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Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry



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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L-aspartic acid-β-hydroxamate


Acute Lymphoblastic Leukaemia


Collision Cross Section


Circular Dichroism


Collision-Induced Dissociation


Capillary Isoelectric Focussing


Drug Product


Drug Substance


Escherichia coli L-asparaginase


Erwinia chrysanthemi L-asparaginase


Electrospray Ionisation


High-Pressure Liquid Chromatography


Scattering Intensity


Ion-Exchange Chromatography


Ion Mobility-Mass Spectrometry


Liquid Chromatography-Mass Spectrometry


Liquid Chromatography-Tandem Mass Spectrometry


2-(N-morpholino)ethanesulfonic acid


Mass Spectrometry


Molecular Weight Cut-Off


Pair-Distance Distribution Function


Pheromone-Binding Protein


Protein Data Bank


Post-Translational Modification


Scattering Vector

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


  1. 1.

    Duval M, Suciu S, Ferster A, Rialland X, Nelken B, Lutz P, et al. Comparison of Escherichia coli–asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer—Children’s Leukemia Group phase 3 trial. Blood. 2002;99(8):2734–9.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Beard MEJ, Crowther D, Galton DAG, Guyer RJ, Fairley GH, Kay HEM, et al. L-asparaginase in treatment of acute leukaemia and lymphosarcoma. Br Med J. 1970;1:191–5.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  3. 3.

    Pieters R, Hunger SP, Boos J, Rizzari C, Silverman L, Baruchel A, et al. L-Asparaginase treatment in acute lymphoblastic leukaemia: a focus on Erwinia asparaginase. Cancer. 2011;117(2):238–49.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  4. 4.

    Salzer WL, Asselin BL, Plourde PV, Corn T, Hunger SP. Development of asparaginase Erwinia chrysanthemi for the treatment of acute lymphoblastic leukemia. Ann N Y Acad Sci. 2014;1329:81–92.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Wriston JC. Asparaginase. Methods Enzymol. 1985;113:608–18.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Aghaiypour K, Wlodawer A, Lubkowski J. Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-Asparaginase. Biochemistry. 2001;40:5655–64.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Lubkowski J, Dauter M, Aghaiypour K, Wlodawer A, Dauter Z. Atomic resolution structure of Erwinia chrysanthemi L-asparaginase. Acta Crystallogr D. 2003;59:84–92.

    Article  PubMed  Google Scholar 

  8. 8.

    Miller M, Rao JKM, Wlodawer A, Gribskov MR. A left-handed crossover involved in amidohydrolase catalysis: crystal structure of Erwinia chrysanthemi L-asparaginase with bound L-aspartate. FEBS J. 1993;328(3):275–9.

    CAS  Article  Google Scholar 

  9. 9.

    Gervais D, Allison N, Jennings A, Jones S, Marks T. Validation of a thirty-year-old process for the manufacture of L-Asparaginase from Erwinia chrysanthemi. Bioprocess Biosyst Eng. 2013;36(4):453–60.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Jenkins N, Murphy L, Tyther R. Post-translational modifications of recombinant proteins: significance for biopharmaceuticals. Mol Biotechnol. 2008;39(2):113–8.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm. 2005;289(1–2):1–30.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544–75.

    Article  PubMed  Google Scholar 

  13. 13.

    Liu DT. Deamidation: a source of microheterogeneity in pharmaceutical proteins. Trends Biotechnol. 1992;10:364–9.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Weitzhandler M, Farnan D, Rohrer JS, Avdalovic N. Protein variant separations using cation exchange chromatography on grafted, polymeric stationary phases. Proteomics. 2001;1:179–85.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Zhao SS, Chen DDY. Applications of capillary electrophoresis in characterizing recombinant protein therapeutics. Electrophoresis. 2014;35(1):96–108.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Yang H, Zubarev RA. Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides. Electrophoresis. 2010;31(11):1764–72.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  17. 17.

