在這項研究中，利用液相層析質譜儀為基礎的蛋白質體分析方法對市售的免疫球蛋白G(Immunoglobulin G, IgG)進行廣泛性之結構鑑定且進一步與藥廠生產的生物相似藥物作一生物相似性分析比較。一開始利用還原性(reduction)及非還原(without reduction)性搭配多種酵素水解消化的策略，並以液相層析串聯式質譜儀(LC-MS/MS)進行胜肽圖譜鑑定(peptide mapping)的方式測定其基本結構序列。透過這個方法我們充分地驗證其一級結構序列 (序列覆蓋率100%)、雙硫鍵連接位址以及轉譯後修飾的發生，其中轉譯後修飾包括醣基化、甲硫胺酸的氧化(Met oxidation)、天冬醯胺的去胺化(Asn deamidation)、N-端麩氨醯胺(Glutamine, E)的環化(pyro-glu)以及C-端賴胺酸(Lysine) 的消去(K clipping)。在早期生產的生物相似藥物中，我們鑑定出其序列包含兩個氨基酸變異(D359E和L361M)，不過在最近生產的批次中已經被修正過來。不僅鑑定生物相似藥物及市售單株抗體之藥物具有相同的16段雙硫鍵，在Fc端的醣基化位址(N300)上也分析出其主要的醣基結構為G0、 G1以及G2，而這兩種產品也存在著相似的醣基分佈。N-端及C-端的修飾在生物相似藥物及市售藥物皆為非常相似；然而甲硫胺酸的氧化(Met oxidation)以及天冬醯胺的去胺化 (Asn deamidation) 這些典型的修飾在兩種產品間存在著微小的差異，可能是由於樣品儲存條件及緩衝溶液成分的不同所造成的。最後成功地利用不標定之定量方法，並搭配T-test統計方法找出市售藥物與生物相似藥物有顯著性差異 (significant difference)之處，以顯示出生物相似藥物與市售藥物之間的相似性及差異性，包括其變異的位址及修飾上的差異。儘管這兩個產品之間不完全相同，但是透過其產品的完整性與關鍵的結構信息，顯示出這兩個產品彼此間確實是具有生物相似性的。總之對於結構複雜的蛋白質藥物而言，液相層析質譜儀 (LC-MS)為基礎的蛋白質體分析是一功能強大的技術。

In this study, liquid chromatography-mass spectrometry (LC-MS)-based proteomics technique was developed to characterize a commercially available IgG1 monoclonal antibody (mAb) product in detail and further compare it with a candidate biosimilar. Multiple enzyme strategy was first developed to couple with mass mapping using LC-MS, and mass sequencing using LC-MS/MS. Through this approach, we were able to fully validate the primary sequence (100% sequence coverage) and the disulfide linkages which form the secondary structure, as well as protein post translational modifications (PTMs) including glycosylation, deamidation pyro-glu formation at the N-terminus, and K clipping at the C-terminus. Furthermore, using label-free quantification method, we were able to deduce the distribution among G0, G1, and G2 N-linked glycosylation at the conserved Fc site of the antibody. The techniques were also used to comprehensively compare the differences between the candidate biosimilar and the commercial mAb. Except two amino acid variants, D359E and L361M, which were intuitively designed by the manufacturer, insignificant differences between the biosimilar and the commercial product were detected, showing these two products were indeed biosimilar to each other. In conclusion, LC-MS based proteomics analysis is a powerful technique to reveal the critical structural complexity of protein drugs.

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