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

Abstract

A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.colil-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.colil-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.

Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry

“…The isotopically unresolved mass envelope of this high-mass molecule is broad (approximately 25 Da at full width at half-maximum), limiting the information provided . However, based on a previous study in which ASNase was intentionally deamidated, it is very plausible that this mechanism is the cause of the mass shift . As deamidation causes a mass increase of 0.984 Da, the observed mass differences indicate an estimated average occurrence of 5 deamidations for the monomer and tetramer, respectively.…”

Thermal sensitivity as a quality control attribute for biotherapeutics: The L‐asparaginase case

“…Deamidated E.coli L‐ASNase was prepared by incubating 1.0 mg/mL L‐ASNase in 1% NH 4 HCO 3 (m/v) for 24 hours, followed by desalting on a PD 10 column. After lyophilization, the protein was dissolved in H 2 O to obtain a concentration of 1 mg/mL . Dry‐heated E.coli L‐ASNase was prepared by heating the E.coli L‐ASNase powder at 80°C for 10 minutes.…”

Design of experiment techniques for the optimization of chromatographic analysis conditions: A review

“…Yao et al. [61] developed a CZE method for the quantification of the Escherichia coli l ‐asparaginase and its acidic variants. The experimental conditions that included pH and concentration of the selected background electrolyte were optimized by a CCD in order to obtain acceptable resolution, migration time, and peak area.…”

“…It is a challenge to the CE for the simultaneous determination of the enantiomeric purity and impurities, but the adoption of DoE enables more efficient development of analysis methods. Yao et al [61] developed a CZE method for the quantification of the Escherichia coli l-asparaginase and its acidic variants. The experimental conditions that included pH and concentration of the selected background electrolyte were optimized by a CCD in order to obtain acceptable resolution, migration time, and peak area.…”