WO1989009643A1

WO1989009643A1 - Novel crosslinked cellulose chromatography media

Info

Publication number: WO1989009643A1
Application number: PCT/US1989/001488
Authority: WO
Inventors: Peter Grandics; Susan Szathmary
Original assignee: Individual
Current assignee: Individual
Priority date: 1988-04-12
Filing date: 1989-04-10
Publication date: 1989-10-19
Anticipated expiration: 1990-10-12
Also published as: AU3539989A

Abstract

A crosslinked cellulose has been developed which retains the highly rigid structure of cellulose upon chemical derivatization in aqueous solution and at high pH and ionic strength. The method of this invention involves a cross-linking process which is carried out in organic solvents. The organic solvent crosslinking medium prevents the hydrogen bonded structure of cellulose fibers from being dissociated yielding fibers which not only retain the original compact and rigid structure of native cellulose but are superior to native cellulose in dimensional stability and swelling characteristics. The crosslinked product can be readily derivatized by ionic, hydrophobic or reactive groups and is suitable for the mobilization of proteins or other biomolecules.

Description

NOVEL CROSSLINKED CELLULOSE CHROMATOGRAPHY MEDIA

BACKGROUND OF THE INVENTION

Crystalline alpha-cellulose has been widely used as a support medium in a variety of chromatographic applications because of its hydrophilic nature and enormous surface area. Cellulose is inexpensive and readily available on an industrial scale. It can be derivatized with all the chemistries having significance in chromatography, thereby attaching affinity ligands, ionizable, hydrophobic, or reactive groups to the support for binding proteins or other bioactive substances.

The most significant limitation on the chromatographic application of cellulose is imposed by the structure of cellulose per se. Even though cellulose is a highly insoluble and rigid material due to an extensive hydrogen bonding between the individual polymeric fibers, the chemical modi ications loosens up this compact structure by interfering with the hydrogen bonds and increase swelling in water. When substituted to a high degree by ionizable groups like carboxymethyl group, cellulose may become a gelatinous material and can be eventually dissolved in water. This type of modifications make cellulose unsuitable for use in column chromatography or batch filtration as it exhibits an extremely high resistance to the flow of aqueous solutions.

To obtain a cellulose-based ion exhange resin suitable for chromatographic applications, the degree of swelling needs to be reduced which can be accomplished by controlling the degree of substitution of the matrix. As decreased levels of substitution reduces swelling, this approach has been explored first since the early 1950s leading to the development of a wide variety of cellulose ion exhange chromatography media which have been widely- used in the field of biotechnology. However, the issues underlying the swelling process have not been addressed. It has been assumed that chemical crosslinking of cellulose fibers would enhance mechanical and flow characteristics of cellulose chromatography media. A generally applicable crosslinking method for polysaccharide chromatography media has been reported by Porath et al. (1971) J. Chromatogr. 60, 167-177. Crosslinking did increase flow rates but swelling of cellulose ion exhange resins in alkali and the dimensional changes of ion exchange resins in high salt have not been eliminated by the crosslinking process. This remains a significant obstacle for fast-flow applications as changes in the hydrodynamic resistance of the resin bed may lead to channel formation or plugging up the column.

No data have been reported to date as to how to crosslink cellulose fibers in such a way that the compact, rigid structure of native cellulose fibers are retained upon chemical modification. Aqueous salt solutions disrupt hydrogen bonding between cellulose fibers leading to partial dissociation of polysaccharide strands which process is further promoted by chemical derivatization. Unless the compact, hydrogen-bonded, "crosslinked" structure of the cellulose fibers is retained, a rigid cellulose chromatographic support which is resistant to alkali or the high ionic strength of elution media cannot be prepared. We have developed a method which permits the preservation of the native structure of cellulose fibers. This objective has been achieved through a novel crosslinking process which stabilizes cellulose fibers in a manner that the undesirable swelling of cellulose ion exchangers in alkali and the dimensional changes of the ion exchange resin bed in high salt is eliminated .while the rigidity of cellulose fibers is increased. SUMMARY OF THE INVENTION

A crosslinked cellulose has been developed which retains the highly rigid structure of cellulose fibers upon chemical derivatization in aqueous solutions and at high pH and ionic strenght. The method of this invention involves a crosslinking process which is carried out in organic solvents. The organic solvent crosslinking medium prevents the hydrogen-bonded structure of cellulose fibers from being dissociated resulting in fibers which not only retain their original, compact and rigid structure of native cellulose but are superior to their native counterpart in dimensional stability and swelling characteristics. The crosslinked product can be readily derivatized by ionic, hydrophobic, or reactive groups and is suitable for the immobilization of proteins or other biomolecules of interest.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the subject invention, a method is provided for a novel type of crosslinking of cellulose chromatography media which retains the native structure and excellent physicochemical characteristics of cellulose fibers. Besides native fibrous (microcrystalline) cellulose, regenerated cellulose in the form of spherical beads or as a microcrystalline powder can be crosslinked by the method. The improved chromatography support can be derivatized with a variety of chemistries, including but not limited to affinity ligands, ion exchange-, hydrophobic-, or reactive groups having the capacity of binding proteins or other biomolecules .

