IL37131A - Stabilized agar product and method for its stabilization - Google Patents

Description

Stabilized agar product and method f ox its stabilization ¾aw»* na»pi a»va -m* ism algae and consists of a mixture of igh molecular poly¬ saccharides which, in extensive hydrolysis, yields galactose as a primary product* Because of its ability to form gels having a high water content, agar has found wide use in the fields of biochemistry and microbiology.

A valuable property of agar is its resistance to microbial " \ attacks.

An agar gel is formed when a hot water solutio of agar Is cooled, when the agar concentration exceeds a certain limit characteristic for different agar types and usually less than 1$ by weight* It is assumed that gels consisting of molecular networks are formed by polysaccharide chains which are held together by hydro¬ gen bonds. ¥hen heated up, the gel dissolves and is hydrolyzed with a strong acid or strong alkali, parti¬ cularly during heating. An agar gel may also be obtained by allowing solid, dry agar particles to swell in water.

However, the resulting product is markedly deficient In those properties considered desirable in the present context.

A number o separation processes in biochemistry are based on the ability of agar gels to bind large quanti¬ ties of water, especially processes based on diffusion of dissolved substances. Such procedures are called gel fil¬ tration or even gel chromatography, molecular exclusion chromatography, molecular sieving chromatography or permeation chromatography. Tills chromatographic technique, adapted for agar, comprises gassing a mixture of substances having different molecular sipes through a bed of agar gel particles saturated with water or an aqueous buffer solution. The substances hereby migrate through he bed at different velocities} high molecular substances pass through the bed more quickly than low molecular ones. The separation range for molecula sieves is determined by the network and the mutual size ratio of the molecules. A gel with low agar concentration may be used for separailfgg high molecular substances mutually, whereas a gel with high agar concentration is more suitable for ractionating substances having smaller molecular dimensions.

The ideal molecular sieve ought to satisfy certain demands. For example, it should not contain charged groups and it should be insoluble and chemically resistant. Common agar is markedly deficient in these respects. - 2a ■ It has now been discovered that agar contains a fraction, agarose, which has a significantly lower content o charged groups than the source material.

Therefore, this fraction has found use as a molecular sieve and has better properties than agar. Agarose has also replaced agar in other fields where it is essential for the gel not to contain large amounts of charged groups chemically bonded in the gel substance.

An additionally improved product phase is obtained according to the U.S. fstent 3,507*851 which describes cross-linking agarose with epichlorohydri . In this way, cross-linked agarose is less soluble than tiie natural agarose and is also more resistant to alkali.

It is exceedingly Important, partloularly for technical applications, to obtain high flow rates through the beds of molecular sieving material, thus allowing for establishment of rapid diffusion equilibria. It Is also extremely important to obtain a well-de ined flow profile through the bed, to ensure high resolution. This is best accomplished by making the particles spherical; this has not been done previously with cross-linked agar gels.

The same properties -which charact erize the ideal molecular sieve are also desirable when the gel is to be used as source material for preparing ion exchangers and other adsorption agents and for preparing enzymes chemically bonded to a gel , and other biologically and chemically active gel materials as well as for preventing convection in a solution, e. g . during preparative electrophoresis . sati sfies The present invention relates to a product which fill's-the demands on molecular sieves better than previously known agar gel products . The invention also relates to a process for making this product with the agar being directly usable as source material without first requiring time-consuming and mat erial -losing fractionating . According to the invention, the agar product may be obtained in the form of spherical .particles having very small adsorption capacity and being insoluble in ■heat™ aael-aikal-t. boil ing 0.5 M sodium hydroxide.

The present invention relates to products of cross-linked agar or agarose with significantly more advantageous properties than previous products and prepared in another manner which is import ant in the present context . The cross -linking may be effected with epihalohydrin, e. g . epibromohydrin or Tne of agar i s known t0 invol ve both epichlorohydrin. ¾*/1?n¾ cross-linking -reaction- w-i-t-h- -ag-ar^ - -both agarose and 4too other polysaccharides in the agar t¾ke.-paj_± . It has been shown that if the cross-linking t akes place in the absence of oxygen and , preferably, in the presence of a reducing agent , e . g . sodium borohydride , the brown coloring and partial decomposing of the agar , which normally occur in reactions in the alkaline medium that is required for the binding of the agar to epichlorohydrin, can be avoided. It has also been shown that cross-linking has- stabilize^ the product to such an extent that it, via alkali treatment, e.g. autoclave treatment one or more times, can "be made more or less sulfate-free. Also, if so desired, said agar product can be practically totally freed of the less desirable adsorption capacity, a great part of which is usually attributed to the product's sulfate content but which can, by the above described treatment, be reduced to zero in most of the substances found in nature without the total sulfur content of the product necessarily having to become zero.

