Ultimate 3000 hplc system

SEC was performed on a Dionex UltiMate 3000 HPLC System using a Waters XBridge Protein BEH SEC 200Å, 3.5-μm column and a diode array detector collecting at 280 nm. Separation for mAb 1 and 3 and the Fc-fusion protein was achieved under native conditions with a 100 mM sodium phosphate, 250 mM sodium chloride mobile phase buffer at pH 6.8. MAb 2 was analyzed with a 10% acetonitrile v/v, 100 mM sodium phosphate, and 250 mM sodium chloride mobile phase buffer at pH 6.8. Results were analyzed using an in-house tool that automatically aligns and chooses peak regions to integrate. The alignment removes time-axis variations using a constrained version of correlation optimized warping,²⁵ in which the detected shifts in each window are robustly fit to a linear function. Integration regions are then selected by automatically identifying the discrete valley minimum between each peak response where a peak-to-valley height was larger than a noise threshold. For SEC, the raw response versus time was used to determine the peak and valley locations.

Separation was carried out using an UltiMate 3000 HPLC system and an Accucore^TM C₁₈ column (2.1 × 100 mm, 1.7 μm, Waters) maintained at 25 °C. The proportions of formic acid (A) and acetonitrile (B) averaged 0.1% (v/v) in the mobile phase. Based on the elution gradient, the analysis was conducted as follows: 0–4 min, 2–12% B; 4–10 min, 12–24% B; 10–20 min, 24–100% B; and 20–28 min, 100% B. The autosampler operated at 4 °C with an injection volume of 10 μL and a flow rate of 0.3 mL/min.
As an important feature of the UPLC-QE-Orbitrap-MS system, positive ion (ESI⁺) and negative ion (ESI⁻) electrospray ion sources were used within the scan range of 66.7 to 1000 m/z. The MS source parameters were set as follows: 35 irregular units of jacket gas flow, 15 irregular units of auxiliary gas flow and 1 irregular unit of purge gas flow. In the MS/MS mode, the impact energy was set to 10%, 20% and 40% of the normalized collision energy. Xcalibur software (version 2.2.42, Thermo Fisher Scientific, Waltham, MA, USA) was used to record and analyze data.