A Study on a Method for Evaluating Glycans in Biopharmaceuticals

Introduction

Many protein-based biopharmaceutical products, typified by antibody drugs, are synthesized in cultured cells derived from eukaryotes such as CHO (Chinese hamster ovary) cells. For this reason, there are inevitably many post-translational modifications to the biosynthesized proteins. Among these, modifications of glycans have gained attention as items for evaluating the quality of biopharmaceuticals since they are associated with the adjustment of protein functions, as well as with the unwanted development of antigenicity depending on their structure. However, there are various technical challenges in evaluating glycans. With O-linked glycans (O-glycan), it is particularly difficult to comprehensively release glycans from protein using enzymes, leading to the use of mainly the following two chemical methods: hydrazinolysis and β-elimination. However, these methods have issues that need to be improved. Hydrazinolysis requires great care since an explosive reagent is handled and therefore is not easy to implement. With the β-elimination method, a peeling reaction where glycans gradually decompose due to a continuous elimination reaction occurs. Conventionally, in analysis of O-glycans using β- elimination, glycans are released so as not to cause a continuous elimination reaction by using the reductive β-elimination method, which involves simultaneous releasing of glycans under alkaline conditions and reducing the root portion of the glycan with a reducing agent. However, this method completely reduces the root portion of the glycan and therefore does not allow labeling such as with fluorescent reagents after releasing glycans, thus limiting the available analysis methods. Also, in analysis by mass spectrometry of glycans obtained in this way, the high sensitivity analysis is not possible because the ionization efficiency of the glycan itself is not high. To address this, a non-reductive β-elimination/fluorescent labeling method is being examined as a method to bind a fluorescent labeling reagent such as 2-AB or PA by not reducing the root of the glycan, but this has not succeeded in significantly suppressing the continuous elimination reaction. Even so, in academic researches where O-glycan was the object of analysis, the existence of by-products due to this peeling reaction has not been problematic enough to hinder researches. However, glycans have to be evaluated for quality control in drugs which are to be administered to the human body, such as biopharmaceuticals, and the question of how to handle the existence of by-products during this evaluation is a major issue. In this article, we report the results of studying a method for releasing O-glycans chemically in which the peeling reaction is suppressed, based on a PMP labeling method.

July 3, 2017 GMT