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Monoclonal Antibody Purification and Technology for Improving Virus Clearance
BioProcessing Network Annual Conference
Brisbane, September 2009
Germano Coppola Technology Transfer Manager
CSL Limited
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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
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CSL Global: Manufacturing Centres of Excellence
Bern, Switzerland
Kankakee, USA
Haemophilia Wound Healing Specialty Products
Broadmeadows, Australia
Marburg, Germany
Immunoglobulins Alpha-1 Proteinase Inhibitor
Plasma Products Technical Innovation
Vaccines Biotechnology
Parkville, Australia
Global Revenue $3.8bn
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Global R&D Pipeline
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CSL360
• A monoclonal antibody (IgG1κ) targeting CD123 (IL-3R α-chain) positive human leukaemic stem cells to be used as an intravenous treatment of acute myeloid leukaemia
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Acute Myeloid Leukemia
• US Incidence 10,500 pa • 18% 5 year survival, often months
• First line therapy = chemo +/- BMT • 80% relapse
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Phase I Development
• Expression system, MCB/WCB
• Upstream/downstream development
• Phase I production (3,000L)
• 15 month program
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Harvest Low pH incubation (viral inactivation)
Protein A chromatography
Anion exchange chromatography
Hydroxyapatite chromatography
Viral filtration
Drug Substance
Ultrafiltration / Diafiltration
CSL360 –Manufacturing Process
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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
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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
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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
IgG Flowthrough Collection
Strip
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
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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
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Areas for Improvement: Viral Filtration Efficacy of Available Viral Filters
Filter Large Virus Clearance (>50nm) Small Virus Clearance (20-30nm)
Pall DV 50 > 6 log PR772 (76-88nm)
Millipore NFR > 6 log Retrovirus (80-130nm)
Asahi Planova 35N > 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
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Viral Filtration: Flux Profile
Time
Flux
L/m²/h
r
30mg/ml (Competitor)
10mg/ml
30mg/ml
5mg/ml
Declining volume flux with increasing protein concentration (Recoveries >98)
Surface Area: 0.001m²
Pressure: 1Bar
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Viral Filtration: Process Economics 5mg/ml vs 10mg/ml vs 35mg/ml
Summary: Processing at 35mg/ml reduces surface area requirements by 30-60%
Mas
s Fl
ux g
/m²
Time (Min)
35mg/ml
10mg/ml
5mg/ml
Target : 10Kg within 3hrs
Conc. mg/ml Area (m²)
5 11 10 7 35 4.5
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Viral Filtration: Clearance Efficacy 5mg/ml vs 35mg/ml
Summary: Efficient viral clearance observed at 5 and 35mg/ml after 4hrs of processing
PP
V L
RV
Protein Concentration
Amount Processed after 4hrs (g/m²)
PPV (LRV)
5mg/ml 1075 4
35mg/ml 2516 >5
5mg/ml
35mg/ml
Time (Min)
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Harvest Low pH incubation (viral inactivation)
Protein A chromatography
Anion exchange chromatography
Hydroxyapatite chromatography
Viral filtration Drug
Substance
Ultrafiltration / Diafiltration
CSL360 –Manufacturing Process
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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
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Observations: Declining Flux Rates/ Filter Fouling Fl
ux L
/m²/h
r
Time (min)
10mg/ml :Recoveries>98%
30mg/ml: Recoveries >98%
10mg/ml: Recoveries 73% 30mg/ml: Recoveries 67%
Process Change – Evaluation of new UF/DF membrane
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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
SEC-HPLC Aggregate content not a reliable predictor
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High Molecular Weight Species – Flow Field Fractionation
Detection by static light scattering at 15º - very sensitive to HMW species
• Membrane: regenerated cellulose, 10KDa MWCO
• Cross-flow gradient 2 mL/min to zero over 15 minutes
VF
UFDF
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Question
Is nanofilter fouling accelerated by the presence of low levels of high molecular species formed during UF/DF which require sensitive detection methods?