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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

Abstract

Purpose

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.

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Abbreviations

AHA:

L-aspartic acid-β-hydroxamate

ALL:

Acute Lymphoblastic Leukaemia

CCS:

Collision Cross Section

CD:

Circular Dichroism

CID:

Collision-Induced Dissociation

cIEF:

Capillary Isoelectric Focussing

DP:

Drug Product

DS:

Drug Substance

EcA:

Escherichia coli L-asparaginase

ErA:

Erwinia chrysanthemi L-asparaginase

ESI:

Electrospray Ionisation

HPLC:

High-Pressure Liquid Chromatography

I(q):

Scattering Intensity

IEX:

Ion-Exchange Chromatography

IM-MS:

Ion Mobility-Mass Spectrometry

LC-MS:

Liquid Chromatography-Mass Spectrometry

LC-MS/MS:

Liquid Chromatography-Tandem Mass Spectrometry

MES:

2-(N-morpholino)ethanesulfonic acid

MS:

Mass Spectrometry

MWCO:

Molecular Weight Cut-Off

P(r):

Pair-Distance Distribution Function

PBP:

Pheromone-Binding Protein

PDB:

Protein Data Bank

PTM:

Post-Translational Modification

q:

Scattering Vector

Rg :

Radius of Gyration

SAXS:

Small-Angle X-ray Scattering

SDS-PAGE:

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

TOF:

Time-of-Flight

UPLC:

Ultra High Pressure Liquid Chromatography

Vc :

Volume of Correlation

WT:

Wild-Type

XIC:

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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Correspondence to David Gervais.

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Supplementary Figure 1
figure10

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
figure11

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
figure12

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

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Supplementary Table 2

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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). https://doi.org/10.1007/s11095-015-1722-2

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KEY WORDS

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