Ion exchange Chromatography Resins For Therapeutic Antibodies (mAbs) Purification

GALAK Chromatography Technology Co., LTD


The basic principle of ion chromatography is based on the interaction between the ionic groups on the surface of the chromatographic packing and the particles carried by the target molecules. Ion exchange (IEX) chromatography is the first step if protein A affinity chromatography is used as the first step in the purification process.

There are many factors that affect the effect of ion exchange chromatography on antibody separation, like the type of ion-exchange resins (cation-exchange or anion-exchange can be further subdivided into strong cation-exchange and weak cation-exchange, strong anion-exchange, and weak anion-exchange), the density of ion groups on the surface of packing and the three-dimensional distribution of ion groups, the type of substrate, porosity and pore size, etc.

The details are as follows.

1. Anion-exchange Chromatography Resins

Anion exchange (AEX) is one of the most common methods for the purification of McAb.

Because the isoelectric point of therapeutic monoclonal antibody is usually high, the buffer solution with pH of 7 ~ 7.5 is suitable. The monoclonal antibody molecules do not interact with the anion-exchange chromatographic packings in any way and are often referred to as the "fluid-through mode". Impurities, such as DNA, host cell proteins and Endotoxin, are negatively charged in this Ph range and interact strongly with the anion-exchange packings to remain on the column.

If a buffer with a higher pH is used, the monoclonal antibody can carry enough negative charge to remain on the anion-exchange column, known as the binding mode. However, too high pH has the potential to cause the monoclonal antibody molecules to mutate, so the binding mode is used less often than the flow-through mode. The anion exchange method can also remove virus in flow-through mode, but its disadvantage is that it is not effective to remove McAb and protein A. In the flow-through mode, the DNA and host cell proteins have been largely removed by the protein A affinity chromatography step, which requires the anion exchange chromatography packing to have a strong adsorption capacity for these impurity molecules, but it doesn't require a very high dynamic load. For McAb that can maintain stability at high Ph, the combination mode may be considered.

Weak-partitioning chromatography (WPC) is a new method to refine McAb with a combination mode. In this method, the target monoclonal antibody (McAb) was firstly bound to anion-exchange chromatographic column with suitable pH buffer, and then the McAb was equally eluted by salt solution with certain conductivity. Because the target monoclonal antibody has a higher isoelectric point than all the impurity molecules, the impurity molecules bind more strongly on the anion exchange chromatography column, i. e. , the target monoclonal antibody is eluted first and the impurity molecules are eluted later in the isoelution. It has been reported in the literature that this method can be used as a refining method to improve the flow-through mode of anion exchange chromatography for removing the defects of the McAb molecular polymer and the protein a molecule, at the same time, it maintains the advantage of flow-through mode to remove DNA, host cell protein and endotoxin.

The following table shows the parameters of some anion exchange chromatographic resins.

Main Parameters Of Anion-exchange Chromatography Resins

ProductsSuppliersLigandSubstrateParticle Size / umPore Diameter / Å
Super Q650 S/M/CTosohquaternary ammoniummethacrylate35/65/1001000
Q Sepharose FFGE Healthcarequaternary ammonium6% cross-linked agarose90n/a
Capto QGE Healthcarequaternary ammonium with aromatic structurehighly cross-linked agarose with dextran surface extender902000
Q Sepharose XLGE Healthcarequaternary ammonium6% cross-linked agarose with dextran surface extendern/an/a
Fractogel EMD TMAE

(M), Hicap

Merck-Milliporetrimethyl ammonium


methacrylate resin with polymeric “tentacles”65800
Unosphere QBio-Radquaternary ammoniumpolymeric120n/a
Q Ceramic Hyper DPallquaternary ammoniumceramic bead filled with ahydrogel50n/a
POROS HQ5ThermoFisherquaternized polyethyleneiminecoated cross-linked poly (styrene-divinylbenzene)501600
Sepromax Q40GALAKtrimethyl ammoniumcoated mono dispersed Cross-linked poly (styrene-divinylbenzene)401000

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2. Cation-exchange Chromatography Resins

Cation exchange (CEX) is also one of the most common methods for the purification of McAb. As mentioned earlier, the IEP of therapeutic MAbs is usually high, so the use of cation exchange in the McAb refining process is in the binding mode rather than the flow-through mode. This characteristic requires that the cation exchange chromatography resin must have excellent selectivity and dynamic loading, these two become the most important index to measure this kind of resin. In many applications, the cation-exchange method has shown better ability to remove host cell proteins, desquamation protein A and high molecular weight monoclonal antibodies than the anion-exchange method, in the current production of the therapeutic monoclonal antibody, macroporous polymer matrix strong cation exchange chromatography resin has been widely and successfully applied. The main reason is that these resins show better selectivity and higher dynamic loading, the researchers noted, it is the nonspecific hydrophobicity of the strong cation exchange chromatographic resins on macroporous polymer substrates that make them more selective. Ge Healthcare later specialized in introducing hydrophobic characteristics into ion-exchange chromatography resins and developed Capto and other mixed chromatography resin to fill this gap. These two important indicators can not be directly predicted by theoretical methods, must be measured by experiment, this is the user must carefully consider.

Like how to get the highest dynamic binding capacity. In the early days, it was generally believed that low buffer conductivity and pH should be the most favorable conditions for the interaction of McAb molecules with cation exchange chromatography resins. Later research proved otherwise. It has been reported that dynamic binding capacity can be increased by increasing the conductivity and Ph of the solution. One explanation is that, at low conductivity and pH, the McAb molecules first bound to the surface and pore of the filler are negatively charged to the later McAb molecules and charge repulsion is serious, resulting in a decrease in the loading; Under the condition of higher conductivity buffer and Higher pH, the repulsion between the negative charges decreases and the dynamic binding capacity increases. GALAK Sepromax S40 shows excellent performance in removing monomeric polymers. A comparison of DBC with POROS HS50 and POROS XS50, Sepromax S40 exhibits a higher DBC at 300 cm/h.

ion-exchange resin purify result
ion-exchange resin comparation

Main parameters for cation-exchange resins

ProductsSuppliersLigandSubstrateParticle Size / umPore Diameter / Å
SP 650 STosohsulfopropyl -SO32-Poly-methacrylate351000
SP 650 MTosohsulfopropyl -SO32-Poly-methacrylate651000
SP 650 CTosohsulfopropyl -SO32-Poly-methacrylate1001000
GigaCap S-650MTosohsulfo -SO32-Poly-methacrylate751000
SP Sepharose FFGE Healthcaresulfopropyl -SO32-6% cross-linked agarose90n/a
Capto SGE Healthcare-SO32- with aromatic structurehighly cross-linked agarose with dextran surface extender902000
SP Sepharose XLGE Healthcaresulfopropyl -SO32-6% cross-linked agarose with dextran surface extendern/an/a
Fractogel EMD SO3Merck-Milliporesulfoisobutyl -SO32-methacrylate resin with polymeric65800
Fractogel EMD SE (M), HicapMerck-Milliporesulfoethyl -SO32-“tentacles”65800
Unosphere SBio-Radsulfo -SO32-polymerIc120700~2000
S Ceramic Hyper DPallsulfo -SO32-ceramic bead filled with hydrogel50n/a
POROS HS5OThermoFishersulfopropyl -SO32-coated cross-linked poly (styrene-divinylbenzene)501600
Sepromax S40GALAKSulfopropyl -SO32-coated mono dispersed cross-linked poly (styrene-divinylbenzene)401000

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