利用質譜技術監控以及鑑定重組蛋白中間產物之雙硫鍵變化
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2016
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Abstract
雙硫鍵是維持蛋白質活性及結構穩定性的主要轉譯修飾官能基。近幾年，固相胜肽合成技術已被廣泛應用於蛋白質藥物生產平台，但仍需透過雙硫鍵重組方能取得含正確結構及活性的產物。因此，一個能快速分析雙硫鍵的工具對於製程開發將有極大助益，也可作為蛋白質藥物必要的品管流程。生物毒素能專一的作用細胞受體，其作用機制是新型藥物開發研究的對象，本研究以固相胜肽合成之心臟蛇毒蛋白 (Cardiotoxin) 為標的，以質譜搭配RADAR軟體分析蛋白重組過程之雙硫鍵變化。藉由之雙硫鍵分析資訊，優化蛋白重組條件，純化出含正確雙硫鍵連結之產物。此外，結構分析與蛋白質活性測試結果，皆顯示重組心臟蛇毒蛋白具有與天然蛇毒蛋白質有相似β摺疊之二級結構與穿透細胞膜之活性。然而，重組過程產生之雙硫鍵錯接蛋白，則呈現不規則結構且不具活性。
整體而言，以質譜搭配RADAR軟體可快速分析重組蛋白的雙硫鍵鍵結位點，雙硫鍵分析資訊可作為蛋白藥物的品管資料。此平台可用於中間產物的分析，藉由雙硫鍵分析資訊優化重組條件，未來可應用於製藥產業對於製造富含多半胱胺酸之生物製劑樣品。
Disulfide bond is one of major post translation modifications for protein skeleton construction and folding maintainess. Recently, using solid phase peptide synthesis (SPPS) to generate peptide drug has been applied in pharmaceutical industry. For cysteine-rich peptides, analysis of correct disulfide bonds can be challenging. In this study, a well-developed platform using mass spectrometry was applied to profile the disulfide linkages of a synthetic peptide toxin, cardiotoxin A3 (composed by 60 amino acids with 4 disulfide bonds), during the refolding process. The bio-active cardiotoxin A3 product with correct folding was obtained by using an optimized dilution refolding method with the presence of GSSG/GSH in weak alkaline condition (phosphate buffer, pH 7.5, containing 1:3 mM ratio of GSSG/GSH). Based on the MS data, we were able to determine the protein fraction that performs exact disulfide linkage patterns as those found in native CTXA3, and optimize the manufacturing condition for peptide production. The further assays proved that the synthetic peptide product exhibited similar biochemical property and cell penetration activity as native one. The study results indicate that bioactive peptides with correct disulfide linkages could be obtained from SPPS approach with appropriate folding condition will be beneficial for the manufacturing of cysteine-rich biologics in biopharmaceutical industry. Eventually, MS-based disulfide analysis platform can provide significant information for protein structure-function study, and be the examination tool for cystein-rich bioproduct qualification.
Disulfide bond is one of major post translation modifications for protein skeleton construction and folding maintainess. Recently, using solid phase peptide synthesis (SPPS) to generate peptide drug has been applied in pharmaceutical industry. For cysteine-rich peptides, analysis of correct disulfide bonds can be challenging. In this study, a well-developed platform using mass spectrometry was applied to profile the disulfide linkages of a synthetic peptide toxin, cardiotoxin A3 (composed by 60 amino acids with 4 disulfide bonds), during the refolding process. The bio-active cardiotoxin A3 product with correct folding was obtained by using an optimized dilution refolding method with the presence of GSSG/GSH in weak alkaline condition (phosphate buffer, pH 7.5, containing 1:3 mM ratio of GSSG/GSH). Based on the MS data, we were able to determine the protein fraction that performs exact disulfide linkage patterns as those found in native CTXA3, and optimize the manufacturing condition for peptide production. The further assays proved that the synthetic peptide product exhibited similar biochemical property and cell penetration activity as native one. The study results indicate that bioactive peptides with correct disulfide linkages could be obtained from SPPS approach with appropriate folding condition will be beneficial for the manufacturing of cysteine-rich biologics in biopharmaceutical industry. Eventually, MS-based disulfide analysis platform can provide significant information for protein structure-function study, and be the examination tool for cystein-rich bioproduct qualification.
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固相胜肽合成, 蛋白質重組, 雙硫鍵, 質譜, 雙甲基化, RADAR, Solid phase peptide synthesis, Refolding, Disulfide Bond, Mass spectrometry, Dimethyl labeling, RADAR