Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis

Kim, Jae-Won; Jones, Laurie; Taylor, Lisa D.; Kannan, Gunasekaran; Jackson, F. J.; Lau, Hollis; Latypov, Ramil F.; Bailey, Bob

doi:10.1016/j.jchromb.2010.05.032

Cited by 29 publications

(16 citation statements)

References 39 publications

(39 reference statements)

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“…Even minor pI differences, charge distribution variations, or structural changes may be sufficient to separate proteins. 15,16 A typical CEX52 chromatogram of an antibody mixture consisting of IgG1-A, IgG1-B, IgG2-B, and IgG2-C is shown in Figure 1(A). It is evident that CEX52 provides complete baseline separation of the different mAbs.…”

Section: Measurement Of Protein Aggregation By Cex52mentioning

confidence: 99%

“…[12][13][14] Previously, we demonstrated the use of native cation-exchange chromatography (CEX) in solving analytical problems associated with charge heterogeneity, covalent modifications, and dimerization in mAbs. 15,16 Here, we continue to exploit the separation capability of CEX to analyze mixtures of fulllength mAbs and their respective Fab and Fc fragments. To the best of our knowledge, this is the first account illustrating the use of CEX as a primary tool for investigating mAb aggregation.…”

Section: Introductionmentioning

confidence: 99%

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The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

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Section: Measurement Of Protein Aggregation By Cex52mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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“…Separate charge profiles of Fab and Fc domains have been obtained using limited Lys-C or papain proteolysis in combination with CEX separation. [57][58][59][60][61] It is known that the variable regions of both LC and HC could contribute to the overall charge heterogeneity of the antibody product, 55,56 and deamidation of LC and HC CDR asparagine residues could cause loss in bioactivity. 44,62 There is, however, no previous report on domain-specific charge profiling of LC and Fd domains.…”

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A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

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“…These truncated fragments were correlated to heavy and light chain peptide maps and sequence identity was confirmation by N-terminal sequencing. Multidimensional analysis of product degradation utilizing both SEC-HPLC and CEX-HPLC followed peptide mapping with MS-ESI detection is shown in work by Lau et al and Kim et al (Lau H., 2010) (Kim, 2010) …”

Section: Purity and Molecular Weight Determinationmentioning

confidence: 99%