Separation of three monoclonal antibody variants
To verify the high separation efficiency of the MCSGP process, the separation of three variants of a MAb has been used as
model system. The three MAb variants contain none, one, and two additional lysine groups at the C-terminal and cannot be separated
with sufficient yield on a preparative resin in a single batch column (Figure 7).
Figure 7. Gradient elution of variants with a shallow gradient on a single column (Merck Fractogel EMD COO, 30m). Dashed
line: summed concentration curve; markers: experimental data from offline analysis of fractions; solid lines: simulation;
blue squares: MAb variant F1; red triangles: MAb variant F2; green circles: MAb variant F3.
Figure 7 shows that the maximum purity of the intermediately eluting MAb variant F2 at 0% yield is approximately 80%. If instead
the MCSGP process is used with the same resin (Fractogel EMD COO, 30m), the yield can be increased to 93% at an even higher
purity (of about 90%) of the MAb variant F2 . Figure 8 compares the analytical chromatograms of the MAb variant F2 purified
with the MCSGP process and the feed mixture.
Figure 8. Analytical chromatograms of MAb variants. Blue: feed mixture; red: MAb variant F2 purified with MCSGP process
A highly pure fraction of the intermediately eluting MAb variant F2 can be obtained using the MCSGP process. If instead only
a single-column process is used, it is impossible to obtain a yield of 93% at a purity of 90% (Figure 7).
Numerical comparison with conventional processes
To quantify the performance gain of the MCSGP process, the process has been compared numerically with the following two conventional
chromatographic processes for the purification of biomolecules:
Figure 9. Multilinear solvent gradient
- single-column batch chromatography with multilinear gradients
- single-column batch chromatography with multilinear gradients and ideal recycling.
The purification of a polypeptide using reversed-phase chromatography has been used as a model system. For this purification
problem, a detailed study has been performed to develop a suitable isotherm and to determine the necessary model parameters.4
Although conventionally linear solvent gradients are applied, recent publications have shown the advantage of nonlinear6 or multilinear gradients.7 In this work, a four-step multilinear solvent gradient (Figure 9) is used for the batch and the ideal recycle process.
To improve the process performance of solvent gradient batch chromatography, product-rich fractions can be recycled to the
feed point. Figure 10 shows the flowsheet for ideal recycle process.
Figure 10. Batch chromatography with ideal recycling