Fig 1.
(a) High purity is often sacrificed at the expense of recovery and vice versa depending on whether the impure fractions containing target products are kept or discarded in a batch elution process. (b) While a classical SMB operation can typically achieve a 2-component separation between the weakly bound species and the target, here we present an advanced SMB approach that can achieve a 3-component separation (right) [18]. The feed, antibody target, strongly bound impurities and weakly bound impurities are illustrated in brown, pink, blue and green respectively. As recycling of impure fractions from the previous step is an important feature of the advanced SMB approach presented here, the relative position of each component is illustrated for each step in 2 representative cycles of the steady state phase. CmAB, CmBC and CmS correspond to different conductivities required to elute the weakly bound impurities, target and strongly bound impurities respectively as outlined in Table 1 and inline dilution was performed with MilliQ water.
Table 1.
Optimised SMB CEX operation parameters applied on three 1.33 mL columns.
Fig 2.
Optimisation of key process parameters for batch CEX capture and subsequent polishing steps.
(a) 96-well plate screening of CEX load conditions. (b) Comparing the effect of different conductivities at pH 6, 2 min residence time (inset) and the effect of different residence times at pH 6, 5 mS/cm on the CEX loading breakthrough curves. (c) CEX AKTA gradient elution profile using 0–1 M NaCl 20 CV gradient elution at pH 6.0. (d) CEX AKTA step elution profile at 15 mS/cm, 20 mS/cm and 35 mS/cm, with the inset illustrating the relative percentage of HMW, LMW and monomeric species as well as the cumulative monomer recovery. (e) A detailed analysis of the profile of different subgroups of HMW, LMW, monomer species as well as HCP obtained using a 0–500 mM NaCl 20 CV gradient at pH 6.0. (f–h) 96-well plate screening of optimal conditions for polishing steps using AEX (f) and HIC resins (g) in FT mode, as well as optimal loading condition for multimodal chromatography resin (h). (i) The monomer and HCP elution profile obtained on the multimodal chromatography resin as a function of different conductivities obtained using 0 – 1 M NaCl 20 CV gradient elution at pH 6.0.
Table 2.
Process parameters of CEX, AEX, HIC and multimodal chromatography batch protocols.
A residence time of 2 min was used for all steps unless otherwise stated.
Table 3.
Evaluation of suitable polishing steps for SMB CEX eluate.
All polishing runs were performed with 1 mL columns.
Table 4.
Head to head comparison of the full purification process utilizing the SMB and batch CEX operations.
All CEX runs were performed with a total of 4 mL resin and all polishing steps with 1 mL resin.