Osterberg T, Fatouros A, Mikaelsson M
Department of Pharmaceutical Technology, Pharmacia & Upjohn AB, Stockholm, Sweden.
Pharm Res. 1997 Jul;14(7):892-8. doi: 10.1023/a:1012199816852.
To develop a stable freeze-dried formulation of recombinant factor VIII-SQ (r-VIII SQ) without the addition of albumin.
Different formulations were evaluated for their protective effect during sterile filtration, freeze-thawing, freeze-drying, reconstitution and long term storage. Factor VIII activity (VIII:C), visual inspection, clarity, solubility, moisture content and soluble aggregates and/ or fragments were assayed.
A combination of non-crystallising excipients (L-histidine and sucrose), a non-ionic surfactant (polysorbate 80) and a crystalline bulking agent (sodium chloride) was found to preserve the factor VIII activity during formulation, freeze-drying and storage. Calcium chloride was included to prevent dissociation of the heavy and light chains of r-VIII SQ. Sodium chloride was chosen as the primary bulking agent since the concentration of sodium chloride necessary for dissolution of r-VIII SQ in the buffer will inhibit the crystallization of many potential cake formers. It was found that L-histidine, besides functioning as a buffer, also protected r-VIII SQ during freeze-drying and storage. A pH close to 7 was found to be optimal. Some potential macromolecular stabilisers, PEG 4000, Haes-steril and Haemaccel, were evaluated but they did not improve the recovery of VIII:C. The freeze-dried formulation was stable for at least two years at 7 degrees C and for at least one year at 25 degrees C. The reconstituted solution was stable for at least 100 hours at 25 degrees C.
The albumin-free formulation resulted in consistently high recovery of VIII:C, very low aggregate formation and good storage stability. The stability of the reconstituted solution makes the formulation suitable for continuous administration via infusion pump. The formulation strategy described here may also be useful for other proteins which require a high ionic strength.
开发一种不添加白蛋白的重组因子VIII-SQ（r-VIII SQ）稳定冻干制剂。
评估不同制剂在无菌过滤、冻融、冻干、复溶和长期储存过程中的保护作用。检测因子VIII活性（VIII:C）、外观检查、澄清度、溶解度、水分含量以及可溶性聚集体和/或片段。
发现非结晶赋形剂（L-组氨酸和蔗糖）、非离子表面活性剂（聚山梨酯80）和结晶填充剂（氯化钠）的组合在制剂、冻干和储存过程中能保持因子VIII活性。加入氯化钙以防止r-VIII SQ重链和轻链解离。选择氯化钠作为主要填充剂，因为r-VIII SQ在缓冲液中溶解所需的氯化钠浓度会抑制许多潜在成饼剂的结晶。发现L-组氨酸除了作为缓冲剂外，在冻干和储存过程中也能保护r-VIII SQ。发现pH接近7时最为适宜。评估了一些潜在的大分子稳定剂，如聚乙二醇4000、羟乙基淀粉灭菌溶液和贺斯，但它们并未提高VIII:C的回收率。冻干制剂在7℃下至少稳定两年，在25℃下至少稳定一年。复溶后的溶液在25℃下至少稳定100小时。
无白蛋白制剂导致VIII:C始终具有高回收率；聚集体形成非常少且储存稳定性良好。复溶溶液的稳定性使该制剂适合通过输液泵连续给药。此处描述的制剂策略可能对其他需要高离子强度的蛋白质也有用。