(See the list of values in Table 1. ) It has also been shown that, under certain conditions with considerably more advantageous results, cross-linking can be attained with bis-epoxides such as l, 3-bis- (2, 3-epoxy-propoxy)butane. Even divinyl sulfone and other cross-linking vinyl compounds can be used for this purpose. It is finally added that chemical compounds which can be converted under the in the reaction conditions and/course of reaction to e.g. epichloro-hydrin or any other of the above given cross-linkers may also be used. Examples of such compounds are l, 3-dichloropropanol-2 (see Example 12) or 2 , 3-dichloropropanol-l (see Example 13) .

By varying the degree of cross-linking and, above all, the nature of the cross-linking molecule^molecular sieves can that ful fil l a desired requirement as to working range for macro be obtained -with--a- e-eHraiia--ext-ea^te--&£--v-ar-i-atele- -fft»l©€¾lar--ΘΧ-molecular separation , , ^ crijding--botnTniar-es, thus with a much hxgher value than what was previously possible. This is illustrated on Fig. 1 which using ^ shows examples of molecular sieving -on cross-linked and hydrolyzed agar. The curves on the figure refer to the follow:-ing; I. Tobacco mosaic virus with a particle weight of about o 2 · 10 and i n- -co-nt i-mro w-s--f© rftt^ with rodl ike shape; II. Adeno-virus Type 3 with a particle weight of about 1.8 · 10 and in spherical form$ III. Poliovirus with a particle weight of about 6.8 · 10 and in spherical form, and IV. Satellite Tobacco Necros Virus with a particle weight of about 2 * 10 and in spherical form.

The products according to the invention may be advantage ously based on swollen gels in bead form and with high water contents. By beginning with a fully swollen in water gel, the degree of cross-linking will be restricted but simultaneously, and in a desirable manner, controllable and reproducible since the gel can be easily penetrated by molecules intended for the cross-linking In most cases, 90% or more water appears to be a suitable water content. It is important that the form of the gel particles is not changed during treatment according to the invention. However, it is also important that by using the method according to the invention, the cross-linked gel be insoluble even at higher temperature and in alkaline solution. Thus, a bed of the product according to the invention can be sterilized by autoclave heating which, in many cases, is of decisive importance. In addition, the insolubility is most advantageous if the agar product is to be used e.g. for purifying pharmaceutical products, especially for injection purposes where contaminated matter, such as antigen substances, must not be released. Furthermore, the agar product remains transparent, colorless or white. This shows that no undesirable disintegration of the agar product occurs during treatment.

In order to further clarify the invention, a number of examples are given below. However, reagents and amounts mention ed may, of course, be varied within the framework of the invention. 37131/2 in preceding examples. A product was obtained which was insoluble and stable in heat and alkali.

Example 5 The cross-linking was effected in the same way as in preceding examples but with epibromohydrin instead of epichloro hydrin. A product with the same appearance is obtained which is insoluble and stable in heat and alkali.

Example 6 The cross-linking was effected in the same way as in Example 3 but with epibromohydrin. A similar product was obtained.

Example 7 The cross-linking was effected in the same way as in Example >+, but with epibromohydrin, A similar product was obtained.

Example 8 The cross-linking was effected according to Example 1 but with 1? 3-bis- (2? 3-epoxypropoxy) -butane instead of epichloro hydrin. This bis-epoxide is, in contrast to epichlorohydrin, miscible with water. A product was obtained which was insoluble and stable in heat and alkali and the original particle form was retained as in the other examples.

Example 9 The cross-linking was effected as in Example 8 but with the water being replaced by a blended solvent of 50% by volume water and 50 by volume ethanol, and with the bis-epoxide less sol uble Ι,Η-bis- (2, 3-epoxypropoxy) -butane which is -M9rja.-slAgii ly s©i bl« in watery than the 1 , 3-anal ogue used in Example 8.

