A new strategy for enhancing the stability of lyophilized protein: the effect of the reconstitution medium on keratinocyte growth factor.
OBJECTIVE
Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF).


METHODS
The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer.


RESULTS
Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein.


CONCLUSIONS
These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.
Zhang
M Z
MZ
Amgen Inc., Department of Protein Chemistry, Amgen Center, Thousand Oaks, California 93120-1789, USA.
Wen
J
J
Arakawa
T
T
Prestrelski
S J
SJ
D000818:Animals; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D005612:Freeze Drying; D006133:Growth Substances / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
C496453:FGF7 protein, human; C496454:Fgf7 protein, mouse; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D011994:Recombinant Proteins; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors