Assessment of Net Charge and Protein–Protein Interactions of Different Monoclonal Antibodies

Lehermayr, Christian; Mahler, Hanns‐Christian; Mäder, Karsten; Fischer, Stefan

doi:10.1002/jps.22506

Cited by 179 publications

(164 citation statements)

References 43 publications

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“…k D has been used to assess the net interaction potential for proteins by several researchers, 21,44,45 and a strong correlation between k D and A 2 has been demonstrated recently. 44 While both k D and A 2 are measured generally over the same concentration ranges, k D may be measured at much lower volumes using microplate based QELS instrumentation and has the potential to be used in high throughput screening. While second virial coefficient values or equilibrium phase transitions are more widely used to measure interaction potentials, we believe that k D is a convenient and reproducible way to assess colloidal stability.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose

James

McManus

2012

J. Phys. Chem. B

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The effect of the sugars sucrose, glucose, and trehalose on the structural and colloidal stability of lysozyme has been investigated using differential scanning calorimetry and quasi-elastic light scattering, respectively. While sugars are known to increase the temperature at which thermal denaturation of protein occurs, it is not clear if, under the same solution conditions, greater colloidal stability is achieved. The measurements were carried out on lysozyme in three different buffer solutions, 0.05 M sodium acetate (pH 4.6), 0.05 M sodium acetate with 5% (w/v) NaCl, and 10 mM sodium phosphate (pH 7.0). The results show that enhancement of structural stability in the presence of sugars is pH, salt concentration, and sugar dependent. Enhancement of colloidal stability in the presence of sugars, while also pH and salt concentration dependent, as expected, only correlates with increases in the structural stability when the solution behavior is not dominated by highly stabilizing electrostatic repulsive interactions. ■ INTRODUCTIONSugars are widely used excipients in the formulation of biotherapeutic products.1−7 Therapeutic proteins are produced and used for the treatment of various human diseases 8 such as insulin for the treatment of diabetes, 9 monoclonal antibodies for rheumatoid arthritis, 10 interferon-α for leukemia, 11 and interferon-β for multiple sclerosis.12 Sugars are generally used as protein structure stabilizers, 2,13 since additional stability is conferred to a protein during lyophilization by the addition of the sugar.14 Two hypotheses have been proposed to describe the stabilizing effect of sugars on proteins during lyophilization; one such hypothesis states that sugar acts as a water substituent and stabilizes proteins by forming hydrogen bonds at specific sites on the surface of the protein.14−16 Another hypothesis, referred to as the "vitrification hypothesis", states that disaccharides form sugar glasses, thereby immobilizing the protein molecules and providing protection against destabilizing reactions. 15,16 Lysozyme is a globular protein with a molecular weight of approximately 14.7 kDa. Lysozyme has been used as a model protein for this study, since its biophysical properties are well understood and the specific contribution of the sugar to its stability over a range of solution conditions can easily be compared with that of the native protein. Lysozyme is a widely studied protein due to its rich phase behavior 17,18 including crystallization, 19 liquid−liquid phase separation, 20−22 and the formation of equilibrium clusters 23−25 and gels. 24 The solution behavior of lysozyme is also well understood in terms of its interaction with salt ions. 26−28Protein stability by itself is a generic term, and it can be thought of in several ways. The stabilization of protein structure against thermal denaturation is referred to as its thermal or structural stability. 37 and presence of excipients. 6 At pH values away from the isoelectric point of the protein, there is an increase in cha...

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Section: ■ Results and Discussionmentioning

confidence: 99%

Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose

James

McManus

2012

J. Phys. Chem. B

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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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“…This technique was classically used in studies of multiple myeloma (4) and is currently used for analyzing the degradation of monoclonal antibodies (14,24,61). IEF has also been used in the study of the humoral response to a number of pathogens (10, 11, 15, 17, 20-22, 30, 37, 39, 45, 46, 50, 51, 55).…”

Section: Discussionmentioning

confidence: 99%

Signature Biochemical Properties of Broadly Cross-Reactive HIV-1 Neutralizing Antibodies in Human Plasma

Sajadi

Lewis

Seaman

et al. 2012

J Virol

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The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

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Assessment of Net Charge and Protein–Protein Interactions of Different Monoclonal Antibodies

Cited by 179 publications

References 43 publications

Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose

Thermal and Solution Stability of Lysozyme in the Presence of Sucrose, Glucose, and Trehalose

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

Signature Biochemical Properties of Broadly Cross-Reactive HIV-1 Neutralizing Antibodies in Human Plasma

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