Bin Yang,Wenhua Li,Hui Zhao,Anling Wang,Yuanjun Lei,Qiuling Xie,Sheng Xiong
Monoclonal antibody (mAb) fragmentation is a widespread issue of protein stability that needs to be carefully monitored for critical mAb quality control during the production process development. This study describes here the discovery and characterization of CHO host cell protease-induced fragmentation of a therapeutic mAb-X in the formulation samples from an early production process. The fragmentation was observed in the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) analysis of mAb-X formulation samples incubated at elevated temperature. Size exclusion liquid chromatography (SEC-HPLC) was used to analyze and collect these cleaved fragments derived from mAb-X. Reversed phase liquid chromatography mass spectrometry (RP-LC-MS) and tandem mass (MS/MS) analysis demonstrated that the fragment was generated mainly due to the hinge region cleavage of mAb-X. The fragmentation rate was characterized in the mAb-X formulation samples at pH from 4.0 to 6.0 using CE-SDS and SDS-PAGE analysis. The percentage of the main fragment increased dramatically from 2.8% to 31.2% as pH decreased from 6.0 to 4.0 at 40 °C for 28 days, which indicated the fragmentation was highly pH-dependent. The SDS-PAGE analysis further verified the pH-dependent property of the framentation of mAb-X. Moreover, the fragmentation was characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. Significant inhibition of mAb-X fragmentation was observed with the addition of pepstatin A to mAb-X formulation samples. These results suggested residual acidic host cell protease(s) in the formulation samples from an early production process caused the fragmentation of mAb-X. To prove evidence, we developed an optimized protein A chromatography to enhance the residual host cell protease(s) removal capability of mAb-X purification process and consequently eliminate the above described cleaved fragment of mAb-X, which further supported the hypothesis that the fragmentation of mAb-X was catalyzed by the residual host cell protease(s) in the formulation samples from the early production process. This case study reiterated that residual host cell protease is a critical quality attribute (CQA) that should be carefully controlled and evaluated to guarantee successful manufacture processes for mAb products.
Xinjian Yin,Yayun Liu,Lijun Meng,Haisheng Zhou,Jianping Wu,Lirong Yang
The active site is the common hotspot for rational and semi-rational enzyme activity engineering. However, the active site represents only a small portion of the whole enzyme. Identifying more hotspots other than the active site for enzyme activity engineering should aid in the development of biocatalysts with better catalytic performance. Glutamate dehydrogenases (GluDHs) are promising and environmentally benign biocatalysts for the synthesis of valuable chiral L-amino acids by asymmetric reductive amination of α-keto acids. GluDHs contain an inter-domain hinge structure that facilitates dynamic reorientations of the domains relative to each other. Such hinge-bending conformational motions of GluDHs play an important role in regulating the catalytic activity. Thus, the hinge region represents a potential hotspot for catalytic activity engineering for GluDHs. Herein, we report semi-rational activity engineering of GluDHs with the hinge region as the hotspot. Mutants exhibiting significantly improved catalytic activity toward several non-natural substrates were identified and the highest activity increase reached 104-fold. Molecular dynamics simulations revealed that enhanced catalytic activity may arise from improving the open/closed conformational transformation efficiency of the protein with hinge engineering. In the batch production of three valuable L-amino acids, the mutants exhibited significantly improved catalytic efficiency, highlighting their industrial potential. Moreover, the catalytic activity of several active site tailored GluDHs was also increased by hinge engineering, indicating that hinge and active site engineering are compatible. The results show that the hinge region is a promising hotspot for activity engineering of GluDHs and provides a potent alternative for developing high-performance biocatalysts toward chiral L-amino acid production.
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