Example 10 The same method was used as in Example 95 but with the difference that the cross-linking was effected in absolute ethanol .

Example 11 The cross-linking was effected as in Example 9 > hut with the difference that the solvent was constituted of $0% by volume ethanol and 50 by volume dioxane.

Example 12 The cross-linking was effected according to Example 3, but the epichlorohydrin was replaced by 1 , 3-dichloropropanol-2. Example 1 The cross-linking was effected according to Example 3, but the epichlorohydrin was replaced by 2, 3-dichloropropanol-l. Example lk The cross-linked gel was prepared according to Example 3, but with the difference that the autoclaving was effected in a solution of 1 M sodium alcoholate in anhydrous ethanol.

Example 1 100 ml swollen bead-shaped agar was equilibrated by washing with 1 M soda solution. 5 ml divinyl sulfone was added in 0 ml soda solution. The gel assumed a milky appearance. The suspension was heated to 50°C for 1/2 hour and the gel was then washed on a filter. A sample was taken out and heated to 100°C. No dissolution could be observed and the gel pellets retained their form. The gel was then transferred into 1 M NaOH solution containing 0. NaBH^. The th&!&y treated gel retained the spherical particle form and a packed bed showed excellent flow properties .

In order to show that the agar beads can be cross-linked, even as liquid particles, and retain their spherical form, the tests described below were carried out. These tests also show that it is not essential to separate the spherical agar particles, after they have been formed, before the cross-linking is effected.

Example 16 500 ml of 6% agar solution in 0.5 M NaOH with 2,5 g NaBH^ was suspended at 75°C in 600 ml ethylene dichloride containing 20 g polyvinyl acetate as a stabilizer. The agitation was controlled so that particles of suitable size (50-250 u) were obtained. 50 ml epichlorohydrin was then added and agitation was continued at 60°C for two hours. After cooling, the gel particles were freed from the emulsifier by careful washing with acetone. The beads were then transferred in water and autoclaved as in Example 1.

To compare the adsorption properties of the new products with that of the source material and the agarose, th-e- tests were carried out as follows s 2 The gel was packed into a bed 0.90 cm in cross section and 2.5 - 3. cm height in chromatography ¾1¾©€δ.* The bed was equilibrated with 0.01 M ammonium acetate buffer pH h.l.

Cytochrome C (0.1% solution in the same buffer) was introduced until the influent and the effluent displayed the same color strength. The bed was washed with buffer solution until no further cytochrome was eluted. Displacement of the adsorbed cytochrome was effected with 0.15 M ammonium acetate buffer, pH +.l. The amount of cytochrome was measured in the eluatej the determination was effected with the adsorptivity 1.5^ cm mg""'" at 280 nm. The gel was washed after each test by 0.5 M sodium hydroxide and then with distilled water until the eluate displayed neutral reaction. The gel was fre^ze-dried and weighed.

In the table, the adsorption capacity for cytochrome C is given, as -well as the sulfur content of some dissimilar agarose gels and agarj in part untreated, in part treated according to the invention (Example 1 ) .

T A B L E Type of gel Adsorption capacity (mg cytochrome C/mg gel) Untreated Treated Untreated Treated Sepharose 2B 0.12*f 0.007 0.179 0.028 Sepharose 0.102 0.005 0.182 0.026 Bio-Gel A® 1. 5 0.080 " 0.008 0.118 0.021 Difco Bacto Agar 0. 2^0 0.060 0. 371 0.0>+9 Sepharose 2B ® Fine Chemicals Bio Rad Lab., Richmond, USA, and Difco Bacto Agar® 6% is made by Difco Lab. Inc., Detroit.

It is to be emphasized that the form stability of the product according to the invention is much greater than that of the agar, or agarose, which is not cross-linked, The products according to the invention retain their form unchanged, even when heated or in an alkaline solution. This contrasts to previously known agar products in pellet form. The present products not only withstand strong alkali and higher temperatures, but can also be freeze-dried, and freeze-dried products may be reswelled to spherical particles by treatment with water.

