Stabilization of proteins by recombinant human gelatins.
Porcine gelatins have been widely used as stabilizers of macromolecular based pharmaceuticals but the mechanism by which they stabilize has not been precisely established. Their variability and immunogenicity, however, make them less than ideal excipients. In this work, we take advantage of the availability of recombinant human gelatins (rhGs) to explore the mechanism by which they may stabilize proteins. Three model recombinant proteins, human serum albumin (HSA), bovine granulocyte colony stimulating factor (bGCSF), and human fibroblast growth factor-20 (FGF-20) that display a range of isoionic points have been selected for this study. The interaction of these model proteins with four different molecular weight rhGs and porcine gelatin was studied using a variety of biophysical techniques including fluorescence, CD and second derivative UV spectroscopy to monitor tertiary and secondary structure as a function of temperature. The 8.5, 25, and 100 kDa rhGs had the greatest effect on conformational and colloidal stability of HSA. The 8.5, 25, and 50 kDa rhGs also increased the T(0) of aggregation of bGCSF and FGF-20. Experiments to probe the mechanism of interaction of model proteins with rhGs suggest that the rhGs might interact with the partially unfolded states of target proteins through a combination of electrostatic and other intermolecular mechanisms to inhibit aggregation.
Thyagarajapuram
Nagarajan
N
Department of Pharmaceutical Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, KS 66049, USA.
Olsen
David
D
Middaugh
C Russell
CR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002626:Chemistry, Pharmaceutical; D002942:Circular Dichroism; D004339:Drug Compounding; D005079:Excipients / Q000737:chemistry*; D005346:Fibroblast Growth Factors / Q000737:chemistry*; D005780:Gelatin / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D008027:Light; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013678:Technology, Pharmaceutical / Q000379:methods; D013696:Temperature
D005079:Excipients; C416003:FGF20 protein, human; D011994:Recombinant Proteins; D012709:Serum Albumin; D016179:Granulocyte Colony-Stimulating Factor; D005346:Fibroblast Growth Factors; D005780:Gelatin