Rapid charge variant analysis of monoclonal antibodies to support lead candidate biopharmaceutical development

Trappe, Anne; Füssl, Florian; Carillo, Sara; Zaborowska, Izabela; Bones, Jonathan

doi:10.1016/j.jchromb.2018.07.037

Cited by 38 publications

(22 citation statements)

References 30 publications

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“… 7–11 At present, criticality (clinical significance) of the attributes is assessed by separating intact proteins on fractions using soft separation techniques, such as ion exchange chromatography (IEX), 12 , 13 hydrophobic interaction chromatography (HIC), 14 , 15 and size exclusion chromatography (SEC) with the goal of having one attribute per fraction. 16–18 An example is that protein molecules with one deamidated asparagine appear as an earlier-eluting peak on cation exchange chromatography (CEX), 19–21 which can be collected as one fraction for further potency testing. 22 However, it is difficult, if not impossible, to cleanly fractionate protein species with only one attribute/modification per chromatographic peak, or to assess criticality experimentally for each attribute.…”

Section: Introductionmentioning

confidence: 99%

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding

Shi

Xiao

Dillon

et al. 2021

mAbs

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Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

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Section: Introductionmentioning

confidence: 99%

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding

Shi

Xiao

Dillon

et al. 2021

mAbs

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“…The using of fast, reliable, and quantitative analytical methods is necessary for this purpose [7]. Charge-based variants have been categorized as acidic, main, and basic species.…”

Section: Methods Of Mab Charge Variant Analysismentioning

confidence: 99%

Charge Variants Analysis of Recombinant Monoclonal Antibodies

Torkashvand¹,

Vaziri

2019

BJSTR

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Recombinant monoclonal antibodies are among the most structurally complex therapeutics with various potential molecular modifications during production processes. Rapid and efficient characterization of these heterogeneities and controlling the upstream and downstream processes for achieving a consistent product is absolutely necessary. In this minireview the importance of characterization of mAb heterogeneity and the current analytical methods for mAb variants will be discussed.

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“…7 As a conventional and nondenaturing technique, ionexchange chromatography (IEX) has been widely used to separate and isolate protein charge variants during protein purification and for subsequent characterization. [22][23][24] Upon the separation of charge variants by IEX, current strategies to determine the effects of modifications on specific charge variant peak involve isolating the peak of interest followed by various mass spectrometric analyses, such as intact mass analysis, peptide mapping, and glycan analysis. 10,25 In addition to being limited by time and resources, this two-step approach may overlook the minor species that do not exhibit distinctive UV peaks and introduce artifacts because of the lengthy sample preparation processes.…”

Section: Introductionmentioning

confidence: 99%

“…A strategy for the direct coupling of MS with IEX involved the application of a pH gradient using volatile salts. 23,24,[29][30][31] Online weak cation exchange (WCX)-MS has been reported for analyzing IgG2 mAbs by using an ammonium hydroxide-based pH gradient. 32 Characterization of digested mAbs and proteins with molecular weights below~30 kDa has also been described by utilizing an ammonium formate and ammonium acetate-based pH and salt gradient.…”

Section: Introductionmentioning

confidence: 99%

“…[40][41][42][43][44] Additionally, CEX has greater resolving power as compared to RP LC and allows complete separation of intact antibody molecules even with one charged modification or a modification leading to partial net charge change. 7,10,23,29,35 With a few exceptions, 45 RP separation of protein charge variants with one modification is typically relatively poor, 46,47 but RP LC-MS is sensitive and easily amenable to top-down analysis. 43,[48][49][50][51] This is because proteins elute in the RP mobile phase in an unfolded and highly charged state, 52 which enables ionization prior to entering the mass analyzer.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Shi

Xiao

Dillon

et al. 2020

mAbs

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2020) Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis, mAbs, 12:1, 1739825, ABSTRACT Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatographytop-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping. ARTICLE HISTORY

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Rapid charge variant analysis of monoclonal antibodies to support lead candidate biopharmaceutical development

Cited by 38 publications

References 30 publications

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding

Charge Variants Analysis of Recombinant Monoclonal Antibodies

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

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