Evaluation of Quenching and Extraction Methods for Nucleotide/Nucleotide Sugar Analysis

Nucleotide sugars are the donor substrates of glycosyltransferases and their availability is known to have an impact on the glycosylation of recombinant proteins including monoclonal antibodies. In addition, the intracellular concentration levels of these metabolites can provide information about the physiological/energetic state of the cell. Therefore, the ability to qualitatively and quantitatively determine the intracellular nucleotides and nucleotide sugars can give valuable insight into the metabolism associated with the glycosylation processes in cells. However, in order to be able to perform a consistent and reliable time specific analysis of these metabolites during a cell culture the metabolism of the cell needs to be stopped immediately at the point of sampling and an efficient extraction needs to be performed. Once the nucleotides and nucleotide sugars are extracted from the cell sample an efficient HPLC method is needed to separate all or most of the metabolites of interest to allow for their identification and quantification. Here, we describe an optimized method for the analysis of the intracellular nucleotide/nucleotide sugar pool in CHO suspension cells which includes protocols for quenching, extraction and HPLC analysis.