Rapid Analysis of Intact Antibody-Drug Conjugates (ADCs) By UPLC Q-TOF MS Wei Jia1*, ChuanFei Yu2*, Kai Gao2 1 Waters corporation, Shang Hai, China; 2 China National institutes for Food and Drug Control, Bei Jing, China INTRODUCTION RESULTS It is generally known that, antibody-drug conjugates (ADCs) are very important tools for targeted therapy. The structure characterization of ADCs is very important for its quality control, but there are many challenges due to its structure complexity. In this work, using QTOF MS with a 12 min long LC method, lots of Zoom in structure information of ADCs can be successfully acquired. Comparing to traditional UV method for drug-antibody ratio (DAR) analysis, and fluorescence The average mass error was 3.2Da (standard deviation 2.35Da) for all 36 peaks (each Dn group contains 4 different MW peaks due to glycosylation), considering the sample’s huge complexity it is an astonishing result. method for antibody glycan analysis, the LC QTOF MS method can give the DAR value and glycan form information simultaneously in a short time, which is very Because there was no deglycosylation procedure, the glycan information, types of glycan and their ratio, can be found in MS data. It was found that, the four main kinds of glycan were G0F&G0F, G0F&G1F, G1F&G1F (or G0F&G2F), G1F&G2F. Their ratios were 19.14%, 36.58%, 33.31%, 10.97% respectively (average value from group D0 to D8). The average mass difference of different Glycosylation peaks in each group was 161.44Da (standard deviation 3.72Da), which number is quite close to the theoretical value to value of Galactose. The good precision of the data is very helpful for judging the glycosylation patterns. important for effective acquiring reliable quality control data of ADCs. METHODS Instrument: Deconvolution by BiopharmaLynx ACQUITY UPLC®, Xevo® G2 QTof UPLC Methods: MassPREPTM Micro desalting column (Waters) H2O with 0.1% FA (phase A), In this work, using Xevo G2 QTof MS with a 12 min long LC method, the MS raw data of Trastuzumab linked DM1 was successfully acquired. Then, the raw data was treated by BiopharmaLynx to get the intact ADC molecular weight values. Primarily, drug-antibody ratio (DAR) and glycan information were shown. There were 9 groups of peak for Trastuzumab due to different number of conjugates. The measured DAR value 3.6 by QTOF is quite similar to 3.5, which was measured by traditional UV method. Zoom in Comparing to traditional UV method for DAR analysis, and fluorescence method for antibody glycan analysis, this robust LC QTOF MS method can give the DAR value and glycan form information simultaneously in a short time, which is very important for effective acquiring reliable quality control data of ADCs. ACN with 0.1% FA (phase B) 0-0.5 min, Phase B 5%, 0.5 mL/min; 0.5-12min, Phase B 5-90%, 0.2 mL/min MS Methods: Full MS range 500m/z-3500m/z Software: BioPharmaLynxTM 1.3 CONCLUSION Basing on the good performance of the benchtop MS system Xevo G2 QTof, the reliable drug-antibody ratio and glycan information can be obtained in a very shot time for antibody-drug conjugates analysis.
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