Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition

Nishi, Hirotaka; Miyajima, Makoto; Nakagami, Hiroaki; Noda, Masanori; Uchiyama, Susumu; Fukui, Kiichi

doi:10.1007/s11095-010-0125-7

Cited by 113 publications

(122 citation statements)

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“…In addition to typical physical (e.g., conformational, colloidal), chemical (e.g., oxidation, deamidation), and interfacial (e.g., freeze-thaw, shear) instabilities, 2 at high concentrations, undesired solution properties such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges. [3][4][5][6][7] RSA (i.e., native, non-covalent, and reversible oligomerization of monomeric species) is typically induced in the crowded environments of high concentrations due to the reduced intermolecular distances and increased probability of molecular collisions. 8 The self-associated species can pose manufacturing and delivery challenges (e.g., high viscosity, clogging of the lines and filters 3,9 ) or cause pain to patients upon administration.…”

Section: Introductionmentioning

confidence: 99%

“…6,7,18,21,23 Potential strategies to mitigate self-association and other undesired high concentration solution properties may include protein engineering approaches, 20,[24][25][26] as well as formulation development and optimization efforts. [5][6][7]9,18,19,21,23,27 While the former can be limited due to the degree of knowledge of sites of interactions, the latter can be time and resource intensive and may not always be fully successful to a desired degree for all antibodies. Historically, a number of computational and analytical screening methods have been used to predict aggregation [28][29][30] and high concentration formulation risks.…”

Section: Introductionmentioning

confidence: 99%

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Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Geoghegan¹,

Fleming²,

Damschroder³

et al. 2016

mAbs

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Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible proteinprotein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Geoghegan¹,

Fleming²,

Damschroder³

et al. 2016

mAbs

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“…8,9 In addition, the RSA of mAbs gives rise to a network of associated higher-order species that can affect the viscoelastic properties of the solution, 10 resulting in increased viscosity, [5][6][7]11 solution turbidity, 12 and, under certain conditions, even phase transitions. 13 The increase in solution viscosity also imposes manufacturing challenges including high back-pressure and clogging of membranes, 2 as well as elevated levels of shear stress during pumping. 14 Reversible-self association of mAbs is generally attributed to weak and transient non-covalent interactions (e.g., hydrogen bonding, electrostatic, hydrophobic, p-p, and van der Waals interactions) between antibody molecules.…”

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

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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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“…Recently, LLPS of several pharmaceutical antibodies have been reported (5)(6)(7)(8)(9). There are five isotypes of mammalian antibodies with distinct Fc regions, including IgA, IgD, IgE, IgG, and IgM.…”

mentioning

confidence: 99%

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

Wang

Lomakin²,

Latypov³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

106

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We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable proetin condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.ntibodies are widely used in research and biotechnology, as well as in medical and pharmaceutical applications. In some cases, concentrated solutions of specific antibodies are required. In particular, monoclonal antibodies (MAb) have become a major category of drugs in the treatment of a variety of diseases (1). In some drug delivery routes e.g., subcutaneous administration, formulations with concentrated antibody solutions are required to achieve therapeutic dosing (2).The physiological functions of antibodies are mostly determined by antibody-antigen and antibody-receptor specific interactions. However, the nonspecific interactions between antibodies (i.e., self-association) in the concentrated antibody solutions can also affect their functions. Nonspecific attractive interactions can cause various forms of condensation, including liquid-liquid phase separation, aggregation, and crystallization. Upon such condensation, antibodies lose their solubility, and may lose their biological activity. Particularly, in pharmaceutical industry, these processes impact storage stability and safety of protein therapeutics thus impeding drug development (3). For example, immunogenicity of some biologics has been attributed to formation of protein aggregates (4). The mechanisms of protein condensation are complex and depend on protein concentration, buffer composition, temperature, etc. Clearly, the factors which affect protein condensation throughout the shelf-life must be understood and controlled to ensure biotherapeutic effectiveness.One important condensation process is liquid-liquid phase separation (LLPS). In LLPS, a homogeneous protein solution spontaneously separates into two coexisting phases with different protein concentrations. This phenomenon takes place upon changing the temperature or other solution conditions, and is reversible. In contrast to aggregation or crystallization, LLPS, while highly sensitive to the average "net" attractive interaction between proteins, are much less sensitive to the distribution pattern and the nature of the "local" interactions on the protein surface. As a result, LLPS exhibits universal features applicable to a variety...

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Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition

Abstract: LLPS was induced in MAb A solution in a low ionic strength condition due to reversible protein self-association mediated mainly by attractive electrostatic interaction among the MAb A molecules in the heavy phase.

Cited by 113 publications

References 44 publications

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

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

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

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