超高效液相色谱串联Qda质谱法检测单抗N糖的研究  

Analysis of N-linked Glycan Profile of Therapeutic Antibodies by UPLC-Qda MS

作　　者：王文波[1] 于传飞[1] 张峰[1] 徐刚领 高凯[1] 王兰[1] 

机构地区：[1]中国食品药品检定研究院单克隆抗体产品室,卫生部生物技术产品检定及标准化重点实验室,北京100050

出　　处：《中国药学杂志》2018年第3期223-227,共5页Chinese Pharmaceutical Journal

基　　金：国家“重点研发计划”生物安全关键技术研发专项资助项目(2016YFC1200904)

摘　　要：目的利用超高效液相色谱串联Qda质谱法对单抗的N糖谱进行定性和定量分析。方法对单抗Fc上的N糖链利用糖苷酶F进行酶切释放,然后用Rapi Fluor-MS染料进行标记,通过Qda质谱对N糖进行糖型鉴别、荧光检测器进行定量检测。结果超高效液相色谱串联Qda质谱法能够准确的对单抗N糖进行定性定量检测,单个糖型的实测m/z值在理论值±0.5内。利用该方法对原研抗CD20人鼠嵌合单抗和生物类似药候选药物的N糖进行分析表明,3个候选药物的N糖谱与原研抗体基本一致,但在部分糖型种类和比例上存在一定的差异,总的岩藻糖修饰糖型比例基本一致。对CHO细胞、NS0细胞和SP2/0细胞表达的单抗N糖进行比对分析显示,3种细胞表达单抗的主要糖型为G0F-N、G0F、Man5、G1Fa、G1Fb和G2F,占总糖型比例的80%以上。相比于NS0细胞和SP2/0细胞,CHO细胞表达抗体糖型种类相对较为简单,共11种糖型。而NS0细胞和SP2/0细胞表达单抗糖型种类和结构较复杂,分别检测到22和26种糖型,其中含有多种复杂的唾液酸和半乳糖修饰糖型,虽然这些糖型所占比例较低,但可能与单抗的体内半衰期和免疫原性相关。结论超高效液相色谱串联Qda质谱法作为快速准确的N糖分析方法,可应用于单抗N糖的质控研究中。OBJECTIVE To analyze the N-glycan profile of therapeutic antibodies by UPLC-Qda MS. METHODS The N- linked glycan in Fc region was released by PNGase F digestion and labeled by RapiFluor-MS, and labeled glycans were analyzed by UPLC-Qda MS. RESULTS The UPLC-Qda MS method showed good accuracy in N-glycan quantitaion and qualification, and the Am/z value of individual N-glyean was in a range of ＋ 0. 5. Three similar biotherapeutic products （SBP） showed a nearly same N-glycan profile with the reference biotherapeutic products （RBP） and the total percentage of fucosylated glycans was comparable, only one fraction of N-glycans had some difference. The major N-glycans of antibodies expressed by CHO cells, NS0 cells and SP2/0 cells were GOF-N, GOF, Man5, G1Fa, G1Fb and G2F, accounting for above 80% of total glycans. Eleven glycoforms were detected in CHO cell expressed antibodies, 22 and 26 glycoforms were detected in NS0 cell and SP2/0 cell expressed antibodies respectively. The N-glycans of NS0 cell and SP2/0 cell expressed antibodies contained more sialylated and galactosylated complex glycoforms, which was related to the antibody half-life in vivo and immunogenicity. CONCLUSION The HILIC UPLC-Qda MS, as a fast and accurate analytical method, can be used in the quality control of N-glycan profile of antibody.

关 键 词：单克隆抗体 N糖基化修饰 Qda质谱 生物类似药 抗体依赖的细胞介导的细胞毒性作用 

分 类 号：R917[医药卫生—药物分析学]