Volume 39, Issue 4 e3343
RESEARCH ARTICLE

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions

Chase E. Herman

Chase E. Herman

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716 USA

Contribution: Conceptualization (equal), Data curation (lead), Formal analysis (lead), ​Investigation (lead), Methodology (lead), Project administration (equal), Validation (lead), Visualization (lead), Writing - original draft (lead), Writing - review & editing (equal)

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Lie Min

Lie Min

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716 USA

Contribution: Data curation (supporting), ​Investigation (supporting), Methodology (supporting), Validation (supporting)

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Leila H. Choe

Leila H. Choe

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716 USA

Contribution: Data curation (supporting), ​Investigation (supporting), Methodology (supporting), Validation (supporting)

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Ronald W. Maurer

Ronald W. Maurer

Biologics Process Development, Bristol Myers Squibb, Massachusetts, 01434 Devens, USA

Contribution: Resources (supporting)

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Xuankuo Xu

Xuankuo Xu

Biologics Process Development, Bristol Myers Squibb, Massachusetts, 01434 Devens, USA

Contribution: Funding acquisition (equal), Project administration (equal), Resources (equal), Supervision (equal), Writing - review & editing (equal)

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Sanchayita Ghose

Sanchayita Ghose

Biologics Process Development, Bristol Myers Squibb, Massachusetts, 01434 Devens, USA

Contribution: Funding acquisition (equal)

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Kelvin H. Lee

Kelvin H. Lee

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716 USA

Contribution: Supervision (equal)

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Abraham M. Lenhoff

Corresponding Author

Abraham M. Lenhoff

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716 USA

Correspondence

Abraham M. Lenhoff, Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA.

Email: [email protected]

Contribution: Conceptualization (equal), Formal analysis (supporting), Funding acquisition (equal), Project administration (equal), Supervision (lead), Writing - review & editing (equal)

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First published: 05 April 2023
Citations: 1

Abstract

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.

CONFLICT OF INTEREST STATEMENT

The authors report no conflict of interest.

DATA AVAILABILITY STATEMENT

Data submitted as Excel file.