N端测序作为单克隆抗体常规放行分析方法的探讨

N terminal sequencing for practical detection of monoclonal antibody

摘要:

旨在探讨N端测序作为单克隆抗体常规放行分析方法的适用性。应用Edman降解法、质量肽图法对两个针对不同靶点的单抗进行N端测序，用肽图法寻找二者的特征性鉴别肽段，用离子色谱、毛细管区带电泳和成像毛细管等点聚焦电泳进行异质性分析。Edman降解法显示两个单抗轻链和重链的15个氨基酸分别完全一致，质量肽图法显示二者轻重链的T1肽段分别完全一致，而肽图法和3种异质性分析方法则可对两个抗体进行有效鉴别。由于人源化或人源单抗序列框架数量较为有限，两个单抗的N末端序列完全相同，运用Edman降解法进行N端测序是否能作为单抗的常规放行分析方法值得进一步商榷，同时上述多种方法可运用于单抗的鉴别分析，并可对其异质性进行控制，较N端测序分析更具有客观性。

Abstract:

Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibody is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.