CONTINUOUS PROCESSING: The Multicolumn Countercurrent Solvent Gradient Purification Process - A continuous chromatographic process for monoclonal antibodies without using Protein A - BioPharm

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CONTINUOUS PROCESSING: The Multicolumn Countercurrent Solvent Gradient Purification Process
A continuous chromatographic process for monoclonal antibodies without using Protein A


BioPharm International


Description of Prototype

Two prototypes of the MCSGP process have been built using commercially available chromatographic equipment from GE Healthcare. In order to realize the process (Figure 3), the following parts from the "Aekta Basic" series have been used for each prototype: three double pumps, one UV detector, one conductivity and pH sensor, and six multiposition valves.4 The system is controlled by Unicorn software. The prototype also includes the possibility of performing cleaning-in-place.

Experimental Results

Purification of a MAb from cCCS using cation-exchange resins

The use of MAbs as therapeutic proteins is one of the strongest growing industry segments. Currently, only a few MAbs are on the market, but hundreds are in the pipelines. Because of the increasing production volumes, chromatographic downstream processing using key purification step Protein A affinity resins is becoming a bottleneck from an economic and engineering point of view.


Figure 4. SEC chromatogram (280 nm) of cCCS (black curve) and pure MAb standard (red line)
Purifying MAbs from cCCS without using Protein A resin is difficult if only single-column chromatographic processes are used. Because the MCSGP process provides a very high separation efficiency, resins which are less specific than Protein A can be used.

The aim of this work was to purify a MAb from a cCCS using the MCSGP process with three columns filled with a commercial, preparative cation-exchange resin (Merck FractoGel SO3-HiCap, M). The supernatant had a low titer of about 0.01 g MAb per liter cCCS and a feed purity of <1% at 280 nm. Figure 4 shows the SEC chromatogram of the cCCS (black line).


Figure 5. SDS-PAGE of MAb from cCCS purified by the MCSGP process and by Protein A
The impurities by far exceed the MAb, as revealed by the comparison with the SEC chromatogram of the pure MAb (red line in Figure 4). Using the MCSGP process (Figure 3), the MAb, being an intermediately eluting product on a cation-exchange resin, can be obtained with high purity from column 2 in the batch mode. The MAb collected from the MCSGP process has been analyzed on a SDS-PAGE and shows comparable purity levels to those of the MAb purified from the same cCCS with Protein A (Figure 5). The recovery of the MAb in the MCSGP process for the shown SDS-PAGE is 95%.

The results of comparable purities of the MCSGP-purified and the Protein A–purified MAb have been double checked with an SEC analysis (Figure 6).


Figure 6. SEC Chromatogram of Protein A–purified MAb (black line) and MCSGP-purified MAb (red line)

From the results above, we can conclude that the highly specific, but expensive Protein A purification step in the downstream processing of MAbs (together with the following batch column cation exchange step), could be replaced by the three-column MCSGP process using comparably cheap cation-exchange resins.


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