Photolytic Labeling To Quantify Peptide-Water Interactions in Lyophilized Solids.
Photolytic Labeling To Quantify Peptide-Water Interactions in Lyophilized Solids.
复制标题
光解标记定量冻干固体中肽-水相互作用。
DOI：
10.1021/acs.molpharmaceut.8b01031
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发表时间：
2019
影响因子：
4.9
通讯作者：
Topp,ElizabethM
中科院分区：
文献类型：
--
作者：
Chen,Yuan;Topp,ElizabethM
冻干肽与水和赋形剂在固体基质中相互作用的探索使用光解标记。将模型肽“KLQ”（Ac-QELHKLQ-NHCH3）与nhs -二嗪（琥珀酰4,4′-氮戊酸酯）共价标记，并将标记的肽（KLQ - sda）配制并在溶液和冻干固体中暴露于紫外线下。固体样品以1:400摩尔比含有以下辅料：蔗糖、海藻糖、甘露醇、组氨酸或精氨酸。在紫外线照射之前，将冻干的固体暴露于不同的相对湿度（RH）环境（8,13,33,45和78%），并测量所得的固体含水量（卡尔菲舍尔滴定法）和玻璃化转变温度（Tg；差示扫描量热法，DSC）。为了开始光解标记，将溶液和固体样品暴露在365 nm的紫外光下30分钟。采用反相高效液相色谱（rp-HPLC）和质谱（MS）对光解标记产物进行定量分析。在冻干固体中，排除氧和使用H218O的研究证实，质量增加18 amu的KLQ加合物的氧来源是与水的反应，而质量增加16 amu的KLQ加合物的氧来源既不是与水的反应，也不是与分子氧的反应。在含有蔗糖或海藻糖的固体中，肽-赋形物加合物随着固体含水量的增加而减少，而肽-水加合物仅在较低RH暴露时增加，然后趋于稳定，这部分符合水替代假说。
Interactions of a lyophilized peptide with water and excipients in a solid matrix were explored using photolytic labeling. A model peptide “KLQ” (Ac–QELHKLQ–NHCH3) was covalently labeled with NHS–diazirine (succinimidyl 4,4′-azipentanoate), and the labeled peptide (KLQ–SDA) was formulated and exposed to UV light in both solution and lyophilized solids. Solid samples contained the following excipients at a 1:400 molar ratio: sucrose, trehalose, mannitol, histidine, or arginine. Prior to UV exposure, the lyophilized solids were exposed to various relative humidity (RH) environments (8, 13, 33, 45, and 78%), and the resulting solid moisture content (Karl Fischer titration) and glass transition temperature (Tg; differential scanning calorimetry, DSC) were measured. To initiate photolytic labeling, solution and solid samples were exposed to UV light at 365 nm for 30 min. Photolytic-labeling products were quantified using reversed-phase high-performance liquid chromatography (rp-HPLC) and mass spectrometry (MS). In lyophilized solids, studies excluding oxygen and using H218O confirmed that the source of oxygen in KLQ adducts with a mass increase of 18 amu are attributable to reaction with water, while those with a mass increase of 16 amu are not attributable to reaction with either water or molecular oxygen. In solids containing sucrose or trehalose, peptide–excipient adducts decreased with increasing solid moisture content, while peptide–water adducts increased only at lower RH exposure and then plateaued, in partial agreement with the water replacement hypothesis.