DE2131139A1 - Stabilized agricultural product and process for its stabilization - Google Patents

Description

Exploaterings Aktiebolaget T.B.F.,

Uppsala, Sweden

Stabilized agar product and process for its stabilization

Agar can be obtained and is made by extracting certain red algae from a mixture of high molecular weight polysaccharides, the main product of which is galactose after extensive hydrolysis. Because Because of its ability to form gels with a high water content, agars are widely used in biochemistry and microbiology. Particularly the resistance of agars to microbes is valued.

When a warm water solution is cooled, the agar gel is formed when the agar concentration exceeds a certain limit characteristic of different agar types, which is usually is less than 1 percent by weight. It is assumed that during gel formation a molecular network is formed in which the polysaccharide chains are held together by hydrogen bonds. When heated, the gel dissolves and becomes with strong acid or strong alkali hydrolyzed, especially when warming up. An agar gel can also be obtained by swelling solid, dry agar particles in water leaves. However, from the current point of view, the product obtained thereby acquires significantly poorer properties.

Most separation processes in biochemistry are based on the ability ' the agäregels to bind large amounts of water, especially processes, which are based on the diffusion of dissolved substances. Such Processes are gel filtration, also called gel chromatography, oil exclusion chromatography, Molecular Segregation Chromatography or Permeation Chromatography. Used on agars, this chromatography insists Technique in which a mixture of substances with different molecular sizes pass through a bed of agar particles

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leaves that are wetted with a water solution, the migrate Substances with different speeds through the bed = The high molecular substances pass the bed faster than the low molecular ones = The working interval for the molecular segregation vdrd is determined by the network and the mutual size ratio of the molecules. A gel with a low agar concentration can be used for the mutual separation of high molecular weight substances while a gel with a high concentration of agar is better for fractionation suitable for substances with smaller molecular dimensions «

The ideal molecular strainer must meet certain requirements. So rope it does not contain any charged groups, for example, and furthermore it is said to be insoluble and chemically resistant »The ordinary agars leaves a lot to be desired in this regard.

It has now been found that the agar contains a fraction, the agarose, with a significantly lower content of charged groups than the starting material. This fraction is therefore as a molecular abseilioi? with better properties than the agars for Am-jenäu.sg gekoamsii. The Agar rose has also replaced the agar in other contexts, when it was requested that the gel should not hold a large amount of charged groups that were chemically bound in the gel substance

A further improved product is obtained according to the American patent 3 50? 851, which is a cross-link of agarose with epichlorohydrin. Agarose bound in this way is less soluble than natural agarose and also more resistant to alkali,

It is of great importance, especially for technical use, that a high current is obtained in molecular separating beds, which the Setting of rapid diffusion equilibria is permitted. It is also of particular importance, a well-defined flow front received by the bed see below, or as close to theoretical piston flow as possible. This is best done by giving the particles a spherical shape. This is at the time with the cross-linked Agar gels didn't happen.

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The properties that characterize the ideal molecular separator are also desirable in those cases where the gel is used as a starting material for the production of ion exchangers or other adsorbents, as well as for the production of enzymes chemically bound to gels or other biologically and chemically active gel materials is intended to prevent convection in a solution, for example in preparative electrophoresis.

The present invention relates to a product which better meets the requirements for molecular absorbers than the agaric products known hitherto. According to the method according to the invention for the production of this product, the agar can be used directly as a starting material without first subjecting it to a time-consuming loss of material with a fractionation process. According to the invention, the agar product is obtained in the form of spherical particles with very low adsorption capacity, which are insoluble in water in heat, even in an alkaline medium,

The present invention relates to cross-linked products Agar or agarose with much better properties than the previous products and made in a different and beneficial way. the Cross-linking can be done with epihalohydrin, for example with epibromohydride or epichlorohydrin. At the cross-linking reactions ! .- ■ it the agar take both agarose and the rest, in the A; ~ are incoming polysaccharides part. It has been shown that if the cross-connection in the absence of oxygen and besides in the presence of a reducing agent, e.g. sodium borohydride, happens, one avoids the brown coloration and partial destruction of the agars, which normally happens in reactions in the alkaline medium, which is required for coupling the agars to the epichlorohydrin. It It has also been shown that the cross-connection stabilizes the agar product in such a way that it can be removed by alkali treatment, e.g. once or several times Treatment in autoclaves, can be made more or less sulfate-free, and if required, almost entirely of the undesirable Adsorptive ability to be freed. For the most part, this becomes common attributed to the sulfate content of the product, however, the treatment mentioned here can affect most of the naturally occurring Substances, to zero, without the total sulfur

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content of the product must necessarily be zero »(cf. the values in Table 1).

