Protein a hitrap hp affinity column

Vectors encoding recombinant mAb 26.4 isoforms were produced as previously described (13 (link), 14 (link)). Briefly, cDNA fragments encoding the 26.4 variable H and L chain regions were codon optimized for mammalian expression and subcloned in-frame with the cDNA sequence of the human IgG1 constant H and L chain regions in pcDNA3.1 expression vectors. In addition to the mAb 26.4.WT, the effector silent variant 26.4.AAAG (L234A, L235A, N297A, P329G) and 26.4.H435A with reduced FcRn binding (15 (link), 16 (link)) were produced. mAb 26.4.WT was also produced containing altered N-glycans, low fucose (LF; 26.4.WT.LF), and high galactose (HG; 26.4.WT.HG). The mAb variants were produced using FreeStyle 293-F cells (Thermo Fisher Scientific, Waltham, MA). Variants with modified N297-linked N-glycans were produced by supplying a fucose decoy, 2-deoxy-2-fluoro-L-fucose, posttransfection to decrease fucosylation, or by adding 5 mM D-galactose (Sigma-Aldrich, Burlington, MA) and expression vector encoding β−1,4-galactosyltransferase 1 to the transfection mix to increase galactosylation. The mAb variants were purified using a Protein A HiTrap HP affinity column (GE Healthcare, Chicago, IL), and their integrity was verified by SDS-PAGE. Fc glycosylation, including galactosylation and fucosylation, was assessed by mass spectrometry.

The venom was a pooled sample obtained from three adult N. sumatrana captured in central Malaysia (Negeri Sembilan) and was supplied by Snake Valley (Seremban, Malaysia).
Resource S ion exchange column and HiTrap Protein A HP affinity column were purchased from GE Healthcare (New Jersey, USA). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate was obtained from Abnova, Taipei, Taiwan. Lichrosphere WP 300 C₁₈ reverse-phase column cartridge was purchased from Merck, New Jersey, USA. iBlot Gel Transfer stacks and iBlot blotting system were supplied by Invitrogen. Sephadex G-25 gel beads and all other reagents were purchased from Sigma – Aldrich (St. Louis, USA) or as stated in the methods.