Ghp acroprep 96 filter plate

Protein A affinity chromatography was used to determine the antibody concentration. For HPLC, we used a Dionex UltiMate 3000 HPLC system equipped with a diode array detector (Thermo Fisher Scientific). Mobile phase A was a 50 mM phosphate buffer with 150 mM NaCl, pH 7.0. Mobile phase B was a 100 mM glycine buffer, pH 2.5. All buffers were filtered through 0.22‐µm filters (GSWP04700; Merck KGaA) and degassed. The system was run at a flow rate of 2.5 ml min⁻¹. We loaded 20 µl of the sample, filtered through 0.2‐µm filters (0.2‐µm GHP AcroPrepTM 96 filter plate; Pall Life Sciences, Ann Arbor, MI), on a POROS A 20 µm Column (2.1 × 30 mm, 0.1 ml; Thermo Scientific). The column was washed with 10 column volumes of mobile phase A, eluted with 20 column volumes of 100% mobile phase B, and re‐equilibrated with 30 column volumes of mobile phase A. The absorbance at 280 nm was measured. We used a similar protein A‐purified IgG1 as the calibration standard. The calibration range was 0.1 to 3 mg mL⁻¹. We evaluated and quantified the results with the Chromeleon^TM 7 software (Thermo Fisher Scientific).

Before testing, the cell culture broth pH was measured. All experiments were performed in 96‐deep‐well plates. Dilutions from 1 to 12 mM of ZnCl₂ were prepared from a 100 mM solution and then added to 0.5 mL of the cell culture broth containing the antibody. After 20 min of incubation at room temperature on the end‐over‐end shaker (Stuart rotator SB3; Cole‐ Parmer, Vernon Hills, IL), the plate was centrifuged at 4000 rcf for 10 min (Centrifuge Heraeus Multifuge X3, Rotor HIGHPlateTM 6000; Thermo Fisher Scientific, Waltham, MA). The supernatant was withdrawn and filtered through 0.2 µm filters (0.2 µm GHP AcroPrepTM 96 filter plate; Pall Life Sciences, Ann Arbor, MI) and analyzed with protein A affinity chromatography (described in Section 2.3).