It has also been shown that under certain conditions one can can achieve beneficial results by cross-linking with Bisepoxides, e.g. 1,3-bis- (2,3-epoxy-propoxy) -butane also use divinyl sulfone and other cross-linking vinyl compounds. Finally, it can be added that you can of course can also use such chemical compounds that under the reaction conditions and the course of the reaction in e.g. epichlorohydrin or another aforementioned cross connector can be converted. As an example of such compounds, 1,3-dichloropropanol-2 (see example 12) or 2,3-dichloropropanol-1 (see example 13).

By varying the degree of cross-linking, mainly the nature of the cross-linking molecule, one can obtain molecular separators with somewhat variable molecular exclusion limits, and with much higher values than was previously possible. This is illustrated in Figure 1 which shows an example of molecular sequencing on 2% crosslinked and hydrolyzed agars. The curves relate to

I Tobacco mosaic virus with a particle weight of around 2. 10

and elongated shape,
II adenovirus type 3 with a particle weight of about 1.8 · 10 and spherical shape,

III poliovirus with a particle weight of approximately 6.8. 10 and spherical shape
and

IV satellite tobacco necrosis virus with a particle weight of approx 2. 10 and spherical shape,

The products according to the invention can advantageously be swollen on Gels are based, in the form of pearls and with a high water content “If you start with a gel that is soaked with mass, it remains The degree of cross-connection can be more limited, however, at the same time as desired can be regulated and reduced by allowing the gel to break down easily allows penetration of the molecules provided for the cross-connections. $ 90 or more of water seems reasonable in most cases

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To be water content. It should be noted that the shape of the Gel particles not changed during the treatment according to the invention. But it is also important that according to the inventive method the cross-linked gel remains insoluble even at higher temperatures and in an alkaline solution. A bed from the inventive The product can therefore be sterilized by warming it up in the autoclave, which is decisive in various cases Should be meaning. The insolubility is also particularly valuable if the agar product is used, for example, for pharmaceutical purification Products are to be used, in particular for injection purposes, where contaminating substances such as antigenic substances must not be released. The agar product also remains transparent, colorless or white, which shows that an undesirable decomposition of the agar product occurs during the treatment.

In order to further illustrate the invention, a number follows below of Ausfükrungsbeispielen, although the reagents mentioned and amounts can be varied as desired within the scope of the invention.

example 1

A suspension of swollen agar or agarose particles, preferably in the form of pearls, which is prepared in a manner known per se from a boaogenic water solution of the current agar or the agar product, whereby even crushed material can be used, in 0.5 M sodium hydroxide solution was mixed with 10 ml of epichlorohydrin added per 100 ml of swollen gel mass. The mixture was heated to 60 ° C. and maintained at this temperature for 2 hours, in a nitrogen atmosphere or in the presence of sodium borohydride with effective shaking. The cross-linked gel was left neutral in water. For hydrolysis of sulfate esters, the cross-linked agar or agarose gel was boiled or autoclaved with an equal volume of 2 M sodium hydroxide solution for 2 hours in the presence of sodium borohydride.

Example 2 *

The cross-connection was carried out according to Example 1, but with The difference is that instead of the waeeer solution, you use a solvent

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-D-

from 50 Vol- $ ethanol and 50 Vol- $ Jo took ο the epichlorohydrin was completely soluble in this geraisch. The resulting cross-linked tr Gel was not soluble in boiling water and was not noticeable Autoclave the change in 1 M sodium hydroxide at 120 C for 1 hour.

