From osmotic second virial coefficient (B₂₂) to phase behavior of a monoclonal antibody

Abstract

Antibodies are complex macromolecules and their phase behavior as well as interactions within different solvents and precipitants are still not understood. To shed some light into the processes on a molecular dimension, the occurring self-interactions between antibody molecules were analyzed by means of the osmotic second virial coefficient (B22 ). The determined B22 follows qualitatively the phenomenological Hofmeister series describing the aggregation probability of antibodies for the various solvent compositions. However, a direct correlation between crystallization probability and B22 in form of a crystallization slot does not seem to be feasible for antibodies since the phase behavior is strongly dependent on their anisotropy. Kinetic parameters have to be taken into account due to the molecular size and complexity of the molecules. This is confirmed by a comparison of experimental data with a theoretical phase diagram. On the other hand the solubility is thermodynamically driven and therefore the B22 could be used to establish a universal solubility line for the monoclonal antibody mAb04c and different solvent compositions by using thermodynamic models.

Influence of macromolecular precipitants on phase behavior of monoclonal antibodies

“…In Figure , the crystallization slot according to George and Wilson defines the range of high crystallization probability. Comparing these conditions with the phase behavior of mAb04c, in Figure , the same picture arises like with salts . Most of these experiments show crystallization within this range, few do not separate and others are crystallizing below this range.…”

“…The higher the net charge of the proteins the higher is the repulsion . This can be confirmed by the B 22 ‐results in pure buffer . From these B 22 ‐results, it can also be shown that the differences in B 22 ‐values for mAb04c in pure buffer from pH 7 to 9 are small, as additional attractive forces like hydrophobic interaction influence the proteins.…”

“…The preparation, storage, and analysis of the samples were conducted according to Rakel et al The composition of the buffer system with five monoprotic acid components and a constant buffer capacity of 10 mM in the pH range of 5–9 was: 10.05 mM MES, 16.6 mM Acetic acid, 8.9 mM MOPSO, 12.3 mM HEPPSO, and 14.4 mM CHES. The required pH‐value was titrated with 4 M NaOH.…”

“…Under the assumption that PEG does not interact with the protein B 22 ‐values in PEG‐solutions were determined and analyzed according to Rakel et al using the Zimm equation . As the refractive index dn / dc of the protein is dependent on the respective PEG molecular weight and its concentration, this value was adjusted for the protein concentration determination with the refractometer.…”

Squeeze flow rheometry as a novel tool for the characterization of highly concentrated protein solutions

“…The parameter characterizes solute‐solute and solute–solvent interactions (Rosenbaum and Zukoski, ). The could be successfully correlated with crystallization slots as well as precipitation points (George and Wilson, ; Rakel et al, ; Ruppert et al, ; Wilson, ). Unfortunately, that could only be shown for ideal dilute solutions.…”