According to the invention* cross-linked and desulfurissed agar cannot be considered a product where cross-linked agarose is included in an otherwise unreacted mass* Accurate examinations indicate that both agaropectines and other polysaccharides in the agar appear to have taken part in the cross-linking process* First, with the treatment according to the above* a arge fraction of the sulfate groups in the agar is removed, whereas most of the earboxyl groups remain* However, under certain circumstances, both these groups can cause adsorption conditions which appear to have a disturbing effect, e.g. during molecular sieving* But* Recording to the present invention, the earboxyl content can be reduced to practically zero, whereby the total adsorption capacity is reduced considerably* By decarboxylation it is possible 1» prepare products with adsorption capacities reduced to 10$ of the values exhibited by those resulting from desulfonation alone* The product is thereby freed, before or after desulfurization, either entirely or by ing i t into a almost completely from earboxyl groups since--.tt-i-s transferred-in-~ hw reactinq t reactive form and then m«de-to--react with strong reducing agents such as metal hydrides, e.g. hydrides of lithium, magnesium, aluminium or boron, or derivatives thereof, bo ranee, etc*, whereby the adsorption capacity of the product is further reduced. thus, particles of cross-linked agar or agar derivatives may be treated with reducing agents such as lithium-aluminium- or hydride or sodium borohydride in a solvent in which the agar or agar derivative is swellable and with which t e hydride does not react or reacts so slowly that the reaction with the agar is not affected.

S e reduction is promoted by elevated temperature. Xt is demanded of this treatment that it will not cause decomposition of the particles or alter their fom.

It has proved difficult to considerably reduce the adsorption capacity by direct treatment of the agar product with LiAlB^ in ether, dioxane, etc. since the solvent plainly fails to penetrate the agar to the required extent. Suitable ways for carrying out the treatment involve the transfer of the agar into a reactive form. A typical method for accomplishing this is by first swelling the particles in water and then washing them with an organic solvent of intermediate polarity, (e.g. n-propano ) followed by dioxane. Other solvents such as tetrahydro-furan may also be used. The agar or agar derivative caa first be converted to ester form, e.g. acetate. In this way, an intermediate product is obtained which swells better in dioxane and other ethers.

Xt has been proved preferable to effect the reduction at elevated temperature, preferably over 80°C. This appears to be a result of the molecular structure opening more at the higher temperature, presumably because the hydrogen bonds break up during polymer forming. In addition, the increase in the solubility and reactivity of the hydrides at high temperature makes the use of high boiling etft-are desirable.

The reduction, e.g. lithium-aluminium hydride treatment, can be efected-elac¼*fly--in agar ox/ agarose* without previous desulfurlzation. In this way, the sulfate groups may be eliminated with simultaneous reduction of the earboxl rous. Of course cross- ned a In the latter case, the adsorption capacity is reduced only by reduction of carboxyl groups. If the reduction is effected on cross-linked acetylated agar or agarose, the remaining acetate groups must be removed by alkaline hydrolysis preferably in the presence of sodium borohydride. To attain satisfactory results, a significant excess of reducing agent is required. By using a quantity of LIAIH which is greater than 1/2 the amount by the weight of/dry agar substance, a product is obtained with a very low adsorption capacity.

In order to further elucidate the invention, two more examples will be given below.

Example 17 300 ml cross-linked, acetylated, 6% agar in bead form was put into a 500 ml round-bottomed flask, and 2 0 ml dioxane was then added. 10 g LiAlH^ was added in portions during careful agitation with a continuous supply of nitrogen gas over the reaction mixture. No reaction or a very weak reaction was obtained. But when the temperature was raised to ·+5°0 , a strong reaction occurred. The mixture was cooled temporarily but then raised to 60°C and kept at this temperature for 1 hour. Then it was raised to 80°C for 2 hours. The reaction was then interrupted with ethyl acetate accompanied by an addition of water, thus cooling the mixture. 1 M hydrochloric acid was added, during refrigeration, until all the deposit was dissolved. The beads were washed quickly with ico-cold 0.1 M hydrochloric acid and then with water.

Deacetylation then took place with 1 M NaOH containing 0.1% NaBH^ at 80°C for 15 minutes. The beads were finally washed with water. 0 The product prepared in this way adsorbed £.01 mg cytochrome C (calculated per mg dry gel) in 0.001 M ammonium formate at pH 3·8 whereas the source material under the same conditions adsorbed 0.12 mg cytochrome C.