Example 5

The cross-connection was carried out according to Example 1 with the difference that the solvent used was absolute ethanol instead of epichlorohydrin. In terms of solubility, the product agreed corresponds to the agar gel obtained in Example 2 "

example k

The cross-connection was in 50 Vol-? O dioxane and 50 Vol- 1 Jo water otherwise the same conditions as in the previous example were carried out. A product was obtained which was insoluble and stable to heat and alkali.

Example 5

The cross-connection was carried out in exactly the same way as in previous example, but with epibromohydrin instead of epichlorohydrin. A product was obtained that was stable in heat and alkali and had the same appearance "

Example 6

The cross-connection was carried out in the same way as in Example 3, but with epibromohydrin. A similar product was obtained.

Example 7

The cross-connection was made in the same way as in example h ; but with epibromohydrin. A similar product was obtained.

Example 8

The cross-connection was carried out according to Example 1, but the epichlorohydrin was exchanged for 1,3-bis- (2,3-epoxy-propoxy) -butane. In contrast to epichlorohydrin, however, this bisepoxide can be mixed in water. A product was obtained which was insoluble and stable to heat and alkali.

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lasted, as in the other examples c

Example 9

The cross-connection was made as in Example 8, one swapped dns water against a mixed solvent of 50 vol- # water and 50 Vol- ^ i ethanol, and the bisepoxide, which is sparingly soluble in water 1, 'f-bis - ^^ - Epoxy-PropoxyJ-Butane was used.

Example 10 The procedure here was the same as in Example 9, but the cross-connection was carried out in absolute ethanol.

Example 11

The cross-connection was made according to Example 91 with the difference, that the solvent consisted of 50 vol- # ethanol and 50 vol- # dioxane ,

Example 12

The cross-connection was made according to Example 3i with the difference that that epichlorohydrin was exchanged for 1,3-dichloropropanol-2.

Example 13

The cross-connection was made according to Example 3, but with epichlorohydrin instead of 2,3-dichloropropanol-1,

Example 1 ^ The cross-linked gel was produced according to Example 3, with the difference that the autoclaving was carried out in a solution of 1 M sodium alcoholate in absolute ethanol.

Example 15

100 ml of swollen bead-shaped agar was washed with M Soda solution brought into equilibrium. 5 μl of divinyl sulfone were used in 50 ml of soda solution. The gel took on a milky appearance. The suspension was heated to 50 C for half an hour and then washed on filter. A sample was taken and heated to 100 C. No disintegration could be perceived, and neither were the gel balls kept their shape. The gel was then immersed in a 1 M NaOH solution

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0.5 $ NaBH, containing, transferred. The gel so treated was retained the spherical particle shape and a bed packed with it showed excellent flow properties.

To show that the agar pearls as well as liquid particles can be cross-connected while maintaining the spherical shape, the following experiments were made, which also show that it is not necessary to separate the spherical particles after their formation in order to then carry out the cross-connection.

Example 16

500 ml of a 6 percent agar solution in 0.5 M NaOH with 2.5 6 NaBH. was suspended at 75 in 600 ml of ethylene chloride containing 20 g of polyvinyl acetate as a stabilizer. The shaking was regulated in such a way that particles of suitable size (50-250, m) were obtained. Then 50 ml of epichlorohydrin were added and the mixture was shaken at 60 for a further 2 hours. After cooling, the gel particles were freed from the emulsifier by careful washing in acetone. The pearls then came into the water and were autoclaved according to Example 1.

To compare the adsorption properties of the new products with those of the starting material and the agarose, the following experiments were carried out:

2
The gel was transferred to a bed of 0.90 cm in cross section and 2.5-3 ₅ 5 era in height chromatograph packed eröhren!. The bed was then equalized with 0.01 M ammonium acetate buffer, pH 4.1. Cytochrome C (0.1 $ solution in the same buffer) was introduced and effluent showed the same color strength. The bed was washed with the buffer solution until no more cytochrome eluted. The absorbed cytoehrome was displaced with 0.15 M ammonium acetate buffer, pH 4.1. The measured

2 - ^ amount of cytochrome in the eluate, where the adsorptivity was calculated as 1.54 cm mg, was 280 run. After each experiment, the gel was washed with 0.5 l sodium hydroxide and then with distilled water until the eluate had a neutral reaction. Pas Gel i-; sjrde freeze = dried and weighed.

