Post-refolding stability considerations for optimization of in-vitro refolding: L-asparaginase as a case study.
L-asparaginase
design of experiment
refolding
stability
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
Apr 2023
Apr 2023
Historique:
revised:
13
12
2022
received:
07
10
2022
accepted:
14
12
2022
medline:
10
4
2023
pubmed:
19
12
2022
entrez:
18
12
2022
Statut:
ppublish
Résumé
L-Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L-asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold-ups during processing, and can result in product loss if stability is an issue, as is the case with L-asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation. In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L-asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)-based optimization was then performed for the refolding step. The net outcome was more than a three-fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L-asparaginase process intermediate was found to be stable for more than a week at room temperature and 2-8°C, while the unoptimized sample was stable at 2-8°C but did not show any activity at room temperature after 72 h. The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L-asparaginase which are labile.
Sections du résumé
BACKGROUND
BACKGROUND
L-Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L-asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold-ups during processing, and can result in product loss if stability is an issue, as is the case with L-asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation.
METHODS AND RESULTS
RESULTS
In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L-asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)-based optimization was then performed for the refolding step. The net outcome was more than a three-fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L-asparaginase process intermediate was found to be stable for more than a week at room temperature and 2-8°C, while the unoptimized sample was stable at 2-8°C but did not show any activity at room temperature after 72 h.
CONCLUSIONS
CONCLUSIONS
The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L-asparaginase which are labile.
Identifiants
pubmed: 36528864
doi: 10.1002/biot.202200505
doi:
Substances chimiques
Asparaginase
EC 3.5.1.1
Urea
8W8T17847W
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2200505Informations de copyright
© 2022 Wiley-VCH GmbH.
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