Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

Tang, Liangjie; Sundaram, Shanmuuga; Zhang, Jingming; Carlson, Ping; Matathia, Alice; Parekh, Babita; Zhou, Qinwei; Hsieh, Ming-Ching

doi:10.4161/mabs.22695

Cited by 57 publications

(53 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The relatively elevated levels of C-terminal lysine heterogeneity of mAb-E was not considered while interpreting our results, as it has been shown previously to have no effect on the pK, stability or conformational dynamics of mAbs. 60,61 Both mAb-A and mAb-E showed »99% purity as measured by SDS-PAGE analysis (data not shown), and greater than 99% monomer content as measured by SEC (see Results). The mAbs were provided at 100 mg/mL and were buffer exchanged into 50 mM sodium phosphate, 150 mM sodium chloride at pH 6.0 or 7.4.…”

Section: Methodsmentioning

confidence: 87%

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Majumdar

Esfandiary²,

Bishop³

et al. 2015

mAbs

View full text Add to dashboard Cite

(2015) Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life, mAbs, 7:1, 84-95, DOI: 10.4161/19420862.2014.985494 To link to this article: https://doi.org/10. 4161/19420862.2014 This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C H 2 and distant segments in the V H , C H 1, and V L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244-254 segment of the C H 2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T onset ) and unfolding (T m 1) temperatures of 7 C and 6.7 C, respectively, for the C H 2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244-254 segment, providing a site-directed mutant example that this segment of the C H 2 domain is an aggregation hot spot in IgG1 mAbs.

show abstract

Section: Methodsmentioning

confidence: 87%

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Majumdar

Esfandiary²,

Bishop³

et al. 2015

mAbs

View full text Add to dashboard Cite

show abstract

“…[24,61] This claim was supported by the fact that the presence or absence of the C-terminal Lys residue does not cause any conformational changes to the antibody's structure. [62] However, a recent study by van den Bremer et al contradicted these findings by reporting that antibody fractions that had undergone complete C-terminal lysine clipping showed a clearly increased CDC activity compared to their counterparts, which still contained lysine residues on both C-termini of the heavy chains. It was then proposed that previous studies may not have been able to identify this link due to a lack of samples containing separated variant fractions rather than mixtures.…”

Section: C-terminal Lysine Clippingmentioning

confidence: 98%

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

View full text Add to dashboard Cite

Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so‐called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half‐life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core‐fucosylation of the N‐glycan and aggregation the effects are clear and should be monitored, but with others such as C‐terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

show abstract

“…[34][35][36] For example, HX-MS has been recently applied to examine the effect of various solution factors and physicochemical structural changes on the local flexibility of mAbs, including salts from the Hofmeister series, 37 pharmaceutical excipients, 38 freeze-thaw cycles, 39 methionine oxidation, 40 asparagine deamidation, 41 antibody-drug conjugation, 42 deglycosylation and glycan modifications, 43 and engineered point mutations. 44 In this study, antibody clusters were characterized by a combination of DLS and chemical cross-linking experiments to determine the effect of solution conditions on the extent of RSA for an IgG1 mAb (referred to as "mAb-C") as a function of mAb-C protein concentration (1-10 mg/mL).…”

Section: Introductionmentioning

confidence: 99%

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Arora

Hickey

Majumdar

et al. 2015

mAbs

View full text Add to dashboard Cite

Volkin (2015) Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody, mAbs, 7:3, 525-539, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the V H domain, the constant domain (C H 2), and the hinge region (C H 1-C H 2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.

show abstract

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

Abstract: (2013) Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry, mAbs, 5:1, 114-125,

Cited by 57 publications

References 23 publications

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Contact Info

Product

Resources

About