The table shows the adsorptive capacity for · cytochrome C as well as

the sulfur content of some different agaros gels and agars indicated, partly untreated and partly treated according to the invention (Example 1).

Gel type

Sepharose 2B '^
Sepharose 6β (Ξ ''
Bio-Gel A <? ^; 1.5 m
Difco Bacto

Table 1 #s Treated 028 Adsorptive capacity 0, 026 (rag cytochrome c / m gel) Untreated 0, 021 Treated untreated 0.179 0, 0 * f9 0.12 * 1- 0.007 0.182 0, 0.102 0.005 0.118 0.080 0.008 0.371 0.2 * f0 0.060

Sepharose 2B ^ 'and Sepharose 6B ^ - are agaroses from Pharmacia Fine

Chemicals AB, Uppsala. Bio-Gel A ¹ ^ is an agarose from Bio Rad Lab., Richmond, USA and Difco Bacto agar * ■ · - '' 6% is from Difco Lab. Inc., Letroit.

Eggs should be emphasized that the dimensional stability of the inventive Product is much larger than with non-cross-linked agar or agarose. So the product according to the invention keeps an unchanged one Shape even when heated in an alkaline solution in contrast to previously known agar products in spherical shape. You can also not only tolerate strong alkali and higher temperatures, They can also be freeze-dried, whereby the freeze-dried products are again transformed into spherical products by treatment with water can be swollen.

According to the invention, cross-linked and sulphurized agars cannot be considered a product in which the cross-linked agarose goes into an otherwise unreacted mass. Exact investigations show that both agar-epectins and others Polysaccharides entering the agars must have participated in the cross-linking process. *

The above treatment removes most of the Sulphate groups of the agaro, while the carboxyl groups to the largest

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Stay back rope. Under certain conditions, however, these can Adsorptxon ratios were carried out, for example on interfering with the straining of molecules.

According to the present invention, however, the carbon monoxide content can also be reduced to practically zero, as a result of which the total adsorptive capacity is significantly reduced even further. Products have been produced with an adsorptive capacity which, by decarboxylation, has been reduced to 10% of the only desulphated products. Here, the product is completely or largely freed from carboxyl groups before or after sulfur removal by converting it into a form prone to reaction and then using strong reducing agents such as metal hydrides, e.g. hydrides of lithium, magnesium, aluminum or boron, their derivatives, etc. Boranes, etc., caused to react, as a result of which the adsorption capacity of the product continues to decrease.

In the following way, particles of cross-linked agar or agar derivatives can be treated with, for example, aluminum or borohydri'l or siaes derivatives thereof, especially lithium aluminum hydride in solvents where the agar or the agar derivative can swell and with which the hydride nods or react so sluggishly ₉ that the reaction with the agar is not disturbed «, Increased temperature promotes the reaction. _{Raan t} demands from this operation that it neither results in the disintegration of the particles nor a change in shape.

It has proven difficult by treating the agar product directly with LiAlH. in ether, dioxane, etc., to lower the adsorption capacity, since the solvent looks! -.- jud does not penetrate the agars to the required extent. Suitable methods for carrying out the treatment include the UeberfiHrc-iang the ägare in a reactive consent form, for example by having initially swell in water, the particles and then washed with an organic solvent of intermedierer polarity, such as n-propanol and then with dioxane, Other solvents, for example tetrahydrofuran, can also be used. You can also transfer the agars or dae Agarederivat in a Esterform, eg acetate. Such

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you get an intermediate that works better in dioxane and others
Ä'tnern swells.

Efi has been found to be suitable for carrying out the reduction at an elevated temperature, preferably above 80 C. This seems to be towards it
are based on the fact that the molecular construction changes at a higher temperature
opens further, apparently by breaking the hydrogen bonds during polymer formation. In addition, the solubility and tendency of the hydrides to react increases at high temperatures. Ethers with a high boiling point are therefore best suited for this.