Example 18 ml 2% cross-linked, desulfurized agar in bead form, according to the above, was swelled in water, washed first with 100 ml n-propanol and then with 200 ml dioxane. 0.5 g LiAlH^ was added to a suspension in 25 ml dioxane. The mixture was heated to 90°C for 20 minutes and then cooled to 0°C when the reaction was interrupted with ethyl acetate and water (ice). The mixture was acidified by the addition of 1 M HC1 to pH 2 and the beads were washed first wth ice-cold 0.1 M HC1 and then with large amounts of water.

The prepared product adsorbed 0.013 m cytochrome C per mg dry gel substance, corresponding to about 10 of the adsorption on the original cross-linked agar.

An agar cross-linked, desulfated and/or decarboxylated according to the above can advantageously serve as a «afe-?i*--in •insoiu-to!e protein-agar derivative/, matri x.

It is known that proteins can be chemically united with insoluble polymers such as cellulose, cross-linked dextran, etc. while either wholly or partially retaining their biologically important properties, enzymatic activity, ability to selectively bind other proteins and other substances, etc. The preconditions are that the chemical fixing is effected under mild conditions ful fil l s and that the polymer fi±3rs certain demands.

Protein-polymer derivatives have already begun to find use in practice and their importance in different fields can be expected to increase rapidly. There are already commercially available enzymes bonded to cellulose or synthetic polymers and they can be used for preparative or analytical, biochemical reactions, e.g. the hydrolysis of esters, breaking down of proteins to peptides and amino acids, oxidation of carbohydrates, etc. Antigen proteins may be bonded to polymers to form adsorbants for corresponding antibodies. Enzyme inhibitors of polypeptide or protein nature can be bonded to polymers to form specific adsorbants for the enzymes against which the inhibitors are directed.

It is obvious that such specific adsorbants provide very effective methods of producing in pure form medically, or in another way biologically active matter found in nature, e.g. for purifying. vaccines. However, all previously known protein polymers are inherent with different deficiencies. According to the present invention, these deficiencies are remedied and an unobjectionable product is produced. Two factors are most important eres a) the structure and properties of the matrix of the polymer used, and b) the method for coupling the protein to the matrix.

It is quite important to choose a suitable polymer for fixing the biologically active protein. This polymer must be shoul d be provided in the chemically inert and mechanically stable. It -ougfet-t©--e.xisi-j-n- form of / particles form which can be effectively and quickly penetrated by proteins. Enzyme-protein complexes and other bio-polymer- protein complexes must be able to form and be dissolved within flattest overall flow profi le. the gel and provide the m»s —■t^eeii- possible ±-94τθΗ.—f-le-w.

It is evident from these requirements that the polymer must be hydrophilic and consist of a macromolecular network of a very open nature. Moreover, the polymer must be able to be brought into a chemically active form which allows chemical coupling of protein under mild conditions.

The/ use -da±_e gel forming polymers have all the shown deficiencies in one or more respects. Cellulose often has, like other synthetic polymers, an unsatisfactory permeability for forming protein-protein complexes. Co-polymers between ethylene and maleic anhydride contain a very high concentration of carboxyl groups whereby the polymer receives strong ionjexchang-ing properties. This is a significant drawback in specific adsorption and also affects enzyme reactions.

Agar also has ionogenic groups. However agarose, one of the polysaccharide components in agar, has a lower concentration of ionogenic groups and is therefore more suitable as a matrix for polymer -bonded proteins. Agar and agarose form mechanically stable gels even when the matrix is of such low concentration that high molecular proteins and even viruses can penetrate the gel. Proteins may be bonded to agar or agarose by e.g. the cyanogen bromide method described in Nature 21*+, 1302(1967) and 215, 1^91 (1 67) or with the oxirane method, Sw-etfctSh- patent No. 3853708 a H na ion 81+ /70. without the simultaneous introduction of ionogenic groups. However, both agar and agarose have the seri-ous drawback of not being completely insoluble. It is assumed that the coherence in the gel is due to hydrogen bridges between the polymer chains. These bridges are dissolved and are rebuilt obviously spontaneously and often, and therefore leakage of polysaccharide occurs continuously. In certain mli¾¾ws suitable for splitting protein-protein complexes which are often formed ■ 'ψ * 37131/2 with hydrogen bonds, the solubility of the agar can be Increased catastroph cally. When protein s fixed, the solubility may Increase even wore. r When separating a substance or a group of substances 1n pure ΛΠΒ, here designated Sj, from a mixture of the components Sj, $2· .· Sj...Sn, separation nvolves removing all the components Sj , where 1 f J. If only Sj forms a complex w th a protein P, Sj can be solated selectively by biospeciflc adsorption followed by desorptlon* Ψ 1s fixed for this purpose to a matrix H; a specific adsorbent 1s formed, here called MP. When MP Is brought Into contact with the mixture S| <·——*» ¾, the Insoluble complex or complexes MPS] are formed which can be liberated from the other components Sj by decanting or washing on a filter* In the next stage, $j Is liberated whereby M 1s regenerate .

