title: 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.
authors.affiliation: Purification Process Development, Amgen Inc., Seattle, WA, USA. gefrohe@amgen.com
authors.forename: Eva
authors.initials: E
authors.lastname: Gefroh
authors.forename: Art
authors.initials: A
authors.lastname: Hewig
authors.forename: Ganesh
authors.initials: G
authors.lastname: Vedantham
authors.forename: Megan
authors.initials: M
authors.lastname: McClure
authors.forename: Alla
authors.initials: A
authors.lastname: Krivosheyeva
authors.forename: Ajay
authors.initials: A
authors.lastname: Lajmi
authors.forename: Yuefeng
authors.initials: Y
authors.lastname: Lu
mesh_terms: D002209:Caprolactam / Q000031:analogs & derivatives* / Q000737:chemistry; D005374:Filtration / Q000295:instrumentation* / Q000379:methods*; D008567:Membranes, Artificial; D011108:Polymers / Q000737:chemistry*; D013395:Sucrose; D047071:beta-Glucans / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D008567:Membranes, Artificial; D011108:Polymers; D047071:beta-Glucans; C009916:nylon 6; D013395:Sucrose; D002209:Caprolactam