Continuous Antibody Capture with Protein A Countercurrent Tangential Chromatography: A New Column-Free Approach for Antibody Purification Andrew L. Zydney Department Head and Walter L. Robb Family Chair Department of Chemical Engineering The Pennsylvania State University Presented at the ECI Conference on Integrated Continuous Biomanufacturing Castelldefels, Spain, October 21, 2013

Continuous Bioprocessing Significant potential opportunities Reduced capital costs / facility requirements Higher productivity Easier scale-up Major technology developments in place Perfusion bioreactors In-line filters Critical challenge is chromatography

Chromatography Options Multi-column periodic counter-current chromatography (PCC) – GE Healthcare Simulated moving bed chromatography (SMB) – Semba, Tarpon, Contichrom Sequential multi-column chromatography (SMCC) – Novasep These approaches typically do not provide truly steady-state operation, potentially leading to variability in product quality

Example: SMB New Developments in Simulated Moving Bed Chromatography Seidel-Morgenstern, Kessler, and Kaspereit Chemical Engineering Technology, 31: 826 (2008)

Objectives Develop and demonstrate a new technology that can provide truly continuous protein purification using available chromatography resins, e.g., Protein A Design criteria: Comparable yield and purity to columns High productivity (10x packed columns) Single use capability (no stainless steel)

Countercurrent Tangential Chromatography - CTC Chromatographic resin (beads) flows as a slurry through a series of static mixers and hollow fiber membrane modules All operations (binding, washing, elution, stripping, equilibration) performed directly on the slurry Countercurrent staging used to reduce buffer and resin requirements, increase product yield and purity

Continuous CTC System True moving bed  Conveyor like process Slurry Tank Binding Washing Elution Stripping Regnera-tion Waste Waste Waste Waste Feed Tank Product Tank True moving bed  Conveyor like process Resin slurry moves counter-currently to buffer in each step

Chromatographic “Stage” Koflo static mixer Provides residence time needed for equilibration in binding and elution steps Excellent radial mixing with minimal pressure drop + Spectrum hollow fiber module Provides complete separation between resin particles and fluid phase High single pass conversion with low pressure losses

Equilibra-tion Buffer Equilibra-tion Permeate Continuous CTC System Centrate Feed Wash 1 Buffer Wash 2 Buffer Elution Buffer Stripping Buffer Equilibra-tion Buffer Binding Wash 1 Wash 2 Elution Stripping Equilibration UF Step Resin Tank Binding Permeate Wash 1 Permeate Wash 2 Permeate Stripping Permeate Equilibra-tion Permeate Product Tank UF Permeate

Countercurrent Staging - Elution Stage 1 Stage 2 Resin Slurry to Strip Washing Resin Slurry from Wash 2nd stage 1st stage pH 3 Elution Buffer Purified mAb

Countercurrent Staging - Elution Stage 1 Stage 2 Resin Slurry to Strip Washing Resin Slurry from Wash 2nd stage 1st stage pH 3 Elution Buffer Purified mAb

Countercurrent Staging - Elution Stage 1 Stage 2 Resin Slurry to Strip Washing Resin Slurry from Wash 2nd stage 1st stage pH 3 Elution Buffer Purified mAb

Countercurrent Staging - Elution Stage 1 Stage 2 Resin Slurry to Strip Washing Resin Slurry from Wash 2nd stage 1st stage pH 3 Elution Buffer Purified mAb

Countercurrent Staging - Elution Stage 1 Stage 2 Resin Slurry to Strip Washing Resin Slurry from Wash 2nd stage 1st stage pH 3 Elution Buffer Purified mAb

Example: 3-Stage Elution Step Concentrated slurry with bound product Elution Buffer Tangential flow filter Static mixer P Static mixer Tangential flow filter R Tangential flow filter Static mixer R P P R Product Concentrated resin slurry

Effect of Staging – Elution Step Number of stages Experimental yield Theoretical yield 1 78 ± 2% 77% 2 94 ± 2% 94% 3 98 ± 1% 98% qp = permeate flow rate qr = retentate flow rate n = number of stages Results for qp / qr = 0.75 From Shinkazh et al., Biotech. Bioeng, 108: 582 (2011)

