Masshunter qualitative analysis program

The sequences of the purified mAbs and respective F(ab’)₂ and Fc fragments were evaluated by LC-MS using a PoroShell 300SB-C8 column (5 µm, 75 × 1.0 mm) on the Agilent HPLC system followed by analysis in the Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies). The composition of the mobile phase A was 99% water, 1% acetonitrile, and 0.1% formic acid, and that of mobile phase B was 95% acetonitrile, 5% water, and 0.1% formic acid. The gradient started with 20% B at 0 min and increased to 85% B at 10 min with the constant flow rate of 50 µL/min. Each sample was subjected to a native run, a reduced run after incubation with TCEP (Sigma), and a deglycosylated run after incubation with TCEP and PNGase F (New England Biolabs). The MassHunter Qualitative Analysis program (version B.06.00, Agilent, Santa Clara, CA, USA) was used to deconvolute the raw data.

Data processing was performed with the Agilent MassHunter Profinder 10.0 software program for deconvolution, alignment, and integration, using the recursive feature extraction (RFE) algorithm. This algorithm performs a deconvolution of the chromatogram and integration of the molecular characteristics present in the samples according to mass and retention time. The data obtained from the deconvolution and integration were filtered by area by calculating the total area for the sample and then the area of each molecular feature. The annotation of the more abundant molecular features obtained was carried out using the CEU MASS MEDIATOR tool (https://ceumass.eps.uspceu.es/ (accessed on 1 October 2021)) [47 (link)], including the Metlin, Kegg, HDMB, and LipidMaps platforms as parameters, and with a tolerance of 10 ppm. Then, MS/MS analyses were performed in order to confirm the identity of the metabolites using MS-DIAL 4.8 (http://prime.psc.riken.jp/compms/msdial/main.html (accessed on 1 October 2021)), in in silico mass spectral fragmentation through CFM-ID 4.0 (https://cfmid.wishartlab.com/ (accessed on October 2021)) and manual MS/MS spectral interpretation using the Agilent MassHunter Qualitative Analysis program (version 10.0, USA).

A Synergi 2.5 μm Fusion-RP 100 Å LC column 100x2mm, particle size 2.5 (Phenomenex) with corresponding guard column was used for all experiments. LC-MS/MS was completed using an Agilent 6410 tandem triple quadrupole mass spectrometer connected to an Agilent 1100 HPLC system with 5 mM ammonium acetate pH 5.3 in water (buffer A) and acetonitrile (buffer B). LC-MS-grade ammonium acetate and acetic acid (Fisher Scientific) were used to generate buffer A, and buffer A was stored as a 1000x stock solution at -20°C and diluted within one week of use. The flow rate was 0.350 mL/min throughout and separation was achieved over a 5 minute gradient from 3 to 5 percent B (S3 Table).
Column oven temperature was held at 35°C throughout the runs. UV traces were monitored at 270 nm and desired analytes were monitored using MRM mode (fragmentor 165 V, dwell time 200 ms, collision energy 20 V). Analytes measured and their MRM parameters are indicated in S2 Table. Note that in cases where only one analyte was being measured, the program did not include the other analytes. Source parameters were gas temp 350°C, gas flow 9 L/min, nebulizer 40 psi, capillary 4000 V (positive ion mode), and the electron multiplier voltage was set to 550 V. Injection volume was 10 μL per injection. All data processing and integration was completed using the Agilent MassHunter Qualitative Analysis program.