Thiol-disulfide interchange in the tocinoic acid/glutathione system during freezing and drying.
Thiol-disulfide interchange in the tocinoic acid/glutathione system during freezing and drying.
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在冷冻和干燥过程中，生育酸/谷胱甘肽系统中的硫醇-二硫化物交换。
DOI：
10.1002/jps.22206
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发表时间：
2010
影响因子：
3.8
通讯作者：
Topp,ElizabethM
中科院分区：
文献类型：
--
作者：
Thing,Mette;Zhang,Jun;Laurence,Jennifer;Topp,ElizabethM
硫醇-二硫键交换(“二硫键干扰”)是蛋白质类药物常见的共价聚集机制。利用烟酸(环-S-半胱氨酸-酪氨酸-谷氨酰胺-天冬氨酸-半胱氨酸(S)；TA(OX))和谷胱甘肽(γ-Glu-Cys-Gly；GSH)，我们以前的工作证明了硫醇/二硫键交换受到冷冻干燥的影响，这种方式与TA(OX)的不可逆和区域选择性损失一致(Zhang等，2009年，药学杂志98/9：3312-3318)。在这里，我们探讨了在TA/GSH体系中，冷冻干燥循环的各个阶段对硫醇/二硫化物交换中的扰动的贡献。在不同辅料存在或不存在的情况下，从磷酸盐缓冲液中共冷冻干燥TA(OX)和GSH，然后用反相高效液相色谱(RP-HPLC)分析TA(OX)和混合二硫化物的生成。研究发现，扰动主要发生在冰冻过程中，在大量的冰被升华除去之前。添加冷冻保护剂(蔗糖)、冷冻保护剂(吐温-20)和快速冷冻只有在冰仍然存在的情况下才会影响产品的分布。通过添加氧化谷胱甘肽(GSSG)降低氧化还原电位对冻干样品和溶液对照的产物分布有不同的影响，但这两种情况都不会增加TA(OX)的保守性。
Thiol-disulfide interchange (“disulfide scrambling”) is a common mechanism of covalent aggregation for protein drugs. Using tocinoic acid (cyclo-S-Cys-Tyr-Ile-Gln-Asn- Cys-(S); TA(ox)) and glutathione (γGlu-Cys-Gly; GSH), our previous work demonstrated that thiol/disulfide interchange is affected by lyophilization in a manner consistent with irreversible and regioselective loss of TA(ox) (Zhang et al., 2009, J Pharm Sci 98/9: 3312–3318). Here, we explore the contributions of stages of the lyophilization cycle to perturbations in thiol/ disulfide interchange in the TA/GSH system. TA(ox) and GSH were co-lyophilized from phosphate buffer in the presence or absence of various excipients, then analyzed for TA(ox) and mixed disulfide products by reverse phase high performance liquid chromatography (rp-HPLC). Perturbations were found to occur primarily during freezing, before significant amounts of ice were removed by sublimation. Addition of a lyoprotectant (sucrose), a cryoprotectant (Tween-20) and flash-freezing influenced the product distribution only while ice was still present. Decreasing the redox potential by the addition of oxidized glutathione (GSSG) affected the product distribution differently in lyophilized samples and solution controls, but in neither case led to increased conservation of TA(ox).