Xiang Tao, Lundell Edwin, Sun Zuping, Liu Hongcheng
Process Sciences Department, Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Oct 15;858(1-2):254-62. doi: 10.1016/j.jchromb.2007.08.043. Epub 2007 Sep 11.
IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.
IgG铰链区的肽键易受水解作用而降解。为研究Fab和Fc对铰链区肽键水解的影响，将一种重组人源化单克隆IgG1抗体、其F(ab')2片段以及一种氨基酸序列与铰链区相对应的模型肽在含有完全蛋白酶抑制剂和EDTA的配方缓冲液中于40℃孵育0、2、4、6和8周。在完整的重组人源化单克隆抗体及其F(ab')2片段的铰链区鉴定出两个主要切割位点，但在模型肽中仅鉴定出一个主要切割位点。完整抗体及其F(ab')2片段的铰链区肽键水解以相当的速率降解，而模型肽降解得更快。得出的结论是，IgG的Fab区而非Fc部分对通过直接水解防止肽键切割有显著影响。在酸性和碱性条件下，铰链区肽键的水解均会加速。
