Monoclonal Antibody Purification and Technology for Improving Virus Clearance

BioProcessing Network Annual Conference

Brisbane, September 2009

Germano Coppola Technology Transfer Manager

CSL Limited

10/9/09

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

10/9/09

3

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

10/9/09

4

Global R&D Pipeline

10/9/09

5

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

10/9/09

6

Acute Myeloid Leukemia

• US Incidence 10,500 pa • 18% 5 year survival, often months

• First line therapy = chemo +/- BMT • 80% relapse

10/9/09

7

Phase I Development

• Expression system, MCB/WCB

• Upstream/downstream development

• Phase I production (3,000L)

• 15 month program

10/9/09

8

Harvest Low pH incubation (viral inactivation)

Protein A chromatography

Anion exchange chromatography

Hydroxyapatite chromatography

Viral filtration

Drug Substance

Ultrafiltration / Diafiltration

CSL360 –Manufacturing Process

10/9/09

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

10/9/09

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

10/9/09

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

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

10/9/09

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

10/9/09

13

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

10/9/09

14

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

10/9/09

15

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

10/9/09

16

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)

10/9/09

17

Harvest Low pH incubation (viral inactivation)

Protein A chromatography

Anion exchange chromatography

Hydroxyapatite chromatography

Viral filtration Drug

Substance

Ultrafiltration / Diafiltration

CSL360 –Manufacturing Process

10/9/09

18

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

10/9/09

19

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

10/9/09

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 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

10/9/09

21

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

10/9/09

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?