2.2. Purification of Non-Deamidated and Single-Deamidated Species

ADC-A contained a mixture of double deamidated, single deamidated, and non-deamidated species. Separation of these materials was performed using Source 15S cation exchange resin (Cytiva, Marlborough, MA, USA). The resin was packed into a 2.2 cm × 20 cm Vantage Column (MilliporeSigma, Burlington, MA, USA) which was operated at 150 cm/h on an Akta Avant (Cytiva, Marlborough, MA, USA). Prior to separation, the resin was sanitized with 1 N sodium hydroxide and then equilibrated with 25 mM acetate pH 5.0. ADC-A was applied to the equilibrated resin at 20 g/L. The loaded resin was re-equilibrated prior to a 20 column-volume gradient elution to 25 mM acetate and 500 mM sodium chloride, at pH 5.0. Fractions were collected during elution and analyzed for protein concentration, purity, and deamidation. As expected, the material eluted from the column according to charge, with double deamidated species eluting prior to the single and non-deamidated materials. There were 60 mg of single deamidated product and 300 mg of non-deamidated product obtained, both with monomeric purities greater than 99.5%. All chemicals were JTBaker USP grade (VWR, Radnor, PA, USA).