WO2002018564A1

WO2002018564A1 - Derivatized potassium silicate supports

Info

Publication number: WO2002018564A1
Application number: PCT/US2001/026761
Authority: WO
Inventors: Jarrod A. Marto; Scott B. Ficarro
Original assignee: University Of Virginia Patent Foundation
Priority date: 2000-08-29
Filing date: 2001-08-28
Publication date: 2002-03-07
Also published as: AU2001288446A1

Abstract

The present invention relates to a microporous support matrix that is cast in place by fusing a silicate containing solution to the walls of a capillary tube along the entire length of the tube. The prepared column is suitable for being cut into multiple fragment lengths and provides supports for bioactive molecules that are to be derivatized onto the support matrix.

Description

Derivatized Potassium Silicate Supports

This application claims priority under 35 U.S.C. § 119(e) to US Provisional Patent Application No. 60/228,642, filed on August 29, 2000, the disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to a method and. composition for immobilizing bioactive molecules onto a chromatographic column. More particularly, the present invention is directed to a fused chromatographic column that can be cut into multiple fragment lengths without disturbing the properties of the column.

Background

High performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS) are two of the most widely used analytical separation techniques. Traditionally, samples are treated in bulk solution with bioactive molecules (enzymes, antibodies, carbohydrates, small molecules, and DNA/RNA) and then introduced into a chromatographic column for further analysis. More recently, a variety of techniques have been used to immobilize the bioactive molecules directly on a chromatographic column so that samples can be introduced directly into the column without having to first treat the sample in a bulk solution. This approach offers several advantages over the traditional bulk solution technique. First because the support provides a large surface area, the ratio of bioactive molecule to target molecule is high, providing for greater efficiency and shorter reaction times when compared to the traditional bulk method. Second, the column may be coupled directly to an HPLC or LC/MS apparatus, minimizing "sample handling" and thereby increasing detection efficiency. Finally, coupling directly to an HPLC or LC MS device allows for automated, high-throughput reaction/analysis systems.

A number of methods for immobilizing bioactive molecules have been developed. Typically, under these approaches the bioactive molecule has been immobilized onto particle-based beads, such as silica, sepharose, polyacrylamide, agarose, methacrylate, and polystyrene beads, and then slurry-packed into a column. Although these methods have proven to be useful analytical techniques, they suffer from a number of limitations. For example, the particle-based beads flow freely in solution; therefore, each column requires a frit to ensure that the beads stay within the column. Because of this limitation, each column must be made individually, thus potentially introducing a source of variability when comparing experimental results obtained using two different columns. Also, the physical characteristics of the slurry- packed column prevent the column from withstanding the increased pressures that are often times encountered in high performance analytical processes.

U.S. patent # 4,793,920, the disclosure of which is incorporated herein in its entirety, describes the formation of microporous supports from solutions of potassium silicate mixed with formamide. When placed inside a tube with a silica- based inner wall, the solution polymerizes to form a silica matrix with a porosity of 3000-5000 angstroms. That patent describes the use of such supports as frits (0.001 - 4 cm long) to support chromatographic column beds. The present invention utilizes potassium silicate cast along the entire length of the column to form a stable, high surface area support, that is easily derivatized for subsequent immobilization of a wide range of bioactive molecules. The design of this invention makes it well suited to be coupled directly to microcapillary high performance liquid chromatography and state-of-the-art liquid chromatography devices. An important advantage of the present invention is that it is particularly well suited for the automated, high-throughput processes that are commonly encountered in modern genomic, proteomic, and drug discovery research.

Definitions As used herein, the term "bioactive molecules" refer to substances that are capable of specific binding to a target compound or exerting a biological effect in vitro and/or in vivo. Bioactive compounds include but are not limited to proteins, carbohydrates, nucleic acids, enzymes, antibodies, pharmaceuticals and the like.

As used herein the term "proteome" relates to a complex mixture of proteins that are derived from a common source, such as an extract isolated from a particular cell or tissue. For example a human proteome represents a mixture of proteins isolated from human cells. The category can be further defined by specifying a particular cell/tissue source for the proteome (i.e. a human myocardial tissue proteome represents all the proteins isolated from human myocardial tissue).

As used herein the term "solid support" relates to a solvent insoluble substrate that is capable of forming linkages (preferably covalent bonds) with soluble molecules. The support can be either biological in nature, such as, without limitation, a cell or bacteriophage particle, or synthetic, such as, without limitation, an acrylamide derivative, agarose, cellulose, nylon, silica, or magnetized particles.

