STRATEGIES TO SUPPRESS AGGREGATION OF RECOMBINANT KERATINOCYTE GROWTH-FACTOR DURING LIQUID FORMULATION DEVELOPMENT
STRATEGIES TO SUPPRESS AGGREGATION OF RECOMBINANT KERATINOCYTE GROWTH-FACTOR DURING LIQUID FORMULATION DEVELOPMENT
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DOI：
10.1002/jps.2600831204
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发表时间：
1994-12-01
影响因子：
3.8
通讯作者：
CHIEN, SL
中科院分区：
文献类型：
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作者：
CHEN, BL;ARAKAWA, T;CHIEN, SL
重组人角质细胞生长因子（rhKGF）是一种相当不稳定的蛋白质，长期储存是一个具有挑战性的问题。在储存过程中，蛋白质在相对较低的温度下展开，并且展开的蛋白质迅速聚集，导致形成大的可见沉淀物。rhKGF的热解折叠显示出类似的模式，即，解折叠之后，随着温度的升高立即发生聚集。由于rhKGF的解折叠和聚集（沉淀）限制了蛋白质的储存寿命，因此通过检查赋形剂对热熔融温度和储存期间蛋白质聚集速率的影响，研究了抑制rhKGF解折叠和聚集的稳定剂。硫酸化多糖和柠檬酸盐被认为是有效的，在增加的熔化温度的rhKGF或防止其聚集。特别是，0.5%（w/v）肝素和高分子量硫酸葡聚糖，和0.5 M柠檬酸盐是非常有效的，减少约50倍的rhKGF聚集率。其他带负电荷的小离子，如磷酸盐，对rhKGF也有中等的稳定作用。对rhKGF聚集途径的机制研究使我们更好地理解了这些分子的稳定作用。增强rhKGF构象稳定性的分子能够有效地抑制rhKGF聚集。
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.