Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products

Abstract

Host cell proteins (HCPs) are considered a critical quality attribute and are linked to safety and efficacy of biotherapeutic products. Researchers have identified 10 HCPs in Chinese hamster ovary (CHO) that exhibit common characteristics of product association, coelution, and age‐dependent expression and therefore are “difficult to remove” during downstream purification. These include cathepsin D, clusterin, galectin‐3‐binding protein, G‐protein coupled receptor 56, lipoprotein lipase, metalloproteinase inhibitor, nidogen‐1 secreted protein acidic and rich in cysteine (SPARC), sulfated glycoprotein, and insulin‐like growth factor‐2 RNA‐binding protein. While the levels of HCPs in the investigated biosimilars were within the acceptable range of <100 ppm, certain “difficult to remove” HCPs were found in the biosimilar samples. This article aims to elucidate the underlying interactions between these “difficult to remove” HCPs and the mAb product. Surface study of rituximab exhibited unstable discontinuous patches of residues on the protein surface that have high propensity to get buried and lower the solvent exposed area. The higher order structure and the receptor binding were not affected, except for one of the biosimilars, owing to extremely low‐HCP levels in its final drug product. Finally, based on the combined experimental and computational data from this study, a probable mechanism of retention for the 10 HCPs is proposed. The results presented here can guide downstream process design or avenues for protein engineering during product discovery to achieve more effective removal of the impurities.

Targeted Host Cell Protein Quantification by LC–MRM Enables Biologics Processing and Product Characterization

“…Nonspecific binding of PLBL2 to protein A/G beads, at high concentrations could also contribute to the observed differences obtained from using the two methods; however, since those differences were not observed in mAb-1′ samples, it is more likely that PLBL2 has interaction with mAb-4 as previously reported . HCP–mAb interactions have also been identified and investigated by several research groups. , However, direct digestion without the HCP enrichment step would provide a full coverage of all HCPs presented. To fully understand the HCP–mAb interaction and the difference between the direct digestion and affinity depletion workflow, continuous study is ongoing to compare the protein profile and quantification of the HCPs enriched by the depletion step to those bound to mAb by both MRM and proteomics-based approaches.…”

“…35 HCP−mAb interactions have also been identified and investigated by several research groups. 42,43 However, direct digestion without the HCP enrichment step would provide a full coverage of all HCPs presented. To fully understand the HCP−mAb interaction and the difference between the direct digestion and affinity depletion workflow, continuous study is ongoing to compare the protein profile and quantification of the HCPs enriched by the depletion step to those bound to mAb by both MRM and proteomics-based approaches.…”

Hybrid Modeling of CHO Cell Cultivation in Monoclonal Antibody Production with an Impurity Generation Module

“…Yet, there are individual differences in characteristics and severity to quality problems among the generated HCPs. These differences can cause additional difficulties in the separation process . For example, the closer the HCP structure to the desired mAb product, the more difficult the separation can become.…”

“High‐risk” host cell proteins (HCPs): A multi‐company collaborative view

“…Downstream processing often includes protein A affinity purification followed by additional polishing steps to further remove aggregates, charge variants, HCPs, T A B L E 1 A comprehensive list of frequently seen HCPs found throughout different processing steps and host cell DNA (Shukla & Thommes, 2010). A literature search performed on the HCPs present after protein A chromatography revealed that many of the same HCPs are found across the biopharmaceutical industry Singh et al (2020). The HCPs identified were similar despite differences in CHO cell lines, upstream processes or downstream processes (Yuk et al, 2015).…”