The reduction, for example a lithium aluminum hydride treatment, can be done directly on cross-linked agar or agarose,
without this being sulphated first. It is thought that some Sulfatg _rv ~~ ^ _r split off, partly carboxyl groups are reduced. It is also possible to treat cross-linked agar or agarose from which sulfate groups have previously been freed by alkaline hydrolysis in a reducing medium. In the latter case, the adsorptive capacity is reduced only by reducing the carboxyl groups. If the reduction is carried out on cross-linked, acetylated agar or agarose, the remaining acetate groups must be removed by alkaline hydrolysis, suitably in the presence of sodium borohydride. To achieve a satisfactory result, a significant excess of reducing agents is required; however, the amount can be reduced significantly if the water content in the reduction system is low. With a quantity of LiAlH, which is greater than half the amount of dry agar substance, a product with a very low adsorption capacity is obtained.

To explain the invention more clearly, some exemplary embodiments are provided here cited.

Example 17

300 ml of cross-linked 5 acetylated 6 percent beaded agars was placed in a 500 ml round bottom flask and 250 ml of doxan was added. 10 g LiAlH. were added in portions with gentle shaking and a continuous supply of nitrogen over the reaction mixture.
There was no or only a very weak response when increasing

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However, when the temperature reached k3 C, there was a vigorous reaction. The mixture was temporarily cooled and then heated to 60 C and maintained at this temperature for 1 hour left and after 2 hours at 80 C ₀ Then, the reaction with ethyl acetate and then with water additive _s interrupted followed by cooling the mixture "During ice cooling, 1 M hydrochloric acid added until any precipitate had dissolved. The beads were quickly washed in ice-cold 0.1 M hydrochloric acid and then in water.

This was followed by deacetylation with 1 M NaOH containing NaBH at 80 ° C. for 15 minutes, after which the beads were washed in water became.

The product thus prepared adsorbed 0.01 mg of cytochrome C (calculated per mg dry gel) in 0.001 M ammonium formate at pH 3j8 while the starting material under the same conditions 0.12 mg of cytochrome C adsorbed.

Example 18

25 ml of 2% cross-linked sulphurized agar in bead form according to the above was swollen in water, washed in 100 ml of n-propanol and then in 200 ml of dioxane. 0.5 g of LiAlH. Was added to a suspension in 25 ml of dioxane mixture was for 20 minutes at 90 ⁰ C is heated, then cooled to 0 ⁰ C, and the reaction was stopped with A'thylacetat and water (ice). They made the mixture by adding 1 M HCl to pH 2 acid, washed the Pearls in ice-cold 0.18 HCl and then in large amounts of water.

The product thus prepared adsorbed 0.013 mg of cytochrome C per mg dry gel substance, which is about $ 10 of adsorption on the original cross-linked agars.

Siae as above querνe ^ baadene, desulphated uaä / or deearboxylated Igar © kasaa as Matrisi in «© iöaliehea PsOteiiiagarsass-i ^ s ^ en advantageous Service do.

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ORIGINAL INSPECTED

It is known that proteins are chemically linked to insoluble polymers like Cellulose, cross-linked dextran, etc. can be linked and all or part of their important biological properties, Their enzymatic activity, the ability to selectively other proteins and other substances, etc. to bind. The suspension this is that the chemical fixation is carried out under gentle conditions and that the polymer meets certain requirements enough.

In practice, one has already used protcin-polymer derivatives started and it is to be expected that their importance will increase sharply in different contexts. There are already cellulose related enzymes or synthetic polymers available for commercial use, cixe to be prepared or analytical, biochemical Reactions, zd>. the hydrolysis of esters, the breakdown of Proteins to peptides and amino acids, the oxidation of carbohydrates etc. can be used. One can attach antigenic proteins to polymers fix to adsorbents for corresponding © antibodies. Enzyme inhibitors polypeptide or protein nature can be combined with polymers to form specific adsorbents for against Inhibitor Directed Enzymes.

