Sephacryl s 200 high resolution column

Saporin-S6 was purified from the seeds of Saponaria officinalis [19 (link)]. Anti-CD20 rituximab/saporin-S6 IT and anti-CD22 OM124/saporin-S6 IT were produced as described in [15 (link),16 (link)]. Briefly, mAbs and saporin-S6 were dissolved in 50 mM sodium borate buffer, pH 9.0 and were derivatized by adding 2-iminothiolane (Sigma-Aldrich, St. Louis, MO, USA), as described in [56 ]. MAbs and the reduced RIP were allowed to react for 16 h at room temperature. The resulting conjugates were separated from RIP homopolymers and free antibody by gel filtration on a Sephacryl S-200 high-resolution column (100 cm × 2.5 cm) (GE-Healthcare, Buckinghamshire, UK), equilibrated, and eluted with phosphate-buffered saline (PBS, 0.14 M sodium chloride in 5 mM sodium phosphate buffer, pH 7.4).
The immunoconjugates were analyzed under non-reducing conditions by SDS-PAGE on a 4–15% PhastGel gradient, and then stained with Coomassie brilliant blue and analyzed, as described in [16 (link)]. Molecular weight markers were from Sigma: myosin (205 kDa), b-galactosidase (116 kDa), phosphorylase B (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa).

prGALNS was purified from a culture medium of a pPIC9-nspGALNS with SUMF1 co-expression culture. Culture medium (~1.7 L) was filtered sequentially through Whatman™ paper No. 1 and 42, and 0.45 and 0.22 μm polyether sulphone membranes (Pall Corp, Port Washington, NY, USA). Permeate was ultra-filtered through a 30 kDa cut-off membrane (Millipore, Billerica, MA, USA), up to reach a final volume of 20 mL, and adjusted to pH 4.5 with 25 mM sodium acetate. Finally, the retentate was dialyzed overnight at 4 °C against 25 mM sodium acetate buffer (pH 4.5) with constant stirring. prGALNS was purified by a two-step chromatography process as previously described for recombinant GALNS produced in E. coli30 . Briefly, a cation exchange chromatography was performed with a Macro-Prep High S support column (Bio-rad, Hercules, CA, USA), equilibrated with 25 mM potassium acetate, pH 4.5, and eluted with a linear gradient of 0–0.5 M NaCl. Fractions with GALNS activity were pooled and applied to a Sephacryl™ S-200 High Resolution column (GE Healthcare, Uppsala, Sweden) with 25 mM potassium acetate, pH 4.5, 10 mM NaCl, in a flow rate of 0.5 mL min⁻¹. Finally, fractions with the highest GALNS activity were pooled and lyophilized. Protein purification was monitored by SDS–PAGE under reducing conditions and GALNS enzyme activity.