In general, the process for achieving the stabilization of the native structure of cellulose fibers is carried out in a nonaqueous medium. This represents the major advantage over conventional crosslinking procedures as the use of a nonaqueous medium promotes hydrogen bonding between the individual cellulose polymeric strands and thus permits the stabilization of the native crystalline structure. The reaction medium may, for example, contain methanol, ethanol, propanol, acetone, dioxane, N,N'-dimethylformamide or dimethylsulfoxide. Generally, polar organic solvents capable of dissolving the crosslinking reagent may be employed.

For use in the present invention, numerous crosslinking reagents may be employed in stabilizing the structure of cellulose fibers. -The crosslinking agents are chemicals that have at least two reactive groups, both of which are reactive with cellulose. Thus, the crosslinking reagent will have the structure R1-A-R2, wherein Ri and R₂ are moieties capable of covalently binding with hydroxyl moieties on cellulose, and A is any group capable of linking Ri and R∑. Examples of Ri and R- moieties include vinylsulfone, epoxy, and aliphatic or aromatic halogenides. Group A eac n with hydro.xyls. Crosslinking agents suitable for use with cellulose are well-known, and any of the conventional agents that are soluble in organic solvents may be used. Such conventional crosslinking agents include divinylsulfone, 1 , 3-dibromo-2-propanol, 1 , 4-butanedioldiglycidyl ether, epichlorohydrine or cyanuric chloride. The concentration of the crosslinking agent is between 1 to 20 percent based on volume with 5 to- 10 percent being most preferred. The crosslinking reaction is catalyzed either by acid or base catalysts. Reagents like ZnClϊ, BF3 , KOH or NaOH may be employed to act as catalyst in the crosslinking reaction. The rection is suitably conducted at temperatures ranging from 0 C to 100 C preferably 20 C to 60 C with room temperature being most preferred. The reaction is carried out with a concentration of solids that is conveniently handled. Suitably, the reaction mixture contains 1 to 50 percent and preferably 10 to 30 percent of cellulose material. The completion of the reaction takes several hours with 16 to 24 h being preferred. After preparation, the crosslinked product is suitably washed to remove reactants and tested for regain and dimensional stability.

Regain is an empirical parameter which relates to the ability of cellulose to swell and is defined as the gram weight of water retained by 1 gram of dried cellulose. The dimensional stability of crosslinked celluloses have been tested in an HPLC instrument at various flow-rates while the pressure in the system has been recorded. This test provides information on the stability of cellulose particles and on the linear flow-rates a cellulose-based chromatography column filling can withstand.

EXPERIMENTAL

The following examples are offered by way of illustration and not by way of limitation. All percents are by weight unless indicated otherwise and all temperatures are centigrade.

EXAMPLE 1

Preparation of crosslinked cellulose chromatography matrix:

Method A: Microcrystalline cellulose (50 g) obtained from Whatman Paper Ltd., Maidstone, Kent, UK, is extensively washed with dry dioxane on a sintered glass filter and transferred into a 1 L three-neck round bottom flask equipped with a teflon impeller stirrer, a thermometer and a 100 ml dropper funnel. Then, 200 ml of dry dioxane and 5 g of epichlorohydrine-BF3 complex mixed in 50 ml of dry dioxane is added over a period of 30 min under continuous stirring. The mixture is allowed to stand overnight at room temperature and then poured on a sintered disk filter funnel and washed with dry dioxane, dioxane/water (1:), and distilled water, respectively.

Method B: Microcrystalline cellulose (50 g) obtained from Whatman Paper Ltd., Maidstone, Kent, UK, is extensively washed with dry methanol on a sintered glass filter and transferred into a 1 L three-neck round bottom flask equipped with a teflon impeller stirrer, a thermometer and a 100 ml dropper funnel. Then, 250 ml of 0.5 M methanolic KOH is added followed by 5 g of epichlorohydrine mixed with 50 ml of dry methanol is added over a period of 30 min under continuous stirring. The temperature is kept around 25-30 C during the addition of reagents and then the mixture is allowed to stand overnight at room temperature. The mixture is poured on a sintered disk filter funnel and washed with dry methanol, methanol/water (1:), and distilled water, respectively. EXAMPLE 2

Introduction of anion exchange groups into the crosslinked chromatography matrix:

The anion exchange derivative is prepared by mixing 25 g of crosslinked cellulose, prepared in accordance with Example 1, with

24 ml of 47% NaOH for 15 min. The matrix was collected by filtration and 5 g of 2-diethylaminoethylchloride hydrochloride and

25 ml of distilled water is added and the mixture is heated to 30 C with stirring. The reaction continued for 1 h. The product was filtered and washed thoroughly with distilled water. The resulting material comprised an effective anion exchange resin.

