Eluting Possibilities with Mixed-Mode Chromatography

Mixed-mode chromatography sorbents and custom ligands aim to optimize protein purification.
May 02, 2007
Volume 31, Issue 5

Changes in the biopharmaceutical industry have led to the need for new chromatographic techniques beyond traditional methods such as hydrophobic interaction chromatography, (HIC), ion exchange chromatography, and affinity chromatography. Protein expression titers and feedstock volumes have increased greatly during the past decade, and fermenter volumes of 12,000–15,000 L are not uncommon.

"Some of the technologies that were developed about 30 years ago are no longer optimal for some of these new challenges," says Sylvio Bengio, scientific communications manager for Pall Life Sciences (East Hills, NY, http://www.pall.com).

Conventional techniques, including the Protein A affinity, still work well. But the cost of these methods is weighing heavily and has led to increasing interest in alternatives, especially new sorbents for mixed-mode chromatography and specially designed ligands.

Improving mixed-mode sorbents

In principle, mixed-mode chromatography is not new. However, researchers now are able to specifically design and finely "tune" the hydrophobic dimension of the mixed-mode mechanism.

Converting a dilute feedstock to a high-purity protein typically involves three steps: a capture step, an intermediate step, and a polishing step, which may involve ion exchange, but there are situations where this is no longer ideal. One example is when processing large volumes of feedstream. Typically, to obtain sufficient capacity with conventional ion exchangers, the conductivity of the feedstock must be adjusted by dilution, sometimes to as much as five times the original volume. Although this practice is feasible at the laboratory scale involving 5- or 10-L volumes, working with a 10,000-L feedstream is another challenge. "Obviously diluting three or four or five times is totally unacceptable because buffer storage volumes create problems," says Bengio. "Ideally, at the capture step, you would like to have a minimal dilution of the feedstock and still get good capacity and recovery in one step; this is exactly what new mixed-mode platforms will do in most cases."

Monoclonals currently represent more than 35% of the proteins in clinical trials, and the current platform technology is affinity chromatography on Protein A. Protein A sorbents are very selective for capturing antibodies from complex feedstreams with high purity in just one step. One of the biggest problems with this ligand, however, is its cost. Some antibody processes may require several hundred kilograms of pure mono clonal for different treatments. "There is strong pressure on the industry to cut purification cost," says Bengio. "Chromatography and especially Protein A sorbents are one of the major contributors to purification cost that the industry wants to cut."

Mixed-mode chromatography also has been used in place of or in combination with HIC processes (i.e., at the initial HIC step, in which case the methods would be orthogonal). Although conventional HIC is well-known, broadly used, and effective at various stages in purification, it requires the addition of high concentrations of lyotropic salt, typically ammonium sulfate, to the feedstock to promote protein binding to conventional HIC resin. Concentrations can range from 1 to 3 M. At the laboratory or small scale, this is usually not a problem. "But in industry where there are thousands of liters of feedstream, adding massive quantities of salt is a nightmare because salt is expensive and creates hardware problems. Furthermore you have to recycle that salt for environmental protection purposes, and this increases the overall cost" says Bengio.

Russell Jones, chromatography marketing manager at Pall Life Sciences, notes that a company using conventional HIC sorbents, is likely to use several tons of lyotropic salt per year. "A process column containing hundreds of liters of conventional HIC sorbent will require the use of significant volumes of buffers containing 1–3 M lyotropic salt, especially over the course of an annual campaign of 30 cycles or more. This will not only have an impact on process cost but also on what you have to do afterward to dispose of the salt in an environmentally friendly way."

Purification methods: old and new

Not only is Protein A expensive, but it leaches and must be removed. Furthermore, the Protein A resin is not stable to 1 M NaOH, which is frequently used for cleaning. (Although GE Healthcare recently introduced "MabSelect SuRe," which is stable in 0.5 M NaOH.) Also, the method requires elution at low pH, which tends to cause aggregation and damage the antibody.

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