Discovery and characterization of CHO host cell protease-induced fragmentation of a recombinant monoclonal antibody during production process development.

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.

Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker.

Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.