Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1998-12-30
2001-05-29
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S413000, C530S416000, C530S427000, C210S198200, C210S635000, C210S656000
Reexamination Certificate
active
06239262
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to displacement chromatography of proteins in hydrophobic interaction and reversed phase liquid chromatographic systems using low molecular weight, surface active-compounds as displacers.
BACKGROUND OF THE INVENTION
Proteins have become commercially important as drug candidates and in other therapeutic applications. One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large scale preparation of proteins, crude products contain not only the desired product but also closely related impurities which are difficult to separate from the desired product. Moreover, biological sources of proteins usually produce complex mixtures of materials.
The term “protein” as commonly understood in the art, and as used herein, refers to peptides having a molecular weight of 10,000 or greater. Peptides having a molecular weight of less than 10,000 are known as polypeptides.
Ion exchange systems chromatographic systems have been used widely for separation of proteins primarily on the basis of differences in charge. Chromatographic systems having a hydrophobic stationary phase offer an alternative basis for separations and have also been widely employed in the purification of proteins. Included in this category are hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC). The physicochemical basis for separation by HIC and RPLC is the hydrophobic effect; proteins are separated on a hydrophobic stationary phase based on differences in hydrophobicity.
Although both HIC and RPLC utilize a hydrophobic stationary phase, the composition of the surface of the hydrophobic or non-polar stationary phase differs. Materials used as a stationary phase in RPLC have what can be considered a “hard” hydrophobic surface, characterized by high interfacial tension between the surface and an aqueous mobile phase. In contrast, the surface of the stationary phase in HIC is composed of a hydrophilic organic layer having weakly non-polar groups attached. This is termed a “soft” surface. In addition, the surface density of hydrophobic groups is generally higher in RPLC systems Man in HIC.
The different surface characteristics of the two systems mandate the use of different mobile phases so that the interaction between the solute and the stationary phase results in a retention time which falls within the range of practical values for the separation. Thus, the mobile phases employed in HIC are typically aqueous with high salt concentrations; most proteins have a higher retention at higher salt concentration. Those used in RPLC are aqueous with organic modifiers, such as acetonitrile and methanol; most proteins have lower retention with higher organic modifier concentrations.
A chromatographic system can be operated in one of two major modes, elution (including linear gradient, step gradient, and isocratic elution) or displacement. The two modes may be distinguished both in theory and in practice. In elution chromatography, a solution of the sample to be purified is applied to a stationary phase, commonly in a column. The mobile phase is chosen such that the sample is neither irreversibly adsorbed nor totally unadsorbed, but rather binds reversibly. As the mobile phase is flowed over the stationary phase, an equilibrium is established between the mobile phase and the stationary phase whereby, depending on the affinity for the stationary phase, the sample passes along the column at a speed which reflects its affinity relative to the other components that may occur in the original sample. The differential migration process is outlined schematically in
FIG. 1
, and a typical chromatogram is shown in FIG.
2
. Of particular note is the fact that the eluting solvent front, or zero column volume in isocratic elution, always precedes the sample off the column.
A modification and extension of isocratic elution chromatography is found in step gradient chromatography wherein a series of eluants of varying composition is passed over the stationary phase. In reversed phase chromatography, step changes in the mobile phase modifier concentration (e.g., acetonitrile) are employed to elute or desorb the proteins.
A schematic illustrating the operation of a chromatographic system in displacement mode is shown in FIG.
3
. The column is initially equilibrated with a buffer in which most of the components to be separated have a relatively high affinity for the stationary phase. Following the equilibration step, a feed mixture containing the components to be separated is introduced into the column and is then followed by a constant infusion of the displacer solution. A displacer is selected such that it has a higher affinity for the stationary phase than any of the feed components. As a result, the displacer can effectively drive the feed components off the column ahead of its front. Under appropriate conditions, the displacer induces the feed components to develop into adjacent “squarewave” zones of highly concentrated pure material. The displacer emerges from the column following the zones of purified components. After the breakthrough of the displacer with the column effluent, the column is regenerated and is ready for another cycle.
An important distinction between displacement chromatography and elution chromatography is that in elution chromatography, desorbents, including for reversed phase chromatography, organic mobile phase modifiers such as acetonitrile, move through the feed zones, while in displacement chromatography, the displacer front always remains behind the adjacent feed zones in the displacement train. This distinction is important because relatively large separation factors are generally required to give satisfactory resolution in elution chromatography, while displacement chromatography can potentially purify components from mixtures having low separation factors. The key operational feature which distinguishes displacement chromatography from elution chromatography is the use of a displacer molecule. In elution chromatography, the eluant usually has a lower affinity for the stationary phase than do any of the components in the mixture to be separated, whereas, in displacement chromatography, the eluant, which is the displacer, has a higher affinity.
Displacement chromatography has some particularly advantageous characteristics for process scale chromatography of biological macromolecules such as proteins. First, displacement chromatography can concentrate components from mixtures. By comparison, isocratic elution chromatography results in product dilution during separation. Second, displacement chromatography can achieve product separation and concentration in a single step. Third, since the displacement process operates in the nonlinear region of the equilibrium isotherm, high column loadings are possible. This allows much better column utilization than elution chromatography. Fourth, displacement chromatography can purify components from mixtures having low separation factors, while relatively large separation factors are required for satisfactory resolution in desorption chromatography.
Preparative chromatography operated in the displacement mode is therefore a potentially attractive method for purifying proteins because of the high resolution and high throughput obtainable. However, the use of high molecular weight displacers has been a deterrent to the implementation of this technology. In contrast, low molecular weight displacers have significant operational advantages as compared to large molecular weight displacers. First and foremost, if there is any overlap between the displacer and the protein of interest, these low molecular weight materials can be readily separated from the purified protein after the displacement process using size-based purification methods, e.g., size exclusion chromatography or ultrafiltration. Furthermore, the relatively low cost of low molecular weight displacers can be expected t
Cramer Steven M.
Shukla Abhinav A.
Sunasara Khurram M.
Heslin & Rothenberg, PC
Low Christopher S. F.
Mohamed Abdel A.
Rensselaer Polytechnic Institute
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