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Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

ABSTRACT: Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small-angle X-ray scattering (SAXS) and complementary biophysical characterization techniques to investigate the conformational flexibility and protein-protein interactions (PPI) of a model mAb molecule under near-neutral and acidic conditions. The study also characterized the interactions between Fab and Fc fragments under the same buffer conditions to identify domain-domain interactions. The results suggest that solution pH significantly influences mAb flexibility and thus could help mAbs remain physically stable by maximizing local electrostatic repulsions when mAbs become crowded in solution. Under acidic buffer conditions, both Fab and Fc contribute to the repulsive PPI observed among the full mAb at a low ionic strength. However, as ionic strength increases, hydrophobic interactions lead to the self-association of Fc fragments and, subsequently, could affect the aggregation state of the mAb.

SUBMITTER: Xu AY

PROVIDER: S-EPMC10561141 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Xu Amy Y AY Blanco Marco A MA Castellanos Maria Monica MM Meuse Curtis W CW Mattison Kevin K Karageorgos Ioannis I Hatch Harold W HW Shen Vincent K VK Curtis Joseph E JE

The journal of physical chemistry. B 20230926 39

Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small- ...[more]

PMID: 37751332

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Project description:Human/humanized IgG4 antibodies have reduced effector function relative to IgG1 antibodies, which is desirable for certain therapeutic purposes. However, the developability and biophysical properties for IgG4 antibodies are not well understood. This work focuses on the head-to-head comparison of key biophysical properties, such as self-interaction and viscosity, for 14 human/humanized, and chimeric IgG1 and IgG4 S228P monoclonal antibody pairs that contain the identical variable regions. Experimental measurements showed that the IgG4 S228P antibodies have similar or higher self-interaction and viscosity than that of IgG1 antibodies in 20 mM sodium acetate, pH 5.5. We report sequence and structural drivers for the increased viscosity and self-interaction detected in IgG4 S228P antibodies through a combination of experimental data and computational models. Further, we applied and extended a previously established computational model for IgG1 antibodies to predict the self-interaction and viscosity behavior for each antibody pair, providing insight into the structural characteristics and differences of these two isotypes. Interestingly, we observed that the IgG4 S228P swapped variants, where the CH3 domain was swapped for that of an IgG1, showed reduced self-interaction behavior. These domain swapped IgG4 S228P molecules also showed reduced viscosity from experiment and coarse-grained simulations. We also observed that experimental diffusion interaction parameter (kD) values have a high correlation with computational diffusivity prediction for both IgG1 and IgG4 S228P isotypes.Abbreviations: AHc, constant region Hamaker constant; AHv, variable region Hamaker constant; CDRs, Complementarity-determining regions; CG, Coarse-grained model; CH1, Constant heavy chain 1; CH2 Constant heavy chain 2; CH3 Constant heavy chain 3; chgCH3 Effective charge on the CH3 region; CL Constant light chain; cP, Centipoise; DLS, Dynamic light scattering; Fab, Fragment antigen-binding; Fc, Fragment crystallizable; Fv, Variable domaing; (r) Radial distribution function; H1 CDR1 of Heavy Chain; H2 CDR2 of Heavy Chain; H3 CDR3 of Heavy Chain; HVI, High viscosity index; IgG1 human immunoglobulin of IgG1 subclass; IgG4 human immunoglobulin of IgG4 subclass; kD, Diffusion interaction parameter; L1 CDR1 of Light Chain; L2 CDR2 of Light Chain; L3 CDR3 of Light Chain; mAb, Monoclonal antibody; MD, Molecular dynamics; PPI Protein-protein interactions; SCM, Spatial charge map; UP-SEC, Ultra-high-performance size-exclusion chromatography; VH, Variable domain of Heavy Chain; VL, Variable domain of Light Chain.

Project description:Spectrophotometric measurements of bilirubin-IX alpha in water and in aqueous/organic solvent mixtures at pH 10.0 as a function of bilirubin-IX alpha concentration (approx. 0.6--400 microM) are consistent with the formation of dimers (KD - 1.5 microM) in dilute (less than 10 microM) aqueous solution and further self-aggregation to multimers at higher concentrations. Added urea (to 10M) and increases in temperature (to 62 degrees C) obliterate the dimer-multimer transition at 10 microM, but added NaCl (to 0.30 M) promotes strong aggregation of dimers over a narrow concentration range, suggesting a 'micellization' phenomenon. Concentrations of dioxan or ethanol greater than 60% (v/v) in water were required to obtain the absorption spectrum of bilirubin-IX alpha monomers, suggesting that both hydrophobic and electrostatic (pi-orbital) interactions are involved in stabilizing the dimeric state in water. Micellar concentrations of sodium dodecyl sulphate induced spectrophotometric shifts in the dimer absorption spectrum of bilirubin-IX alpha consistent with progressive partitioning of bilirubin-IX alpha monomers into a relatively non-polar region of the micelles and allowed a deduction of the apparent critical micellar concentration that closely approximated the literature values. The pattern of bilirubin IX alpha association with bile salts is complex, since the absorption spectrum shifts hypsochromically below and bathochromically above the critical micellar concentration of the bile salts. Consistent with these observations, bilirubin IX alpha appears to bind to the polar face of bile salt monomers and to the polar perimeter of small bile salt micelles. At higher bile salt concentrations some-bilirubin-IX alpha monomers partition into the hydrophobic interior of the bile salt micelles. Our results suggest that under physiological conditions the natural conjugates of bilirubin-IX alpha may exhibit similar physical chemical properties in bile, in that dimers, highly aggregated multimers and bile salt-associated monomers may co-exist.

Dataset Information

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Publications

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

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