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

Abstract

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 log 10 reduction) of β-glucan detected in the two batches of mAb A microfiltration permeates (MFP) and 99.99% (3.9 log 10 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.

Beta-glucan contamination of pharmaceutical products: How much should we accept?

“…Beta-glucans have also been described in antibody products in development for potential administration to humans, notably those produced in yeast cells or cultured with yeast-derived additives [ 68 ].…”

Development of downstream processing to minimize beta‐glucan impurities in GMP‐manufactured therapeutic antibodies

“…Beta‐glucans are produced by a range of prokaryotic and eukaryotic organisms, and both insoluble and soluble forms have been used therapeutically for many years, especially in oriental medicine . Beta‐glucans have also been reported to originate from cellulose‐based filters, cell culture raw materials, and formulation buffers used in pharmaceutical processing …”

“…8,9 Beta-glucans are produced by a range of prokaryotic and eukaryotic organisms, and both insoluble and soluble forms have been used therapeutically for many years, especially in oriental medicine. [10][11][12] Beta-glucans have also been reported to originate from cellulose-based filters, [13][14][15][16][17] cell culture raw materials, 18 and formulation buffers used in pharmaceutical processing. 15,17 Beta-glucan contamination of pharmaceutic products may be suspected when an "out of specification" result is received following endotoxin testing.…”

Control of leached beta‐glucan levels from depth filters by an improved depth filtration flush strategy

“…5 Since a major component of traditional depth filter media is cellulose fibers, this is an entry point for beta-glucan impurities into the product stream in an antibody manufacturing process. [6][7][8][9] Other sources of beta-glucans in biotherapeutic manufacturing processes include buffer raw materials, such as sucrose, and cell culture raw materials, mainly yeast-derived hydrolysates. 6 In typical monoclonal antibody (mAb) processes, depth filters are often used in the primary clarification step to remove solids and to prepare the solution for the subsequent purification steps.…”

“…These glucose polysaccharides are found in bacteria, fungi, and in the cell wall structure in plants, or more specifically, cellulose 5 . Since a major component of traditional depth filter media is cellulose fibers, this is an entry point for beta‐glucan impurities into the product stream in an antibody manufacturing process 6‐9 . Other sources of beta‐glucans in biotherapeutic manufacturing processes include buffer raw materials, such as sucrose, and cell culture raw materials, mainly yeast‐derived hydrolysates 6 …”