Monoclonal Antibody Purification and Technology for Improving Virus Clearance

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1 Monoclonal Antibody Purification and Technology for Improving Virus Clearance BioProcessing Network Annual Conference Brisbane, September 2009 Germano Coppola Technology Transfer Manager CSL Limited

2 Outline CSL Limited Monoclonal Antibody CSL360 Downstream Process & Viral Clearance for CSL360 Viral Clearance Improvement IEX Chromatography Viral Clearance Improvement Viral Filtration Summary: Viral Clearance Efficacy Observation: Filter Flux Decay & Cause 2

3 CSL Global: Manufacturing Centres of Excellence Parkville, Australia Broadmeadows, Australia Marburg, Germany Bern, Switzerland Kankakee, USA Vaccines Plasma Products Haemophilia Immunoglobulins Alpha-1 Proteinase Biotechnology Technical Innovation Wound Healing Specialty Products Inhibitor Global Revenue $3.8bn 3

4 Global R&D Pipeline 4

5 CSL360 A monoclonal antibody (IgG 1 κ) targeting CD123 (IL-3R α-chain) positive human leukaemic stem cells to be used as an intravenous treatment of acute myeloid leukaemia 5

6 Acute Myeloid Leukemia US Incidence 10,500 pa 18% 5 year survival, often months First line therapy = chemo +/- BMT 80% relapse 6

7 Phase I Development Expression system, MCB/WCB Upstream/downstream development Phase I production (3,000L) 15 month program 7

8 CSL360 Manufacturing Process Harvest Protein A chromatography Low ph incubation (viral inactivation) Anion exchange chromatography Drug Substance Ultrafiltration / Diafiltration Viral filtration Hydroxyapatite chromatography 8

9 Process Viral Clearance Required Efficacy Regulatory Requirements (EMEA) Process should include at least two effective (> 4 log) orthogonal viral clearance steps Effective clearance of both enveloped and non-enveloped viruses 9

10 CSL360 Process Viral Clearance STAGE MVM MuLV Protein A 3.47 >5.34 Low ph VI NE >4.48 Anion Exchange 1.87 >3.98 Hydroxyapatite NE >1.22 Viral filtration NE >4.56 Total 5.34 >19.58 NE: Not Effective LVR < 1.0 log 10

11 Areas for Improvement: Anion Exchange Chromatography Purpose : Polishing Step: Remove CHOP, DNA, Aggregates and Leached Protein A Viral Clearance: Dependant on virus properties, process ph & conductivity and resin condition - Curtis et al 2003 CSL360: IgG1: PI = Process: Performed in Flow Through mode Conditions: Resin Q- Sepharose FF 25mM Tris + 100mM NaCl ph 7.5, 11-13mS/cm 25mM Tris + 10mM NaCl ph 7.5, 4-5mS/cm IgG Flowthrough Collection 11 Strip

12 Anion Exchange Chromatography Efficacy 12mS/cm vs 5mS/cm Target 12mS 5mS Recovery >95% >95% CHOP 40-60% reduction > 90% reduction Virus MuLV > 4 Log >4 Log Virus MVM 2 Log >4 Log Summary: Reducing conductivity significantly improved clearance of MVM 12

13 Areas for Improvement: Viral Filtration Efficacy of Available Viral Filters Filter Large Virus Clearance (>50nm) Small Virus Clearance (20-30nm) Pall DV 50 Millipore NFR Asahi Planova 35N > 6 log PR772 (76-88nm) > 6 log Retrovirus (80-130nm) > 6 log BVDV (80-130nm) Pall DV 20 > 6 log PR772 (76-88nm) > 3 log PP7 Bacteriophage (26nm) Millipore NFP > 6 log Retrovirus (80-130nm) > 4 log øx-174 Bacteriophage (26nm) Asahi Planova 20N > 6 log BVDV (80-130nm) > 4 log Parvovirus (18-26nm) Evaluation: CSL360 & Asahi Planova 20N 13

14 Viral Filtration: Flux Profile 5mg/ml Flux L/m²/hr 10mg/ml 30mg/ml 30mg/ml (Competitor) Time Surface Area: 0.001m² Pressure: 1Bar Declining volume flux with increasing protein concentration (Recoveries >98) 14

15 Viral Filtration: Process Economics 5mg/ml vs 10mg/ml vs 35mg/ml Mass Flux g/m² 35mg/ml 10mg/ml 5mg/ml Time (Min) Conc. mg/ml Area (m²) Target : 10Kg within 3hrs Summary: Processing at 35mg/ml reduces surface area requirements by 30-60%

16 Viral Filtration: Clearance Efficacy 5mg/ml vs 35mg/ml 35mg/ml PPV LRV 5mg/ml Time (Min) Protein Concentration Amount Processed after 4hrs (g/m²) PPV (LRV) 5mg/ml mg/ml 2516 >5 16 Summary: Efficient viral clearance observed at 5 and 35mg/ml after 4hrs of processing

17 CSL360 Manufacturing Process Harvest Protein A chromatography Low ph incubation (viral inactivation) Anion exchange chromatography Drug Substance Viral filtration Ultrafiltration / Diafiltration Hydroxyapatite chromatography 17

18 Process Viral Clearance Stage MVM MuLV OLD NEW OLD NEW Protein A > Low ph VI NE NE >4.48 >5.41 IEX 1.87 >6.93 >3.98 >3.99 Hydroxyapatite NE NE >1.22 >1.58 Viral filtration NE >5.33 >4.56 >4.55 Total 5.34 >15.13 >19.58 >18.14 NE: Not Effective 18

19 Observations: Declining Flux Rates/ Filter Fouling Flux L/m²/hr 10mg/ml :Recoveries>98% 30mg/ml: Recoveries >98% 10mg/ml: Recoveries 73% 30mg/ml: Recoveries 67% Time (min) Process Change Evaluation of new UF/DF membrane 19

20 Observations: Declining Flux Rates/ Filter Fouling Cause: Product Quality? Declining Flux Rate Sample (30mg/ml) Test: SEC-HPLC TSK G3000SWXL Pre-Filtration Post-Filtration Monomer Content Dimer Content Aggregate Content Rapid Filter Fouling Sample (30mg/ml) Test: SEC-HPLC TSK G3000SWXL Pre-Filtration Post-Filtration Monomer Content Dimer Content Aggregate Content SEC-HPLC Aggregate content not a reliable predictor

21 High Molecular Weight Species Flow Field Fractionation Detection by static light scattering at 15º - very sensitive to HMW species UFDF VF Membrane: regenerated cellulose, 10KDa MWCO 21 Cross-flow gradient 2 ml/min to zero over 15 minutes

22 Question Is nanofilter fouling accelerated by the presence of low levels of high molecular species formed during UF/DF which require sensitive detection methods? 22