We had to completely re-think the purification steps from start to finish in order to minimise time delays and eliminate the potential for losses. The affinity capture step worked reasonably well, but had to be optimised to maximise binding to the resin, and minimise elution volumes. This avoided the need for concentration (and the associated losses) prior to the size exclusion step. However, on larger size exclusion columns the protein was unnaturally retarded, and the yield was significantly less than expected. Moving to a smaller column length, increasing the salt concentration to counter-act ionic interactions, and doing shorter/faster runs allowed more of the protein to elute in an active, monomeric state. This set up required smaller load volumes, but this was accommodated by having the smaller elution volumes off the affinity resin.
The proportion of aggregated to monomeric protein increased with time (seen over a 6 hour period), as was clearly observed on the elution profiles from the size exclusion column (see UV elution profile below).
Adding glycerol to the running buffer also limited aggregation, and had the added bonus of stabilising the protein sample during the freeze/thaw process, allowing the protein to retain its monomeric state and good biological activity. This was confirmed by analytical size exclusion chromatography and activity assays.