Protein A chromatography resin lifetime—impact of feed composition

Abstract

Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This article focuses on the impact of feed composition on the performance of a typical agarose-based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and nonhistone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC-MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb, and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss, or impact on product quality was seen to vary depending on the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin aging as a basis for improved process understanding. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:412-419, 2018.

Chromatography process development aided by a dye‐based assay

“…These aggregate‐like structures may have occluded the resin pores, preventing further protein uptake, as observed by limited permeation of the dye. Similar structures, primarily deposited on the resin surface with protruding protein aggregates and interacting with other beads, have also been visualized with scanning electron microscopy . Additionally, fluorescence measurements in a plate reader demonstrated approximately 4‐fold higher levels of fluorescence when using heat‐denatured instead of native BSA solution, which was comparable to the manufacturer's positive (aggregated lysozyme) control (Fig.…”

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

“…However, from the downstream perspective, cell viability is only relevant when the amount of RPC and DNA increases, which only occurs during cell lysis. This could also be of significant relevance for the yield, longevity, and overall performance of the subsequent protein purification strategies . In general, reducing the soluble impurity level can simplify early‐stage downstream processing .…”

“…Although the HCP content is regarded as a critical quality attribute of the final product, the implementation of HCP as a routine process variable in mammalian cell culture has not been reported to date. Nevertheless, for integrated or continuous processes, monitoring and control of HCP content could be of particular importance, such as in avoidance of fouling or blocking in chromatography resins during downstream operations.…”

Analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse

“…Among the different tools, fluorescence is the only one that can be used for online monitoring of resin fouling. In a previous publications and , we have described the various studies performed to understand the mechanism of the fouling process (also include studies to understand the impact of different feed materials on resin fouling ), monitoring the foulant deposition with number of cycles together with the strategy to control the fouling process. When performing numerous subsequent cycles in column mode, there can be a gradual buildup of contaminants on the chromatography resin, causing fouling.…”