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.

(1→3)-ß-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.

Large-scale capture and partial purification of plasmid DNA using anion-exchange membrane capsules.

The demand for larger quantities of high-purity plasmids continues to increase. Substantial quantities of plasmid DNA are needed to support viral and plasmid-based gene-therapy programmes, including drug delivery, preclinical and clinical studies and production of DNA vaccines. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of plasmid DNA is becoming more widespread. Development of an efficient capture-unit operation for very large plasmid DNA molecules from these large lysate volumes can present a considerable challenge. Use of conventional chromatographic beaded media for plasmid capture is problematic, owing to their restrictive pores, which limits access of the large DNA molecules to only those binding sites on the beads' outer surface. Anion-exchange membranes, on the other hand, have large convective pores that allow the plasmid DNA to readily access all of the membrane's anionic binding sites, even at very high flow rates. The convenience of anion-exchange membranes, configured in ready-to-use capsules, can greatly simplify large-scale plasmid purification strategies. The effectiveness of membrane-based technology for the capture of a pCAT reporter plasmid from large volumes of clarified Escherichia coli lysate was demonstrated. The captured and eluted plasmid pool was found to have been concentrated 10-fold with a reduction in endotoxin of four orders of magnitude.