    Hao P, Ren Y, Datta A, Tam JP, Sze SK. Evaluation of the effect of trypsin digestion buffers on artificial deamidation. J Proteome Res. 2015;14(2):1308–14. doi:10.1021/pr500903b.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Farnan D, Moreno GT. Multiproduct high-resolution monoclonal antibody charge variant separations by pH gradient ion-exchange chromatography. Anal Chem. 2009;81(21):8846–57.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Rea JC, Moreno GT, Lou Y, Farnan D. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. J Pharm Biomed Anal. 2011;54(2):317–23.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Han M, Guo A, Jochheim C, Zhang Y, Martinez T, Kodama P, et al. Analysis of glycosylated type II interleukin-1 receptor (IL-1R) by imaged capillary isoelectric focussing (i-cIEF). Chromatographia. 2007;66:969–76.

    CAS  Article  Google Scholar 

  21. 21.

    Sosic Z, Houde D, Blum A, Carlage T, Lyubarskaya Y. Application of imaging capillary IEF for characterisation and quantitative analysis of recombinant protein charge heterogeneity. Electrophoresis. 2008;29:4368–76.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Gandhi S, Ren D, Xiao G, Bondarenko P, Sloey C, Ricci MS, et al. Elucidation of degradants in acidic peak of cation exchange chromatography in an IgG1 monoclonal antibody formed on long-term storage in a liquid formulation. Pharm Res. 2012;29:209–24.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Khawli LA, Goswami S, Hutchinson R, Kwong ZW, Yang J, Wang X, et al. Charge variants in IgG1: isolation, characterisation, in vitro binding properties and pharmacokinetics in rats. MAbs. 2010;2(6):613–24.

    PubMed Central  Article  PubMed  Google Scholar 

  24. 24.

    Gervais D, O’Donnell J, Sung M, Smith S. Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi L-asparaginase. Process Biochem. 2013;48(9):1311–6.

    CAS  Article  Google Scholar 

  25. 25.

    Gervais D, Foote N. Recombinant deamidated mutants of Erwinia chrysanthemi L-asparaginase have similar or increased activity compared to wild-type enzyme. Mol Biotechnol. 2014;56(10):865–77.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Gervais D, King D. Capillary isolelectric focussing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures. Anal Biochem. 2014;465:90–5.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Harms E, Wehner A, Jennings MP, Pugh KJ, Beacham IR, Röhm KH. Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts. Protein Expr Purif. 1991;2:144–50.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Derst C, Henseling J, Röhm KH. Probing the role of threonine and serine residues of E coli asparaginase II by site-specific mutagenesis. Protein Eng. 1992;5(8):785–9.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Pringle SD, Giles K, Wildgoose JL, Williams JP, Slade SE, Thalassinos K, et al. An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom. 2007;261:1–12.

    CAS  Article  Google Scholar 

  30. 30.

    Sivalingam GN, Yan J, Sahota H, Thalassinos K. Amphitrite: a program for processing travelling wave ion mobility data. Int J Mass Spectrom. 2013;345:54–62.

    Article  PubMed  Google Scholar 

  31. 31.

    Glatter O, Kratky O. Small-angle x-ray scattering. London: Academic; 1982. Chapter 28.

  32. 32.

    Feigin LA, Svergun DI. Structure analysis by small-angle x-ray and neutron scattering. New York: Plenum Press; 1987.

    Book  Google Scholar 

  33. 33.

    Rambo RP, Tainer JA. Characterising flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers. 2011;95(8):559–71.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  34. 34.

    Receveur-Brechot V, Durand D. How random are intrinsically disordered proteins? A small-angle scattering perspective. Curr Protein Pept Sci. 2012;13(1):55–75.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  35. 35.

    Durand D et al. NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers. J Struct Biol. 2010;169(1):45–53.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Rambo RP, Tainer JA. Accurate assessment of mass, models and resolution by small-angle scattering. Nature. 2013;496:477–81.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  37. 37.

    Tian X, Langkilde AE, Thorolfsson M, Rasmussen HB, Vestergaard B. Small-angle x-ray scattering screening complements conventional biophysical analysis: comparative structural and biophysical analysis of monoclonal antibodies IgG1, IgG2, and IgG4. J Pharm Sci. 2014;103:1701–10. doi:10.1002/jps.23964.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  38. 38.

    Garst AD, Heroux A, Rambo RP, Batey RT. Crystal structure of the lysine riboswitch regulatory mRNA element. J Biol Chem. 2008;283(33):22347–51.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  39. 39.

