Demonstrating β-glucan Clearance in CHO- and Yeast-Produced Monoclonal Antibodies during Downstream Purification Processes

Biologics production using yeast or CHO with Yeastolate as cell culture additives often introduces β-glucan, which could potentially pose immunogenicity risk, if not adequately removed. Although a previous study has shown the effective clearance of Yeastolate-derived β-glucan by Protein A chromatography, the clearance pattern of yeast cell derived β-glucan remains unknown. In this study, we characterized the β-glucan clearance patterns during downstream processing of three monoclonal antibody (mAb) products, one mAb fragment from Pichia pastoris (mAb A) and two full mAbs from CHO expression system (mAb B and mAb C), by Glucatell assay. We demonstrated effective β-glucan clearance in both small (100 L) and large scale (5000 L) batches of mAb A as well as in one batch of mAb B. Protein A purification step removed an average of 97.74% (1.7 log10 reduction) of β-glucan detected in the two batches of mAb A microfiltration permeates (MFP) and 99.99% (3.9 log10 reduction) of β-glucan detected in mAb B clarified culture fluid harvest (HCCF). Residual β-glucans post Protein A purification in the two batches of mAb A were further removed by the two polishing chromatography steps (94.76% reduction on average). Residual β-glucan measured in the mAb A and mAb B drug substance ranges from 7.8 to 19 pg/mg, which is unlikely to alter physiological concentrations significantly in healthy adults when administered with typical intravenous doses. However, in mAb C, after almost complete removal (99.99%) by Protein A purification step, β-glucan level increased more than 20 fold in Viral Filtration (VF) product sample, indicating that it can be introduced from materials used in downstream process, such as cellulose-based filters and membranes. Our study results suggest that although β-glucan can be cleared by Protein A and other chromatographic steps such as AEX, monitoring β-glucan clearance during downstream process development remains very important to identify and avoid potential contaminations to the drug substance.