Development of a capillary zone electrophoresis method to quantify E. coli L-asparaginase and its acidic variants

Han Yao, Jana Vandenbossche, Cari E. Sänger-van de Griend, Yorick Janssens, Cristina Soto Fernández, Xiaolong Xu, Evelien Wynendaele, Govert Willem Somsen, Rob Haselberg, Bart De Spiegeleer*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.coli L-asparaginase (L-ASNase) and its acidic variants. During the initial method development, a variety of experimental conditions were screened. Subsequently, a Design of Experiments (DoE) was used to optimize the pH and concentration of the selected background electrolyte (BGE) containing both TRIS and boric acid. Optimization was performed taking into account both the separation efficiency of L-ASNase and its acidic variants as well as overall method robustness. A repeatable separation between E.coli L-ASNase and its acidic variants was achieved on a bare fused silica capillary in combination with a BGE consisting of both 400 mM TRIS and boric acid. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness. The recovery for L-ASNase was 97.9–104.4% with a precision RSD of 1.5–3.2%, while the recovery of impurities was 92.1–109.8% with a RSD of 1.7–4.6%. The quantification limit was 1.9% (m/m). Moreover, the CZE-UV method was applied to determine the degradation rate in the presence of ammonium bicarbonate, confirming the suitability of the method. The degraded, partially charged L-ASNase was evaluated for its in-vitro enzymatic activity showing an insignificant different enzyme activity compared to the unmodified sample.

Original languageEnglish
Pages (from-to)83-91
Number of pages9
JournalTalanta
Volume182
DOIs
Publication statusPublished - 15 May 2018

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Keywords

  • Acidic impurities
  • Capillary zone electrophoresis
  • Design of experiments (DoE)
  • l-asparaginase

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