Long- and Short-Range Electrostatic Interactions Affect the Rheology of Highly Concentrated Antibody Solutions

Chari, Ravi; Jerath, Kavita; Badkar, Advait; Kalonia, Devendra S.

doi:10.1007/s11095-009-9975-2

Cited by 154 publications

(149 citation statements)

References 39 publications

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“…This behaviour is consistent with the increase in the k D value for 5E when adding sodium chloride. Previous studies have found a strong correlation between increased aggregation rates and electrostatic selfassociation [13,55,80,99], which might also explain the increased aggregation of 5E.…”

Section: The Patch-charged Mutantsmentioning

confidence: 82%

“…However, care must be taken when engineering in charged mutations. If the protein net charge is close to zero, an anisotropic charge distribution can cause protein self association [13,14,71,99], which has been correlated with increased aggregation propensity [55,80]. To avoid increasing protein charge anisotropy, charged mutations should carry the same sign as the net charge on the corresponding protein scaffold [31,100].…”

Section: Introductionmentioning

confidence: 99%

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The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

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Section: The Patch-charged Mutantsmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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“…The foregoing moderate viscosity reductions of inorganic salts suggest that electrostatic interactions (Chari et al, 2009), in general, were not of a major importance in concentrated protein solution's viscosity. Therefore, we hypothesized that hydrophobic protein-protein interactions instead were predominantly responsible for the reversible protein aggregates in concentrated solutions creating resistance to flow and, in turn, leading to high solution viscosity.…”

Section: Resultsmentioning

confidence: 99%

Hydrophobic salts markedly diminish viscosity of concentrated protein solutions

Klibanov

2010

Biotech & Bioengineering

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Reducing viscosities of concentrated solutions of therapeutic proteins is important for their subcutaneous and intravenous delivery. Although inorganic salts and optimizing the pH were previously reported to dramatically lower the viscosity of a monoclonal antibody solution, herein we have determined these effects not to be general. Separately, we have found that hydrophobic ionic excipients, both anionic and cationic, substantially decrease the viscosity of concentrated (300-400 mg/mL) aqueous solutions of bovine serum albumin and γ-globulin. The more hydrophobic the excipient, the greater its viscosity-lowering effect is. With cationic ones, the concomitant contribution of the counter-ion broadly follows the chaotropic order. The most potent excipients lower the viscosity over fourfold to levels far below the 50 cP threshold for subcutaneous injections. The observed viscosity reductions are rationalized in terms of three-dimensional transient protein networks formed in concentrated solutions due to hydrophobic and, to a lesser extent, ionic interactions. These reversible protein aggregates are responsible for strong resistance to flow in concentrated protein solutions and hence their high viscosity; hydrophobic ions apparently effectively compete for these interprotein interactions, thereby giving rise to less viscous solutions.

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“…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%

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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Long- and Short-Range Electrostatic Interactions Affect the Rheology of Highly Concentrated Antibody Solutions

Cited by 154 publications

References 39 publications

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Hydrophobic salts markedly diminish viscosity of concentrated protein solutions

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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