Improvement in accuracy and specificity of high-throughput sialic acid assay

Abstract

The efficacy of many therapeutic proteins is influenced by their sialic acid content. Optimizing and consistently controlling the qualitative attributes of proteins produced in bioprocesses would benefit significantly from a high-throughput method (HTM) for measuring the sialic acid content. Previously, we developed an HTM that could rapidly analyze many crude culture samples in parallel. Here, we used high-throughput protein purification and denaturation to improve the accuracy and specificity of the HTM. With these modifications, the current HTM can accurately, precisely, rapidly (70 min), and specifically analyze 80 crude culture samples in parallel. It requires only 30 μL crude sample, and has a quantitation limit of 1 μM sialic acid. Its accuracy has been tested on five different types of glycoproteins. Moreover, we found that sample protein denaturation is crucial to ensure complete cleavage of sialic acid by sialidase. The current HTM can be used for many applications in cell line and bioprocess development.