title: Strategies to suppress aggregation of recombinant keratinocyte growth factor during liquid formulation development.
abstract: Recombinant human keratinocyte growth factor (rhKGF) is a fairly unstable protein, posing a challenging problem for long-term storage. During storage, the protein unfolds at relatively low temperatures and the unfolded proteins aggregate rapidly, leading to the formation of large visible precipitates. Thermal unfolding of rhKGF displays a similar pattern, i.e., unfolding is followed immediately by aggregation as the temperature is increased. As the unfolding and aggregation (precipitation) of rhKGF limit the storage life of the protein, a search for stabilizers to suppress rhKGF unfolding and aggregation has been done by examining the effects of excipients on thermal melting temperature and on the rate of protein aggregation during storage. Sulfated polysaccharides and citrate are found to be effective in increasing the melting temperature of rhKGF or preventing its aggregation. In particular, 0.5% (w/v) heparin and high molecular weight dextran sulfate, and 0.5 M citrate are highly effective, decreasing the rates of rhKGF aggregation by about 50-fold. Other negatively charged small ions, such as phosphate, also have moderate stabilizing effects on rhKGF. A mechanistic study of the aggregation pathway of rhKGF has led to a better understanding of the stabilizing effects of these molecules. Molecules which enhance rhKGF conformational stability are capable of effectively suppressing rhKGF aggregation.
authors.affiliation: Department of Pharmaceutics and Drug Delivery, Amgen Inc., Thousand Oaks, CA 91320.
authors.forename: B L
authors.initials: BL
authors.lastname: Chen
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: E
authors.initials: E
authors.lastname: Hsu
authors.forename: L O
authors.initials: LO
authors.lastname: Narhi
authors.forename: T J
authors.initials: TJ
authors.lastname: Tressel
authors.forename: S L
authors.initials: SL
authors.lastname: Chien
mesh_terms: D000818:Animals; D002021:Buffers; D002460:Cell Line; D055598:Chemical Phenomena; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002627:Chemistry, Physical; D002942:Circular Dichroism; D004355:Drug Stability; D004356:Drug Storage; D005079:Excipients / Q000737:chemistry; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006133:Growth Substances / Q000737:chemistry*; D006361:Heating; D006493:Heparin / Q000737:chemistry; D015603:Keratinocytes / Q000187:drug effects; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011134:Polysaccharides / Q000494:pharmacology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride; D013431:Sulfates / Q000494:pharmacology
chemical_list: D002021:Buffers; D005079:Excipients; C496454:Fgf7 protein, mouse; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D011134:Polysaccharides; D011994:Recombinant Proteins; D013431:Sulfates; D051523:Fibroblast Growth Factor 7; D012965:Sodium Chloride; D005346:Fibroblast Growth Factors; D006493:Heparin