Harris R J, Murnane A A, Utter S L, Wagner K L, Cox E T, Polastri G D, Helder J C, Sliwkowski M B
Department of Medicinal and Analytical Chemistry, Genentech, Inc., So. San Francisco, CA 94080.
Biotechnology (N Y). 1993 Nov;11(11):1293-7. doi: 10.1038/nbt1193-1293.
A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.
一种针对人表皮生长因子受体-2胞外结构域的重组抗体经过了详细的结构表征。通过回收两种胰蛋白酶肽形式证明了重链的异质性，在残基376处，一种含有谷氨酰胺，另一种含有预期的酪氨酸。随后的实验表明，Y376Q变体是在将抗体重链和轻链基因转染到中国仓鼠卵巢细胞过程中产生的。纯化抗体中Y376Q变体的水平（范围：27%至1%）与细胞年龄成反比。对建立的细胞系进行亚克隆，并通过对细胞提取物的聚合酶链反应分析和对纯化抗体的蛋白质分析发现其具有异质性。10%的亚克隆产生高水平的Y376Q变体，而90%的亚克隆产生仅具有预期重链序列的抗体。本报告证明了肽图谱作为评估重组细胞系遗传异质性的灵敏工具的实用性。
