Fragmentation of a recombinant monoclonal antibody at various pH
Fragmentation of a recombinant monoclonal antibody at various pH
复制标题
DOI：
10.1007/s11095-008-9606-3
复制
发表时间：
2008-08-01
影响因子：
3.7
通讯作者：
Liu, Hongcheng
中科院分区：
文献类型：
--
作者：
Gaza-Bulseco, Georgeen;Liu, Hongcheng
目的。为了确定直接水解和β消除的相对重要性，抗体铰链区断裂的两种常见机制，以及不同PH下保守的N-连接寡糖对抗体断裂的影响。重组单抗在40℃不同pH的缓冲液中孵育5周。用体积排阻色谱(SEC)测定碎裂程度。用液相色谱质谱(LC-MS)分析SEC组分，确定特定的裂解部位。酸性和碱性pH对直接水解度有促进作用，而在pH为7及以上时，β-消除有助于铰链区的碎裂。此外，随着样品pH从9降低到5，铰链区的主要肽键水解点向C末端方向移动。在pH为4时，主要裂解部位移出铰链区，定位于CH2结构域。在pH值为5~9的范围内，低聚糖不影响铰链区的碎裂，但在pH值为4时，低聚糖可减缓CH2结构域的碎裂。PH值对抗体的裂解程度、部位和机制有影响。低聚糖仅在pH为4时影响裂解速率。
Purpose. To determine the relative importance of direct hydrolysis and beta-elimination, two common mechanisms of antibody hinge region fragmentation, and the impact of the conserved N-linked oligosaccharides in affecting antibody fragmentation under various pH.Methods. A recombinant monoclonal antibody was incubated in buffers of various pH at 40 degrees C for 5 weeks. The level of fragmentation was measured using size-exclusion-chromatography (SEC). The specific sites of fragmentation were determined by analyzing SEC fractions using liquid chromatography mass spectrometry (LC-MS).Results. Direct hydrolysis was accelerated by acidic and basic pH, while beta-elimination contributed to hinge region fragmentation at pH 7 and above. In addition, a shift of the major peptide bond hydrolysis sites in the hinge region towards the C-terminal direction with the decrease of sample pH from 9 to 5 was observed. At pH 4, the major cleavage site shifted outside the hinge region and was localized in the CH2 domain. Oligosaccharides did not affect hinge region fragmentation in the pH range of 5-9, however, at pH 4 oligosaccharides slowed down fragmentation in the CH2 domain.Conclusions. Antibody fragmentation level, sites and mechanisms were affected by pH. Oligosaccharides only affected the rate of fragmentation at pH 4.