As used herein the term"casting the fusible material" refers to the process of converting a fusible material from a liquid (colloidal suspension) to a solid. As used herein the ternϊ'fusible material" refers to any material that is capable of undergoing a transition from a liquid "sol" state to a solid form (i.e. a sol- gel material).

As used herein a column that is "suitable for cutting" refers to a column that can be cut into multiple fragment lengths while maintaining the integrity of the column (i.e the fragmented columns continue to function in a manner consistent with the parent uncut column).

The term "linked" refers to a connection between two groups and includes covalent, ionic, hydrogen bonds, hydrophilic and hydrophobic interactions that bind two entities to one another. A "linker" is a molecule or group of molecules attached to a substrate and spacing a bioactive material from the substrate. Linkers may further supply a labile linkage that allows the bioactive material to be detached from the substrate. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties and enzyme-cleavable groups.

Brief Summary of Invention

This invention describes the fabrication of fused silica capillary columns that contain a silica based support matrix cast along the entire length of the chromatographic column. To prepare this invention, tubing of arbitrary internal diameter and length is filled with a fusible material comprising a silica component. The fusible material is then cast to form a microporous support matrix throughout the entire length of the fused silica tubing. Because the initial tubing can be arbitrarily long, the resultant column can be readily cut to an appropriate length dictated by the end-user's volume and/or sample requirements. The remaining column length is then stored for future use. Presumably, the performance characteristics of the individual columns will be extremely consistent because they originate from a common precursor. These fused silica columns are designed to couple directly to microcapillary-scale high performance liquid chromatography separations, and are also ideally suited for state-of-the-art liquid chromatography-mass spectrometry analyses.

Detailed Description of the Invention The present invention is directed toward a fused silica chromatographic column containing microporous silica based supports, and methods of preparing and using such columns. In particular, the present invention is directed to a fused silica chromatographic column having a length of at least 10 cm, and more preferably a length ranging from about 100 to about 500 cm or more, that is immobilized on the inner wall of a tube along substantially the entire length of the tube. The column is prepared, by adding a fusible material to a tube, wherein the tube has a coating on the inner wall of the tube that forms covalent bonds with the fusible material upon casting of the fusible material. In one embodiment, the invention comprises a silica tube and a fused microporous silica support matrix cast along the entire length of the tube. In accordance with one embodiment, potassium silicate is cast along the entire length of fused silica tubing to form a mechanically stable, high surface area microporous support, which is easily derivatized for subsequent immobilization of a wide range of bioactive molecules. These columns are designed to couple directly to microcapillary high performance liquid chromatography (HPLC) separations and state-of-the-art liquid chromatography mass spectrometry. In addition, the configuration detailed herein is ideally suited for automated, high-throughput processes commonly encountered in modern genomic, proteomic, and drug discovery research.

To prepare one embodiment of the present invention, fused silica tubing of arbitrary internal diameter and length is filled with a solution of potassium silicate and formamide. Upon heating, this solution polymerizes in-situ to form a microporous, silica-based support matrix throughout the entire length of fused silica tubing. After appropriate chemical preparation, a variety of molecules may be covalently attached, forming microcapillary columns suitable for any number of automated, high throughput processes, including enzymatic digestion, affinity purification and combinatorial chemistry. The microporous support provides both high surface area and mechanical stability and is ideally suited for automated, continuous flow, high throughput applications. Because the initial fused silica tubing can be arbitrarily long, the resultant column can be readily cut to an appropriate length dictated by the end-user's volume and/or sample requirements. The remaining column length is simply stored for future use. In principal, an initial or precursor column many meters in length can be cut into many individual columns, each only 5- 10 centimeters long. Presumably, performance characteristics would be extremely consistent across individual columns since they originate from a common precursor.

Furthermore, as noted above the column can be modified to include bioactive molecules attached to the silica support matrix, and these bioactive molecules can then in turn react with target substrates contained within a sample that is passed through the column. The bioactive molecules can be linked directly or indirectly to the solid support and in one embodiment the bioactive agent is bound through a linker. The column can also be directly coupled to a HPLC or LC/MS apparatus allowing for analysis and identification of the sample with a minimum amount of sample handling and greater efficiency.

