Stable formulations of recombinant human growth hormone and interferon-gamma for microencapsulation in biodegradable microspheres
Stable formulations of recombinant human growth hormone and interferon-gamma for microencapsulation in biodegradable microspheres
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DOI：
10.1023/a:1016063109373
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发表时间：
1996-10-01
影响因子：
3.7
通讯作者：
Jones, AJS
中科院分区：
文献类型：
--
作者：
Cleland, JL;Jones, AJS
目的。蛋白质控释制剂的成功开发要求蛋白质在制造过程中不变性。主要目的是开发两种重组人蛋白，人生长激素(RhGH)和干扰素-γ(rhIFN-Gamma)，在高蛋白浓度(>；100 mg/mL)下稳定在通常用于微胶囊的有机溶剂、二氯甲烷和乙酸乙酯中。筛选了几种辅料，以获得每种蛋白质的最大溶解度。这些配方(水溶液、冷冻干燥球磨、喷雾干燥或等电沉淀物)然后通过在有机溶剂中乳化并回收到多余的缓冲液中快速筛选。用悬浮在有机溶剂中的固体蛋白进行额外的筛选，然后用过量的缓冲液回收。用天然大小排阻色谱(SEC-HPLC)和圆二色谱(CD)测定天然蛋白的回收率。采用水包油(W/O/W)或水包油固体(S/O/W)法将所选制剂包埋在聚乳酸-聚乙醇酸微球中。用SEC-HPLC法、CD法和生物测定法分析了在生理条件下孵育的微球释放的初始蛋白。在快速筛选方法中，给出的处方的稳定性与PLGA微球的包封期的稳定性有很好的相关性。含有吐温20或80的重组人生长激素制剂可使天然蛋白得到塔式回收，而海藻糖和甘露醇制剂(磷酸盐缓冲液，pH 8.0)则可完全回收天然重组生长激素。其他添加剂如羧甲基纤维素、明胶和葡聚糖70不是有效的稳定剂，聚乙二醇对重组人生长激素有一定的稳定作用。将海藻糖/重组人生长激素(1：4质量比)和甘露醇/重组人生长激素(1：2质量比)制剂(磷酸二氢钾缓冲液，pH 8.0)冷冻干燥，分别重组为200和400 mg/mLrhGH，并将其包裹在PLGA微球中。蛋白质是从这些微球中以其天然状态释放出来的。重组人生长激素的冻干制剂产生了类似的结果，表明海藻糖和甘露醇具有稳定蛋白质的能力。含有吐温20或聚乙二醇的喷雾干燥(空气和冷冻干燥)制剂产生的小固体颗粒重组人生长激素在乙酸乙酯中稳定，但在二氯甲烷中不稳定。用重组人干扰素-γ(137 mg/mL琥珀酸缓冲液，pH 5.0)也得到了类似的结果，在有机溶剂中，甘露醇和海藻糖都能稳定蛋白质，导致天然的重组人干扰素-γ从PLGA微球中释放。这种快速筛选方法可以开发稳定的浓缩蛋白质溶液或固体蛋白质制剂，这些蛋白质制剂可以成功地包裹在PLGA微球中。所观察到的辅料通过优先水化蛋白质来稳定这些蛋白质的功能，而在干燥状态下(例如海藻糖)可能通过水取代在蛋白质表面产生保护性涂层来稳定蛋白质，但对其他蛋白质的研究应该进一步深入了解包埋过程中蛋白质稳定的这种机制。
Purpose. The successful development of controlled release formulations for proteins requires that the protein not be denatured during, the manufacturing process. The major objective was to develop formulations that stabilize two recombinant human proteins, human growth hormone (rhGH) and interferon-gamma (rhIFN-gamma), at high protein concentrations (>100 mg/mL) in organic solvents commonly used for microencapsulation, methylene chloride and ethyl acetate.Methods. Several excipients were screened to obtain the maximum solubility of each protein. These formulations (aqueous, lyophilized milled, spray dried, or isoelectric precipitate) were then rapidly screened by emulsification in the organic solvent followed by recovery into excess buffer. Additional screening was performed with solid protein that was suspended in the organic solvent and then recovered with excess buffer. The recovery of native protein was determined by native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The selected formulations were encapsulated in polylactic-coglycolic acid (PLGA) microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water (S/O/W) methods. The initial protein released from the microspheres incubated at physiological conditions was analyzed by SEC-HPLC, CD, and biological assays.Results. The stability of a given formulation in the rapid screening method correlated well with stability during encapsulation in PLGA microspheres. Formulations of rhGH containing Tween 20 or 80 resulted in tower recovery of native protein, while trehalose and mannitol formulations (phosphate buffer, pH 8.0) yielded complete recovery of native rhGH. Other additives such as carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, respectively, and then encapsulated in PLGA microspheres. The protein was released from these microspheres in its native state. Lyophilized formulations of rhGH yielded analogous results indicating the ability of trehalose and mannitol to stabilize the protein. Small solid particles of rhGH generated by spray drying (both air and freeze-drying) formulations containing Tween 20 or PEG were stable in ethyl acetate, but not methylene chloride. Similar results were also obtained with rhIFN-gamma (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and trehalose were observed to stabilize the protein during exposure to the organic solvents resulting in the release of native rhIFN-gamma from PLGA microspheres.Conclusions. The rapid screening method allowed the development of stable concentrated protein solutions or solid protein formulations that could be successfully encapsulated in PLGA microspheres. The excipients observed to stabilize these proteins function by preferential hydration of the protein, and in the dry state (e.g., trehalose) may stabilize the protein via water substitution yielding a protective coating around the protein surface, Studies of other proteins should provide further insight into this mechanism of protein stabilization during encapsulation.