Zeba 7 k mwco spin desalting column

C9-depleted serum and purified C9 standard were generated using affinity chromatography. Human serum was obtained from the Australian Red Cross Blood Service under QIMRB human ethics approval P2352. Serum from five healthy donors was pooled, diluted 1:1 with PBS then sterile filtered (0.22 µm). A 20 mL sample of the pooled serum was injected onto a 1 mL PBS-equilibrated HiTrap NHS-Activated HP affinity column (Cytiva, North Ryde, Australia) coupled to a monoclonal anti-C9 antibody [26 ]. C9-depleted serum fractions were collected in the flow-through and depletion confirmed by ELISA before pooling. The column was thoroughly washed with PBS before C9 was eluted with 5 mL of 0.1 M glycine, pH 2.5 and neutralized by addition of 0.5 mL of 1.5 M Tris, pH 8.8. Eluted C9 was desalted and buffer exchanged vs. PBS using a Zeba 7 K MWCO spin desalting column (Thermo Fisher Scientific) and concentrated using an Amicon ultra 10 K MWCO centrifugal filiter unit (Merck Millipore, Bayswater, Australia). C9 purity was assessed by SDS-PAGE and Western blot using a commercial anti-C9 antibody (ab17931, Abcam, Melbourne, Australia). Protein concentration of serum purified C9 was determined by BCA assay (Thermo Fisher Scientific).

All electrolyte solutions contained Optima™ LC/MS Grade water (Thermo Scientific PN W6500) and or Optima™ LC/MS Grade ACN (Fisher Chemical PN A955) solvents. Anolyte was an aqueous solution of 1% formic acid (ACROS Organics™ PN 27048). Catholyte was an aqueous solution of 1% diethylamine >99.5% Reagent Grade (Sigma Aldrich PN 47216) and the mobilizer was a 1% formic acid, 50% ACN, and 49% water.
A 500 mM cathodic spacer solution containing Free Base l‐Arginine ≥ 99.5% (Arg) (Sigma Aldrich PN 11009–100G‐F) was prepared by dissolving 0.870 mg of Arg powder into 10 mL of Optima™ LC/MS Grade water. A 200 mM anodic spacer solution containing Free Acid Iminodiacetic Acid 98% (IDA) (Sigma Aldrich PN 220000–500G) was prepared by dissolving 0.270 mg of IDA powder into 10 mL of Optima™ LC/MS Grade water. Spacer solutions were stored at room temperature.
Peptide amino acid sequences initially reported by Shimura were synthesized and individually dissolved in LC/MS grade water at 5 mg/mL to act as internal pI markers 19.
Prior to desalting, lyophilized mAb material was reconstituted with MS grade water. All mAb samples were desalted with a 0.5 mL Zeba™ 7K MWCO spin desalting column (Thermo Fisher Scientific PN 89882).