It is obvious that such specific adsorbents Opening up very effective ways to purify medicinal or Naturally occurring substances that are otherwise biologically effective, e.g. for cleaning vaccines. All previously known protein polymers however, had various disadvantages. E according to the invention, however, this is remedied and you bring a flawless Product comes about where two factors are essential, namely

a) Structure and properties of the polymeric matrix used and

b) Type of coupling of the protein to the matrix.

It is therefore extremely important that you have a suitable polymer for Fixing the biologically active protein chooses. This polymer must be chemically inert and mechanically stable. It is said to exist in the form of particles that are effectively and quickly pene-

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let trier «The Enzyme-Protein Coapiexe and other Biopolyierkom · = Plexes must be able to form and dissolve in the gel, and one as possible allow ideal piston flow.

It is clear from these claims that the polymer should be hydrophilic and consist of a macromolecular network of a very open nature should exist. The polymer should also be brought into a chemically active state that a chemical coupling of the protein undor gentle conditions allowed.

The previously used, gelbildesbn polymers have all DER one way or another to mangling suffered ,, cellulose ₃ soi-ste many aadere synthetic polymers have an incomplete person '^ - r ^ bin.iocvsur formation of protein-ProteinkoHplesten ,. Copolymers ίΐ -.- i'-r-hsi: Italy and maleic anhydride contain a dM ^ chsns h -; -. ·; 'Z- ^ ez, - tration of' carboxyl groups, making the polyamide rigid. ϊ.οι: - ϊ ~ ξλ :: τ --- 'receives exchanging properties, which svjh Sfaeliteil in csr- 3; .- 3Π-1_.1οίί: -ΐΓ: Adsorption are also due to enzyme reactions. eiiv-jivh-sn *

The agar also contains ionogenic groups. Bas Agare ~ e _; ei ^ e the Poi ^ sacciiaridekoaponentea in the agar ?, has isös ~ se ?: zir? E :. ^! , -r ^I r?, ST-r-- ^; -Concentration of ionogenic groups and © igaet therefore see bz? - ^: S3V au? / Matrix for polymer-bound Proteia © = Agar® and agarose form stable gels even if the mass is such a nisdr-i ^ iii Eoase tration is present that high molecular weight PEOeins and even "ΰίνβιι Ln a * s gel can penetrate" You can connect proteins with agare or agaros-3, g = B, aaeh der bromeyanaethodsg like ia Mature 2i4, 1J02 ^ 196?) uad 215, 1 ^ 91 (1967) besehriebes, oüer nach, the oxirane cathode, Swedish patent application 8 * 5-3 / 70, olms that with it ioaogenic groups are introduced on the track side. Both agar © as from ': ilgar-ose half indec = - sen a serious disadvantage, si © aiad sees that it is entirely possible to assume that the content of the compound is produced by the hydrogen bubbles between the polysaccharides ge know milieus that are suitable for splitting · νοη protein complexes, which are often formed with hydrogen compounds, the solubility of the * agar can increase catastrophically. When fixing the

10 9 8 5 3/1099 original m ^

- ι ρ—

Protein can further increase its solubility.

The production of a substance or substance class, here with

S. denotes, rsus a device of the components S, Sp ..... S

S is done by removing all components S where i / j is will. If only S. forms a complex with a protein P, one can

J
Isolate S. in principle by a biospecific adsorption with subsequent desorption. For this purpose, P is fixed on a matrix M, whereby a specific adsorbent is formed, here symbolically referred to as MP. If MP is brought into contact with the mixture S., .., .., £, the insoluble complex or the complex MPS, which is formed by decanting or washing on filters of the rest, is formed

3 ·

Components S. can be exempted. The next step should clear S.

^Mps -i κ MP + S.

where MP is regenerated.

S. is not infrequently very strongly bound to MP, especially in high-grade Specificity. The dissociation of S. can therefore constitute the critical moment in this form of the scrambling cleaning process. Both S. and MP should retain their properties.

The protein P must not be denatured, because then the adsorption capacity get lost. The dissociation can often take place with so-called chnotropic ions, e.g. rhodanide and iodide in high concentration, for example 3 M solutions. Here, however, dissolve A {* are and agarose on a large scale.