Sj Is often quite strongly bonded to MP particularly where a high degree of specificity exists. Therefore, dissociation or releasing of S can constitute the critical moment 1n this form of specific purification. Both Sj and HP shall retain their properties. The protein Ψ mus not be denatured since the adsorption capacity would then be lost. Dissociation can often occur with so-called chaotrop c Ions, e.g. su foc anate or Iodide In high concentrations, e.g. 3 M solutions, but agar and agarose are then dissolved 1n great amounts.

However, according to the present Invention, an agar product can be prepared with most Insignificant solubility and practically total trackin In 1onogen1c groups, since cross-Unking within agar has now been achieved with a special method. It has also been shown that these cross-Hn ngs do not prevent the fixing of different types of proteins to the cross-linked agar -with/known per se^methodsj. Thus e.g. a suitable coupling can be attained with the cyanogen bromide or oxirane methods, and even with other methods. The protein may e.g. be an enzyme whereby an "insoluble enzyme" is formed which, at the same time, is a specific adsorbant for all inhibitors and antibodies directed against the enzyme, as well as other substances which build complexes with the enzyme. Also, suitable organic chains may first be bonded to the agar. The protein is then coupled to these chains with the intention of preventing steric defects. Since practically all proteins P can form antibodies, a complex pair PS. may always be found which in principle fills J the requirements. The protein P fixed at the cross-linked agar can be included in a protein complex of a higher order, e,g, in a virus or in a cell particle, such as a ribosome or a cell membrane fragment. The protein may also be composite, e.g. glycoprotein.

There is no sharp distinction between proteins and polypeptides. In the present application, the word "protein" is used in a general sense and oven embraces polypeptides having high molecular weight, e.g. over ^000.

According to the invention, protein-agar products have been prepared which « . such media ^ ^ 1) are insoluble in /aqueous soi¾iti©i¾s wfeea?e proteins are not hydrolyzed, 2) can be penetrated by proteins, polysaccharides, viruses and other high molecular matter during the forming of reversible molecular complexes in the gel, 3) can be treated with substances such as iodides, carbamicf and guanidine without agar poly- saccharides, with or without protein, being dissolved from the gel phase.

The protein-agar product is preferably prepared from agar, in bead form, which has been cross-linked and desulfated and, if desired, decarboxylated as described above. A protein-agar so prepared forms rapid-filtering beds where ionogenic groups disturbing the adsorption and enzyme reactions are eliminated from the native agar. The permeability in the protein-agar particles can be regulated by the concentration of agar in the water-swelled particles during the cross-linking.

It is particularly important that the agar matrix does not contain ionogenic groups, as adsorption and desorption shall take place with very low salt concentrations. Atf ion exchange adsorption can, under such conditions, completely spoil the biospecific fractionating and reduce the exchange to zero. The protein-agar 1 s insolubility is especially importait since the substances to be purified are for clinical use, e.g. for intravenous injection when immune reactions may occur. -Leakage of enzymes, antigens, antibodies, etc. can jeopardize the analytical use of polymer-bonded proteins* All these complications are avoided with the use of the present invention.

In order to further elucidate and illustrate the invention, its application will be described in the examples below. Example 19 1 liter^swollen, cross-linked, . desulfated agarose beads according to the above was mixed in with 20 g cyanogen bromide dissolved in 50 ml water. After 2 minutes, 2 moles NaOH was added to pH 11-11.5. After 9 minutes, the gel was washed with about 2.51 0.5 M NaHC0 at 2i+0C. 50 ml of a solution containing 10 g concanavalin A dialyzed against 0.05 M sodium acetate buffer, pH 6 * 0 , was added to the activated gel and the suspension was agitated for 2 hours at pH 8.0. The concanavalin-agarose gel was washed consecutively with 2 1 of each of 0 , 5 M NaHCO^ buffer at pH 8.0 , distilled water, 0. 3 M sodium formate buffer at pH 3.0 and 1 M with respect to NaCl, 0.05 M sodium acetate buffer at pH 6.0 containing 0.02$ NaN-. and 0.001 M Mg+2 and Ca+2. The azide is added to prevent bacterial growth .

