化学
色谱法
高效液相色谱法
大小排阻色谱法
单克隆抗体
毛细管电泳
十二烷基硫酸钠
抗体
生物化学
酶
生物
免疫学
作者
Wenhua Li,Bin Yang,Dongmei Zhou,Jun Xu,Wenjia Li,Wen-Chen Suen
标识
DOI:10.1016/j.jchromb.2017.02.019
摘要
Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) has been widely used to separate and quantify monoclonal antibody (mAb) fragments and impurities. Two fragments with different masses smaller than monomer were observed in the CE-SDS analysis of a recombinant IgG1 mAb treated at 40 °C for 28 days. Reversed phase liquid chromatography mass spectrometry (RP–LC–MS) and trypsin digestion peptide mapping followed by LC–MS/MS analysis were employed to conclusively demonstrate that these antibody fragments were the degradation products derived from a Ser–Ser peptide bond cleavage at the heavy chain complementarity determining region (CDR). The total percentage of the cleaved antibody heavy chain was increased from 1.1% in the control sample to 4.6% in the forced degradation sample stored at 40 °C for 28 days by using CE-SDS analysis. Native size exclusion liquid chromatography (SEC-HPLC) was used to analyze the fragment profile of this treated mAb but did not reveal fragments smaller than the monomer. However, a small fragment was detected using denaturing size exclusion liquid chromatography (dSEC-HPLC) and was confirmed to be the N-terminal fragment of the heavy chain cleaved at the CDR region using RP–LC–MS analysis. These results suggested that this N-terminal fragment may be closely associated with the rest of the cleaved antibody molecule through non-covalent bond interactions. Moreover, when dSEC-HPLC and RP–LC–MS were used to analyze the aggregate and monomer samples individually collected from SEC-HPLC, both samples contained the same amount of the cleaved N-terminal fragment. These results suggested that the CDR region cleaved fragments likely not be involved in aggregates formation of this antibody. This case study reiterated that a combination of orthogonal analytical methods plays a crucial role in comprehensively analyzing and characterizing antibody fragmentation.
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