The column used in this invention can be of arbitrary diameter and length as defined by the size of the tube that is used to load the fusible material. In general, the inner diameter of the column will typically be approximately 250 μm, while the length of the column will typically range from about 50 cm to about 1000 cm, and more preferably the column will be about 100 cm. If the column is being used for liquid chromatography, the inner diameter of the tube ranges from about 10 μm to about 1000 μm while the length ranges from about 50 cm to about 1 m. The fused silica tubing dimensions listed above may be readily scaled to provide column volumes and sample capacities appropriate for preparative applications. Additionally, because the column can be easily cut, this method can be used to make a precursor column of an indeterminate length (i.e. greater than 1 m in length). Depending on the end user's specific needs, an individual column can be cut from the precursor column and the remainder stored for subsequent use. This method offers an improvement over individual preparations because the precursor column will yield individual columns that are extremely consistent with one another because they came from the same precursor column. Casting microporous potassium silicate supports inside fused silica capillary columns represents one embodiment of the present invention. In an alternative embodiment, the derivatized microporous supports described herein are fabricated on various forms of silica. For example, recent efforts aimed at miniaturized, high throughput analyses have led to so-called "lab on a chip" procedures, in which virtually all sample manipulation (isolation, separation, detection, etc.) occurs in small channels, tens to hundreds of microns in diameter, etched in silica wafers. This approach requires an absolute minimum' of sample handling, and may eventually provide the ultimate in rapid, high sensitivity analysis. Derivatized potassium silicate supports represent an ideal means to implement sample manipulation within these small channels.

The tubing used in the present invention preferably comprises a fused silica component at least on the inner wall of the tube so that upon loading of the fusible material, the material can bind to the inner surface of the tube and form a mechanically stable, high surface area microporous support matrix. Likewise the fusible material that is selected preferably contains a silica component so that it can bind to the inner wall of the tube. Methods for selecting the proper tubing and fusible material such that the fusible material binds to the tube and forms a mechanically stable microporous support matrix are well known to those skilled in the art. In one embodiment of the present invention, the fusible material comprises formamide and potassium silicate. In another embodiment of the invention, the fusible material further comprises water and ethanol and the silica component of the fusible material is tettaethoxysilane. Both of these embodiments are examples of a class of chemical reactions known as sol-gel processes, which produce silica-based support matrixes suitable for use in the present invention. There are several methods for loading the fusible material into the tube that are well known to those skilled in the art. One method involves depositing the fusible material directly into an open end of the tube using a pipette, syringe or other appropriate means. Alternatively the material can be deposited into the tube through the use of positive or negative pressure. In one embodiment, the method for depositing the fusible material into the tube involves using a so-called "helium bomb". With this method, the fusible material is loaded into an eppendorf tube. The eppendorf tube, with the fusible material inside of it, is then attached to a supply of pressurized helium. When the pressurized helium is released into the eppendorf tube, the fusible material is forced out of the eppendorf tube and deposited into the tube that is to be used to make the chromatographic column.

Sufficient amounts of the fusible material are deposited into the tube so the material occupies the entire length of the column or at least substantially the entire length of the column. The term "substantially the entire length of the column" as described herein means that, when using a column that is at least 10 cm long, no more than 1 cm at either end of the column is left unoccupied by the fusible material before it is cast. Alternatively, column is considered substantially filled when no more than 10% of the column is left unoccupied before casting.

Casting the tube involves fusing the fusible material to the inner wall of the tube so that the fusible material forms a solid microporous support matrix. Methodologies for casting the fusible material are well known to those skilled in the art. In one embodiment, the fusible material that is selected is a heat fusible material and the method for casting the tube further comprises the step of heating the fusible material inside the column. In another embodiment, the fusible material is mixed with a base and cast using a base catalyzed reaction. The speed of the base catalyzed reaction can be increased by heating the fusible material inside the column. The fusible material is heated at a temperature in the range of about 90° C to about 110^° C for a period in the range of about 45 minutes to about 75 minutes.

In accordance with one embodiment of the present invention, the solid microporous support matrix is used as a size restrictor and separates out the various components of a sample based on their size. Alternatively, bioactive molecules can be linked to the microporous support using standard techniques known to the skilled practitioner. Depending on the bioactive molecule that is immobilized onto the column, a wide variety of reaction products can be obtained by passing samples through the column and analysis of these products provides a set of information unique to each sample.

To prepare a microporous support that contains bioactive molecules linked to the support, a support is selected that contains various reactive groups already linked to the support. Microporous supports that have been functionlized with reactive groups, such as hydroxyl or amino functionalities are commercially available. Bioactive molecules can derivatized onto such microporous supports by covalently bonding the active molecule directly onto the amino and/or hydroxyl groups of the microporous support. Alternatively the bioactive material can be bound though a linker. The specific bioactive molecule to be linked to the support will be chosen according to the end user's specific needs but can be selected from the group including, but not limited to, carbohydrates, nucleic acids, enzymes, antibodies, antigens, and other small molecules.

