrombogenic activity in Australian manufactured immunoglobulins PPB13 May 2013 Sharon Vyas*, Thanae Malinas, Joseph Bertolini, Maria Panayi, Nunzio Mancuso CSL Behring, Camp Rd, Broadmeadows, Victoria, Australia

2 CSL Behring, Australia Immunoglobulin portfolio IVIG SCIG Hyperimmune IVIG Hyperimmune IMIG 2

3 History of Thromboembolic Events in IgG Recent increases in Thromboembolic events (TEE) observed with the administration of specific lots of a competitive IVIG Factor XIa identified as the major procoagulant contaminant Ph.Eur. Monograph (0918/0338) revised to institute measures to control the content of thrombosis generating agents in immunoglobulin preparations Manufacturing process has the capability to remove thrombosis generating agents The product does not exhibit thrombogenic (procoagulant) activity 3

4 CSL Response CSL has not seen any increase in TEE signals from Australian immunoglobulin products to date. CSL proactively initiated an evaluation on the thromboembolic potential of IVIG/SCIG products. 4

5 Assessment of CSL Products Scope of study To evaluate the risk of potential thrombosis generating agents in IVIG & SCIG products. To demonstrate compliance with the updated immunoglobulin monograph requirements Investigation of two IgG product manufacturing processes 1. Chromatographic manufacturing process IV & SC 2. Cohn manufacturing process IV 5

6 Experimental Design Process mapping Full characterisation of the removal of FXIa and FXI antigen throughout the manufacturing processes Identification of critical process steps that remove FXIa and FXI antigen QbD studies Spiking studies to investigate process capacity Additional studies to explore process parameters and demonstrate robustness Evaluation of final product lots 6

7 Assays used for Quantification Assays established in-house for evaluating procoagulant activity of IgG Non-activated Partial Thromboplastin Time (NaPTT ) - FXI deficient plasma Thrombin Generation Assay (TGA) time to peak, peak height & area under curve Factor XI ELISA - antibody specific for Factor XI antigen Factor XIa - Chromogenic Assay 7

8 Chromatographic Products Intragam P - 6% IVIG Evogam - 16% SCIG 8

9 Chromatographic Manufacturing Process Cryosupernatant SNI Delipidated SNI Diafiltered SNI Euglobulin depleted SNI Crude IgG Conc. pure IgG Low ph incubation Pasteurised IgG Virus filtered IgG Intragam P IV 6% Evogam SC 16% 9

10 Chromatographic Process Mapping Factor XIa (ng) per (g) protein Identification of steps in the Chromatography process that removes Factor XIa 0.0 Cryosuper SNI Delipidated SNI Diafiltered SNI Euglobulin depleted Crude IgG Pure IgG Final Product 10

11 Chromatographic Process Mapping 200 Identification of steps in the Chromatography process that removes Factor XI antigen Factor XI (µg) per (g) protein Cryosuper SNI Delipidated SNI Diafiltered SNI Euglobulin depleted Crude IgG Pure IgG Final Product 11

12 Chromatographic Manufacturing Process Cryosupernatant SNI Delipidated SNI Diafiltered SNI Low ph incubation step can also reduce FXIa Euglobulin depleted SNI Crude IgG Reduction of FXIa across pasteurisation Conc. pure IgG Low ph incubation Pasteurised IgG Virus filtered IgG Intragam P IV 6% Evogam SC 16% 12

13 QbD Studies Pasturisation Spiking studies to demonstrate capacity of pasteurisation to eliminate Factor XIa/XI Robustness evaluated by studying ph design space Low ph Incubation Spiking experiments conducted to demonstrate capacity of low ph step Factor XIa/XI assessed at various timepoints at 0 C, 4 C and 27 C 13

14 Pasteurisation Capacity & Robustness Spiked with 200 ng/ml Factor XIa Factor XIa Factor XI antigen Factor XIa concentration (ng/ml) ph 4.5 ph 4.8 ph 5.1 Factor XI concentration (ng/ml) ph 4.5 ph 4.8 ph Pasteurisation time (hours) Pasteurisation time (hours) 14

