A complete single-use manufacturing process of monoclonal antibodies: a case study Jakob Liderfelt, Janne Simola, Annika Forss, Gustav Rodrigo, Karin Torstensson, Tomas Björkman, Kjell Eriksson, Hans J Johansson, and Andreas Muranyi GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden First presented in October 2009
Introduction This presentation describes a monoclonal antibody process that was run in pilot-scale. The process is based on technology suitable for flexible manufacturing. The upstream process was operated using single-use, cell reactor technology. In the downstream process prepacked chromatography columns and a chromatography system with a single-use flow path were used. Crossflow filtration steps were carried out using pre-wetted and presanitized membranes and a single-use filtration system. 2 /A Complete Single Use Manufacturing
Introduction A two-step chromatography process was developed using an alkaline stable protein A medium followed by a multimodal anion exchanger. A downstream yield of over 80% and specified purity levels were achieved. 3 /A Complete Single Use Manufacturing
Materials and Methods Cell culture was performed in a WAVE BIOREACTOR, at a volume of 100L. Clarified cell culture supernatant containing a monoclonal antibody (MAb) was obtained by centrifugation followed by normal flow filtration. 4 /A Complete Single Use Manufacturing
Materials and Methods The pilot-scale run was performed using an ÄKTA ready chromatography system with ReadyToProcess columns. The disposable crossflow filtration steps were designed using ReadyToProcess hollow fibers, and ReadyCircuit assemblies with disposable pressure sensors. The system was assembled using ReadyMate disposable aseptic connectors. To determine aggregate content, size exclusion chromatography (Superdex TM 200) was used. Leached protein A and host cell protein (HCP) were quantified by commercial ELISA kits. 5 /A Complete Single Use Manufacturing
Materials and Methods 6 /A Complete Single Use Manufacturing
Results Capture step 7 /A Complete Single Use Manufacturing
Results Capture step The capture step was run in three cycles. The average yield was 96%, the aggregate level in the eluate was 10 %, and HCP was 15 ppm. The chromatogram is an overlay of two cycles showing high consistency between two runs. A virus inactivation step at ph <3.6 was performed before normal flow filtration (NFF) with ULTA pure HC. Capture step description Load: 30 g MAb/L medium Buffers: Equilibration/wash: 20 mm NaPO 4, 150 mm NaCl, ph 7.4 Intermediate wash: 20 mm NaPO 4, 500 mm NaCl, ph 7.4 Elution: 20 mm Na-citrate, ph 3.7 Residence time: 4 minutes (8 minutes during elution) 8 /A Complete Single Use Manufacturing
Results 1st crossflow filtration step 9 /A Complete Single Use Manufacturing
Results Polishing step 10 /A Complete Single Use Manufacturing
Results 2nd crossflow filtration step 11 /A Complete Single Use Manufacturing
Results Process summary 12 /A Complete Single Use Manufacturing
Summary A MAb process was scaled up to 100 L cell culture volume, in a single-use format, and compared to a traditional stainless steel format. The single-use process employed WAVE Bioreactor, ÄKTA ready system, ReadyToProcess columns, ReadyCircuit assemblies, and ReadyToProcess hollow fibers. A high degree of flexibility is demonstrated by the fact that only one chromatography system and one crossflow filtration system was used. The product quality, i.e., purity, and yield achieved in the ReadyToProcess process matched that of the traditional stainless steel process; but with a substantial reduction in process time. 13 /A Complete Single Use Manufacturing
Acknowledgments www.gelifesciences.com GE, imagination at work and GE Monogram are trademarks of General Electric Company. ÄKTA, ÄKTAexplorer, HiTrap, HiScreen, MabSelect SuRe, Capto, PreDictor, ReadyCircuit, ReadyMate, ReadyToProcess, Superdex, ReadyMate, ULTA, WAVE BIORECTOR are trademarks of GE Healthcare companies. ReadyMate is covered by US patent number 6,679,529 B2 owned by Johnson & Boley Holdings, LLC and licensed to GE Healthcare companies. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. 2009-11 General Electric Company - All rights reserved. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden First presented in October 2009 14 /A Complete Single Use Manufacturing