Principal Scientist Boehringer Ingelheim Pharma Chappaqua, New York
Biocatalysis is often employed as an alternative to chemical catalysis in the production of New Chemical Entities (NCEs) due to enzymes' inherent selectivity, their ability to function under mild conditions, and the sustainable manufacturing processes they offer. The removal of these enzymes after reactions is essential and determining the residual enzymes is an obstacle that hinder the widespread adoption of biocatalysis. Spectrophotometric assays based on commercial kits were commonly used to determine residual total proteins. Other methods such as protein binding assays and Mass Spectrometry were also proven as suitable method to analyze the trace level of residual proteins. There are a few challenges remained in establishing a suitable method for a specified Active Pharmaceutical Ingredients (APIs). Generally, these assays are primarily designed for quantifying proteins in aqueous solutions, which make it challenging when adapting them for low-water-solubility APIs by combining organic solvents and water buffers due to strong matrix effects from APIs and specific organic solvents. Moreover, some colorimetric assays may not be suitable for highly colored API solutions, limiting their applicability in some cases. Additionally, binding assay and mass spectrometry also requires sophisticated and time-consuming method development. Overall, none of these assays could be used universally. In this work, we summarized our recent analytical approaches in the projects that involves the biocatalysts. Furthermore, we developed a universal method for controlling residual protein in NCEs. This method uses commercially available labeling reagents followed by chromatography analysis. The method is compatible with organic and water mixed buffer. With the HPLC separation, the protein signal is enhanced for more precise measurements, eliminating the interference from small molecules with a linearity detection range from 100 to 1000 ppm of the target enzyme in the API.
Learning Objectives:
Describe analytical techniques for detecting and quantifying residual enzymes in small molecule APIs, including HPLC, ELISA, LC-MS/MS, and protein staining methods.
Challanged of current common analytical method including interference from API during Spectrophotometric and other assays.
A novel method for controlling residual protein in NCEs was developed. This method uses commercially available labeling reagents followed by chromatography analysis with a linearity detection range from 100 to 1000 ppm of the target enzyme in the API.