15 Pasteurisation Capacity & Robustness Spiked with 800 ng/ml Factor XIa Factor XIa Factor XI antigen Factor XIa concentration (ng/ml) ph 4.5 ph 4.8 ph 5.1 Factor XI concentration (ng/ml) ph 4.5 ph 4.8 ph Pasteurisation time (hours) Pasteurisation time (hours) 15

16 Pasteurisation Capacity & Robustness Spiked with 3000 ng/ml Factor XIa Factor XIa Factor XI antigen Factor XIa concentration (ng/ml) ph 4.5 ph 4.8 ph 5.1 Factor XI concentration (ng/ml) ph 4.5 ph 4.8 ph Pasteurisation time (hours) Pasteurisation time (hours) Greater rate of reduction at ph 4.8 & 5.1 across all spiking levels Factor XIa reduction of >90% after 3 hr and >99% after 10 hr at ph 4.8 and

17 Low ph Incubation: Factor XIa 240 ng/ml FXIa spike 2000 ng/ml FXIa spike Factor XIa concentration (ng/ml) Factor XIa concentration (ng/ml) % reduction in Factor XIa at 27 C after 14 days No significant effect at 0 C or 4 C 17

18 Low ph Incubation: Factor XI Antigen 240 ng/ml FXIa spike 2000 ng/ml FXIa spike Factor XI concentration (ng/ml) Factor XI concentration (ng/ml) No significant effect on Factor XI antigen levels at 0 C, 4 C or 27 C 18

19 Chromatographic Final Product Data Product FXIa (ng/ml) NaPTT (sec) 1:10 dilution Intragam P IVIG Evogam SCIG TGA (pass/fail) < Pass < Pass < Pass < Pass < Pass < Pass < Pass 19

20 Cohn Products CMV 6% IVIG Tetanus 6% IVIG 20

21 Cohn Manufacturing Process Cryosupernatant SNI Fraction II+III DF and conc SNIII Virus filtered bulk Low ph incubation Final product CMV IVIG Tetanus IVIG 21

22 Cohn Process Mapping Factor XIa (ng) per (g) protein Identification of steps in the Cohn process that removes Factor XIa 0 Cryosuper SNI Fraction II+III DF conc SNIII Final Product 22

23 Cohn Process Mapping 400 Identification of steps in the Cohn process that removes Factor XI antigen Factor XI (µg) per (g) protein Cryosuper SNI Fraction II+III DF conc SNIII Final Product 23

24 Cohn Process Critical Process Step Significant Factor XIa/XI reduction observed with the generation of SNIII from Fraction II+III DoE approach to examine the design space 24

25 Fraction II+III to SNIII- DoE Contour Map - Effect of ph/ethanol on level of Factor XIa ph > 5.35 improved FXIa reduction in SNIII Ethanol not a significant factor at higher ph 25

26 Cohn Final Product Data Product CMV IVIG Tetanus IVIG FXI antigen (ng/ml) FXIa (ng/ml) NaPTT (sec) 1:10 dilution TGA < Pass <6.25 < Pass <6.25 < Pass <6.25 < Pass <6.25 < Pass 26

27 Conclusion Established that the IVIG & SCIG manufacturing processes can effectively partition and inactivate Factor XIa/XI Inactivation of Factor XIa evident over pasteurisation in Chromatographic process Pasteurisation capacity and robustness demonstrated Removal of Factor XIa evident with low ph incubation and generation of SNIII in Cohn process Better understanding of process design space via QbD approach Final product data demonstrates low Factor XIa and lack of procoagulant activity Risk of TEE is minimal for CSL Behring Australian IVIG & SCIG products 27

28 Thanae Malinas Joseph Bertolini Maria Panayi Pauline Pearce Nunzio Mancuso Innocent Bekard Acknowledgment Thank you for your Attention 28

29 Verification of the removal of thrombogenic activity in Australian manufactured immunoglobulins PPB13 May 2013 Sharon Vyas*, Thanae Malinas, Joseph Bertolini, Maria Panayi, Nunzio Mancuso CSL Behring, Camp Rd, Broadmeadows, Victoria, Australia