Abstract: This work is to establish a HPLC-peptide mapping method for specific identification of anti-human CD52 monoclonal antibody（CD52 mAb）. After denatured by guanidine hydrochloride and reduced by DTT,the free cysteine sulfhydryl of CD52 mAb was alkylated. Then digestion buffer was replaced by ultrafiltration,trypsase was digested,and the digestion reaction was terminated. HPLC conditions were as：An Agilent HPLC column（Eclipse XDB-C18 4.6×250 mm 5μm）adopted using water solution（containing 0.1% trifluoroacetic acid）as the mobile phase A and acetonitrile solution（containing 0.1% trifluoroacetic acid）as the mobile phase B;gradient elution of mobile phase A and B;detection wavelength 214 nm,30℃. The conditions for mass spectrometry were as：Data were obtained with positive ionization for 135 min;TOF MS+ scan range in 350-1 500 Da,Product Ion+ scan range in 100-1 500 Da,mass spectrometer resolution 40 000,and Exceeds in 150 Cps. The peptide fragments corresponding to heavy chain CDR1,CDR3 of CD52,and light chain CDR1 of CD52 were identified by mass spectrometry. The specific verification by HPLC-peptide mapping showed that the detection result was not affected by excipients and heterologous antibody. The results from precision verification demonstrated that the RSD% of the target peak area was in 1.7%-3.8% and the RSD% of relative retention time of the target peak was 0.1%-0.2%,which were less than 5% of acceptable standards. The results of durability indicated that the conditions of 3 μg trypsase,37℃ and 18 h were the optimal. In a sum,by HPLC-peptide mapping CD52 mAb may be qualitatively identified based on the CDR related peptides. The validation results of methodology reveal that the established method is appropriate to specific identification of anti-human CD52 mAb and can be used for quality control and batch release test.

Key words: monoclonal antibodies, peptide map analysis, HPLC, mass spectrometry