Differences between reversible (self-association) and irreversible aggregation of rHuG-CSF in carbohydrate and polyol formulations

Severe immunogenic and other debilitating human disorders potentially induced by protein aggregates have brought this phenomenon into the focus of biopharmaceutical science over the past decade. Depending on its driving forces, the process induced in the model protein rHuG-CSF may be either reversible or irreversible, resulting in the assembly of self-associated protein species or irreversible aggregates of various final morphologies. The aim of our work was to investigate the correlation between irreversible and reversible aggregation and the protective effect of non-specific formulation stabilisers, selected from the group of carbohydrates and polyols including trehalose, xylitol, cellobiitol, turanose, cellobiose, leucrose, lactitol, lyxose, and sorbitol, against both irreversible protein aggregation and reversible self-association processes of the rHuG-CSF. The formation of irreversible aggregates was thermally induced and evaluated using differential scanning calorimetry and size-exclusion chromatography. As opposed to the irreversible aggregation process, the process of self-association was induced by the agitation experiment by directly augmenting the protein solution contact surfaces. Absence of statistical connectivity between different stabilisers’ ability to inhibit self-association or aggregation reactions indicates that these are two distinct physicochemical processes with different formulation stabilizing outcomes.Reaction mechanism of thermally induced aggregation observed in the study was in line with published literature data, while the reaction mechanism for self-association process was postulated. The postulate has been verified experimentally by isothermal calorimetry and agitation set of experiments conducted after size-exclusion chromatography and asymmetrical flow field-flow fractionation separation of monomeric, dimeric, trimeric, oligomeric, and large self-associated forms detected on multi-angle light scattering, fluorescence, UV, and refractive index detectors. Besides defining the mechanism and kinetic of self-association in stabilized rHuG-CSF formulations, special attention was also paid to the shifts and ranks of the free energy of the aggregation or self-association transition states.

Graphical abstractSolid line-reaction coordinate diagram of rHuG-CSF irreversible aggregation. N* and N2∗ represent transition state species, and DGNN∗‡ activation free energy of aggregation. A2 represents aggregation-competent intermediate, irreversible species responsible for reaction irreversibility and larger aggregates formation (Am). Relative to the protein native state (N), doted arrows represent shifts in the free energy when carbohydrate, or polyol stabilizers or tungsten (W) were added. Dotted line- reaction coordinate diagram of rHuG-CSF reversible aggregation. Na∗ represent transition state specie. Aan+1, Aan+2 and (•••) represent aggregation-competent intermediates responsible for larger self-associated form formation (Aam) and reaction reversibility. Doted arrows next to Na∗ represent shifts in the free energy when carbohydrate, or polyol stabilizers were added (Na∗ shifts are significantly smaller relative to N* shifts).Figure optionsDownload full-size imageDownload as PowerPoint slide