Introduction and clearance of beta‐glucan in the downstream processing of monoclonal antibodies

Abstract

β-Glucan process-related impurities can be introduced into biopharmaceutical products via upstream or downstream processing or via excipients. This study obtained a comprehensive process-mapping dataset for five monoclonal antibodies to assess β-glucan introduction and clearance during development and production runs at various scales. Overall, 198 data points were available for analysis. The greatest β-glucan concentrations were found in the depth-filtration filtrate (37-2,745 pg/ml). Load volume correlated with β-glucan concentration in the filtrate, whereas flush volume was of secondary importance. Cation-exchange chromatography significantly cleared β-glucans. Furthermore, β-glucan leaching from the Planova 20N virus removal filter was reduced by increasing the flush volume (1 vs. 10 L/m 2 ). β-glucan concentrations after filter flush with 10 L/m 2 were consistently <10 pg/ml. No or only limited β-glucan clearance was attained via ultrafiltration/diafiltration (UF/DF). However, during the first run with monoclonal antibody (mAb) 4, β-glucan concentration in the UF/DF retentate was 10.8 pg/mg, potentially due to β-glucan leaching from the first run with a regenerated cellulose membrane. Overall, β-glucan levels in the final mAb drug substance were 1-12 pg/mg. Assuming high doses of 1,000-5,000 mg, a β-glucan contamination at 20 pg/mg would translate to 20-100 ng/dose, which is below the previously suggested threshold for product safety (≤500 ng/dose). K E Y W O R D S depth filtration, downstream processing, monoclonal antibody, process-related impurities, β-glucan 1 | INTRODUCTION β-Glucans are large polysaccharides with varied chemical structures in which the β-D-glucose monomers are frequently linked by) glycosidic bonds. They occur naturally in the cell walls of bacteria, yeast, cereals, seaweed, and fungi, and have widely varying molecular weights that can range from thousands to millions of Daltons. 1-3 β-(1,3)-D-Glucans are becoming increasingly recognized as pharmaceutical contaminants with immunomodulating properties that have the potential to cause infusion reactions; 1,4 they are commonly described as innate immunity-modulating impurities or process-related impurities (PRI). 1,[4][5][6] During pharmaceutical production of therapeutic proteins such as monoclonal antibodies (mAbs) by mammalian cell culture processes, there are many sources for β-glucan contamination. 1 These sources include cellulose-based filters or membranes, and fungal