The IEX process is reversible and the ion exchanger itself can be regenerated or “loaded” by washing it with an excess of the ions that will be exchanged. For example, IEX is commonly used in water purification to produce soft water, whereby calcium and magnesium cations are exchanged against sodium and hydrogen cations.

The same principles of exchanging ions apply to protein, DNA or viral purification using IEX. The amphoteric nature of all proteins makes the IEX method useful because the net charge of any protein depends on the pH of the environment. The protein’s net charge is based on the positive and negative charges scattered across the surface according to the location and orientation of its amino acid residues. How these varying charges balance out to an overall positive or negative charge is expressed as the protein’s net charge. The pH value at which point the net charge is neutral is called the isoelectric point (pI); above the pI, the protein will be negatively charged, and below it, positively charged (Table 1, pg. 12). This simple fact is exploited by IEX such that all proteins will bind at some pH to either an anionic or cationic exchanger.

Since this binding is reversible, the elution buffer (eluent) is used to recover components from the matrix by altering their net charges. These fractions can be analysed either online or collected and evaluated later.

Although the science behind the technique is complex, the actual steps are relatively simple in theory (Fig. 1, pg. 12) — including column equilibration, sample application (and adsorption), column washing, elution of bound molecules, column regeneration and re-equilibration. These are common steps to all chromatographic processes utilizing resin-based reusable columns.

Isocratic elution makes use of a constant mobile phase elution buffer, whereas gradient elution changes the relative affinity by changing the conditions (i.e., pH or salinity) in a stepwise or continuous manner.

Displacement elution uses a substance with greater affinity for the stationary phase to kick out the bound molecules (similar to competitive binding). This introduces a new substance to the entire process and is not ideal when trying to purify materials for further use.

While there are many types of common chromatographic materials, the major classes of ion exchangers are cation and anion, which contain functional groups that can undergo reactions with cations and anions, respectively. Selecting the functional group relies on three main criteria:

1)     Net charge of the molecules of interest
2)     Molecular weight of the solutes
3)     Type of solvent system

For most applications, the ultimate goal is to achieve a “pure” product in as few purification steps as possible. This reduces costs and time required for a purification process, while retaining reproducibility. Traditional IEX resins pack into columns (Fig. 2a, pg. 10), and their capacity to bind proteins depends on the size and distribution of pores and the electrically charged molecular groups across the surface of each bead (Fig. 2b, pg. 10).

Membrane Adsorbers

IEX membrane chromatography is a technology that has recently been developed and it has some distinct advantages over traditional resins. The most significant effect of utilizing a membrane is the convective flow process through the macroporous adsorber compared to a resin. The chemical ligands are immediately available to bind any passing solute. Mustang® IEX membranes have 0.8-mm convective pores, thereby permitting faster flow rates without loss of binding. This, in turn, allows the researcher to increase the speed of purification or the volume of their process.

Pore size becomes even more limiting when trying to purify large molecules. Plasmid DNA, viruses and large proteins have low binding capacities to traditional resins because these particles are often too large to diffuse into the resin’s pore structure. Therefore, binding is limited to the active chemistry groups that are on the outer surface of the bead, accounting for less than 10% of the stated binding capacity of commonly used resins. The large pores in a macroporous membrane create channels for immediate availability of all molecules. Supercoiled plasmid DNA can be isolated and purified from bacterial lysate using membrane chromatography. This may prove to be very useful with the growth in gene therapy products and the need for larger quantities and very pure supercoiled plasmid material.

This limitation in current column chromatography technologies applies to the process of purifying any large molecule. Proteins over 500 kD — such as IgM and thyroglobulin, or other gene therapy products such as viruses — are challenging. The high resolution and prepacked format of membrane chromatography columns makes them ideal wherever there is a need for fast, efficient and reproducible isolation of these biological products. This benefit is further realized when evaluating the dynamic binding capacity and determining that membrane chromatography can offer up to 50% better binding capacity than analytical resins (15-mm particles), even with the higher suggested flow rates (Fig. 3).

Finally, any chromatographic column must be able to give reproducible results when operated appropriately. From the initial column pouring to the selection and preparation of the mobile phase to the final selection of pressure to be applied, one must consider what the matrix material is and how to best handle it. Air bubbles can cause problems with decreased flow rates and resolution.

Another advantage of membrane chromatography is that the capsules arrive preassembled. They have been engineered for high flow rates and maximum binding capacity. Air bubbles trapped inside a resin column may destroy the structure of the conventional column or at the least, decrease resolution. Even if an air bubble is introduced, it can be eased out of a membrane adsorber by an increase in pressure without any loss in resolution. In addition, no resin fines, which may clog the column, are produced. The polyethersulfone base material on which the chemistries are linked in a Mustang adsorber system has been commercially used in many macrofiltration products and has proven to be robust to pressure changes, pH variations and have a low non-specific absorptive profile. This gives us confidence that it will produce the best results possible in a chromatographic process.

In summary, membrane adsorber chromatography can be used to replace many traditional IEX applications. The binding capacities are comparable to traditional resins, while the flow rates are 10 to 100 times faster. One is therefore not limited by the sample size but rather by the true binding capacity of the chosen membrane absorber capsule. Using membrane adsorber technology, viral, protein and plasmid purification processes can be accelerated without loss of purity. This will reduce process times and costs, and will also speed R&D and product development. Membrane IEX chromatography gives us another tool in both the lab and the production plant to allow us to reach our isolation and purification goals faster and with greater cost effectiveness.

Yardenah Brickman, PhD is senior manager, Scientific and Technical Support, at the Life Sciences division of Pall (Canada) Ltd. (Mississauga, ON). After completing her undergraduate degree at York University (Toronto,ON), Brickman completed her PhD at the University of Melbourne (Melbourne, Australia) in 1996. She then managed a neurological research laboratory at the Alfred Hospital in Melbourne before returning to Canada in 1998. Upon her return she worked on a post-doctoral research project with funding from the Medical Research Council (now CIHR). Brickman provides assistance and expertise for a wide range of laboratory and pilot-scale applications, membrane chromatography, and nucleic acid affinity membranes. She can be reached at 905-542-0330; e-mail: ardenah_brickman@pall.com”>yardenah_brickman@pall.com.