Liu J, Zhao H, Volk K J, Klohr S E, Kerns E H, Lee M S
Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492, USA.
J Chromatogr A. 1996 May 31;735(1-2):357-66. doi: 10.1016/0021-9673(95)01054-8.
Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.
使用毛细管电泳（CE）和毛细管液相色谱（CLC）对单克隆抗体嵌合体BR96和相应的阿霉素（DOX）免疫缀合物进行了比较肽图谱分析。已开发出一种独特、高度灵敏且具有选择性的方法，该方法结合了紫外吸收和激光诱导荧光（LIF）检测，并应用于包括抗体和缀合物以及相关药物和缀合连接物的酶消化物的研究。基于抗癌药物DOX产生天然荧光的独特特性建立了分析方法。利用氩离子激光488 nm的激发波长，使用腙连接子与单克隆抗体缀合的DOX的检测限可达阿托摩尔水平。基于模型肽缀合物的成功缀合和分析开发了相关方法。通过CE和CLC结合在线紫外和LIF检测对单克隆抗体BR96及其免疫缀合物的酶消化物进行图谱分析，从而得到用于结构分析的独特指纹图谱。采用二维LC-CE方法，通过LIF检测的CE对来自LC的缀合肽-DOX物种进行进一步分析。混合物中含药物的肽片段易于检测，可使用其他互补分析技术进一步表征。