According to the invention, however, one can use an agar product with insignificant Establish solubility and practically without ionogenic groups by using a special process in the agar Establish cross connections. It has also been shown that these cross-connections are not an obstacle when it is true, according to known ones Methods to fix various proteins to the cross-linked agars. So you can e.g. after the Cyanogen bromide or oxirane method, as well as in other ways, bring about such a coupling. The protein can e.g. be an enzyme, whereby an "insoluble enzyme" is formed, which at the same time is a special » specific absorbent makes up for everyone

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ORIGINAL INSPECTED

set up inhibitors and antibodies as well as other substances that complex with the enzyme. You can also start with the agare connect suitable organic chains to which the protein is then coupled in order to prevent steric disturbances. Because practical all proteins that form P, antibodies are always a complex pair PS.

to find that in principle meets the requirements. That to the cross-linked Agar-fixed protein P can be included in a higher-order protein complex, e.g. in a virus or in a cell particle, for example khibosome or a cell fragment. The protein can also be composed, e.g., a glycoprotein.

There is no sharp distinction between proteins and polypeptides. In the present application, "protein" is used in a broad sense, this definition even includes polypeptides with a high molecular weight, e.g. from over 4000.

According to the invention it has been possible to produce protein agar products, the

1) are not soluble in water solutions in which proteins are not hydrolyzed,

2) of proteins, polysaccharides, virus and other high molecular weight. Substances can be penetrated with the formation of reversible molecular complexes in gels, and

3) with substances that split hydrogen bonds such as iodides, Carbamides and guanidine can be treated without removing agar polysaccharides with or without protein from the gel phase to be triggered.

Protein agar products are suitably prepared from agar in the form of pearls, which have been cross-linked and desulphated and possibly decarboxylated as described above. Proteins agar produced in this way forms fast- filtering beds, so that ionogenic groups which interfere with adsorption and enzyme reactions are excluded from the native agar »Permeability in protein agar particles, Ban can regulate cross-connections by concentrating the agars in the water-seen particles.

ORIGINAL INSPECTED

If adsorption and desorption are to take place at a very low salt concentration, It is particularly important that the agar matrix does not contain any ionogenic groups «A non-specific ion exchange adsorption can endanger the entire biospecific fractionation under such conditions, and the yield becomes zero. the Insolubility of the protein agars is particularly important when the substances to be purified are intended for clinical use, e.g. for intravenous injection when immune reactions can occur. Leakage of enzymes, antigens, antibodies etc. can affect the analytical Endanger the use of polymer-bound proteins. The present invention can avoid all such complications will.

In order to illustrate the invention and to explain it in more detail, its application is described in the form of exemplary embodiments,

Example 19

1 liter of swollen, cross-linked, desulphated agarose pearls aloud 20 g of cyanogen bromine dissolved in 50 ml of water were added to the above. After 2 minutes, 2 M NaOH was added, pH 11-11.5. After 9 minutes the gel was washed with about 2.5 1 0.5 M NaHCO- at + ^ ° C.

500 ml of a solution of 10 g Concanacalin A and against. 0.05 M sodium acetate buffer, pH 6.0, dialyzed, was added to the activated gel and the suspension stirred for 2 hours at pH 8.0. The concanavaline agarose gel was then washed successively with 2 1 each of 0.5 M NaHCO., Buffer, pH 8.0, distilled water, 0.3 M sodium formate buffer, pH 3.0, and 1 M with regard to NaCl, 0.05 M sodium acetate buffer , pH 6.0 containing 0.02 # NaN and 0.001 M Mg ⁺² and Ca ⁺² .

A 6.3 x 1 cm bed was then packed from the one prepared as above Protein gel and a solution of glycogen, 2 mg / ml, was introduced onto the bed at a rate of 5 ml per hour. To about 10 hours the bed was saturated for glycogen. The bed was washed with a buffer, whereupon the eluate was added one after the other was completely freed from glycogen, by the negative of the orcinol reaction it could be proven that no carbohydrate from the gel licked. The bed was washed in 0.1 M sodium formate buffer, pH 3> 0i

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about 35 mg of glycogen was displaced from the bed »

In this example the matrix M consists of cross-linked agaroeedas protein P from concanavalin and S. from glycogen, S. kanu also a

y y

other polyglucan or a glycose containing glycoprotein.