Experimental System Clarified cell culture fluid (Fujifilm Diosynth) Monoclonal antibody product POROS® MabCapture A resin – Life Technologies 45 µm diameter particles, Protein A ligand MidiCros® hollow fiber modules - Spectrum Lab 0.5 µm PES membranes, 1 mm ID, 200 cm2 area Static mixers – Koflo Corportation 29 cm length, 1 cm ID

Critical Filtrate Flux Feed: 10% slurry, 100 mL/min

Critical Filtrate Flux Feed: 10% slurry, 100 mL/min Critical flux corresponds to 80% conversion using 10% slurry

Critical Filtrate Flux Feed: 10% slurry, 100 mL/min Critical flux corresponds to 80% conversion using 10% slurry System design: 7.5% slurry 75% conversion Extra safety limit enables stable operation for long times

CTC Process using Protein A Operation Number of stages Buffer pH Mixed Binding 2 -- 7.4 7.6 Wash 1 3 20 mM Na2HPO4 + 0.5 M NaCl 7.1 7.5 Wash 2 20 mM Na2HPO4 7.2 7.0 Elution 40 mM Citrate 3.2 3.3 Strip 10 mM HCl + 0.1 M NaCl 2.0 2.5 Equilibration 8.1

Multiple Runs Run mAb (g/L) mAb Load Time Feed Flow Rate (L/hr) mAb Load per Resin 1 – feasibility 1.2 16 g 3 hr 4.5 190 g/L 2 – long time 0.72 8 g 24 hr 0.45 470 g/L 3 – high titer 4 hr 230 g/L

Run 1 - Pressure Profiles 12 Stable operation Pressure <10 psi Laminar flow All plastic tubing and connectors 10 8 Pressure, P (psig) 6 4 2 0.5 1.0 1.5 2.0 2.5 Elapsed Time, t (hr)

Run 1- mAb Purification >95% yield SEC Profiles >98% purity Productivity of 63 g mAb/L resin/hr (10x packed column) No detectable protein aggregates No detectable changes in resin SEC Profiles Elution Pure mAb CCCF Absorbance Elution Time, t (min)

Host Cell Protein (ppm) Run 1 - mAb Purification Sample Host Cell Protein (ppm) Clarified Harvest 675,000 CCTC System 1,200 Packed Column 2,800 Host cell protein measured relative to mAb via ELISA HCP level in CCTC system 2x lower than packed column Yield >95%, purity >98% Similar levels of high MW to purified (reference) mAb

Run 2 – Product Profile Steady-state with respect to product concentration and impurity profile Long time operation possible For t > 12 hr hollow fiber modules had to be replaced due to bacterial growth

Run 2 – HCP levels HCP level remains constant throughout 24 hr run >95% purity Productivity of 19 g mAb/L resin/hr (reduced due to low titer feed)

Host Cell Protein (ppm) Run 3 - mAb Purification Very low HCP level due to use of high titer feed (4.5 g/L) spiked with purified mAb >99% purity Productivity of 52 g mAb/ L resin / hr 2.6 cycles / hr for resin Sample Host Cell Protein (ppm) 2 hr 310 3 hr 345 4 hr 382 HCP measured via ELISA relative to mAb

Advantages of CCTC System Continuous operation with high productivity All resin used at all times Steady-state operation with respect to product concentration and impurity profiles No columns / packing Reduced labor costs and validation Greater flexibility in multi-product facilities Disposable flow path if desired Potential for single-use systems Ideal for production of clinical batches

Future Opportunities Use of smaller resin particles Much better mass transfer  less residence time needed in binding and elution steps Lower hold-up volume  greater productivity No issues with pressure drop for slurry flow Direct integration with perfusion bioreactor Opportunity for continuous steady-state processing Dramatic improvements in overall productivity

Summary Countercurrent tangential chromatography (CCTC) for mAb purification Continuous and steady-state operation demonstrated for 24 hr Purity and yield comparable to packed column Countercurrent staging reduces resin requirements while increasing product yield and purity Low pressure operation  opportunities for disposable single-use flow path Modular design for enhanced flexibility

Acknowledgements Oleg Shinkazh Boris Napadensky Achyuta Teella Founder and President, Chromatan Boris Napadensky VP of Engineering, Chromatan Achyuta Teella Senior Scientist at Chromatan, Post-doc at Penn State Travis Tran Associate Scientist, Chromatan Gary Brookhart Senior Research Scientist, Fujifilm Diosynth

Funding / Support