Multipronged approach to managing beta‐glucan contaminants in the downstream process: Control of raw materials and filtration with charge‐modified nylon 6,6 membrane filters

Abstract

(1→3)-β-D-Glucans (beta-glucans) have been found in raw materials used in the manufacture of recombinant therapeutics. Because of their biological activity, beta-glucans are considered process contaminants and consequently their level in the product needs to be controlled. Although beta-glucans introduced into the cell culture process can readily be removed by bind-and-elute chromatography process steps, beta-glucans can also be introduced into the purification process through raw materials containing beta-glucans as well as leachables from filters made from cellulose. This article reports a multipronged approach to managing the beta-glucan contamination in the downstream process. Raw material screening and selection can be used to effectively limit the level of beta-glucan introduced into the downstream process. Placement of a cellulosic filter upstream of the last bind-and-elute column step or effective preuse flushing can also limit the level of contaminant introduced. More importantly, this article reports the active removal of beta-glucan from the downstream process when necessary. It was discovered that the Posidyne(®) filter, a charge-modified nylon 6,6 membrane filter, was able to effectively remove beta-glucans from buffers at relatively low pH and salt concentrations. An approach of using low beta-glucan buffer components combined with filtration of the buffer with a Posidyne membrane has been successfully demonstrated at preparative scale. Additionally, the feasibility of active removal of beta-glucan from in-process product pools by Posidyne membrane filtration has also been demonstrated. Based on the data presented, a mechanism for binding is proposed, as well as a systematic approach for sizing of the Posidyne filter.

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

“…Sucrose is commonly used in formulation buffers for mAbs due to its stabilizing ability and protein aggregation‐reducing properties . However, it has been reported that when sucrose is harvested and refined, it is coprocessed with beta‐glucans and previous reports have shown beta‐glucans to have entered pharmaceutical products through the addition of sucrose …”

“…Posidyne® filters are nylon‐based filters used for the removal of endotoxins in the manufacturing process of biologics. They have recently been reported to also remove beta‐glucan contaminants in small scale manufacturing . Filtration of 50 mL of 250 g/L sucrose through a small scale Posidyne® filter resulted in efficient removal of beta‐glucans, with levels reduced from 1,740 pg/mL to below 25 pg/mL (Table ).…”

“…However, Planova™ 20N has been validated by the supplier (Asahi Kasei) to leach ≤200 pg/mL of beta‐glucan. Furthermore, a study by Gefroh et al comparing virus filters, identified the Planova™ 20N filter as one that shows minimal leaching of beta‐glucans.…”

“…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 …”

Impact of Residual Impurities and Contaminants on Protein Stability

“…A common class is leachables from chromatography resins, such as protein A and Strep‐Tactin from their respective affinity columns. β‐glucans can leach out from cellulose filters and also come from raw materials used in the manufacturing process . The potential nonmetallic leachables from product‐contacting material surfaces during protein DS manufacturing was recently evaluated.…”