This article presents the multicolumn countercurrent solvent gradient purification (MCSGP) process, which uses three chromatographic
columns, and incorporates the principle of countercurrent operation and the possibility of using solvent gradients. A MCSGP
prototype has been built using commercial chromatographic equipment. The application of this prototype for purifying a MAb
from a clarified cell culture supernatant using only a commercial, preparative cation exchange resin shows that the MCSGP
process can result in purities and yields comparable to those of purification using Protein A. The second application example
for the MCSGP prototype is the separation of three MAb variants using a preparative weak cation-exchange resin. Although the
intermediately eluting MAb variant can only be obtained with 80% purity at recoveries close to zero in a batch chromatographic
process, the MCSGP process can provide 90% purity at 93% yield. A numerical comparison of the MCSGP process with the batch
chromatographic process, and a batch chromatographic process including ideal recycling, has been performed using an industrial
polypeptide purification as the model system. It shows that the MCSGP process can increase the productivity by a factor of
10 and reduce the solvent requirement by 90%.
The increasing production volumes of biomolecules, especially therapeutic proteins, and a rising cost pressure from the market,
has engendered a strong interest in the chromatographic purification step in the downstream processing of biomolecules.1–2 This is because the chromatography step is often irreplaceable and also a major cost driver in downstream downstream.
The generic purification problem to be solved, particularly in the area of chromatographic bioseparations, is a separation
of the feedstock into three fractions: the early eluting, weakly adsorbing impurities; the desired product(s), and the late
eluting, strongly adsorbing impurities. A literature overview of chromatographic three-fraction separation processes can be
found elsewhere.3 Conventionally, the generic three-fraction purification problem is solved using batch column chromatography, often incorporating
a linear variation of the eluting composition with respect to the elution time (also called solvent gradient chromatography).
The column effluent is collected in several consecutive, small portions and some of these portions ideally contain the desired
product with an average purity being higher than the required one, thereby making up the product fraction. To increase the
yield of solvent gradient chromatography, product-rich portions collected from the column effluent, which do not fulfill the
purity specifications, can be recycled to the feed point. The advantages of such a recycle have been discussed elsewhere in
Besides batch solvent gradient chromatography and its derivative including a recycle, the multicolumn countercurrent solvent
gradient purification (MCSGP) process is a third suitable process developed recently to perform three-fraction separations
of biomolecules.3–5 This process involves three columns and continuously purifies a complex feed mixture into three fractions, thereby incorporating
Conventional multicolumn processes such as simulated-moving-bed (SMB) chromatography cannot be used for the purification of
biomolecules, because an SMB can only perform a binary separation and solvent gradients can not be implemented properly. SMB
chromatography is therefore more suitable for a binary separation of small molecules.
This work explains the principle of the MCSGP process and shows experimental results from the MCSGP prototype for purifying
a MAb from clarified cell culture supernatant (cCCS) without using protein A resin, and a separation of three MAb variants.
A numerical comparison between the conventional batch chromatography process and the MCSGP process with respect to yield and
process productivity is also performed for a polypeptide purification. The Pareto curves obtained from the optimization procedure
are compared and the performance superiority of the MCSGP process is explained.
Principle of the MCSGP Process
The generic problem in the chromatographic purification of biomolecules can be simplified to the chromatogram as shown in
Figure 1. Simplified chromatogram of generic chromatographic purification problem (time axis from right to left); S: strongly
adsorbing, late eluting impurities; P: product; W: weakly adsorbing, early eluting impurities.