Vydac® Process Scale Up of Protein & Polypeptides Application Note

Calculating the Desorbing Solvent or Salt Concentration 

Adsorption of proteins and polypeptides on reversed-phase columns is an all-or-nothing phenomenon. Their separation virtually always requires a gradient. Each polypeptide has a specific “desorption concentration” — the solvent concentration at which the polypeptide releases from the stationary phase and moves rapidly off the column.

In general, the shallower the gradient (D solvent concentration/D mobile-phase volume), the better the resolution of closely eluting polypeptides. In analytical and bench-scale prep, where the objective is to resolve many peaks, it is customary to run shallow gradients over a wide range of solvent concentrations. In process chromatography, however, the objective is usually to achieve maximum purification and throughput for a single target molecule.

The Recommended Strategy

(1) Load the sample in as dilute a solution as practical at a solvent concentration 10% to 20% below the desorption concentration for the target molecule. This minimizes aggregation but assures adsorbtion to the stationary phase.

(2) Elute the column using a very shallow gradient in the region of desorption of the target molecule. This shallow gradient can be preceded by a more rapid ramp to a solvent concentration just below the desorption concentration.

(3) Once the target molecule has eluted, ramp rapidly to a much higher solvent concentration to remove latereluting contaminants, then back to the starting concentration for loading the next sample. Determining the desorption concentration for a target molecule is an important step in designing a process purification. It is not as trivial as it may seem. Due to system delay volumes, the programmed gradient usually does not directly reflect the concentration at which a polypeptide is released. The following procedure will give the correct value.