Approximately 100 ng of intact mAb was injected onto a Waters Acquity I-Class UPLC Protein BEH C4 1 x 50 mm, 1.7 mm, part no: 186005589) using Acquity I-Class UPLC (Waters Corp.) at a flow rate of 0.250 mL/min. The column was kept at 70°C. Mass spectrometry grade HPLC solvents (0.1% Formic acid and B: 100% ACN in 0.1% Formic acid) were purchased from VWR (part no: LC 452-1, LC 441-1). The column effluent was introduced into a Waters Vion Qtof mass spectrometer via electrospray ionization using a spray voltage of 2.0 kV and cone voltage of 120 V. The resulting chromatographic peaks were integrated to generate summed mass spectra, which were deconvoluted by maximum entropy method (MaxEnt1) using Unifi software (Waters) to yield observed masses. The mass spectrometer was calibrated with NaCsI (Waters, part no: 700002646-8) within 1 ppm mass accuracy. As a secondary control, molecular weight of a commercial monoclonal antibody (Waters, part no: 186006552) was confirmed (within <30 ppm) prior to the experiment. The molecular mass data were interpreted using the theoretical mass of the mAb sequence considering modifications such as disulfides, N-terminal pyroglutamate and various glyco-forms. The glycol-forms were quantified using the relative ion currents.
Vion qtof mass spectrometer
Manufactured by Waters Corporation
The Vion Qtof mass spectrometer is a high-resolution, quadrupole time-of-flight mass spectrometer designed for accurate mass measurements and qualitative analysis. It features a high-performance ion source, a quadrupole mass analyzer, and a time-of-flight mass analyzer. The Vion Qtof provides precise mass determination and detailed structural information for a wide range of analytical applications.
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Lab products found in correlation
2 protocols using vion qtof mass spectrometer
1
Monoclonal Antibody Characterization by UPLC-MS
2
Synthesis and Characterization of Organic Compounds
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All commercial chemicals were purchased from Sigma Aldrich and were used as received. The reactions were monitored by thin-layer chromatography (TLC) carried out on silica gel plates using UV light as visualizing agent. Column chromatography was performed with silica gel (spherical, particle size 40 μm). NMR spectra and high-resolution mass spectroscopy (HMRS) were recorded at the n2STAR facility (Koç University). The 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a Bruker Avance III Ultrashield (500 MHz) spectrometer and were analyzed using Topspin. NMR chemical shifts are reported in parts per million with the solvent resonance as the internal standard (CDCl3, 1H: δ 7.26 ppm, 13C: δ 77.16 ppm; DMSO d6, 1H: δ 2.50 ppm, 13C: δ 39.52 ppm). Coupling constants (J) are reported in Hertz (Hz). Multiplicities are reported using the following abbreviations: s = singlet, brs = broad singlet, d = doublet, dd = double doublet, ddd = double double doublet, dt = double triplet, t = triplet, m = multiplet. The HMRS analyses were performed on a Waters Vion QTOF mass spectrometer.
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