Carboxylate-Dependent Gelation of a Monoclonal Antibody

Esue, Osigwe; Kanai, Sonoko; Liu, Jun; Patapoff, Thomas W.; Shire, Steven J.

doi:10.1007/s11095-009-9963-6

Cited by 31 publications

(20 citation statements)

References 45 publications

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“…12 It has been recognized that ion-protein binding can modulate the charge properties of proteins and promote aggregation and self-association. [25][26][27][28] Assuming that salt has a significant effect on protein stability, an increase or decrease in the extent of mAb aggregation as a function of salt concentration can be expected. To investigate the effect of salt on antibody aggregation, 70 C heat treatment was applied to protein solutions supplemented with 0.1M and 1.0M NaCl.…”

Section: Temperature-induced Mab Aggregationmentioning

confidence: 99%

“…This adds to the number of examples where protein-protein interactions influence macroscopic properties of the system such as viscosity, opalescence, gelation, and so forth. 25,27,50 Many cases exist that illustrate the tendency of mAbs to participate in these phenomena. 3,51 In contrast to the largely irreversible non-native aggregation discussed above, interactions involved in phase separation tend to be weak, highly reversible, and transient in nature.…”

Section: Phase Separation Of Mabsmentioning

confidence: 99%

“…Although the exact nature of these interactions is currently unknown, this finding illustrated the importance of Fab regions in mAb phase separation, corroborating a number of recent reports. 25,27,28 …”

Section: Phase Separation Of Mabsmentioning

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: Temperature-induced Mab Aggregationmentioning

confidence: 99%

Section: Phase Separation Of Mabsmentioning

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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“…Solution properties such as pH, ionic strength, temperature and excipients are selected to maintain physical and chemical stability and to ensure long product shelf life. 1 Information about stability gathered in early formulation studies is also important to ensure that the manufacturing process is compatible with the product. Biochemical properties of the antibody such as net charge, charge distribution and surface exposed hydrophobic patches are some of the important factors influencing physical stability in dilute solutions.…”

Section: Introductionmentioning

confidence: 99%

“…Aggregation, precipitation, crystallization, liquid-liquid phase separation, opalescence and high viscosity are all manifestations of self-interactions that can be promoted by high antibody concentration. 1,[5][6][7][8][9][10][11] The different outcomes are due to the nature of the forces dominating the interactions (electrostatic, hydrophobic, short range attraction) and specific solution conditions. 4 These phenomena differ fundamentally from rapid heat-or chemically-induced aggregation or precipitation in that the intermolecular interactions can be reversed by changing the solution conditions such as pH, ionic strength, temperature or excipients, suggesting that a native or near-native protein conformation is maintained.…”

Section: Introductionmentioning

confidence: 99%

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

Casaz

Boucher²,

Wollacott

et al. 2014

mAbs

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(2014) Resolving selfassociation of a therapeutic antibody by formulation optimization and molecular approaches, mAbs, 6:6, 1533-1539, DOI: 10.4161/19420862.2014 To link to this article: https://doi.org/10. 4161/19420862.2014 A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of selfassociation, formation of a semi-solid gel or "gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6 C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1 C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.

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