Mechanism of Reversible Self-Association of a Monoclonal Antibody: Role of Electrostatic and Hydrophobic Interactions

Esfandiary, Reza; Parupudi, Arun; Casas‐Finet, José R.; Gadre, Dhanesh; Sathish, Hasige A.

doi:10.1002/jps.24237

Cited by 92 publications

(100 citation statements)

References 33 publications

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“…Esfandiary et al have recently shown that the same mAb can form a monomer-trimer-hexamer equilibrium under similar solution conditions (at 1-10 mg/mL at room temperature). 45 Nonetheless, as shown in this work, notable differences in the extent of RSA are observed by solution viscosity and HX-MS measurements at 5 vs. 60 mg/mL of mAb-C.…”

Section: Solution Viscosity As a Function Of Protein Concentration Tsupporting

confidence: 53%

“…These DLS results are consistent with those reported previously by Esfandiary et al for the same antibody molecule formulated under similar solution conditions. 11,45 To further assess the effect of salt on reversible self-association (RSA) of mAb-C, we used chemical cross-linking to measure the effect of sulfate on the extent of RSA at pH 7. In the presence of 300 mM sodium sulfate, higher molecular weight bands at »300 kDa and »450 kDa were observed (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These analytical limitations were encountered with DLS and SV-AUC measurements of mAb-C under different solution conditions as described in this work and as reported previously. 11,45 In the present study, we correlate solution effects on the RSA of mAb-C, as determined by DLS and chemical crosslinking at lower mAb-C concentrations, to solution viscosity measurements at higher protein concentrations. We then directly mapped the local regions of mAb-C involved at the interface of RSA at the higher protein concentration using HX-MS, an analytical tool that has been widely used to map interfaces of protein-protein interactions.…”

Section: Discussionmentioning

confidence: 99%

“…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. As the pH was changed from 6 to 8, the tendency of mAb-C to self-associate increased, possibly due to overall decrease in electrostatic repulsive interactions.…”

Section: Discussionmentioning

confidence: 99%

“…The probable involvement of histidine side chain residues in the RSA of this mAb was demonstrated indirectly by DLS studies (vs. solution pH and composition) as reported recently. 11,45 The homology model indicates that one of these histidines is highly solvent exposed (see Fig. 7C).…”

Section: Discussionmentioning

confidence: 99%

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

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et al. 2015

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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: Solution Viscosity As a Function Of Protein Concentration Tsupporting

confidence: 53%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%