Quantitation of host cell proteins in biopharmaceuticals from chinese hamster ovarian and vero cell lines using capillary electrophoresis western blots.
2D Western coverage
Biopharmaceutical reagent validation
Capillary electrophoresis
Enzyme-linked immunosorbent assays
Host cell proteins quantitation
Simple Western
Journal
Journal of pharmaceutical and biomedical analysis
ISSN: 1873-264X
Titre abrégé: J Pharm Biomed Anal
Pays: England
ID NLM: 8309336
Informations de publication
Date de publication:
05 Sep 2023
05 Sep 2023
Historique:
received:
26
01
2023
revised:
12
04
2023
accepted:
24
04
2023
medline:
12
6
2023
pubmed:
20
5
2023
entrez:
19
5
2023
Statut:
ppublish
Résumé
Quantitation of host cell proteins (HCPs) is essential in the process of preparation of many biological and vaccine products. Common methods of quantitation include the widely applied enzyme-linked immunosorbent assays (ELISAs), mass spectrometry (MS) and other orthogonal assays. Prior to using these techniques, critical reagents need to be evaluated, for example, antibodies need to be assessed for HCP coverage. Percent of HCP coverage is often established by denatured 2D Western blot. However, ELISAs measure the amount of HCP only in a native state. There are limited studies linking reagents validated by 2D-Western to ensure adequate coverage in the final ELISA. ProteinSimple's newly developed capillary Western blot technology allows for separation, blotting, and detection of proteins in a semi-automated and simplified format. Capillary Westerns are similar to slab Westerns, with the added benefit of being quantitative. Here we outline the capillary Western method that links the 2D Western coverage and ultimately ELISAs for more efficient HCP quantitation. This study describes the development of the capillary Western analytical method to quantitively evaluate HCPs in Vero and Chinese Hamster Ovarian (CHO) cell lines. The amount of CHO HCPs decreases as the sample is purified as expected. Using this approach, we determined that the detected Vero HCPs amount was similar irrespective of denatured (capillary Western) versus native assay format (ELISA). This new method can also be potentially employed to quantitatively assess the anti-HCP antibody reagent coverage used in commercial HCP ELISA kits.
Identifiants
pubmed: 37207489
pii: S0731-7085(23)00189-9
doi: 10.1016/j.jpba.2023.115420
pii:
doi:
Substances chimiques
Proteins
0
Biological Products
0
Types de publication
Journal Article
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
115420Informations de copyright
Copyright © 2023 Pearson. Published by Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cara Pearson, Yi Wang, Eman Alkurdi, Young Zhang, Kuo Yin, Richard R. Rustandi, and John W. Loughney report financial support was provided by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.