Reagents and solvents used were obtained as at least reagent grade from commercial sources and used without further purification unless noted otherwise. Moisture or air-sensitive reactions were conducted under an argon atmosphere in an oven-dried (120 °C) glass apparatus. The solvents were removed under reduced pressure using standard rotary evaporators. Flash chromatography was carried out on a Biotage Isolera One (Charlotte, NC); while analytical thin-layer chromatography (TLC) was performed using precoated TLC silica gel 60 F254 aluminum sheets purchased from EMD (Gibbstown, NJ) and visualized using UV light. Reaction monitoring, compound characterization, and purity analysis were performed using a 1260 Infinity/6420 Triple Quad (Agilent Technologies, Inc., Santa Clara, CA) with a Supelco Discovery HS C18 column (Sigma-Aldrich). All the compounds were identified to be at least 98% pure by UV. Compounds of interest were analyzed by high resolution MS (HRMS) using an Agilent 6230 ESI-TOFMS (Santa Clara, CA). 1H NMR spectra were obtained on a Varian Mercury 400 (Varian, Inc., Palo Alto, CA). 13C HNMR spectra were obtained on a Varian VX 500 equipped with a Varian XSens 2 channel NMR Cold Probe (Varian, Inc., Palo Alto, CA). The chemical shifts are expressed in parts per million (ppm) using suitable deuterated NMR solvents.
Agilent 6230 esi tofms
Manufactured by Agilent Technologies
Sourced in Canada
The Agilent 6230 ESI-TOFMS is an electrospray ionization time-of-flight mass spectrometer. It is designed to accurately measure the mass-to-charge ratio of ionized molecules.
Automatically generated - may contain errors
4 protocols using agilent 6230 esi tofms
1
Analytical Techniques for Compound Characterization
2
Analytical Techniques for Compound Characterization
3
Intact Mass Measurement of Proteins
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The pooled fraction at a 0.4 mg ml−1 concentration was buffer exchanged into 0.1% formic acid using 10 kDa MWCO centricon (Pall Corporation, USA). Intact mass measurements on these fractions were made on AdvanceBio RP mAb C4 (4.6 × 100 mm, 5 μm, Agilent Technologies) column operated at 80 °C using Agilent 1290 Infinity Quaternary LC system coupled online to Agilent 6230 ESI-TOF–MS. 5 μg sample was loaded on the column and separated using a 35 min linear gradient from 2 to 60% B at a flow rate of 0.5 ml min−1. Detection was performed by monitoring UV absorption at 280 nm and TIC was recorded for 1000–6000 m/z. MS spectra were calibrated in the positive ion mode before analysis. The capillary gas temperature/voltage (Vcap) was set to 350 °C and 5500 V, respectively, and the fragmentor voltage (Vfrag) was 400 V. The MS spectra were deconvoluted using the maximum entropy (MaxEnt) algorithm with resolution of 20,000 and 8 iterations.
4
Characterization of Bevacizumab Charge Variants
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The subunit analysis of the bevacizumab was carried out by separating the light chain and heavy chain. The sample preparation involved solubilizing the isolated charge variants (50 µg) with 30 µl of 6 M guanidine followed by the addition of 3 µl of 0.5 M DTT to a final concentration of 50 mM. The samples were reduced by incubation at 55 °C for 1 h and finally equilibrated to room temperature before subjecting to LC–MS analysis43 (link). LC–MS analysis consisted of separating the light chain and the heavy chain of bevacizumab charge variants on an Agilent AdvanceBio RP mAb C4 (4.6 × 100 mm, 5 μm, Agilent Technologies) column operated at 60 °C using Agilent 1290 Infinity Quaternary LC system coupled online to Agilent 6230 ESI-TOF–MS. The mobile phase used for the separation consisted of buffer A (water + 0.1% formic acid) and buffer B (Acetonitrile + 0.1% formic acid) at a linear gradient from 2%B to 65%B in 30 min. MS spectrum was acquired in the mass range 500–3000 Da in positive mode. Other MS parameters included capillary gas temperature/voltage (Vcap) was set to 250 °C and 4500 V, respectively, and the fragmentor voltage (Vfrag) was 280 V. The MS spectra were deconvoluted using the maximum entropy (MaxEnt) algorithm with resolution 20,000 and 9 iterations.
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