EXAMPLE 3

Introduction of cation exchange groups into the crosslinked ^• chromatography matrix:

The cation exchange derivative is prepared by mixing 25 g of crosslinked cellulose, prepared in accordance with Example 1, with 70 ml of distilled water and 40^' ml of 47% NaOH for 10 min. The matrix was collected by filtration and 50 ml isopropylalcohol and 2 g sodium monochloroacetate is added and the mixture is heated to 80 C with stirring for 30 min. The product was filtered and washed thoroughly with distilled water. The resulting material comprised an effective anion exchange resin.

EXAMPLE 4

Introduction of reactive groups into the crosslinked chromatography matrix :

The reactive group is introduced into the support by stirring 25 g of crosslinked support, prepared in accordance with Example 1, with 20 ml of 1 M NaOH, 4 ml epichlorohydrine and 100 mg NaBH₄ at room temperature for 4 h. The activated resin is recovered by filtration, washed with distilled water to neutrality, with 50 ml of acetone and then with distilled water to remove acetone.

EXAMPLE 5

Introduction of hydrophobic groups into the crosslinked chromatography matrix:

The hydrophobic group is introduced into the support from Example 4 by stirring 25 g of activated support with 20 ml of 0.1 M NaHCO3/acetone (1:1), pH 8.5 , containing 0.5 ml of n-hexylamine for 4 h. The resin is recovered by filtration, washed with distilled water/acetone (1:1) and then with water to remove acetone. The resulting material comprises a-cellulose substrate with hydrophobic n-hexylamine moieties covalently bound thereto.

EXAMPLE 6

Coupling of protein to the activated, crosslinked matrix:

Immunoglobulin (mouse IgG) is coupled to the activated support by stirring 25 g of activated resin with 20 ml of 0.1 M NaHCO₃, pH 8.0, containing 0.2 g of antibody at 4 C for 16 h . The coupled resin is recovered by filtration, washed with 0.1 M NaHCO3 and the residual reactive groups are inactivated by incubation in a 0.1 M ethanolamine, pH 8.5, for 4 h. The coupled resin is stored in phosphate-buffered saline at 4 C.

ANALYTICAL METHODS

1. Regain: A cellulose sample is placed in 10 times of the resin volume of either distilled water or 0.5 M NaOH and allowed to stand for 10 rain. The material is filtered and the alkali-treated sample is washed until the pH of the supernatant falls to 8. The samples are then transferred into Gooch crucibles, moistened with water and placed in centrifuge cups. The centrifuge is spun at approximately 3000xg for 60 min and the water content of the samples is measured after drying overnight at 105 C.

Water regain=Weight of water/Weight of dry sample

2. Dimensional stability: A slurry of cellulose sample is packed into an HPLC column by pump packing and the resin bed is washed with distilled water at a flow rate of 1 ml/min. Water is then pumped through the column at increasing flow-rates and the pressure in the system is monitored and plotted against the flow-rates.

DISCUSSION

Current and past efforts to retain the rigidity of microcrystalline cellulose upon derivatization to chromatography media have not been successful since in the strongly alkaline crosslinking reaction, that is carried out in an aqueous medium, cellulose polymeric strands dissociate as the extensive hydrogene bonding responsible for the strong association of individual fibers is disrupted. Therefore, the fibers undergo extensive swelling with the coincident separation of polymeric strands which are then crosslinked randomly in a process preserving the swollen, disorganized structure rather than the initial, compact crystalline structure of alpha-cellulose.

The subject invention is aimed at rectifying these shortcomings and introduces a crosslinking method which prevents swelling and even improves upon the initial rigidity of microcrystalline alpha-cellulose. The method is based upon crosslinking cellulose in a nonaqueous medium with the omission of extreme pH conditions. The resulting product swells in NaOH to a lower degree than the inital cellulose and exhibits much lower swelling than its conventionally-crosslinked counterpart which is reflected by the regain values (Table 1).

Table 1. Swelling of cellulose chromatography supports

Test cellulose cellulose cellulose

(native) (conventionally- (crosslinked crosslinked) by invention)

Regain H₂0 0.51 2.2 0.48

g/g 0.5 M NaOH 0.84 3.5 0.52 evidences the extreme stability of the cellulose chromatography matrix of subject invention. Native microcrystalline cellulose, conventionally crosslinked cellulose and a crosslinked cellulose prepared by our method has been compared in an HPLC apparatus. Columns of equal dimensions have been packed and run at various flow-rates while the pressure in the system has been recorded (Table 2) .

