Platform downstream processes in the age of continuous chromatography: A case study Mark Brower BioProcess Technology & Expression Bioprocess Development Kenilworth, NJ Integrated Continuous Biomanufacturing Castelldefels, Spain October 2013

Batch Stainless / Single Use Batch Stainless Continuous Single Use Enabled PROCESS INTENSIFICATION Next Generation Transition to Future Concepts To meet increasing global demands requires…

6H 18H 24H 30H 36H 42H 48H 54H 60H 12H Primary Recovery (Centrifugation / MF + DF) Bulk Purification Protein A Chromatography Viral Inactivation (Low pH Hold) DNA / HCP / Viral Adsorption Anion Exchange Chromatography Variant and Aggregate Clearance Cation Exchange Chromatography Viral Filtration Nanofiltration Concentration / Buffer Exchange Microfiltration / Diafiltration Bioburden Reduction Sterile Filtration mAb Downstream Purification Bulk Formulation Fine Increased flexibility Reduced footprint Reduced capital spend Better resource utilization

Continuous Processing Vision - 2,000L SUB* Overall DSP Time Cycle is Dictated by the Longest Step Other Steps are Lengthened to Compensate S U B* Depth /BRF Filtration Surge Bag BioSMB Protein A Single-Use Centrifugation Surge Bag p p BRF p p pp Viral Filtration Surge Bag Surge Bag Formulation: BRF/DiaF Continuous UF Anion Exchange Membrane p p Polishing Step Continuous Viral Inactivation AEXMAEXM BRF Surge Bag *Single-Use Bioreactor

SUB Harvest Bag DF / BRF SU Centrifuge SMB Protein A Viral Inactivation AEX Membrane Mixed Mode SPTFF Continuous Processing Case Study mAb 1 - Non-platform

MCC for Bind & Elute Applications Eq Waste 2 nd pass Depleted Feed 2 nd pass Wash Elute Prod uct Feed Strip Wash 2 C1 C5 C2 C6 C3 C7 C4 C8 Switch Time Methods based on batch process Loading, washing, elution, CIP carried out simultaneously Flexibility in loading zone

CEX CMCC Load Zone Design  2 methods designed to maximize time in the elution zone  Wash 1 in parallel 8 columns (shorter / continuous feed)  Wash 1 in series 6 columns (longer / discontinuous feed) W1 Feed 2 nd Pass W1 Feed2 nd Pass Longer residence time in the elution zone Similar column cycling compared with protein A Productivity 3.7X batch process

SMB Transformation of Platform CEX Step 1.2cm x 3cm pre-packed columns Poros HS Adsorbent q batch =50mg/mL Feed = 11-13g/L 2 different load zone configurations Good agreement between experimental and theoretical capture efficiency CMCC loading was 60-73mg/mL at high yield >95% 3.7 x Specific Productivity Design Equations* *Miyauchi and Vermeulen (1963)

Aggregate Clearance – Wash in Series Configuration Effect of column height investigated 1.2 x 3.4cm, 1.2 x 6.8cm, 0.5 x 20cm Feed aggregation varied (low and high) Six 1.2 x 3.4cm columns for MCC 4 th cycle fractionation (20 fractions per column pooled) Similar pre-peak observed in batch and MCC Process Similar pool aggregate levels observed Little difference observed at different column heights

Integration of MCC CEX into Continuous DSP - 100L platform harvest VI BioSMB Protein A BRF pp Viral Filtration Surge Bag Surge Bag Formulation: BRF/DiaF Continuous UF p p BioSMB CEX AEXMAEXM BRF Surge Bag S U B* Depth /BRF Filtration Surge Bag Surge Bag p p BRF p p CRITICALITYCRITICALITY Continuous UF pH

STDEV(%) Between Columns =1.01% Continuous CEX Performance 16 Overlaid CEX Elution Profiles AEXM Effluent Feed Column

Average Yield DNA* [ppm] HCP [ppm] Res. ProA [ppm] % Monomer Centrifugation97.3%N/S DF/BRF98.6%30,515383,300N/S Protein A SMB98.1%N/S Viral Inactivation100%21, % Anion Exchange Membrane 98.8%<LOQ % Cation Exchange Chromatography 84.2%<LOQ605<LOQ99.2% SPTFF99.5% <LOQ99.0% Overall77.9% <LOQ99.0% Continuous Processing Case Study mAb 2 - Platform Mass Balance = 93%

DSP Productivity Enhancement StepContinuous Protein A Chromatography [g/(L·h)] 3.1 Cation Exchange Chromatography [g/(L·h)] 3.7 Overall [g/day] ~3x MCC steps enjoy a modest specific productivity increase Other steps suffer from lower specific productivities because they are slowed to accommodate the incoming flow rate The overall DSP will be 2-4x more productive (g/day) by operating in parallel (dependent on Protein A column sizing)

Conclusions and Future Work A platform cation exchange step was transformed into a MCC process –3.6X specific productivity increase –Maintained consistent aggregate separation performance compared to the batch process –Integrated into continuous DSP top reflect platform operation with 84% yield at the 100L scale –Matched cycles with protein A step Interface CEX step with continuous viral filtration Scale up process to 2000L in 24hours

Acknowledgements BTE –Ying Hou –David Pollard Analytical Support –Joe Fantuzzo –John Troisi –Jun Heo Fermentation Support –Patty Rose –Chris Kistler –Rachel Bareither Protein Purification Process Development –Nihal Tugcu –Thomas Linden –Marc Bisschops –Steve Allen

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