Identification of Process-Related Impurities and Corresponding Control Strategy in Biocatalytic Production of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid Using Sucrose Phosphorylase.

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an ideal substitute for l-ascorbic acid because of its remarkable stability and improved biological activity, which can be easily applied in cosmetic, food, and medicine fields. However, impurity identification and control are significant procedures during the manufacturing of AA-2G. This study assessed a manufacturing routine of AA-2G synthesized by sucrose phosphorylase (SPase). First, three unknown process-related impurities were observed, which were further identified as 3-O-α-d-glucopyranosyl- l-ascorbic acid (impurity I), 2-O-α-d-glucopyranosyl-l-dehydroascorbic acid (impurity II), and 13-O-α-d-glucopyranosyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (impurity III), respectively. Second, a comprehensive formation pathway of impurities was elucidated, and specific strategies corresponding to controlling each impurity were also proposed. Specifically, the content of impurity I can be reduced by 50% by fine tuning reaction conditions. The impurity II-free purification process was also achieved by applying a low concentration of alkali. Finally, a semi-rational design was introduced, and a single mutant L343F was obtained by site-directed mutagenesis, which reduced impurities I and III by 63.9 and 100%, respectively, without affecting the transglycosylation activity. It is expected that the reported impurity identification and control strategies during the AA-2G production will facilitate its industrial production.