Abstract
Monoclonal antibodies (mAbs) are often needed and applied in high concentration solutions, >100 mg/mL. Due to close intermolecular distances between mAbs at high concentrations (~10–20 nm at 200 mg/mL), intermolecular interactions between mAbs and mAbs and solvent/co-solute molecules become non-negligible. Here, EPR spectroscopy is used to study the high-concentration solutions of mAbs and their effect on co-solvated small molecules, using EPR “spin probing” assay in aqueous and buffered solutions. Such, information regarding the surrounding environments of mAbs at high concentrations were obtained and comparisons between EPR-obtained micro-viscosities (rotational correlation times) and macroscopic viscosities measured by rheology were possible. In comparison with highly viscous systems like glycerol-water mixtures, it was found that up to concentrations of 50 mg/mL, the mAb-spin probe systems have similar trends in their macro- (rheology) and micro-viscosities (EPR), whereas at very high concentrations they deviate strongly. The charged spin probes sense an almost unchanged aqueous solution even at very high concentrations, which in turn indicates the existence of large solvent regions that despite their proximity to large mAbs essentially offer pure water reservoirs for co-solvated charged molecules. In contrast, in buffered solutions, amphiphilic spin probes like TEMPO interact with the mAb network, due to slight charge screening. The application of EPR spectroscopy in the present work has enabled us to observe and discriminate between electrostatic and hydrophobic kinds of interactions and depict the potential underlying mechanisms of network formation at high concentrations of mAbs. These findings could be of importance as well for the development of liquid-liquid phase separations often observed in highly concentrated protein solutions.
Highlights
Cellular environments are densely crowded with several components like ions, peptides, proteins and small molecules
In addition to studying spin probes in buffered aqueous solutions of Monoclonal antibodies (mAbs), we study the spin probes and proteins in pure water to have a system in which the fundamental interactions without added salts/buffers can be studied
We describe data obtained from simulation of the EPR spectra for each spin probe interacting with mAbs in water and buffer
Summary
Cellular environments are densely crowded with several components like ions, peptides, proteins and small molecules In such compact media, the protein concentration can rise up to a total concentration of 300–400 mg/mL which in turn can affect the function and stability of the respective proteins [1]. Challenges arise due to non-ideal solution behavior at such concentrations, like phase separation, aggregation or gelation, which all can negatively affect several steps during protein manufacturing and impair shelf life of the drug product. Detailed reviews about such perturbed behavior of high concentration solutions can be found elsewhere [6,7,8,9,10,11,12]. Some diseases are caused as results of high concentration of proteins and are related to protein-protein self-interaction (PPI), solution behavior and crowding effects like cataract [13,14] and neurodegenerative disease [15,16]
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