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.

中文翻译：

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确定固定化青霉素G酰基转移酶产生的头孢克洛残留蛋白的整合策略。

在制药工业中，通过环境上可持续的酶促过程替代半合成的β-内酰胺类抗生素（SSBA）的常规化学合成路线的趋势正在不断增长。这些具有诸如减少溶剂和废物处理的成本以及由于更少的反应步骤而节省时间的优点。青霉素G酰基转移酶（PGA）是一种工业上重要的酶，主要用于催化SSBA的合成。在这项研究中，我们使用三种不同的分析方法确定了与PGA相关的残留蛋白质含量的综合策略，这是最终产品中至关重要的质量问题。头孢克洛被用作SSBA的代表性实例。高效液相色谱与荧光检测（HPLC-FD）结合使用，可以对PGA残留蛋白和其他低分子量（MW）杂质进行常规分析，具有很高的检测特异性和灵敏度，与Bradford分析和微流体蛋白芯片电泳相媲美。但是，这后两种方法分别在定量和定性分析方面都比较出色，应被视为HPLC-FD方法的必要辅助方法。通过这三种方法的结合，在来自两个不同制造商的四个（总共13个）头孢克洛批量样品中检测到痕量残留蛋白，主要蛋白MW约为63 kDa。这表明较高MW的PGA亚基倾向于在终产物中持续存在。