Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody.
Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody.
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揭示重组人源化单克隆抗体中的糖化热点。
DOI：
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发表时间：
2008
影响因子：
7.4
通讯作者：
Kathleen Francissen
中科院分区：
文献类型：
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作者：
Boyan Zhang;Yi Yang;Inn H. Yuk;R. Pai;P. McKay;C. Eigenbrot;M. Dennis;V. Katta;Kathleen Francissen
生物技术公司和监管机构正在追求蛋白质治疗剂的每一个细节的完整表征，作为减轻产品质量相关安全问题风险的手段。在重组人源化单克隆抗体（称为rhuMAb）的表征过程中，电喷雾质谱分析表明轻链高度糖化。使用硼酸盐亲和色谱分离的糖化和未糖化材料，使用胰蛋白酶肽图谱和串联质谱进行充分表征。使用该抗体的单蛋白数据库的自动SEQUEST搜索和匹配肽的质谱的广泛手动研究，在高度糖化（>53%）样品中观察到轻链和重链中的多个暂定糖化位点。通过使用Edman降解分析和MALDI-TOF/TOF质谱法对分离的糖化肽进行广泛的序列分析，鉴定了主要糖化位点，并确认为轻链上的赖氨酸49。rhuMAb与其他12种重组单克隆抗体的序列比对和rhuMAb Fab部分的计算机模拟表明，位于轻链第二互补决定区（CDR 2）附近的赖氨酸残基49的异常高水平糖化，是由于轻链上CDR 1中天冬氨酸残基31在催化Amadori重排中的空间邻近效应。
Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.