Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(β-acetylglucosaminyl)- l-asparaginase case study.
HCP ELISA assay
HCP identification and quantitation by LC-MS/MS
N-(4)-(β-acetylglucosaminyl)-
l-asparaginase
hitchhiking behavior
host cell proteins
risk assessment
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
11 2020
11 2020
Historique:
received:
04
12
2019
revised:
01
04
2020
accepted:
13
07
2020
pubmed:
25
7
2020
medline:
3
2
2022
entrez:
25
7
2020
Statut:
ppublish
Résumé
Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(β-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.1.26) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS-HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.
Identifiants
pubmed: 32706388
doi: 10.1002/bit.27514
pmc: PMC7689792
doi:
Substances chimiques
Antibodies, Monoclonal
0
Proteins
0
Recombinant Proteins
0
Asparaginase
EC 3.5.1.1
Glucosamine
N08U5BOQ1K
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
3368-3378Informations de copyright
© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.
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