Process for separating a substance from a mixture

Details Process for separating a substance from a mixture Process for separating a substance from a mixture

1999-03-03

2001-06-26

Therkorn, Ernest G. (Department: 1723)

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

C210S656000, C210S198200, C210S502100

Reexamination Certificate

active

06251278

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to chromatographic material which when covalently coupled to an appropriate affinity ligand can be used for the separation and/or detection of biological and other materials by way of high performance affinity chromatography (HPAC) or other separation or detection techniques.
BACKGROUND OF THE INVENTION
The coupling of various ligands to solid supports to form chromatographic agents reportedly has been accomplished by using relatively short, low molecular weight linkers or relatively long, high molecular weight linkers which inherently are hydrophilic because of their ionic properties or hydrophobic because of their non-ionic and nonpolar nature.
U.S. Pat. No. 4,532,232 to Larsson, et al. and U.S. Pat. No. 4,406,792 to Glad, et al. each disclose inter alia the coupling of a ligand to silica by way of the short length linker -glycidoxypropyltrimethoxy silane.
U.S. Pat. No. 4,415,665, to Mosbach, et al., reports the coupling of a substituted adenosine 5′-monophosphate to silica particles pretreated with 2,2,2-trifluroethanesulfonyl chloride or to porous silica gel substituted with —(CH
2
)
3
—O—CH
2
—CH(OH)—CH
2
OH— and pretreated with 2,2,2-trifluroethanesulfonyl chloride prior to coupling. Mosbach also reports the use of sulfonyl chlorides to couple specific proteins and a substituted adenosine 5′-monophosphate to polysaccharide derivatives, e.g., agarose and cellulose.
U.S. Pat. No. 4,431,554 to Atkinson, et al., reports the coupling of various organic dyes to silica by use of inter alia -glycidoxypropyltrimethoxy silane alone or in conjunction with a bifunctional aliphatic linker of relatively short length, e.g., 1,6 diamino hexane.
U.S. Pat. No. 4,177,038 to Biebricher, et al. states that low molecular weight polyethyleneglycol 200 can be used to couple biological material to a solid support by using organic diisocyanates to couple one end of the glycol to biological material and the other end to a cellulose support.
Biebricher also reports an example involving porous glass pellets, wherein -aminopropyltrimethoxy silane is first coupled to the porous glass pellets. Thereafter, the silane treated silica is reacted with tetramethylene diisocynate and then coupled to 3-aminophenyl boronic acid.
The coupling of ligands to solid support through specific water soluble or hydrophilic linkers is reported in U.S. Pat. No. 3,715,278 to Miller and U.S. Pat. No. 4,352,884 to Nakashima, et al. In Miller, a copolymer of ethylene and maleic anhydride was coupled to calcium silicate particles pretreated with -aminopropyltrimethoxy silane. The enzyme subtilisin was then coupled through the side chain carboxylic groups of the coupled polymer.
In Nakashima, et al., a copolymer of a hydrophilic acrylate or methacrylate and an unsaturated carboxylic acid or amine reportedly was used to coat the surface of a solid support apparently to minimize the non-specific adsorption of the support. Bio-active material was then coupled directly to the carboxyl or amino groups directly by carbodiimide condensation or indirectly through e-aminocaproic acid or diaminoheptane.
A significant disadvantage in the chromatographic agents of Miller and Nakashima, however, are the free carboxylic acid and amino groups of the copolymers which may remain after coupling of bio-active material. These chromatographic agents therefore may have significant residual ion exchange properties which may result in undesirable adsorption or adversely effect the properties of the bound bio-active material.
Other chromatographic agents which may have residual ion exchange properties and thus suffer from these same disadvantages include those disclosed in: U.S. Pat. No. 4,210,722 to Silver (copolymer containing &bgr;-hydroxyalkylamine); U.S. Pat. No. 4,415,663 to Symon, et al. (polyamine impregnated support); U.S. Pat. No. 4,132,596 to Meiller, et al. (cross-linked polymer containing tertiary amino groups or quarternary ammonium salts); U.S. Pat. No. 3,888,864 to Cleeland, Jr. et al. (linkage of aminoalkylethers of opium alkaloids to carboxylated latex particles); U.S. Pat. No. 4,451,568 to Schneider, et al. (copolymers of acrylic acid or derivatives thereof, e.g. aminoalkylmethacrylates); U.S. Pat. No. 4,438,196 to Lantero, Jr. (polyamine adsorbed on activated granular carbon); U.S. Pat. No. 4,610,962 to Takagi, et al. (regenerated cellulose fiber treated with polymer containing pendant carboxylic anhydrides); and U.S. Pat. No. 4,581,337 to Frey, et al. (latex particles coated with water insoluble copolymer treated with second copolymer having a polyetherpolyamine linker).
In addition, the use of various hydrophobic linkers have been reported. See for example, U.S. Pat. No. 4,071,409 to Messing, et al. (polymeric aromatic isocyanate linkers) and U.S. Pat. No. 4,007,089 to Smith III (asymmetric bifunctional saturated or unsaturated hydrocarbons as linker).
Many of the above described chromatographic agents are not suitable for chromatographic separations which utilize high linear flow rates (e.g., greater than about 0.1 c/min) and/or application of relatively high pressures (e.g., greater than about 200 psi). Thus, for example, derivatives of polysaccharides, e.g., cellulose, sepharose, agarose, etc., are not useful in such applications since they are relatively compressible.
Further, many solid supports which may be used in such high velocity/high pressure chromatography applications, e.g., cross-link sulfonated polystyrene, have demonstrated undesirable non-specific adsorption.
Accordingly, it is an object of the present invention to provide chromatographic material which is substantially free of reversible non-specific adsorption.
Further, an object of the present invention is to provide chromatographic material with substantially improved pH stability.
Still further, an object of the present invention is to provide a chromatographic material having improved binding kenetics.
Finally, it is an object herein to provide chromatographic material which is substantially non-compressible as evidenced by high linear flow rates greater than approximately 0.1 cm/min.
SUMMARY OF THE INVENTION
The present invention comprises chromatographic material having the general formula S-B-X, where S is a substantially non-compressible solid support, B is a binding group and X is a substantially non-ionic hydrophilic spacer. This chromatographic material is formed by covalently coupling the solid support through the binding group to the spacer.
The invention also comprises chromatographic material having the general formula S-B-X-Y-L where S, B and X are as previously described, L is an affinity ligand, and Y is a coupling group. The chromatographic material is formed by covalently coupling S-B-X as described and coupling said ligand to said spacer X through the coupling group Y.
The invention further comprises chromatographic material having the general formula S-B-X-Y′ where S, B and X are as previously described and Y′ is an activated coupling group. This chromatographic material is formed by covalently coupling S-B-X to Y′. This chromatographic material may then be used to covalently couple an affinity ligand L which is reactive with the activated coupling group to form the chromatographic material having the general formula S-B-X-Y-L.
In addition, the invention also comprises a process for the chromatographic separation of a substance from a mixture. The mixture containing the substance is first contacted with the chromatographic material described herein containing a ligand having an affinity for the substance of interest. The chromatographic material is thereafter washed with a solution to remove non-binding species of the mixture from the chromatographic material followed by the passing of an eluting solution through the chromatographic material to recover the bound substance.
The invention also comprises a process for the isolation and detection of at least one substance in a mixture utilizing the chromatographic material herein. The mixture containing the subs

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