A 6.3 x 1 cm bed was then packed by the protein gel, prepared according to the above, and a solution of glycogen, 2 mg/ml, introduced at a rate of 5 ml per hour. After about hours, the bed had been saturated with respect to glycogen. The bed was washed with buffer, whereby the eluate gradually became totally free from glycogen. As the orcinol reaction was negative, it could be ascertained that no carbohydrate leaked out of the gel. The bed was washed with 0.1 M sodium formate buffer at pH 3- 0 whereby about 35 mg glycogen was displaced from the bed.

In this example, the matrix M was cross-linked agarose, the protein P was concanavalin and S. was glycogen. S. can also be another polyglucane or a glycoprotein containing glycose.

Example 20 100 mg bis-epoxide cross-linked, desulfated agar beads (dry weight 6% regarding matrix content) was suspended in k ml distilled water and was activated with k ml BrCN solution, 25 m BrCN/ml, for 6 minutes at pH 11 and at room temperature. The activated gel was washed, with cold 0.1 M sodium solution. mg ribonuclease A was dissolved in 5 l--&o4iBhM sodi ¾¾ΙΌcarbonate solution and was allowed to react with the activated gel at +-h°C for 30 hours. The coupling product was carefully washed. The ribonuclease-agar gel contained 82 mg protein per gram gel. The enzyme was tested against different ribonucleic acid preparations. The activity was in the interval 25 - 50% of the free enzyme. This is a very satisfactory result considering the high molecular weight of the substrate and the fact that ribonucleic acid is a linear polymer, thus making permeability more difficult.

Example 21 ml epichlorohydrin cross-linked, desulfated, bead-shaped agar was washed on a filter first with dimethyl sulfoxid and then with water. The gel was activated with BrCN in the same way as in Examples 1 and 2. 50 ml antilymphocyte globulin (ALG) was dissolved in 20 ml 0.5 NaHCO^ solution and added to the activated gel. The coupling was carried out in a refrigerat ed room for 2k hours. The ALG agar was then carefully washed with 0.5 M NaHCO^, 0.1 M NaAc buffer at pH 3.9, 0.05 M Tris-HCl buffer at pH 8.5 and 0.05 M sodium phosphate buffer at pH 7.5. All the buffer solutions were 1 M with regard to NaCl. The gel was packed into a column. ml sonicate of human lymphocytes was introduced in the column. After washing with phosphate buffer, pH 7· 5, the ad- Nal sorbed material was desorbed with 20 ml 1 M DfaJ in the phosphate buffer. The solution was concentrated and analyzed. No carbohydrates were released from the column. The desorbed material was examined wa¾h a cytotox test and was found to consist of immunoactive leucocyte fragments.

Example 22 8 ml settled, 1.3-dichloropropanol-2 cross-linked, desulfated, and LiAlH^ reduced agar beads were treated with ml 1 M NaOH containing 20 mg NaBH^ and 2 ml 1.3-butanediol-diglycidyl ether under agitation at room temperature for 6 hours. The gel was then washed with distilled water and 0.2 M bi sodium hydrocarbonate buffer, pH 9.0. 100 mg soyabean trypsin inhibitor (STI) dissolved in ml sodium -h-yd½carbonate buffer at pH 9.0 was added to the gel which was then allowed to stand at room temperature during agitation for 20 hours. The gel was first with the carbonate buffer and then with 0.1 M glycine buffer containing 1 M NaCl, at pH 3.0.

The gel was then transferred in 0.05 M Tris-HCl, pH 7. 8, containing 0.5 M NaCl and 0.02 M Ca +2 and packed in a column.

A solution containing 0.2 mg/ml commercial trypsin was allowed to pass the bed. Inactive material passed through undelayed.

The gel absorbed 3. mg active trypsin, which could be el used petto¼-?&4©d with 0.05 M glycine-HCl, pH 3.0 , containing 0.5 M NaCl and 0.02 M Ca+2.