When using this method of derivatization, the hydroxyl and amino groups on the microporous support are typically "protected" from reactivity and the groups must be activated before the bioactive molecule is bonded to these groups. Methods for activating the amino and hydroxyl groups of the microporous support are well known to those skilled in the art. Activation involves adding or removing certain functional groups from the hydroxyl and/or amino groups. These activations are most often accomplished via chemical means. In one preferred embodiment, hydroxyl group activation is accomplished by passing 3-aminopropyl-tri-ethoxy silane over the column. The 3-aminopropyl-tri-ethoxy silane adds an amine group onto the hydroxyl groups of the support matrix. In another preferred embodiment, amino group activation is accomplished by passing gluteraldehyde over the column. The gluteraldehyde adds a functional group containing an aldehyde onto the amino groups of the support matrix.

The bioactive molecules can also be derivatized onto the column using a ligand binding interaction. Briefly, a ligand binding interaction refers to a binding interaction between a ligand and its binding partner. Typically, a ligand is a small molecule and the binding partner is a binding protein or protein fragment. Suitable binding interactions include those interactions between ligands and their binding partners having affinity constants greater than about 10⁸. Representative examples of suitable ligand binding pairs include valphosphanate/carboxypeptidase A, cytostatin/papain, biotin/streptavidin, and riboflavin/riboflavin binding protein.

Selection of the bioactive molecule to be immobilized onto microporous support will depend upon the nature of the analysis to be performed. Methodologies for binding compounds to reactive groups present on the support are well known to those skilled in the art. Because of the large number of different molecules that can be immobilized onto the column, a wide variety of reaction products can be obtained by passing samples through the column. Accordingly, analysis of these products by subsequent separation and identification provides a set of information unique to each sample and useful for identifying the sample molecule, probing the sample molecule's primary structure, or evaluating the sample molecule's reactivity towards a particular enzyme or antibody.

In one embodiment of the present invention, the bioactive molecules immobilized onto the microporous support include enzymes which digest proteins or peptides. Such enzymes may be broadly referred to as peptidases, and include enzymes such as various aminopeptidases, carboxypeptidases, and endopeptidases (e.g., trypsin).

In another embodiment of the present invention, the immobilized molecules are enzymes which modify proteins or peptides by adding various groups to the protein or peptide. Such modifying enzymes include glycosylating enzymes that add sugar moieties (e.g., galactosyltransferase, fucosyltransferase, and mannosyltransferase) and phosphorylating enzymes that add phosphate groups (e.g., MAP kinase, protein kinase C and ERK).

In a further embodiment of the present invention, the immobilized molecules are enzymes which modify proteins or peptides by removing certain groups. Such modifying molecules include enzymes which remove sugar moieties from glycoproteins (e.g., glycosidases, galactosidase, fucosidase, and mannosidase) and phosphate groups from phosphoproteins (e.g., protein phosphatase I and calcineurin). In a further embodiment of the present invention, the immobilized molecules are antibodies. The antibodies immobilized on the microporous support can be one or more specific monclonal antibodies or a polyclonal mixture. The columns containing immobilized antibodies will be used in accordance with one embodiment to extract a particular compound from a sample that contains a complex biological mixture.

In one embodiment of the present invention a method is provided for identifying proteins contained in a sample. The method for identifying an unknown protein comprises the steps of: 1) using the present invention to prepare a derivatized potassium silicate column immobilized with a peptidase; 2) coupling this column directly to a reversed phase microcapillary HPLC column, such that the resulting enzymatically digested peptides exit the column and are retained on the chromatographic bed; 3) gradient elution of the enzymatically digested peptides from the HPLC column directly into a mass spectrometer; and 4) determination of the primary amino acid sequence based on tandem mass spectrometry data, either via manual interpretation or computerized database search algorithms.

In addition, multiple columns with different bioactive molecules may be combined in series. For example, a derivatized potassium silicate column immobilized with a specific antibody may be used to extract a certain a protein population from a complex biological mixture. After the column is washed to remove unbound and non-specific bound material, the antibody bound protein is then released from the antibody using standard techniques and subjected to further analysis. For example the immobilized antibody column can be coupled to a second column that comprises an immobilized peptidase, with the resulting peptides (and thus proteins) identified as described above.