Table 2. Pressure vs. flow-rate characteristics of cellulose supports

Pressure (psi)

Flow-rate Native Crosslinked Crosslinked cellulose cellulose cellulose

(ml/min) (conventional) (invention)

2.0 210 290 95

3.0 320 460 210

4.0 460 640 360

5.0 610 800 490

6.0 760 940 590

7.0 900 1120 700

8.0 1070 750

9.0 - 840

10.0 __ 900

The data clearly demonstrate that with the conventionally crosslinked cellulose support backpressure builds up rapidly in the system resulting in clogging of the column at a flow-rate lower than that of native cellulose. This is indicative of the less organized structure of conventionally crosslinked cellulose. On the other hand, cellulose crosslinked by the method of subject invention exhibits at increasing flow-rates the lowest pressure drop which is even lower than that of its native counterpart. This observation suggests that the invented method increases upon the rigidity of the cellulose matrix.

We have also investigated the swelling properties of cellulose media upon introduction of ion exchange groups (Table 3). Celluloses crosslinked conventionally or by the method of subject invention have been derivatized with DEAE-groups.

Table 3

Test DEAE-cellulose DEAE-cellulose conventional invention

Regain (g/g) H₂0 2.3 2.1

0.5 M NaOH 4.3 1.9

When DEAE functionalities are introduced into conventionally crosslinked cellulose, alkali treatment significantly increases the regain value resulting in swelling of the resin which can lead to plugging up the column or formation of channels in the resin bed. This does not permit in situ sterilization with NaOH. However, ion exchange resin prepared by using the support of this invention does not swell in the NaOH sterilant, thereby eliminating the concern of expansion or shrinkage of column filling. This is a significant advantage as sanitary operation requires in situ sterilization of chromatography media.

Claims

What is claimed is:

1. A method of crosslinking cellulose for use as a chromatography material , comprising the steps of: combining cellulose with an organic solvent, a crosslinking reagent having the formula Rι-A-R₂, a catalyst selected from ZnCl₂, BF3, KOH, and NaOH to form a reaction mixture and reacting said cellulose in said reaction mixture for a period of time sufficient to crosslink said cellulose; and separating said crosslinked cellulose from said reaction mixture.

2. The method of Claim 1, wherein the native configuration and structure of said cellulose is maintained during said reacting step without substantially sweling said cellulose.

3. The method of Claim 1, further comprising the step introducing an anion and cation exchange group onto said crosslinked cellulose.

4. The method of Claim 1, further comprising the step introducing hydrophobic group onto said crosslinked cellulose.

5. The method of Claim 1, further comprising the step introducing reactive groups, capable of covalently binding biomolecules, onto said crosslinked cellulose.

6. The method of Claim 5, wherein said biomolecule is a protein.

7. The method of Claim 5, wherein said biomolecule is an antibody.

8. The method of Claim 5, wherein said biomolecule is an enzyme.

9. The method of Claim 5, wherein said biomolecule is a peptide.

10. The method of Claim 1, wherein said crosslinked cellulose has a water regain of less than 1, wherein water regain is calculated by soaking said crosslinked cellulose in water for 10 minutes, centrifuging for 60 minutes at 3000xg, and drying for 10 hours at 105 C, then dividing the weight of the retained water by the weight of the sample prior to soaking.

11. The method of Claim 10, wherein said water regain is no greater than that of native cellulose.

' 12. The method of Claim 10, wherein said crosslinked cellulose has a water regain of less than 1.5, wherein NaOH regain is calculated ' by soaking said crosslinked cellulose in 0.5 M NaOH for 10 minutes, washing until the material is at pH 8, centrifuging for 60 minutes at 3000xg, and drying for 10 hours at 105 C, then dividing the weight of the retained water by the weight of the sample prior to soaking.

13. The method of Claim 12, wherein said NaOH regain is no greater than that of native cellulose.

14. The method of Claim 1, wherein the pressure required to achieve a given flow rate through a column packed with said crosslinked cellulose is no greater than the pressure reqired to achieve said flow rate through- the same column packed with native microcrystalline cellulose.

15. A crosslinked cellulose matrix, comprising cellulose that has been crosslinked in an organic solvent to achieve a water regain to NaOH of 1+ 0.2 g/g.

16. The matrix of Claim 15, having a ratio of water regain to NaOH regain less than that of the native cellulose from which said matrix was prepared.

17. The matrix of Claim 15, wherein the ratio of water regain to NaOH regain is less than 1 +_ 0.2.

18. The matrix of Claim 15, wherein the organic solvent crosslinking medium contains less than 30% water.