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Issue 11, 2012
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Improvement in accuracy and specificity of high-throughput sialic acid assay

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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.

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

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Publication details

The article was received on 03 May 2012, accepted on 27 Aug 2012 and first published on 29 Aug 2012


Article type: Paper
DOI: 10.1039/C2AY25448D
Citation: Anal. Methods, 2012,4, 3565-3569
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    Improvement in accuracy and specificity of high-throughput sialic acid assay

    L. R. A. Markely, K. N. Jonnalagadda, M. Sinacore, T. Ryll and S. Prajapati, Anal. Methods, 2012, 4, 3565
    DOI: 10.1039/C2AY25448D

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