title: Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK. dave.gervais@portonbiopharma.com.
authors.forename: David
authors.identifier: http://orcid.org/0000-0002-4996-3217
authors.initials: D
authors.lastname: Gervais
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.
authors.forename: Darryl
authors.initials: D
authors.lastname: King
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kanda
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Foote
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.
authors.forename: Lucy
authors.initials: L
authors.lastname: Elliott
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.
authors.forename: Phillip
authors.initials: P
authors.lastname: Brown
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, Darwin Building Room 101A, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Natacha O
authors.initials: NO
authors.lastname: Lee
authors.affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, Darwin Building Room 101A, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Thalassinos
authors.affiliation: Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Pizzey
authors.affiliation: Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Rambo
authors.affiliation: ChELSI Institute, Dept of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Minshull
authors.affiliation: ChELSI Institute, Dept of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Dickman
authors.affiliation: Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.
authors.forename: Stuart
authors.initials: S
authors.lastname: Smith
mesh_terms: D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000592:standards; D001215:Asparaginase / Q000302:isolation & purification* / Q000592:standards; D002853:Chromatography, Liquid; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D011487:Protein Conformation; D053838:Scattering, Small Angle; D053719:Tandem Mass Spectrometry
chemical_list: D000970:Antineoplastic Agents; D001215:Asparaginase
keywords: L-asparaginase; SAXS; deamidation; ion mobility; pH-induced conformational change