重组单抗药物的肽图分析

Peptide Mapping of Recombinant Monoclonal Antibody Pharmaceuticals

建立了重组单抗药物的肽图分析方法。在变性条件下向抗体溶液加入还原剂,打开抗体内部所有交联的二硫键,再加入烷基化试剂封闭所有的自由巯基,使抗体分子在溶液中以游离伸展肽链的形式存在。加入胰蛋白酶,将充分伸展的肽链酶解成小的肽段。用反相高效液相色谱层析分析肽图谱。对连续3批中试产品及理化测定对照品进行肽图分析,各样品均能酶解完全,批间肽图谱一致。该方法实用有效,适于进行重组单抗等结构复杂的大分子蛋白药物的肽图检查分析。

A method was established for peptide mapping of recombinant monoclonal antibody pharmaceuticals.In denaturing conditions,reducing reagent was added to recombinant antibody solution,all the crosslinked disulfide bonds were broken,and then alkylation reagent was added to block the free sulfhydryl residues,the resultant antibody molecules stayed in the solution as dissociated and extensively stretched peptide chains.The trypsin was added to the solution and the antibody was digested to small peptide fragments.Reversed phase HPLC was used to separate e the peptide fragments.The in-house reference and three consecutive batches of recombinant antibodies manufactured in pilot scale were analysed,all the samples could be digested completely and the peptide map profiles were consistant with each other.The method for peptide mapping was effective in proteins such as recombinant antibodies with complex structure.