Example 1 A derivatized potassium silicate column was prepared by mixing potassium silicate (Kasil #1, PQ Corporation, Valley Forge, PA) with formamide in a mass ration of 6: 1. The mixture is vortexed for 30 seconds and then centrifuged for 1 minute. The supernatant is transferred to a fresh tube, and then pressure loaded into fused silica capillary tubing. Flow is stopped once the potassium silicate solution is observed exiting the end of the fused silica capillary tubing. The column is heated in an oven at 100 degrees Celsius for 1 hour. Example 2

A derivatized potassium silicate column immobilized with trypsin was prepared by casting the microporous support matrix as described in Example 1. Hydroxyl group activation is performed by passing a solution of 3-aminopropyl-tri- ethoxy silane (5% in toluene) over the column for several hours. The column is then rinsed with water for 2 hours. Terminal amino group activation is performed by passing gluteraldehyde (10% in 50 mM sodium phosphate buffer, pH = 8) over the column for several hours. The column is then rinsed with 50 mM sodium phosphate buffer, pH = 8, for 2 hours. Enzyme coupling reagent is prepared by adding 200 micrograms of sequencing grade trypsin (Promega, Madison, WI) and 100 micrograms of sodium cyanoborohydride to 1 milliliter of 50 mM sodium phosphate buffer, pH = 8. This solution is passed through the column overnight. Finally, the column is rinsed with 0.1% acetic acid for 30 minutes. At this point the column may be stored for future use or smaller lengths may be cut and used depending on the end- user's particular volume and/or sample capacity requirements.

Claims

Claims:

1. A process of making a fused chromatographic column within a tube, said tube comprising an inner and outer wall, and the process comprising the steps of:

* providing a fusible material that binds to the tube upon casting, wherein said fusible material comprises a silica component; depositing a sufficient quantity of the fusible material in the tube to substantially fill the tube; and casting the fusible material.

2. The process of claim 1, wherein the fusible material comprises active groups, selected from the group consisting of hydroxyl and amino groups, linked to the silica.

3. The process of claim 2, wherein a bioactive molecule is linked to the fusible material.

4. The process of claim 3, wherein the bioactive molecule is an enzyme.

6. The process of claim 3, wherein the bioactive molecule is a nucleic acid.

7. The process of claim 1, wherein the fusible material is a heat fusible material and the step of casting the column comprises heating the material.

8. The process of claim 1, wherein the fusible material further comprises a base and the step of casting the column comprises a base catalyzed reaction.

9. The process of claim 8, wherein the casting of the fusible material to the column is further catalyzed by heating the fusible material.

10. The process of claim 1, wherein the fusible material comprises potassium silicate and formamide and the inner wall of said tube comprises a silica-based material.

11. The process of claim 1 , wherein the column has a length of at least 0.5 m and is suitable for cutting.

12. A process of making a fused silica chromatographic column within a tube, said tube comprising an inner and outer wall, wherein the process comprises the steps of: providing a fusible material comprising formamide and potassium silicate, wherein said potassium silicate has been functionalized with a protected active group, and said fusible material binds to the inner wall of the tube upon casting of the fusible material; depositing a quantity of the fusible material in the tube so that the chemically fusible material occupies substantially the entire length of the tube; casting the fusible material; activating said protected active groups; binding a bioactive molecule to said active groups.

13. The process of claim 12, wherein protected active group is selected from the group consisting of hydroxyl and amino groups.

14. The process of claim 13, wherein the bioactive molecule is selected from the group consisting of proteins and carbohydrates.

15. The process of claim 14, wherein the bioactive molecule is an enzyme.

16. The process of claim 13, wherein the bioactive molecule is a nucleic acid.

17. A fused silica chromatographic column having a length of at least 10 cm and comprising a silica-based support; and a tube having an inner wall and an outer wall, wherein said silica-based support is immobilized on the inner wall of said tube and along substantially the entire length of the tube.

18. The chromatographic column of claim 17, wherein said silica-based support comprises potassium silicate and said inner wall comprises a silica-based material.

19. The chromatographic column of claim 18, wherein said silica-based support is functionalized with hydroxyl or amino groups.

20. The cliromatographic column of claim 18, further comprising a bioactive molecule bound to said potassium silicate.

21. A fused silica column comprising: a potassium silicate-based support immobilized along substantially the entire length of a tube; said silica-based support being functionalized with compounds selected from the group consisting of hydroxyl and amino groups.