Monoclonal Antibody Purification and Technology for Improving Virus Clearance BioProcessing Network Annual Conference Brisbane, September 2009 Germano Coppola Technology Transfer Manager CSL Limited
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
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
Global R&D Pipeline 4
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
Acute Myeloid Leukemia US Incidence 10,500 pa 18% 5 year survival, often months First line therapy = chemo +/- BMT 80% relapse 6
Phase I Development Expression system, MCB/WCB Upstream/downstream development Phase I production (3,000L) 15 month program 7
CSL360 Manufacturing Process Harvest Protein A chromatography Low ph incubation (viral inactivation) Anion exchange chromatography Drug Substance Ultrafiltration / Diafiltration Viral filtration Hydroxyapatite chromatography 8
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
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
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 = 8.5-9.0 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
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
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
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
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 5 11 10 7 35 4.5 15 Summary: Processing at 35mg/ml reduces surface area requirements by 30-60%
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 1075 4 35mg/ml 2516 >5 16 Summary: Efficient viral clearance observed at 5 and 35mg/ml after 4hrs of processing
CSL360 Manufacturing Process Harvest Protein A chromatography Low ph incubation (viral inactivation) Anion exchange chromatography Drug Substance Viral filtration Ultrafiltration / Diafiltration Hydroxyapatite chromatography 17
Process Viral Clearance Stage MVM MuLV OLD NEW OLD NEW Protein A 3.47 1.94 >5.34 2.61 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
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
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 99.52 99.51 Dimer Content 0.37 0.40 Aggregate Content 0.08 0.09 Rapid Filter Fouling Sample (30mg/ml) Test: SEC-HPLC TSK G3000SWXL Pre-Filtration Post-Filtration Monomer Content 98.9 99 Dimer Content 1.0 1.0 Aggregate Content 0.13 0.04 20 SEC-HPLC Aggregate content not a reliable predictor
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
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