Capto q

Purification of 18/12/TxM was from clarified CHO cell culture supernatant by the following steps. Supernatant was loaded onto a MabSelect SuRe (from Cytiva, Marlborough, MA, formerly GE Healthcare) equilibrated with 4 volumes of 50 mM Tris 1M NaCl, pH 7.0, followed by washing the column with 4 volumes of the same buffer. The column was further washed with 4 volumes of 0.1M sodium citrate, pH 5.0 to remove contaminating proteins. The 18/12/TxM protein was eluted off the column with 0.1M glycine-HCl, pH 3.5. The eluted protein was then adjusted to pH 3.6 with 1M citric acid and incubated at room temperature for 1 h to inactivate viruses. A 1M Tris base was then added to this sample to bring the pH to 4.2 and then loaded onto a Capto Q (from Cytiva) column equilibrated with 4 volumes of 50 mM sodium acetate, pH 4.2, followed by washing the column with the same buffer until A280 reached to baseline. Protein peak (containing 18/12/TxM) of the Capto Q column flow through during sample loading and a wash step was collected and then formulated in 0.1M glycine and 0.2M sorbitol, pH 4.0. Sodium dodecyl sulfate (SDS) PAGE and size-exclusion chromatography–high-performance liquid chromatography (SEC-HPLC) analysis indicated that 18/12/TxM is at least 90% pure, with approximately 70% of protein existing as a dimer and rest as oligomers.