Assessment of antibody fragmentation by reversed-phase liquid chromatography and mass spectrometry
Assessment of antibody fragmentation by reversed-phase liquid chromatography and mass spectrometry
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DOI：
10.1016/j.jchromb.2008.10.015
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发表时间：
2008-12-01
影响因子：
3
通讯作者：
Lundell, Edwin
中科院分区：
文献类型：
--
作者：
Liu, Hongcheng;Gaza-Bulseco, Georgeen;Lundell, Edwin
抗体在铰链区和其他区域的片段化。通过反相（RP）液相色谱和质谱（LC-MS）研究pH对抗体片段化水平和模式的影响。在40 ° C下在不同pH的缓冲液中孵育10周的重组单克隆抗体的铰链和铰链以外的区域中观察到广泛的片段化。易于水解的肽键主要位于靠近环结构或在环结构中的结构域-结构域界面周围。的网站以及水平的肽键水解的缓冲液pH值的影响。在协议与以前的研究结果时，只有铰链区片段进行监测，pH 6是最佳的减缓小丑抗体片段的区域以外的铰链。它还表明，与分子排阻色谱（SEC）随后进行馏分收集和质谱鉴定相比，RPLC-MS分析可以更好地评估重组单克隆抗体的易感区域。(C)2008 Elsevier B. V.保留所有权利。
Antibody fragmentation in the hinge region and other regions. and the impact of pH on the level and pattern of antibody fragmentation were investigated by reversed-phase (RP) liquid chromatography and mass spectrometry (LC-MS). Extensive fragmentation was observed in the hinge and in regions other than the hinge of a recombinant monoclonal antibody that was incubated in buffers of various pH at 40 degrees C for 10 weeks, Peptide bonds that were susceptible to hydrolysis were located mainly around the domain-domain interfaces close to or in the loop structures. The sites as well as the level of peptide bond hydrolysis were affected by the buffer pH. In agreement with previous findings when only the hinge region fragmentation was monitored, pH 6 was optimal for slowing clown antibody fragmentation in regions other than the hinge. It also demonstrated that analysis by RPLC-MS provided a better assessment of the susceptible regions of recombinant monoclonal antibodies than size-exclusion chromatography (SEC) followed by fraction collection and mass spectrometry identification. (C) 2008 Elsevier B.V. All rights reserved.