Maity Haripada, Karkaria Cyrus, Davagnino Juan
Biopharmaceutical Process Sciences, CuraGen Corporation, 322 East Main Street, Branford, CT 06405, USA.
Int J Pharm. 2009 Aug 13;378(1-2):122-35. doi: 10.1016/j.ijpharm.2009.05.063. Epub 2009 Jun 6.
This study discusses the effect of key factors like containers, buffers and the freeze (controlled vs. flash freezing) and thawing processes on the stability of a therapeutic protein fibroblast growth factor 20 (FGF-20). The freezing profiles monitored by 15 temperature probes located at different regions in a 2-L bottle during freezing can be grouped into three categories. A rapid drop in temperature was observed at the bottom followed by the top and middle center of the bottle. The freeze-thawing behavior in a 50 ml tube is considerably uniform, as expected. Among phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), citrate and histidine (each containing 0.5 M arginine-sulfate) buffer systems, a minimum pH change (0.4 pH unit vs. approximately 1.7 pH unit) was observed for the phosphate buffer system. Thawing in a 50 ml tube at room temperature standing resulted in a significant phase separation in citrate, histidine and HEPES buffers; however, phase separation was least in the phosphate buffer system. These phase separations were found to be temperature dependent. No effect of Polysorbate 80 on freeze-thawing of FGF-20 was observed. Significant concentration gradients in major buffer components and protein concentration were observed during freeze-thawing in a 2-L bottle. The segregation patterns of the various components were similar with the top and bottom layers containing lowest and highest concentrations, respectively. In the formulation buffer no pH gradient was formed, and the precipitation of FGF-20 during thawing at the top layer was related to an insufficient amount of arginine-sulfate and the precipitation at the bottom layer was due to a salting out effect. The precipitate generated during thawing goes into solution easily upon mixing whole solution of the bottle and the various gradient formations do not cause any irreversible change in structure, stability and isoform distribution of FGF-20. Comparison of slow freezing and flash freezing data suggests that the gradients in excipient and protein concentrations are mainly formed during thawing.
本研究探讨了容器、缓冲液以及冷冻（控制冷冻与快速冷冻）和解冻过程等关键因素对治疗性蛋白成纤维细胞生长因子20（FGF - 20）稳定性的影响。在冷冻过程中，通过位于2升瓶不同区域的15个温度探头监测到的冷冻曲线可分为三类。观察到瓶底部温度快速下降，随后是顶部和中部中心。正如预期的那样，50毫升试管中的冻融行为相当均匀。在磷酸盐、HEPES（4 -（2 - 羟乙基）- 1 - 哌嗪乙磺酸）、柠檬酸盐和组氨酸（每种均含有0.5 M精氨酸 - 硫酸盐）缓冲系统中，磷酸盐缓冲系统的pH变化最小（0.4个pH单位，而其他约为1.7个pH单位）。在室温下静置于50毫升试管中解冻时，柠檬酸盐、组氨酸和HEPES缓冲液中出现了显著的相分离；然而，磷酸盐缓冲系统中的相分离最少。发现这些相分离与温度有关。未观察到聚山梨酯80对FGF - 20冻融的影响。在2升瓶中冻融过程中，主要缓冲成分和蛋白质浓度出现了显著的浓度梯度。各成分的分离模式相似，顶层和底层分别含有最低和最高浓度。在制剂缓冲液中未形成pH梯度，解冻时FGF - 20在顶层的沉淀与精氨酸 - 硫酸盐量不足有关，而在底层的沉淀是由于盐析作用。解冻过程中产生的沉淀物在将瓶中整个溶液混合后很容易重新溶解，并且各种梯度形成不会导致FGF - 20的结构、稳定性和异构体分布发生任何不可逆变化。缓慢冷冻和快速冷冻数据的比较表明，辅料和蛋白质浓度的梯度主要在解冻过程中形成。
