Dionex ultimate 3000 dad detector

Analytical protein A chromatography was performed using a TSK 5PW protein A column (Tosoh) with an inner diameter of 4.6 mm and a length of 3.5 cm. The column was connected to a Dionex U3000 (Thermo Fisher) equipped with a Dionex Ultimate 3000 DAD detector (10 mm pathlength) and a Dionex WPS‐3000 TSL Micro Autosampler. The column was equilibrated (30 mM potassium phosphate, pH 7.5, 150 mM NaCl) at 2 mL/min and 10 or 50 μL of filtered (0.2 μm, Millipore) sample was injected. After a wash step, the bound protein was eluted using 0.01 M HCl. The UV absorbance was monitored at 280 and 300 nm. For antibody peak determination, the 280 or 300 nm antibody peak was integrated and compared with a calibration curve to calculate mAb concentration. For purity determination, the flow through signal at 280 nm and the antibody peak were used to calculate the percentage of signal corresponding to the mAb.

Optical rotations were obtained on a JASCO P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were recorded on a Thermo Scientific Dionex Ultimate 3000 DAD detector, and IR spectra were taken on a Nicolet NEXUS 470 spectrophotometer as KBr disks (Thermo Fisher Scientific, Waltham, MA, USA). ¹H and ¹³C NMR, DEPT, and 2D NMR spectra were recorded on an Agilent 500 MHz DD2 (Agilent Technologies Inc., Santa Clara, CA, USA) using TMS as an internal standard. HRESIMS spectra were measured on a Bruker Impact HD microTOF Q III mass spectrometer (Bruker, Rheinstetten, Germany) using the standard ESI source. UHPLC-MS was operated using a Thermo Scientific Dionex Ultimate 3000 system coupled with the Bruker amazon SL Ion Trap mass spectrometry, controlled by Hystar v3.2 and Chromeleon Xpress software. A Thermo Scientific™ Acclaim™ C₁₈ column (2.1 × 100 mm, 2.2 μm) was used. The mobile phase consisted of H₂O and acetonitrile (ACN), both containing 0.1% formic acid. Semipreparative HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) was performed using an ODS column (Bruker ZORBAX SB-C₁₈, 9.4 × 250 mm, 5 μm, 3 mL min⁻¹). Vacuum-liquid chromatography (VLC) was carried out over silica gel H (Qingdao Marine Chemical Factory, Qingdao, China).

All chromatographic methods were carried out by UltiMateDionex 3000 SD. For preliminary evaluation of possible metabolites, the chromatograph was equipped with a Dionex UltiMate DAD 3000 detector (ThermoFisher Scientific, Milan, Italy). The latter parameters are summarized in Table 5.
The LS-MS method was performed on the aforementioned chromatographic system associated with a (HESI-II) Q Exactive Plus mass spectrometer (ThermoFisher Scientific^®). For separating the tentative metabolites, the octadecylsilane column was used with the mobile phase, which consisted of 0.1% formic acid in dist. water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The analysis was conducted by gradient elution with a constant 0.3 mL/min flow rate of 0–1.5 min, 5–10% B; 1.5–13 min solvent B linearly increased to 60% followed by 6 min isocratic elution; 19–23 min 60–95% B and finished with 95% solvent B an isocratic elution for 2 min for 25 min.