Editing MEP Resin (section)

== Optimization<br> ==

Antibody Purification – General Considerations<br>• Antibody binding and optimization<br>– Antibody-containing sample can be loaded directly onto MEP HyperCel AcroSep columns without preliminary desalting or concentration. Capacities of ≥ 20 mg/mL are generally obtained. Prior to sample loading, equilibrate the column with PBS or buffer, with pH and salt concentration equivalent to sample.<br>– During preliminary studies, it is recommended that loading be conducted at a low to moderate flow rate of 0.2-0.5 mL/min. Maximum capture efficiency is<br>achieved when the residence time is around 5-7 min. <br>– Check the pH. For most antibodies, maximum capacity is obtained when pH is between 7.4 and 9.5. Binding capacity is generally independent of pH in this<br>range <br>– Salt concentration. Although the majority of antibodies do not require the addition of salt beyond physiologic concentrations (~150 mM, Figure 2) for<br>good binding, this is not true for all antibodies. If binding is below the stated capacity and increased capacity is required, consider adding an additional<br>200-400 mM salt to promote binding. There will be a concomitant increase in impurity binding as well, but the increase in antibody binding is generally larger Elute bound antibody using a dilute buffer at pH 4. Buffers typically used include 50 mM sodium acetate or 20 mM sodium citrate (2-10 CV). For highly<br>pH sensitive proteins, pH up to 5.8 can be used, but elution may be less efficient. Elution will be more efficient in the absence of salt.<br>– All eluted antibodies should be neutralized as quickly as possible after elution in order to maintain the structural integrity.<br>– To improve purity of eluted antibody, elution optimization is recommended. A step-elution sequence using buffers of decreasing pH (e.g. 5.5, 5.0, 4.5, 4.0,<br>and 3.0) is effective. 50 mM sodium acetate or 20 mM sodium citrate are typically used as elution buffers. Elution at each step should be continued for<br>2-4 CV, or until complete elution of an observed peak is accomplished. Based on analysis of collected fractions, a second step-elution experiment at narrower pH increments or at alternative values may be useful. Ultimately, the elution procedure would be simplified to a three-step sequence. First, at the highest pH, elution of basic impurities would be accomplished. Second, at the optimized, intermediate pH, the target protein would be recovered. Fin<br>ally, at the lowest pH in the sequence, acidic impurities, aggregates, and misfolds would emerge. Depending on the characteristics of the acidic and hydrophobic impurities, this final step may be conducted at pH 3.0. It is useful to conduct a final wash at pH 3.0 to elute any remaining impurities, if the column is to be regenerated for use with additional samples.<br>