Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis

King, Cory; Patel, Rekha; Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Gu, Zhenyu; Liu, Hongcheng

doi:10.1016/j.jchromb.2018.03.049

Cited by 25 publications

(19 citation statements)

References 42 publications

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“…When measured by differential scanning calorimetry, the fragment antigen binding (Fab) fragment with the deamidation product, isoaspartate (isoAsp), is less stable compared to Fab with the original Asn residue [22]. Deamidation increases the molecular weight of mAbs by 1 Da and generates acidic species [2,12,13,20,21,22,31,32]. Variants containing deamidation products are less hydrophobic than those with the original Asn residues [20,32].…”

Section: Asn Deamidationmentioning

confidence: 99%

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Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

Beck

Liu²

2019

Antibodies

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Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.

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Section: Asn Deamidationmentioning

confidence: 99%

“…Deamidation increases the molecular weight of mAbs by 1 Da and generates acidic species [2,12,13,20,21,22,31,32]. Variants containing deamidation products are less hydrophobic than those with the original Asn residues [20,32]. Deamidation does not impact in vivo clearance [21,26].…”

Section: Asn Deamidationmentioning

confidence: 99%

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

Beck

Liu²

2019

Antibodies

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“… 6 As one of the main criteria for attribute criticality assessment, the efficacy of therapeutic proteins is measured as binding strength to the therapeutic target and duration in human circulation (pharmacokinetics). 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.…”

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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“…This observation suggests that local chemical or structural changes, in addition to changes in global antibody properties, caused by these chemical modifications may induce changes in the surface hydrophobicities of the mAbs in different ways and lead to distinct HIC retention behaviors. 66 For Met oxidation, our mass spectrometry analysis revealed that the oxidation levels of the 2 mAbs were similar in the Fc regions but markedly different in the Fab regions (Table 1), which suggests that the Fab region is primarily responsible for the observed differences in hydrophobicity for the Metoxidized samples. For Trp oxidation, the oxidized samples for the 2 mAbs display markedly different HIC retention behavior despite the fact that both mAbs primarily undergo Trp oxidation at one site in the CDRs, which is different for each mAb.…”

Section: Discussionmentioning

confidence: 84%