Biopharmaceutical Process Sciences, CuraGen Coporation, 322 East Main Street, Branford, CT 06405, USA.
Curr Pharm Biotechnol. 2009 Sep;10(6):609-25. doi: 10.2174/138920109789069297. Epub 2009 Sep 1.
The purpose of this study was to dramatically enhance the solubility (> 400 fold) and stability of a therapeutic protein (Fibroblast Growth Factor 20) and to perform detailed biophysical characterization for the optimization of its formulation. The solubility of FGF-20 strongly depends on pH, arginine concentration and anions present in a buffer system. In the absence and presence of arginine, solubility was higher at lower pH (5 < or = pH < or = 6) and then decreased steadily with a minimum solubility at around pH 6.3 and plateaus at around pH 7.5 respectively. For a given pH, the protein was most soluble in arginine-sulfate. The solubility of FGF-20 increases with an increase in arginine-sulfate concentration for a given pH. However, a salting out effect was observed at higher arginine-sulfate concentration. Polysorbate-80 did not have any striking effect on solubility and no effect on thermal stability, but it significantly prevented the loss of protein under agitated conditions. Thermal stability of FGF-20 measured by DSC was increased with an increase in arginine-sulfate concentration (at least up to 0.5M). A sturdy dependence of thermal stability on pH was observed with about a 15 degrees C increase in T(m) (melting temperature) at pH 7.0 in comparison to pH 5.0. From the DSC data, approximate stability curves were generated and cold denaturation temperatures were predicted. Denaturant induced unfolding studies provided better insight of FGF-20 in different solution conditions in terms of structure and stability than the DSC data. An inverse relationship of solubility and thermal stability was observed in the pH range of 5.0 to 8.5 at a fixed arginine concentration and is consistent with Linderstrom-Lange's smeared model. A direct correlation between solubility and thermal stability was observed at different arginine concentrations for a fixed pH. The effect of arginine on the solubility and stability of FGF-20 was dominated by the preferential binding interaction.
本研究的目的是极大地提高一种治疗性蛋白（成纤维细胞生长因子 20）的溶解度（> 400 倍）和稳定性，并对其进行详细的生物物理特性分析，以优化其配方。FGF-20 的溶解度强烈依赖于 pH 值、精氨酸浓度和缓冲体系中的阴离子。在不存在和存在精氨酸的情况下，较低 pH 值（5 <= pH <= 6）时溶解度更高，然后随着 pH 值约为 6.3 时溶解度稳步下降，并分别在 pH 值约为 7.5 时达到平台。对于给定的 pH 值，在精氨酸-硫酸盐中蛋白质的溶解度最高。在给定 pH 值下，FGF-20 的溶解度随精氨酸-硫酸盐浓度的增加而增加。然而，在较高的精氨酸-硫酸盐浓度下观察到盐析效应。聚山梨醇酯 80 对溶解度没有显著影响，也没有对热稳定性产生影响，但它能显著防止在搅拌条件下蛋白质的损失。通过 DSC 测量的 FGF-20 的热稳定性随精氨酸-硫酸盐浓度的增加而增加（至少在 0.5M 时增加）。在 pH 7.0 时与 pH 5.0 相比，观察到热稳定性对 pH 值的强烈依赖性，T(m)（熔点）增加了约 15°C。从 DSC 数据中生成了近似稳定性曲线，并预测了冷变性温度。与 DSC 数据相比，变性剂诱导的展开研究提供了更好的关于 FGF-20 在不同溶液条件下的结构和稳定性的见解。在固定精氨酸浓度的 pH 范围 5.0 至 8.5 内，观察到溶解度和热稳定性之间的反比关系，这与 Linderstrom-Lange 的弥散模型一致。在固定 pH 值的不同精氨酸浓度下，观察到溶解度和热稳定性之间的直接相关性。精氨酸对 FGF-20 的溶解度和稳定性的影响主要由优先结合相互作用决定。
