Erwinia chrysanthemi L-asparaginase (ErA) is an enzyme commonly used in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). Biopharmaceutical products such as ErA must be monitored for modifications such as deamidation, typically using ion-exchange chromatography (IEX). Analysis of clinical-grade ErA using native IEX resolves a number of enzymatically-active, acidic variants that were poorly characterised.
ErA IEX variants were isolated and fully characterised using capillary electrophoresis (cIEF), LC-MS and LC-MS/MS of proteolytic digests, and structural techniques including circular dichroism, small-angle X-ray scattering (SAXS) and ion-mobility mass spectrometry (IM-MS).
LC-MS, MS/MS and cIEF demonstrated that all ErA isolates consist mainly of enzyme lacking primary-sequence modifications (such as deamidation). Both SAXS and IM-MS revealed a different conformational state in the most prominent acidic IEX peak. However, SAXS data also suggested conformational differences between the main peak and major acidic variant were minor, based on comparisons with crystal structures.
IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.
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Acute Lymphoblastic Leukaemia
Collision Cross Section
Capillary Isoelectric Focussing
Escherichia coli L-asparaginase
Erwinia chrysanthemi L-asparaginase
High-Pressure Liquid Chromatography
Ion Mobility-Mass Spectrometry
Liquid Chromatography-Mass Spectrometry
Liquid Chromatography-Tandem Mass Spectrometry
Molecular Weight Cut-Off
Pair-Distance Distribution Function
Protein Data Bank
- Rg :
Radius of Gyration
Small-Angle X-ray Scattering
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
Ultra High Pressure Liquid Chromatography
- Vc :
Volume of Correlation
Extracting Ion Current
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ACKNOWLEDGMENTS AND DISCLOSURES
The authors would like to thank Roger Hinton, Head of Development & Production for making facilities and funds available for this work, and Trevor Marks, Head of Process and Analytical Development Group, for facilitating these studies. Further thanks go to the entire Development & Production team at PHE Porton. MJD acknowledges support from the Biotechnology and Biological Sciences Research Council UK [BB/M012166/1]. NOL is funded by a BBSRC iCASE award BB/L015382/1.
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Gervais, D., King, D., Kanda, P. et al. Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry. Pharm Res 32, 3636–3648 (2015). https://doi.org/10.1007/s11095-015-1722-2
- ion mobility
- pH-induced conformational change