以胜肽圖譜法鑑定重組人類胰島素及胰島素相似物製劑   全文下載

Identification of Recombinant Insulin Analogues by Peptide Mapping Method

人類胰島素是由51個胺基酸組成的多?類荷爾蒙，是重要的糖尿病治療藥物。目前除了以基因工程技術製造的人類胰島素外，另有藥廠開發出多種胰島素類緣物（insulin analogues），提供速效型和長效型等不同的藥理功能。當今歐美藥典已陸續收錄各類胰島素製劑之分析方法，惟中華藥典尚未加以收錄。由於胰島素類緣物通常僅修改一至三個胺基酸，與人類胰島素結構差異不大，許多分析方法無法有效鑑別此細微差異，因此本計畫以胜?圖譜技術（peptide mapping），對國內市售胰島素製劑進行分析方法開發及品質評估研究。胜?圖譜技術係利用蛋白質內切?（Endo-proteinase）處理胰島素檢品，針對特定胺基酸位置進行專一性水解，被切割成為多?片段的胰島素檢品，再以高效液相層析儀（High Performance Liquid Chromatography, HPLC）進行分析，建立該檢品的胜?圖譜，此方法除可藉由與標準品比對來評估該製劑品質是否合乎規範外，並能有效鑑別不同的胰島素成分。本研究依歐、美藥典中之胰島素製劑分析方法篇章作準則，經修改後設計以一貫的實驗流程分析所有的胰島素檢品，期能快速穩定的完成品質鑑別。我們建立六種胰島素之胜?圖譜（包括Insulin Human 、Lispro 、Aspart 、Detemir 、Glargine和Glulisine），並取部分市售胰島素製劑與標準品進行比對，結果顯示各製劑之胜?圖譜與其標準品表現一致，顯現產品品質合乎標準。在可獲得標準品的情況下，胜?圖譜法不僅可鑑別蛋白質序列中單一胺基酸置換，亦能分辨二胺基酸序列互換的變化，實屬快速便捷又有效的分析方法，日後可進一步開發本方法用於生長激素或顆粒細胞群落刺激因子等基因工程製劑的檢驗，以因應日漸成長的生物學名藥後市場品質評估需求。"

Insulin human, containing 51 amino acids, is a small polypeptide hormone that regulates blood glucose homeostasis. Patients with insulin-dependent diabetes mellitus require insulin therapy through the administration of exogenous insulin to avoid ketoacidosis. Utilizing various genetic engineering techniques, pharmaceutical companies have developed a variety of rapid- or long-acting insulin analogues. Analytical methods for various types of insulin and insulin analogues are gradually being included in the United States Pharmacopeia (USP) and the European Pharmacopeia (Ph. Eur.), but not yet in the Chinese Pharmacopeia. Usually these insulin analogues only differ by 1 to 3 amino acids, which is too subtle to distinguish by most of analytical methods currently available. In this study a peptide mapping technique was employed to screen insulin analogues for quality assessment. Peptide mapping is capable of identifying single amino acid changes resulted from events such as errors in the reading of complementary DNA sequences or point mutations. Here we analyzed 6 insulin preparations including insulin human, insulin lispro, insulin aspart, insulin detemir, insulin glargine and insulin glulisine using peptide mapping analysis. The peptide fingerprints of the insulin products we tested all corresponded well to those of the standard materials. Our peptide mapping method is more accurate in identifying the subtle differences between the insulin analogues than chromatography is. We concluded that peptide mapping is a valuable initial screening tool for quality assessment of insulin analogues. In the future, we intend to continue to develop this technology for post-marketing surveillance of other biopharmaceuticals and biosimilars, such as somatropin, erythropoietin and G-CSF.