Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907.
Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907.
J Pharm Sci. 2020 Jan;109(1):709-718. doi: 10.1016/j.xphs.2019.04.024. Epub 2019 Apr 26.
Excipients used in lyophilized protein drug products are often selected by a trial-and-error method, in part, because the analytical methods used to detect protein-excipient interactions in lyophilized solids are limited. In this study, photolytic labeling was used to probe interactions between salmon calcitonin (sCT) and excipients in lyophilized solids. Two diazirine-derived photo-excipients, photo-leucine (pLeu) and photo-glucosamine (pGlcN), were incorporated into lyophilized solids containing sCT, together with an unlabeled excipient (sucrose or histidine) at prelyophilization pH values from 6 to 9.9. Commercially available pLeu was selected as an ionizable photo-excipient and amino acid analog, while pGlcN was synthesized as an analog of sugar-based excipients. Photolytic labeling was induced by exposing the solids to UV light (365 nm, 30-60 min), and the resulting products were identified and quantified with liquid-chromatography mass spectrometry. The distribution of photo-reaction products was affected by the photoreactive reagent used, the type of unlabeled excipient, and the solution pH before lyophilization. When other components of the solid were identical, the extent and sites of labeling on sCT were different for pGlcN and pLeu. The results suggest that ionizable and nonionizable excipients interact differently with sCT in lyophilized solids and that photo-excipients can be used to map these interactions.
在冷冻干燥蛋白药物产品中使用的赋形剂通常是通过反复试验的方法选择的，部分原因是用于检测冷冻干燥固体中蛋白质-赋形剂相互作用的分析方法有限。在这项研究中，光解标记被用于探测鲑鱼降钙素（sCT）与冷冻干燥固体中的赋形剂之间的相互作用。两种二氮烯衍生的光致赋形剂，光亮氨酸（pLeu）和光氨基葡萄糖（pGlcN），与未标记的赋形剂（蔗糖或组氨酸）一起在冷冻干燥前的 pH 值为 6 到 9.9 的范围内被掺入含有 sCT 的冷冻干燥固体中。市售的 pLeu 被选为可离子化的光致赋形剂和氨基酸类似物，而 pGlcN 则被合成作为糖基赋形剂的类似物。通过将固体暴露于 365nm 的紫外光（30-60 分钟）来诱导光解标记，并用液相色谱-质谱法对所得产物进行鉴定和定量。光反应产物的分布受所用光致反应试剂、未标记赋形剂的类型和冷冻干燥前溶液的 pH 值的影响。当固体的其他成分相同时，pGlcN 和 pLeu 对 sCT 的标记程度和部位不同。结果表明，可电离和非电离赋形剂在冷冻干燥固体中与 sCT 相互作用方式不同，光致赋形剂可用于绘制这些相互作用。
