Herman, Chase E.Min, LieChoe, Leila H.Maurer, Ronald W.Xu, XuankuoGhose, SanchayitaLee, Kelvin H.Lenhoff, Abraham M.2023-06-222023-06-222023-04-05Herman, CE, Min, L, Choe, LH, et al. Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions. Biotechnol. Prog. 2023;e3343. doi:10.1002/btpr.33431520-6033https://udspace.udel.edu/handle/19716/32937This is the peer reviewed version of the following article: Herman, CE, Min, L, Choe, LH, et al. Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions. Biotechnol. Prog. 2023;e3343. doi:10.1002/btpr.3343, which has been published in final form at https://doi.org/10.1002/btpr.3343. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited. © 2023 American Institute of Chemical Engineers. This article will be embargoed until 04/05/2024.Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.en-USaggregatesdigest methodhost-cell proteinsprotein A chromatographyproteomicsArticle