All publications and patent applications referenced herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
1. Field of the Invention
The invention relates to external gradient chromatography. More particularly, the invention relates to novel methods for the separation of charged molecules such as proteins according to their isoelectric points (pI's) and includes the systems and buffering compositions employed for separating such charged molecules.
2. Description of the Related Art
Chromatofocusing is a form of anion exchange chromatography that was first described by Sluyterman and co-workers (see e.g., L. A. AE Sluyterman and O. Elgersma, Journal of Chromatography A, 150(1), 1978, 17-30; L. A. AE Sluyterman and J. Wijdenes, Journal of Chromatography A, 150(1), 1978, 31-44; L. A. AE. Sluyterman and J. Wijdenes, Journal of Chromatography A, 206(3), 1981, 429-440; L. A. AE. Sluyterman and J. Wijdenes, Journal of Chromatography A, 206(3), 1981, 441-447; L. A. AE. Sluyterman and C. Kooistra, Journal of Chromatography A, 470(2), 1989, 317-326). In chromatofocusing, a retained pH gradient is automatically generated inside a chromatographic column as a Polybuffer species or other buffering species in the elution solution titrates functional groups on the surface of a weak anion exchanger resin.
Numerous variations of the original Sluyterman approach to chromatofocusing have been published. Most of these techniques are based on a strategy of pre-equilibrating a weak ion exchange column with a starting buffer at one extreme of an intended internal pH gradient. A sample of charged molecules, typically proteins, is introduced at the column entrance. At the initial pH, all of the molecules to be separated have a charge opposite that of the ion exchange resin and thus bind immediately to it. In the discussion below, the molecules to be separated are referred to as proteins, but that should be construed as a preferred aspect. The resin is then perfused with a solution consisting of multi-component buffers at a desired final pH. Due to variations in the binding affinity of the buffering species for the weak ion exchange resin, a retained pH gradient is automatically created inside the column. The gradient on a weak anion exchange resin is an ascending (as opposed to descending on a weak cationic exchange resin) gradient in pH along the length of the column, but a monotonically descending (ascending for weak cationic exchange resin) pH gradient in time. The separation process begins when the final pH buffer enters the column and causes immediate release of a protein because its pH is below the isoelectric point (pI) of that bound protein. The protein then travels through the column until it reaches a pH in an evolving retained pH gradient that is higher than the protein's pI whereupon the protein rebinds to the ion exchange resin. The elution of the protein continues when the monotonically descending pH of the eluent front again reaches the protein's pI and the protein ceases to bind to the ion exchange resin. The protein once again travels down the column until it encounters a pH higher than its pI and again rebinds. This process is continuously repeated until the protein emerges from the column at its pI. Any protein molecule lagging behind the main band will be at a pH such that it has a charge of the same sign as the buffering groups bound to the ion exchange resin. This situation causes the protein to move down the column more quickly than the band itself due to the electrostatic repulsive forces between the identically charged protein and the ion exchange resin-bound buffering groups. Likewise, a protein molecule diffusing ahead of the main band will experience an increase in binding affinity for the ion exchange resin and will consequently move slower than the main band. The total result is a powerful focusing effect that, under optimal conditions, allows separations of proteins whose pI's differ by as little as 0.02 pH units.
The effective range of pH of the most widely used chromatofocusing technique utilizes commercially available special Polybuffers (e.g. Pharmalyte 8-10.5, Polybuffer 96 and Polybuffer 74) and weak exchange resins (e.g. Mono P, PBE94 and PBE 118) and is about 3 pH units, typically in the ranges 9 to 6 or 7 to 4. The Polybuffers are expensive and bind strongly enough to proteins to make their removal from the purified protein a significant problem. As a result, this potentially extraordinarily valuable fast purification technique has been limited to a laboratory purification technique that finds little practical application in bulk industrial protein purification.
Several groups have tried to overcome these limitations with relatively simple buffer solutions as eluents but still employing either specially designed or commonly available weak anion exchanger resins. The most successful of these systems (Logan et al, Biotechnology and Bioengineering, 62(2), 1999) utilizes a two component elution buffer of common, easily removed buffer components useful down to a pH as low as 5.0 to create an evolving retained gradient in a weak anionic column with an initial pH as high as 9.5. This system works well enough over the pH range described to show that high volume chromatofocusing is feasible. There is, however, no external control of the gradient generated in Logan's method.
The few publications that have urged the use of external gradients and a small number of inexpensive buffers in combination with weak anionic columns report a maximum effective pH gradient range of 3.5 units, which is 20% less then that reported by Logan et al. (see e.g., Yansheng Liu and David J. Anderson, Journal of Chromatography A, 762(1-2), 1997, 207-217; Yansheng Liu and David J. Anderson, Journal of Chromatography A, 762(1-2), 1997, 47-54; Lian Shan and David J. Anderson, Journal of Chromatography A, 909(2), 2001, 191-205; Ronald C. Bates et al., Journal of Chromatography A, 890(1), 2000, 25-36; Xuezhen Kang et al., Journal of Chromatography A, 890(1), 2000, 37-43; Douglas D. Frey et al., U.S. Pat. No. 5,851,400); Jan Walther-Rasmussen and Niels Høoby, Journal of Chromatography B, 746(2), 2000, 161-172).
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