    Guo W, West JM, Dutton AS, Tsuruta H, Kantrowitz ER. Trapping and structure determination of an intermediate in the allosteric transition of aspartate transcarbamoylase. Proc Natl Acad Sci U S A. 2012;109(20):7741–6.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  40. 40.

    Heck AJR. Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods. 2008;5:927–33.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Thalassinos K, Pandurangan AP, Xu M, Alber F, Topf M. Conformational States of macromolecular assemblies explored by integrative structure calculation. Structure. 2013;21:1500–8.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  42. 42.

    Zhou M, Morgner N, Barrera NP, Politis A, Isaacson SC, Matak-Vinkovic D, et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science. 2011;334:380–5.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  43. 43.

    Hilton GR, Thalassinos K, Grabenauer M, Sanghera N, Slade SE, Wyttenbach T, et al. Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry. J Am Soc Mass Spectrom. 2010;21:845–54.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Marklund EG, Degiacomi MT, Robinson CV, Baldwin AJ, Benesch JLP. Collision cross sections for structural proteomics. Structure. 2015;23:791–9.

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Homer RB, Allsopp SR. An investigation of the electronic and steric environments of tyrosyl residues in ribonuclease A and Erwinia carotovora L-asparaginase through fluorescence quenching by caesium, iodide and phosphate ions. Biochem Biophys Acta. 1976;434:297–310.

    CAS  PubMed  Google Scholar 

  46. 46.

    Minton NP, Bullman HMS, Scawen MD, Atkinson T, Gilbert HJ. Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene. Gene. 1986;46:25–35.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Illarionova NO, Petrov LN, Olennikova LV, Roshchin SN, Pasechnik VA, Haliapin BD, et al. Investigation of the L-asparaginase secondary structure in a wide pH region. Mol Biol Mosc. 1980;14:951–5.

    CAS  Google Scholar 

  48. 48.

    Mezentsev YV, Molnar AA, Gnedenko OV, Krasotkina YV, Sokolov NN, Ivanov AS. Oligomerisation of L-asparaginase from Erwinia carotovora. Biochem Mosc Suppl Ser B Biomed Chem. 2007;1(1):58–67.

    Article  Google Scholar 

  49. 49.

    Wojtasek H, Leal WS. Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes. J Biol Chem. 1999;274:30950–6.

    CAS  Article  PubMed  Google Scholar 

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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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Corresponding author

Correspondence to David Gervais.

Electronic supplementary material

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Supplementary Figure 1

Tandem MS/MS spectra of peptide unmodified peptide TGNGIVPPDEELPGLVSDSLNPAHAR (885.7868 m/z) [M+3H]3+ and deamidated peptide TGDGIVPPDEELPGLVSDSLNPAHAR (886.1136 m/z) [M+3H]3+. The abundant ions are highlighted. The mass shift of 1 Da for the b-series ions identified between unmodified and deamidated forms of the peptide, confirm the position of deamidation at N3. Also lack of mass shift in y-series ions identified also rules out deamidation and positions N21 and R26. Inset shows the isotope distribution of the parent ion. (GIF 182 kb)

Supplementary Figure 2

LC-MS/MS Isotope distribution profile for the unmodified peptide and deamidated peptide highlighting the corresponding 1 Da shift in mass. The experimental isotope profiles were compared to the theoretical isotope profiles of the peptides to confirm deamidation. (GIF 110 kb)

Supplementary Figure 3

Guinier plots of SAXS data from analyses at 1.25mg/mL concentration. (GIF 121 kb)

High Resolution Image (TIFF 61 kb)

High Resolution Image (TIFF 43 kb)

High Resolution Image (TIFF 41 kb)

Supplementary Table 1

(DOCX 41 kb)

Supplementary Table 2

(DOCX 44 kb)

Supplementary Table 3

(DOCX 43 kb)

Supplementary Table 4

(DOCX 36 kb)

Supplementary Table 5

(DOC 44 kb)

Supplementary Table 6

(DOC 43 kb)

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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).

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  • deamidation
  • ion mobility
  • L-asparaginase
  • pH-induced conformational change
  • SAXS