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Primary Recovery & Harvest Processes for non-mAb Recombinant Proteins

Authored and Presented by: Cameron T. Phillips, Niket Bubna, Ph.D., David Chang and Sigma S. Mostafa, Ph.D

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Primary Recovery & Harvest Processes for non-mAb Recombinant Proteins

  1. 1. Primary Recovery & Harvest Processes for non-mAb Recombinant Proteins Non-mAb proteins present challenges during primary recovery and harvest processes due to their unique physicochemical properties which may be incompatible with platform harvest clarification processes. From a CDMO perspective, the increasing number of non-mAb proteins entering clinical trials has resulted in the need for a streamlined workflow to develop non-mAb harvest processes to meet manufacturing timelines. Using two case studies, we demonstrate a workflow to develop a primary recovery and harvest process for non-mAbs which presented challenges in recovery and product quality. The process was then scaled to 2000-L scale for manufacturing. Abstract Cameron T. Phillips, Niket Bubna, David Chang and Sigma S. Mostafa KBI Biopharma, Durham, NC References Conclusions Acknowledgments We thank Sigma Mostafa for support and guidance, and express our gratitude to Lynwel Cunanan and James Hamlin for their help. Primary recovery and harvest processes for two challenging non-mAb proteins were developed for 2000 L scale. Depth filters from several different filter media types were tested, but cellulose depth filters had the best performance for recovery and product quality. Final processes for both molecules included single-use centrifugation step using kSep 400, then scaled to 2000 L with the kSep 6000. Yield was increased from near 0% to > 90% for non-mAb 1, while bioactivity was significantly improved for non-mAb 2 up to 200 L scale. Scale-up to Manufacturing Primary recovery and harvest of cell culture processes typically consist of centrifugation, depth filtration, or a combination of both. Centrifugation of cell culture material removes large particles and cellular debris whereas depth filters can be used for removal of cell debris and some non- specific process impurities. Typical product recovery for mAbs are > 95%. During primary recovery of two non-mAb molecules, significant product loss and loss of bioactivity was observed. Manufacturer Material Filter Type Retention Range (μm) Millipore HC Dual Layer Cellulose/DE D0HC C0HC B1HC A1HC F0HC X0HC 0.5 – 9 μm 0.2 – 2 μm 0.1 – 0.7 μm 0.1 – 0.5 μm 0.1 – 0.4 μm < 0.1 μm Millipore HC Single Layer Cellulose CE15 CE20 10 – 15 μm 5 – 11 μm Millipore Polypropylene Polygard (10 um) Polygard (5 um) Clarigard (3 um) Clarigard (1 um) 10 μm 5 μm 3 μm 1 μm Pall Profile II Polypropylene Profile II 200 Profile II 100 Profile II 50 20 μm 10 μm 5 μm Pall Ultipor Glass Fiber GF Plus (10 um) GF Plus (6 um) 10 μm 6 μm Pall Supracap 100 Cellulose P900 PDK5 8 – 20 μm 1.5 – 20 μm Sartorius Sartoclear Cellulose with inorganic filter aids PB1 C8HP 4 – 11 μm 4 μm Sartorius Sartoclean Cellulose Acetate CA GF 0.8 – 3 μm 0.8 – 3 μm Sartorius Sartopure Glass Fiber Glass Fiber Polypropylene GF plus GF PP3 1.2 μm 1.2 μm 20 μm GE Healthcare Glass Fiber ULTA Capsule GF 5 μm Initial depth filter screening was conducted at bench-scale with different filter media across multiple vendors and pore sizes. Top filters were chosen based on product recovery, product quality of the filtrate, and ease of manufacturing. Introduction Results from Harvest Process Development Experimental Plan Depth Filter Screening Plan Initial harvest material was generated in 3 L glass reactors for initial depth filter screening studies. After identifying top filters based on recovery and product quality analysis, the process was scaled to pilot (50 and 200 L) using single-use vessels. Primary recovery process was evaluated with top filters, with and without single-use centrifuge (kSep 400). Based on pilot-scale results, the process was then scaled to 2000 L SUB for manufacturing. Harvest Process Lock Recovery/Bioactivity analysis Choose top filters Molecule Problem non-mAb 1 0% recovery with platform process non-mAb 2 Low product bioactivity with platform process + Single-use centrifuge Depth filtration Depth filtration Depth filter screening Jackalope non-mAb 1 non-mAb 1 non-mAb 2 non-mAb 2 non-mAb 2 non-mAb 2 Bench-scale depth filter screening 3 L 50 L cGMP 2000 L MFG Pilot-scale (50 L / 200 L) harvest process development Choose top filters for scale-up non-mAb 1, platform process non-mAb 2, platform process No product recovery non-mAb 1 non-mAb 2 without kSep with kSep non-mAb 2 Pre-kSep turbidity Post-kSep turbiditySartorius kSep single-use continuous centrifuge Harvest Process Lock Low product quality non-mAb 1 Yield increased from ~0% to >90% non-mAb 2kSep Scalability (non-mAb 1) 90% reduction in turbidity from 50 L to 2000 L Slight decrease in yield at 2000 L (>80%) Bioactivity data unavailable Top filters Top filters Top filters Top filters Top filtersTop filters Millipore Millistak Singh, Nripen. “Clarification technologies for mAb manufacturing processes: Current state and future perspectives.” Biotechnology and Bioengineering (2016). Jungbauer, Alois, and Nikolaus Hammerschmidt. "Integrated continuous manufacturing of biopharmaceuticals." Kleinebudde, P., Khinast, J., Rantanen, J., Eds (2017). Roush, David J., and Yuefeng Lu. "Advances in primary recovery: centrifugation and membrane technology." Biotechnology progress (2008). filter schematic kSep schematic