Affinity Chromatography

1. Affinity Chromatography Audrey Cheung Michael Kwong Marc-Andre Lynch Image source: Bio-Rad Laboratories

2. Function in process • Removal of contaminant proteins • Utilizes affinity of protein-A for the Mab to purify the product. • High efficiency – 90% • Protein-A effective for monoclonal antibody purification.

3. How it works • Equilibrate – add mixture to batch to bind target molecule (Mab) • Wash out mixture – rid of impurities • Add wash buffer • Remove wash buffer • Add elution buffer • Remove elution buffer containing target molecule • Regeneration of original conditions Image source:Wikipedia

4. Alternatives • Protein G – can have higher affinity for certain molecules however it is generally more expensive. • Protein L - used for purifying monoclonal antibodies from impurities. Can be ordered as ready-to-use prepacked devices for isolation and purification of immunoglobulin. • Melon Gel Support - binds non-antibody serum proteins (ex. albumin and transferrin) while allowing the antibody to flow through in a mild buffer. Offers a gentle way to purify antibodies from serum. Source: www.pierce.net

5. Current design • Parameters of interest for optimization: Volume load, mass load, flow rate, resolution, recovery, bed configuration, purity and cost.

6. Parameter investigation • Difficult to vary the yield likely due to an already high affinity for Mab. • Inverse relationship between batch time and linear velocity. • Effect of linear velocity on Mab/acetic acid ratio found to be negligible. • Increase in column height resulted in slight increase in batch time. • Increase in height decreased Mab/acetic acid ratio – consistent with theory that columns tend to be shorter and wider in scale-up process.