Comparative Effects of pH and Ionic Strength on Protein–Protein Interactions, Unfolding, and Aggregation for IgG1 Antibodies

Şahin, Erinç; Grillo, Adeola O.; Perkins, Melissa D.; Roberts, Christopher J.

doi:10.1002/jps.22198

Cited by 202 publications

(202 citation statements)

References 49 publications

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“…10 For example, Pathak et al demonstrated that the presence of reversibly associated clusters at high protein concentrations contributed to an increase in solution viscosity. 52 Similar trends in viscosity in response to changes in solution conditions that we describe here for mAb-C have been reported for other IgG1 mAbs, where the extent of viscosity increased in a concentration-dependent manner with increasing ionic strength 11,18,30,53,54 and solution pH, 11,55 related to elevated levels of protein RSA due to charge shielding effects. [56][57][58] The isoelectric point (pI) range of mAb-C is basic (pI»9.1-9.4), 45 and therefore, the overall surface charge of mAb-C is expected to be positive at neutral pH.…”

Section: Discussionsupporting

confidence: 82%

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

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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.

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Section: Discussionsupporting

confidence: 82%

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

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“…In addition to effector functions and stability and biophysical characteristics, analytical and regulatory aspects are important selection criteria for such monoclonal antibodies (mAbs) destined for clinical use. Further, it is well known that during manufacturing, purification, formulation, and storage, the isotypes behave differently in terms of their sensitivity to pH or temperature, aggregation propensity, and susceptibility to degradation (11)(12)(13). With regard to monitoring or predicting stability, thermal stability is increasingly accepted as a convenient surrogate measure of global stability.…”

mentioning

confidence: 99%

Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability

Peters

Smales

Henry

et al. 2012

Journal of Biological Chemistry

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Background: IgG1 and IgG4 have different inter-light chain-heavy chain disulfide bond (DSB) arrangements. Results: IgG4 mutants with an IgG1-like DSB and a S241P hinge mutation showed increased Fab thermal stability and reduced DSB heterogeneity compared with IgG4 WT. Conclusion: Fab domain thermal stability and DSB heterogeneity of IgG4 can be improved. Significance: Such engineered IgG4 molecules offer potential advantages during therapeutic antibody production.

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“…Although the ordinate intercept of a Debye plot for protein solutions is predicted to underestimate M 2 , this potential deficiency of static light scattering for the measurement of protein molecular mass in aqueous solution has only resurfaced recently [10,11,23]. Ordinate intercepts signifying A 1 values greater than unity have been reported for buffered aqueous…”

Section: (20)mentioning

confidence: 99%

Rigorous analysis of static light scattering measurements on buffered protein solutions

Wills

Winzor

2017

Biophysical Chemistry

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Comparative Effects of pH and Ionic Strength on Protein–Protein Interactions, Unfolding, and Aggregation for IgG1 Antibodies

Cited by 202 publications

References 49 publications

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

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

Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability

Rigorous analysis of static light scattering measurements on buffered protein solutions

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