抗CD52单克隆抗体HPLC-肽图分析方法的建立

Establishment of HPLC-Peptide Mapping Method for the Characterization of Anti-CD52 Monoclonal Antibody

建立高效液相色谱(HPLC)-肽图分析方法,用于抗人CD52单克隆抗体的专属性鉴别。抗CD52单抗样品经盐酸胍变性、DTT还原,释放出的游离半胱氨酸残基进行烷基化。超滤置换酶切缓冲液后进行胰蛋白酶酶切并终止。色谱条件:采用Eclipse XDB-C18 4.6×250 mm 5μm(Aglient)色谱柱,0.1%TFA水溶液与0.1%TFA乙腈溶液为流动相,梯度洗脱,检测波长为214 nm,柱温为30℃;质谱条件:分析时长135 min;检测方式正离子,TOF;MS+扫描范围350-1 500 Da;Product Ion+扫描范围100-1 500 Da;质谱分辨率40 000;Exceeds,150 Cps。CD52单抗重链CDR1、CDR3、轻链CDR1对应肽段由质谱鉴定出。HPLC-肽图方法专属性验证显示辅料制剂及异种抗体对检测结果无干扰;精密度验证结果显示目标峰的峰面积RSD%均在1.7%-7.6%之间。且目标峰的保留时间RSD%均在0.1%-0.2%之间,小于5%的可接受标准;耐用性结果显示,3μg胰蛋白酶、37℃和18 h的酶切条件是最合适的样品处理条件。基于CDR相关肽段鉴别的HPLC-肽图分析方法可定性鉴定出抗CD52单抗,方法学验证结果显示该方法适用于抗人CD52单抗的专属性鉴别并可用于质量控制及批检验放行。

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 conditionswere as :An Agilent HPLC column(Eclipse XDB-C18 4.6×250 mm 5μm)adopted using water solution(containing 0.1% trifluoroaceticacid)as the mobile phase A and acetonitrile solution(containing 0.1% trifluoroacetic acid)as the mobile phase B;gradient elution ofmobile phase A and B;detection wavelength 214 nm,30℃. The conditions for mass spectrometry were as :Data were obtained with positiveionization for 135 min;TOF MS+ scan range in 350-1 500 Da,Product Ion+ scan range in 100-1 500 Da,mass spectrometer resolution40 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 byexcipients 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 resultsof durability indicated that the conditions of 3 μg trypsase,37℃ and 18 h were the optimal. In a sum,by HPLC-peptide mapping CD52 m Abmay be qualitatively identified based on the CDR related peptides. The validation results of methodology reveal that the established method isappropriate to specific identification of anti-human CD52 mAb and can be used for quality control and batch release test.