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, C(H)2, and C(H)3 domains of a human immunoglobulin G(1) (IgG(1)), 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 C(H)2 domains were native-like, and (iv) the C(H)3 domains were involved in the aggregation process.
Souillac
Pierre O
PO
Pharmaceutical Sciences and Technology group, Biogen Idec, 14 Cambridge Center, Cambridge, Massachusetts 02142, USA. pierre.souillac@biogenidec.com
D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D019791:Guanidine; D007074:Immunoglobulin G / Q000737:chemistry*; D008623:Mercaptoethanol; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea
D006023:Glycoproteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D008623:Mercaptoethanol; D014508:Urea; D019791:Guanidine