LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

Abstract The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > ...

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Autores principales:	Sumit Kumar Singh, Deepak Kumar, Himanshu Malani, Anurag S. Rathore
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/95c2af03819a449dadb3673598c9d62f
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spelling	oai:doaj.org-article:95c2af03819a449dadb3673598c9d62f2021-12-02T14:16:06ZLC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity10.1038/s41598-020-79541-22045-2322https://doaj.org/article/95c2af03819a449dadb3673598c9d62f2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79541-2https://doaj.org/toc/2045-2322Abstract The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.Sumit Kumar SinghDeepak KumarHimanshu MalaniAnurag S. RathoreNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sumit Kumar Singh Deepak Kumar Himanshu Malani Anurag S. Rathore LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
description	Abstract The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.
format	article
author	Sumit Kumar Singh Deepak Kumar Himanshu Malani Anurag S. Rathore
author_facet	Sumit Kumar Singh Deepak Kumar Himanshu Malani Anurag S. Rathore
author_sort	Sumit Kumar Singh
title	LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
title_short	LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
title_full	LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
title_fullStr	LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
title_full_unstemmed	LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
title_sort	lc–ms based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/95c2af03819a449dadb3673598c9d62f
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LC–MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

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