Host cell protein clearance during protein a chromatography: Development of an improved column wash step

Abstract

in Wiley InterScience (www.interscience.wiley.com).Host cell protein (HCP) contaminant clearance is a significant concern during downstream process development for biopharmaceuticals. Protein A chromatography as a capture step for monoclonal antibodies and Fc fusion proteins can clear a large proportion of these impurities from cell culture harvest. Nevertheless, remaining levels of this process-related impurity class do present significant constraints on the rapid development of effective and robust polishing steps. Conventionally, an intermediate pH wash is employed between column loading and elution to minimize HCP levels after Protein A chromatography. A significant mechanistic finding presented in this work is that HCP contaminants that persist following Protein A capture predominantly comprise species that associate with the product in preference to direct interaction with the chromatographic resin. This suggests that the development of improved column wash techniques to maximize HCP clearance ought to focus on disrupting protein-HCP interactions rather than Protein A-HCP interactions. A higher wash pH to preserve product -Protein A binding along with the use of additive combinations to disrupt interactions between HCPs and the product are investigated. This strategy was successfully applied to develop a broadly applicable wash condition that has the potential for eliminating the need for product specific optimization of wash conditions. A combination of 1 M urea and 10% isopropanol in the wash buffer were successfully applied as a platform wash condition for Protein A chromatography. Use of this (and other similar) wash conditions are anticipated to aid the rapid development of effective downstream processes for monoclonal antibodies and Fc fusion proteins resulting in their rapid introduction into clinical trials.

Host cell proteins in biologics development: Identification, quantitation and risk assessment

“…It was found that the HCP clearance factor in the mAb-spiked samples was significantly lower than the mAb-free culture but very close to the typical mAb-containing sample. This result indicated that sub-populations of HCPs can co-purify with the mAb of interest, Shukla and Hinckley (2008) also reported similar findings recently.…”

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

“…During purification it is well established that small amounts of host cell protein (HCP), including intracellular proteins, will co‐purify with the production biopharmaceutical 5. The presence of HCPs in a final drug preparation is referred to as a process‐related impurity,6 and such impurities may potentially affect immunogenicity, the production of anti‐drug antibodies, and drug efficacy and/or safety.…”

“…Hence, bacterial HCPs have the potential to influence the immunogenicity of the biotherapeutic by having adjuvant‐like activity. HCP contamination is also an issue for mammalian cell expression systems 6, 12, 13. However, non‐mammalian HCPs are more likely to pose a risk in patients, compared with their mammalian counterparts.…”

Hydroxocobalamin association during cell culture results in pink therapeutic proteins

“…4 Host cell proteins, however, have been observed to co-purify due to protein interactions. 5 Similarly, vitamins in cell culture media have shown high affinity Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins.…”