Each system was run according to its respective purification and desalting protocols. Purified and desalted protein concentration was determined by SDS-PAGE with UV detection. Post-desalting yield and purity of protein were nearly identical for both systems while the dedicated system purification was capable of a nearly threefold increase in throughput without the complex setups required by the FPLC system.
Results were evaluated with respect to yield, buffer consumption, processing time, and method scalability. Conductivity measurements of the dedicated system were taken in situ while those for the dialysis required sample removal and measurement with a calibrated conductivity meter.
Both procedures successfully desalted the protein sample with equivalent recovery of protein >90%. Desalting by the dedicated system was 8–11 times faster than dialysis and consumed 1/20 the amount of buffer. Concentration of dialyzed protein was twice that of the dedicated system’s gel filtration product, an advantage which must be weighed against the dedicated system’s superior throughput, greatly reduced buffer consumption, and automated, scalable capability.
The manual system was selected for its ability to replicate dedicated system affinity and desalting column volumes, 1 mL and 10 mL, respectively. Results indicate that the dedicated automated system has higher protein yields and superior reproducibility compared to the manual method. Protein product purity of both systems is equivalent.
The dedicated system’s ability to automate the collection of purification data, an option not available with the manual system, simplifies evaluation of the effectiveness of the purification. Unlike the automated system, the manual method requires the user to remain continually on site to apply buffer and collect fractions. It also requires manual transfer of the purified protein to the desalting column and subsequent centrifugation. The dedicated system executes both affinity purification and desalting without user intervention.