In another study by Safavy et al.[25], the progress and the extent of the PTX-C225 conjugation reaction were followed by MALDI-MS. (ADC). The loading value of a drug on the conjugate is CCND3 defined as the average number of moles of that drug attached to a monoclonal antibody. The loading value is considered as the major quality feature of an ADC since it specifies the payload amount that reaches the tumor cells and can straightly alter safety and efficacy of the conjugate[1-8]. The UV/VIS spectroscopic analysis of the ADC is known to be the easiest procedure to determine this feature. The maximum absorbance values of the UV/VIS spectra of the drug and the antibody should be different to implement this procedure. The concentrations of mAb and drug can be calculated separately by solving two equations at the same time using the ADCs measured absorbance and the mAbs extinction coefficients at 280 nm and the drug at its max. Then the molar ratio can be determined, which refers to the moles of drug per mole of antibody. It is necessary to integrate the portion of the drug to the measured absorbance at 280 nm and any protein quota to the measured absorbance at the drug max[9-12]. The reliability of the spectroscopic method can also be confirmed by applying orthogonal techniques such as radiometric[13] and chromatographic[11] methods. According to the chemistry used for the drug-to-antibody conjugation, various methods have been introduced to determine the drug-to-antibody ratio (DAR). In the case of lysine amide Rebaudioside D conjugation, it would be difficult to separate conjugates by chromatography because of their high heterogeneity. Evidence has shown the application of mass spectrometry for the analysis of these ADCs[14]. UV MALDI-TOF method was one of the first instances of ADCs characterization by mass spectroscopy in the early 1990s in which a comparison was made between the mass spectra of intact conjugated mAbs and the related parent monoclonal antibodies. The results of this method were not desirable in terms of low mass accuracy for large molecules, and due to limited resolution, it could not supply resolution of Rebaudioside D various forms of ADCs with different drug loads. However, the mass change of the peak centroids was used to define the average DAR, and the peak configuration was applied to model the distribution[15]. LC-MS with electrospray ionization coupled to time-of-flight (TOF) or triple quadrupole mass detectors were used by previous investigations such as those focusing Rebaudioside D on the analyses of T-DM1 (trastuzumab-MCC-DM1) and thio-trastuzumab-DM[16], huN901-SPP-DM[17], and C242-DM4[18]. These techniques yield more stringent mass and resolution than can be gained using MALDI. The goal of this study was to compare the DAR values acquired from UV spectroscopy with the related values resulted from intact mass measurement by MALDI-TOF/TOF method. Rebaudioside D Actually, we attempted to show that in cases where ESI-TOF-MS is not available, intact mass measurement of conjugates by MALDI-TOF/TOF mass spectroscopy could be a reliable technique to calculate the DAR values of conjugates. For this purpose, three different linkers with different masses and length sizes (Table 1), including SMCC, SM(PEG)2, and SM(PEG)12, were applied to conjugate DM1 drug molecule to the trastuzumab antibody. Table 1 Physical properties of used linkers Molar0.1% solution Molarmilli molar =
Where MWConjugate, MWTrastuzumab, MWDM1, and MWLinker are the molecular weights of each trastuzumab conjugate, trastuzumab, DM1, and linker, respectively, and 115 is the molecular weight of the N-Hydroxysuccinimide leaving group of.