Cordoba Armando J, Shyong Bao-Jen, Breen Deirdre, Harris Reed J
Late Stage Analytical Development, Genentech Inc., South San Francisco, CA, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Apr 25;818(2):115-21. doi: 10.1016/j.jchromb.2004.12.033.
Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.
单克隆抗体（MAb）的液体制剂通常在铰链区的木瓜蛋白酶切割位点附近发生片段化，产生Fab和Fab+Fc形式。本研究的目的是调查这种片段化是否由蛋白酶引起。将四种密切相关的单克隆抗体交换到含有NaCl的pH 5.2醋酸盐缓冲液中，并在-20℃、5℃、30℃或40℃下储存1个月。片段化产生的尺寸排阻色谱（SEC）峰级分通过电喷雾质谱分析以鉴定切割位点。还研究了蛋白质抑制剂或宿主细胞蛋白对片段化的影响。所有四种抗体的片段化程度相同，发生在重链铰链区的Ser-Cys-Asp-Lys-Thr-His-Thr序列中。由于Asp-Lys键切割产生的片段显示出两种相差18 Da的形式。一种具有以Asp结尾的铰链区序列的合成肽在孵育后未显示片段化或18 Da的损失。蛋白酶抑制剂不影响切割速率或改变片段化位点。降解不受宿主细胞蛋白含量的影响。片段化似乎是一个动力学过程，不是由低水平的宿主细胞蛋白酶引起的。
