(603k) Protein Disaggregation By Thermal Cycling

Protein aggregation is inevitable in biopharmaceutical processing. Extensive processing required during synthesis and purification of biopharmaceutical products exposes these protein molecules to various kinds of stresses which destabilize them and cause them to aggregate. Factors that can directly affect protein stability include the solution pH, protein concentration, ionic strength, buffer type, temperature, pressure, exposure to gas-liquid interface, phase change (e.g. freeze-thawing) and presence of chemical additives. The native structure of a protein is only marginally more stable than the unfolded structure and it can therefore be perturbed quite easily. Unfolding and refolding of proteins occur continuously and the partially unfolded intermediates formed during such processes are highly susceptible to forming aggregates. These aggregates are non-native, i.e. there is significant alteration in secondary and tertiary structure and therefore loss in biological activity. Protein aggregates are generally lack therapeutic activity and could potentially be immunogenic, i.e. could elicit severe immunological reactions. We developed a thermal-cycling technique for simultaneous disaggregation of protein oligomers and refolding of the released monomer. Monoclonal antibodies of the immunoglobulin G (or IgG) class form stable non-native oligomers through Fab-Fab interactions. Monoclonal antibody samples containing known amounts of oligomers were exposed to rapid heating and cooling cycles using a laboratory thermal-cycler. The heating phase of the thermal-cycle released the partial unfolded monomer and the rapid cooling phase led to protein refolding and minimized the probability of protein re-aggregation. Orthogonal analytical techniques such as size exclusion chromatography, hydrophobic interaction chromatography, and circular dichroism spectroscopy were used to assess the extent of oligomer disaggregation and refolding.