Separation of three monoclonal antibody variants
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.
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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 8. Analytical chromatograms of MAb variants. Blue: feed mixture; red: MAb variant F2 purified with MCSGP process
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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.
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
Figure 9. Multilinear solvent gradient
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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:
- 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.
Figure 10. Batch chromatography with ideal recycling
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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.
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