Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product.
Bioreactors
Cell Culture Techniques
/ instrumentation
Cells, Cultured
Chromatography, Reverse-Phase
Coagulants
/ isolation & purification
Culture Media
/ metabolism
Factor IX
/ isolation & purification
Humans
Protein Conformation
Protein Processing, Post-Translational
Proteomics
Quality Control
Recombinant Proteins
/ isolation & purification
Spectrometry, Mass, Electrospray Ionization
Tandem Mass Spectrometry
Workload
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
23 03 2021
23 03 2021
Historique:
received:
26
06
2020
accepted:
18
02
2021
entrez:
24
3
2021
pubmed:
25
3
2021
medline:
11
8
2021
Statut:
epublish
Résumé
Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
Identifiants
pubmed: 33758337
doi: 10.1038/s42003-021-01903-x
pii: 10.1038/s42003-021-01903-x
pmc: PMC7988164
doi:
Substances chimiques
Coagulants
0
Culture Media
0
Recombinant Proteins
0
Factor IX
9001-28-9
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
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