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

Gervais, David; King, Darryl; Kanda, Patrick; Foote, Nicholas; Elliott, Lucy H.; Brown, Phillip J.; Lee, Natacha O.; Thalassinos, Konstantinos; Pizzey, Claire; Rambo, Robert P.; Minshull, Thomas C.; Dickman, Mark J.; Smith, Stuart W.

doi:10.1007/s11095-015-1722-2

Cited by 12 publications

(8 citation statements)

References 47 publications

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“…However, such isolated variants often contain more than one PTM, so care must be taken during interpretation of the results. Multiple, orthogonal analytics may be required to fully understand the changes present in the protein, such as recently reported for L‐asparaginase …”

Section: Impact Of Deamidation On Protein Biopharmaceuticalsmentioning

confidence: 99%

“…Multiple, orthogonal analytics may be required to fully understand the changes present in the protein, such as recently reported for L-asparaginase. 80 Deamidation may be expected to have a deleterious impact on biopharmaceutical protein function. Certainly this appears to be the case for many proteins including recombinant human stem cell factor.…”

Section: Impact Of Deamidation On Protein Biopharmaceuticalsmentioning

confidence: 99%

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Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Gervais¹

2015

J of Chemical Tech & Biotech

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Understanding of product-related variants, such as variants with post-translational modifications, is an important part of biopharmaceutical development. Deamidation is a common post-translational modification occurring in biopharmaceutical proteins, affecting L-asparagine (Asn) and to a lesser extent, L-glutamine (Gln) residues. The rate of deamidation reactions are influenced by factors including protein structure (primary, secondary and higher structure), temperature and pH. In the vast majority of cases, deamidation is undesirable in biopharmaceuticals, and may lead to potential changes in protein structure, function, stability and immunogenicity. Measurement and characterisation of deamidated biopharmaceutical variants may be challenging, particularly with regard to quantitation of the two L-aspartate isoforms that are created, L-aspartic acid (Asp) and isoaspartate (isoAsp). Deamidation may occur intracellularly or during biopharmaceutical manufacture and storage, and must be understood, minimised and controlled, particularly in a regulatory context. Process control strategies that have been employed to date include alterations to fermentation steps, additives to cell cultures, chromatographic separation of charge variants and protein engineering to remove deamidation-prone Asn residues. However, the impact of deamidated forms of biopharmaceuticals should also be thoroughly studied, as they may not necessarily represent deleterious changes to the function of the molecule or the quality of the final product. This mini-review provides a summary of the current understanding of the origins, control and measurement of deamidation during biopharmaceutical development.

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Section: Impact Of Deamidation On Protein Biopharmaceuticalsmentioning

confidence: 99%

Section: Impact Of Deamidation On Protein Biopharmaceuticalsmentioning

confidence: 99%

Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Gervais¹

2015

J of Chemical Tech & Biotech

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“…Structural changes were compared with those revealed by ion mobility analysis. Panels A and B were adapted with permission from figures from references 54 and 57, respectively.…”

Section: Figurementioning

confidence: 99%

“…Overall the results show that IMS-MS is a useful tool for monitoring protein stability upon ligand binding and it is also suggested that such combined measurements could be very useful in the pursuit of drug development. In other studies, IMS-MS measurements (Figure 3A) were combined with small angle x-ray scattering (SAXS) spectroscopy to study enzymatic variants and provided structural information that was not typically obtained for ion exchange chromatography isolates [54]. Other experiments combined fluorescence spectroscopy and gas-phase dimer-induced fluorescence self quenching with IMS-MS measurements to study the structures of small peptides and ubiquitin [55].…”

Section: Introductionmentioning

confidence: 99%

Magnifying ion mobility spectrometry–mass spectrometry measurements for biomolecular structure studies

Majuta

Maleki

Karanji

et al. 2018

Current Opinion in Chemical Biology

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Ion mobility spectrometry-mass spectrometry (IMS-MS) provides information about the structures of gas-phase ions in the form of a collision cross section (CCS) with a neutral buffer gas. Indicating relative ion size, a CCS value alone is of limited utility. Although such information can be used to propose different conformer types, finer details of structure are not captured. The increased accessibility of IMS-MS measurements with commercial instrumentation in recent years has ballooned its usage in combination with separate measurements to provide enhanced data from which greater structural inferences can be drawn. This short review presents recent outstanding developments in scientific research that employs complementary measurements that when combined with IMS-MS data are used to characterize the structures of a wide range of compounds.

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“…60 A further example of pH-induced changes to HOS was studied in the anti-leukaemic enzyme Erwinia chrysanthemi L-asparaginase (ErA). Minor HOS changes were detected using SAXS and IM-MS. 68 Initially, these conformational changes were detected simply as fully-functional acidic variants using ion-exchange chromatography (IEX), and were assumed to be deamidated enzyme variants. Further investigation using tryptic-digest LC-MS showed that deamidation was absent, but SAXS ( Fig.…”

Section: Relationship Between Biopharmaceutical Manufacture and Hos Cmentioning

confidence: 99%

Higher‐order structure and conformational change in biopharmaceuticals

Orphanou¹,

Gervais²

2018

J of Chemical Tech & Biotech

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The higher order structure (HOS) of protein biopharmaceuticals is critical for biological and pharmacological function. Slight changes in HOS can impact product efficacy and quality, and therefore must be characterised. The technology and techniques available for HOS characterisation has advanced in recent years. Although methods such as the low‐resolution technique circular dichroism (CD) and the labour‐intensive but high‐resolution technique X‐ray crystallography may still be routinely used to assess HOS, other techniques, such as mass spectrometry (MS) with hydrogen‐deuterium exchange (HDX) and small‐angle X‐ray scattering (SAXS) are becoming more commonplace. Post‐translational modifications (PTMs) may have a large impact on HOS, potentially resulting in undesirable biopharmaceutical variant formation, as well as aggregation. In addition to PTMs, factors such as pH, temperature, stabilising agents and polymers have the potential to induce alterations in protein HOS. A proposed HOS assessment strategy is presented in this review to ensure the robustness of biopharmaceuticals during full process development. Where changes in HOS do occur, there may be a risk of immunogenic response in patients, so regulatory authorities now require comprehensive characterisation, risk assessment and understanding of biopharmaceuticals during development, manufacture and storage. This enables quality controls to be put in place to mitigate or avoid conditions where conformational changes are known to occur, and ensures that the quality, safety and efficacy of biopharmaceutical proteins are not compromised. © 2018 Society of Chemical Industry

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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: IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.

Cited by 12 publications

References 47 publications

Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Magnifying ion mobility spectrometry–mass spectrometry measurements for biomolecular structure studies

Higher‐order structure and conformational change in biopharmaceuticals

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