MALDI-TOF质谱准内标法及其用于rhTPO复杂糖一致性研究

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)准内标法准确测定重组人血小板生成素(rhTPO)仿制药和原研药中4类糖修饰的相对含量,并进行样本间和批间一致性比较。结合多种切糖酶逐步切除rhTPO的N-糖、唾液酸和O-糖;以牛血清白蛋白(BSA)为标准品,与样本非混合点靶;采用MALDI-TOF质谱准内标法检测,分别获得各样本完整含糖分子量及不同层次切糖后分子量;通过分子量差值计算4类糖修饰的相对含量;最后比较仿制药与原研药样本间和批间一致性。以BSA多电荷峰对rhTPO切糖前后的MALDI-TOF质谱测定值进行准内标法校正,BSA多电荷峰之间相对标准偏差(RSD)均≤0.075%,批间RSD为0.001%~0.004%,方法误差均0.01%。rhTPO仿制药N-糖、O-糖唾液酸、O-糖和糖化糖的相对含量分别约为24.6%、2.9%、9.0%和5.1%,批间一致性良好,且总糖含量与文献值一致;rhTPO原研药N-糖、O-糖唾液酸、O-糖和糖化糖的相对含量分别约为25.6%、2.9%、7.9%和3.5%,总糖含量较文献值偏低。与rhTPO原研药相比,仿制药糖基化糖相对含量基本一致,糖化糖含量有差异。rhTPO糖化糖研究未见文献报道,其差异是不同厂家rhTPO之间差异的重要因素,主要由发酵工艺差异导致。

MALDI-TOF MS with Quasi-internal Calibration Method and Its Study on Batch-to-Batch Consistency in Complex Glycan of rhTPO

The relative contents of four kinds of glycan forms in the inovator and biosimilar products of recombinant human thromboplastin (rhTPO) were accurately determined using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) with quasi-internal calibration,the consistencies between products and among batches were evaluated.The N-glycan,sialic acid and O-glycan of rhTPO were removed off with different endoglycosidase,respectively,bovine serum albumin (BSA)used as the standard was pointed target separate with the sample.The molecular weights of the intact glycoproteins and the different forms of deglycosylated proteins were determined by MALDI-TOF MS with quasi-internal calibration.The glycan contents in samples were calculated by the molecular weight differences among different deglycosylated samples.Finally,the consistency of the inovator and biosimilar products were evaluated.The multi-charge peaks of BSA were used for the quasi-internal calibration of MALDI-TOF MS.And their calibration relative standard deviations(RSDs) were all no more than 0.075％,and the RSDs among different calibrations were among 0.001％-0.004％.The method deviations away from the sequence molecular weight were less than 0.01％.For the biosimilar drug,the relative contents of N-glycan,sialic acid,O-glycan,and glycation were 24.6％,2.9％,9.0％ and 5.1％,respectively,and the batch-to-batch consistency was quite good.The total glycan contents were consistent with those reported in the literature.For the innovator drug,the relative contents of N-glycan,sialic acid,O-glycan and glycation were 25.6％,2.9％,7.9％ and 3.5％,respectively.The total glycan content was lower than that reported in the literature.The relative contents of glycosylation between the innoventor products and the biosimilar products were almost the same but the relative contents of glycation showed some differences.Glycation of rhTPO has not been reported up-to-now,the differences of the relative contents of glycation might be the important factors for the different rhTPO manufacturers,the glycation differences were mainly caused by the differences of fermentation process.