Establishment And Application Of Analytic Platform Of Physicochemical Characteristics For Therapeutic Monoclonal Antibodies

In recent decades,therapeutic monoclonal antibodies(to be called "mAb drugs" for short)have achieved significant curing effects including cancer,immune system diseases and organ transplantation rejection,owing to its high specificity,high affinity and low side effects.Moreover,mAb drugs always account for a half of the global top 10 best-selling drugs in recent years.The outstanding clinical performances,as well as excellent economic benefits,made mAb drugs undoubtly the focus and the hot spot of pharmaceutical drugs worldwide.Meanwhile,researchers are engaged in validating new techniques,developing new mAbs targeting various targets,and broadening their indications,to expand the application potentials of mAb drugs.As the development and manufacture of mAb drugs are gaining momentum,how to ensure the safty and effectivity of mAb drugs would be a big question for the regulators of setting quality control standards.The general chapter "Pandects of Therapeutic Monoclonal Antibodies for Human Use"(2015 Ch.P.)is mainly concerning six aspects of mAb drugs’ quality analysis as identification and consistency analysis including purity and related substances(size varients,charge varients,product related impurities,and process related impurities),glycosylation,potency,contents and others(osmolality,insoluble particles,abnormal toxicity,modified mAbs,etc.).They are the basic requirements raised by the Chinese drug regulatory authorities,however there is no specific methods.Our laboratory is experienced in mAb drugs’ quality analysis.Several platform techniques have been established in our lab,including bioactivity analysis platform,charge heterogeneity analysis platform,and mass spectrometry characterization platform.On this basis,as well as taking USP general principle <129> and Medicines Compendium "Rituximab" for references,our study further improves mAb drugs’ physical and chemical properties analysis platform in our lab.This platform is consisted of mAb identification,separation and determination of size and charge variants,and glycosylation modification analysis of mAb drugs.Then using these methods,seven batches of adalimumab injections were analyzed,and the comparabilities of original drugs and two biosimilars of rituximab were revealed as well.The results showed that these platform methods were suitable for the physical and chemical properties analysis of mAb drugs,and might be reference methods for mAb drugs’ quality control.Methods Qualification of USP General Principle <129>: There are 4 methods recorded in USP 40 General Principle <129>,including SEC-HPLC for the determination of monomers and high molecular weight substances(HMWS),CE-SDS for the determination of low molecular weight substances(LMWS),CE and HPLC for the analysis of N-glycosylation,and ion chromatography for the evaluation of sialic acid in mAbs.These methods were verified through system suitability tests and applied in 4 mAbs so as that the methods qualification was finished in our lab.Meanwhile,a whole column imaging detection-capillary isoelectric focusing(WCID-CIEF)method was established as a charge heterogeneity analysis method,and a UPLC method,which was more sensitive and stable,was investigated for the determination of sialic acid.These methods were applied in 4 mAb drugs and the method generality were verified as well.CQAs Analysis of Adalimumab in Physicochemical Properties: Several main physicochemical properties of adalimumab,including N-glycosylation,sialic acid level,size variants and charge variants were analyzed by the verified platform techniques established in the former study.Furthermore,their differences in post-translational modifications were investigated by using UPLC-Q TOF MS.The results showed that 7 batches of adalimumab were stable in charge heterogeneity and post-translational modifications,and no additional post-translational modifications appeared during the storage period.Preliminary Comparability Analysis of Rituximab Original Drug and Its Biosimilars: The physicochemical properties of rituximab original drug and two biosimilars were analyzed and compared by the validated platform methods as well as those recorded in USP Medicines Compendium "Rituximab".The results revealed that all CDR regions were identified by peptide mapping and their primary structures were basically consistent.Their pI values all met the requirements of 9.1~9.5,but there were some differences in acid(pI<main peak)and basic(pI>main peak)varients.This conclusion was consistent with the result of ion exchange chromatography(IEC)method.However,CIEF and IEC methods have different separation mechanisms.As a result,the relative quantitation results of charge varients in these two methods were not always the same.The N-glycosylation analysis showed that the main glycoforms in three rituximabs were G0 F and G1 F,and their relative contents in biosimilar A were more equivalent to the original drug.The total contents of glycoforms containing Fuc or terminal Gal in biosimilar B were significant less than those in the original drug,which inferred that biosimilar B might be glycoengineered.The sialic acid analysis by UPLC showed that the Neu5 Gc levels in three samples were relative low(<LOD),and the Neu5 Ac level in the original drug was much higher than its two biosimilars.The results of size varients revealed that the monomers content in the original drug were higher than those in the biosimilars.In biosimilar B,there was another obvious peak whose molecular weight was slightly smaller than that of the main peak,which suggested that the main peak might suffer partial degradation in biosimilar B.