The trypsin was adsorbed ¾o the STI gel in a 1 ; 1 ratio. The cross linking of the agar gel thus does not affect the capacity to form reversible complexes.

Example 23 A raw extract of bovine pancreas powder (from the pancreas of a cow) was introduced into a bed of STI gel according to Example 22. Ko trypsin activity passed the column before 50 ml extract was introduced. The gel was washed with Tris-HCl-buffer according to Example 22 and 0.1 M NaAC/HAc buffer, pH k . 5, was introduced, the cymotrypsin being displaced. The pH was then lowered by eluting -with glycine buffer according to Example 22. 3.5 mg pure trypsin was displaced from the column. The nn adsorption was significantly far less than for a similar bed based on commercial agarose which was not cross-linked, desulfated and reduced.

The gel could be used an unilimted number of times without a noticeable reduction of the adsorption capacit . No releasing of the trypsin inhibitor could be ascertained.

The bed, as in Examples 19 to 22, had excellent flow properties.

Claims (1)

1. What we claim is Agar product for separation in such as for molecular as a stabilizer in electrophoresis or as inert source material for preparing well defined ion exchangers or specific adsorbents of ent as gel matrices for fixing enzymes or other chemical or for other similar characterized in that the agar fractions prepared from agar preferably are practically completely insoluble in water and strong 1 due to and if desired are practically free entirely or partially free from earboxyl thereby haying a very low absorption capacity for basic mattery Product according to claim characterized in that the agent is or epibromohydri Product according to claim characterized in that the agent is a Product according to claim characterized in that the agent is divinyl eulfone or another linking vinyl Agar product according to claims 1 to characterized in that it contains no or few earboxyl and if no being the product of aotlvization followed by reaction with strong reducing such as hydrides of boron or derivatives having a very decreased adsorption according that a product is as source Product according to claim 5 or claim characterized in that the source material used is a agar derivative consisting of an ester of a lover acetic and that if the product of reduction with and of removal of the remaining acetyl groups alkaline hydrolysis in presence of sodium Agar product according to claim characterized in that the particulate part of agar or agar product is with epihalo divinyl or is with substances that gives rise to suc compounds under the prevailing reactive conditions that is deeulfated optionally that adapted to be chemically coupled to a protein component which in turn may be included in a reversible molecular complex or Agar product according to claim characterized in that the protein is an or an enzyme product according to claim characterized in that the protein is an immunoglobulin or corresponding product according to claim characterized in that the protein is a lectin or other carbohydrate binding 37 ί 2 Method of preparing agar products according to claims 1 to characterized in that the or agarose particles preferably in the form of homogeneously swollen obtained from a homogeneous aqueous are treated in an aqueous slurry with or analogically reacting dlvinyl sulfones or other vinyl whereby the reagent if be dissolved in an organic solvent and the reaction is carried out in an alkaline medium in the absence of if in the presence of a strong reducing the sulfate content in the product may then be lowered by alkaline also under or reducing until the adsorption capacity for basic matter has decreased to or the desired and that the product is then optionally Method according to claim characterized in that the reagent is dissolved in organic solvents such as Method according to claim o claim characterised in that the gel concentration of the mixture when the takes place can vary between 20 and Method according to claims 1 to characterized in that the agents occurs in an alkaline medium at elevated Method according to claim characterized in that the alkali concentration is between and 37 according to claim claim in that the is preferably kept between Method according to claims to characterized in that the reaction time for the exceeds 1 Method according claims 2 to in that the sulfur content after the is lowered by extraction and hydrolysis by boiling the agar gel in a solution in an environment and repeating this boiling or in the presence of sodium borohydrlde if the initial sulfur content was Method according to claim characterized that decreased adsorption capacity achieved by wholly or substantially freeing the agar product from groups by transferring into a reactive and then reducing strong reducing agents such as hydrides of boron or derivatives such as hydride Method according to claim characterized in that the reducible form obtained by first swelling the primary product in water and then washing first a suitable organic and then with Method according to claim 20 or claim that the organic solvent used is ether or an aky ether with a high boHlng according to claims 20 to characterized in that the water content in the system before the tion is reduced to practically Method according to claims 20 to characterized in that the reduction is effected at high preferably 80 Agar product for separation substantially as hereinbefore Method of preparing agar product for separation substantially as hereinbefore Box Attorneys for Applicant 2δ insufficientOCRQuality