生物共軛物在選擇性和動力學障礙方面遇到許多挑戰，而這些挑戰很少被描述。我們應用質譜搭配蛋白質體學的技術來顯示在殘基的尺度下的連接子共軛效果，發現在多重接合蛋白共軛物中採用動力學控制的還原胺化（KRA）的聚共軛方法與常見的經由N-羥基琥珀酰亞胺酯（NHS）進行SN2加成共軛方法相比更為優越。在相等的化學計量比下，無論延長的反應時間如何，兩個反應的共軛產率都非常低。因此我們比較過量連接子，在慢動力學條件下（8℃和pH 5.8）以及在席夫鹼形成階段的RA反應速率決定步驟和NHS反應之間進行比較。對於α-乳白蛋白（〜14.2 kDa）和Herceptin IgG（〜150 kDa），RA的結合產率顯示出比NHS反應高2-5倍。鑑定出通過兩個反應實現的共軛位點不同。對於平均每個蛋白所具有共軛連接子（LPR）〜4的多共軛物，RA的共軛位點分佈比NHS反應更均勻。此外，由於水解，NHS反應只能持續1-2小時。相反，RA反應遵循偽一級動力學，α-乳清蛋白和Herceptin IgG速率常數分別為0.06 、0.04 LPR / hr。通過將接頭試劑逐漸添加到硼酸鹽預混的IgG溶液中，進一步控制動力學，以提高共軛產率和位點均一性。在用100x過量連接劑進行4小時滴定的情況下，使用比NHS反應少2-3倍的試劑可得到主要連接在Herceptin IgG的2-3個溶劑暴露的胺位點上的四個連接子。已顯示通過KRA偶聯的Herceptin IgG保留了針對靶向細胞的更高階結構和藥物功效。與其他特定的共軛方法相比，KRA有望以更低的成本實現同質的多共軛。

Bioconjugation encounters many challenges in selectivity and kinetic obstacle which are rarely characterized. Here we applied mass spectrometry and proteomics technique to characterize amine conjugations at residue level and demonstrated a superior poly-conjugation method using kinetically controlled reductive amination (KRA) compared to the most commonly used SN2 reaction by N-hydroxysuccinimide ester (NHS). Under equal stoichiometry, the conjugation yield was extremely low for either reactions regardless of extensive reaction time. The comparability was carried out under excess linker with a slow kinetics condition (8℃and pH 5.8) and between the Schiff base formation-limited RA step and NHS reaction. The conjugation yield of RA was shown to be 2-5 times higher than that of NHS reaction for α-lactalbumin (~14.2 kDa) as well as Herceptin IgG (~150 kDa). The conjugation sites achieved by two reactions were identified to differ. For poly-conjugation with conjugated linker per protein (LPR) ~4, RA achieved a more homogeneous distribution of conjugation sites than those by NHS reaction. Moreover, NHS reaction could only last for 1-2 hours due to hydrolysis. In contrast, RA reaction follows pseudo-first order kinetics with a rate constant determined to be 0.06 and 0.04 LPR/hr for α-lactalbumin and Herceptin IgG, respectively. The kinetics was further manipulated to increase the conjugation yield and site homogeneity through gradual addition of linker reagents to the borate-premixed IgG solution. Under 4-hr titration with 500x excess linkers, four linkers mainly conjugated on the 2-3 solvent-exposed amine sites of Herceptin IgG were achieved using 2-3 times less reagents than NHS reaction. The conjugated Herceptin IgG by KRA was shown to conserve higher order structure and drug efficacy towards targeting cells. Compared to other specific conjugation methods, KRA holds great promises to achieve homogenous poly-conjugations with less cost.

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