Two continuous modes of SMBC useful for affinity purification are step and isocratic mode. The Step-SMBC mode establishes independent zones, each having a unique buffer condition for target capture, sorbent washing/contaminant removal, target elution, and column regeneration. The number of columns residing within a zone can be adjusted depending on the capacity and volumes required to achieve the desired separation.
The zones accomplish “steps” analogous to those in SC affinity protocols, but operated in a continuous cycle. The Step-SMBC mode is ideal for protein A or protein G purification of monoclonal antibodies. Figure 2 shows protein A purification of a monoclonal antibody from concentrated tissue culture fluid using POROS® MabCapture™ A resin.
Antibody recovery was 95%, purity was greater than 99%, and productivity of 200 mg/hr was achieved with 8 x 1 mL columns at 900 cm/h flow rate. With 8 x 5 mL columns and 2,000 cm/h flow rate, the productivity from a 1 g/L titer of monoclonal antibody culture fluid could exceed 2.8 g/h. These results represent significant reductions in column size, process time, and buffer consumption versus a single column protocol.
The Isocratic-SMBC mode utilizes a single eluent, and operating parameters (flow rates, switch times) are adjusted to preferentially slow the target protein migration through the matrix relative to nontarget proteins (raffinate). The countercurrent separation with the programmed advance of the product collection stream enables continuous peak shaving of the target molecule for optimum purity. Figure 3 shows IMAC purification of His-tagged proteins from bacterial cell lysates using Isocratic-SMBC. For three human kinase substrates, purity averaged 16% greater than that obtained by SC purification, and productivity averaged >60 mg/hr using 8 x 1.0 mL IMAC columns.