Integrative strategy to determine residual proteins in cefaclor produced by immobilized penicillin G acylase

There is a growing trend in the pharmaceutical industry towards substituting conventional chemical synthesis routes of semi-synthetic β-lactam antibiotics (SSBAs) through environmentally sustainable enzymatic processes. These have advantages such as cost reduction in terms of solvent and waste treatment and time saving owing to fewer reaction steps. Penicillin G acylase (PGA) is an industrially important enzyme that is mainly used to catalyze the synthesis of SSBAs. In this study, we established an integrative strategy using three different analytical methods for determining the PGA-associated residual protein content, which is a critical quality issue in the end product. Cefaclor was taken as representative example of SSBAs. High-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) allowed the routine analysis of PGA residual proteins and other low molecular weight (MW) impurities with high detection specificity and sensitivity, comparable to those of the Bradford assay and microfluidic protein chip electrophoresis. However, these latter two methods were superior for quantitative and qualitative analysis, respectively, and should be regarded as necessary adjuncts to the HPLC-FD method. By combining the three methods, trace levels of residual proteins were detected in four (out of 13) cefaclor bulk samples from two different manufacturers, with a major protein MW of ∼63 kDa. This suggests that the higher MW PGA subunit tends to persist in the end product. The integrative determination strategy described here can be used to evaluate SSBA bulk samples and monitor the process of SSBA manufacturing by enzymatic methods, especially in terms of inter-batch consistency and process stability.