Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.
Anal Chem
; 93(23): 8161-8169, 2021 06 15.
Article
em En
| MEDLINE
| ID: mdl-34032423
ABSTRACT
Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissorbatos
/
Produtos Biológicos
Tipo de estudo:
Prognostic_studies
/
Risk_factors_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos