Biophysical Characterization of Insoluble Aggregates of a Multi-Domain Protein: An Insight into the Role of the Various Domains

Insoluble (visible) aggregates of a homodimer fusion glycoprotein, consisting of the first extracellular domain of a human protein, fused to the hinge, CH2, and CH3 domains of a human immunoglobulin G1 (IgG1), were observed during early formulation development. The soluble fraction of the fusion protein was compared to the visible aggregates by various biophysical techniques such as intrinsic and ANS fluorescence emission, reducing and nonreducing SDS-PAGE, equilibrium folding and refolding experiments in urea and guanidine hydrochloride in the absence and presence of mercaptoethanol. Significant differences were observed between the visible aggregates and the supernatant. Partial unfolding of the aggregated molecules was detected by intrinsic and ANS fluorescence. Using urea and guanidine hydrochloride unfolding/solubilization and refolding curves, it was possible to extrapolate that (i) the aggregates were not covalently linked but tightly associated, (ii) the fused domains of the protein were unfolded but not involved in the aggregation process, (iii) the CH2 domains were native-like, and (iv) the CH3 domains were involved in the aggregation process. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association.