Example 20

100 bisepoxidquerverbundene, desulfated Agareperlen mg (dry weight, 6% ira respect to matrix content) were suspended in B ml of distilled water and with k ml BrCfI "solution, 25 rag BRCK / ml, 6 minutes at pH 11 and room temperature activated. The activated gel was washed with cold 0.1 M sodium hydrogen carbonate solution.

30 mg of ribonucleas A were dissolved in 5 nil 0.1 M sodium hydrogen carbonate solution and ^{react with the activated GsI at + V 3} G for 30 hours. The ribonucleas aga rule contained 82 mg of protein per gram of gel in the interval 25-50 $ of the free ensym, an extremely satisfactory result, in view of the high molecular weight of the substrate and the fact that the rhibonucleic acid is a thin polymer, which makes permeability difficult.

Example 22

35 ml of epichlorohydrin cross-linked, desulphated, bead-shaped agars were washed on filters with dimethyl sulfoxide and then with water = the gel was activated with BrCN, in a manner similar to that in Examples 19 and 20. 50 ml of antilymphocyte globulin (ALG) were dissolved in 20 ml of 0 , 5 NaHCO, solution and added this to the activated gel. The coupling was carried out in a cooling room for Zh hours, after which the ALG agars were treated with 0.5 M NaHCQ_, 0.01 M NaAc buffer, pH 3 , 9, 0.05 M Tris-HCl buffer, pH 8.5 and 0.05 M sodium phosfate buffer, pH 7.5, carefully dried. All buffer solutions were 1 M in Begug on NaCl. The gel was packed in a column.

5 ml of human lymphocyte zonicate was introduced onto the column. After washing with a phosphate buffer, pH 7.5, it was desorbed adsorbed material with 20 ml of 1 M NaI in the phosphate buffer The solution was concentrated and analyzed. No coal came out of the column

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hydrate triggered. The desorbed material was examined with the Cytotox test and found to be composed of immunoactive leukocyte fragments.

Β; ispiel 22

8 .T.I sedimented, cross-linked with 1,3-dichloropropanol-2, desulphated and with LiAlH. Reduced agar beads were treated with 10 ml of 1 M NaOH containing 20 mg of NaBH. and 2 ml of 1,3-butanediol diglycidyl ether with shaking for 6 hours at room temperature. The gel was then washed with distilled water and 0.2 M sodium hydrogen carbonate buffer, pH 9.0.

100 mg soybean trypsin inhibitor (STI) in 10 ml sodium hydrogen carbonate buffer fer, pH 9 »0, was added to the gel. The gel was then shaken at room temperature for 20 hours. The gel was then washed with hydrogen carbonate buffer and then with 0.1 M glycine buffer, pH 3.0, containing 1 M NaCl.

The gel was transferred to 0.05 M Tris-HCl, pH 7.8 containing of 0.5 M NaCl and 0.02 M Ca and packed it in a column. Thereupon A solution containing 0.2 mg / ml commercial trypsin was allowed to pass through the bed. The inactive material flowed through without delay. 3.5 mg of active trypsin was taken up by the gel and could with 0.05 M glycine HCl, pH 3.0, with a content of 0.5 M NaCl and 0.02 M Ca, to be displaced.

The trypsin adsorbed into the STI-Gel in a molar ratio of 1: 1. the Cross-linking of the agar gel did not affect the ability to form a reversible complex.

Example 23

A crude extract of bovine pancreas powder (from the pancreas of cows) was introduced onto a bed of STI-Gel according to Example 22. Trypsin activity was imperceptible until 50 ml of extract was introduced. They washed the gel with Tris-HCl buffer according to Example 22 and 0.1 M NaAc / HAc buffer, pH k, 5 has been introduced, wherein the chymotrypsin could be displace. The pH was then lowered by elution with glycine buffer according to Example 22. Thereby

ι η $ 9 cr ./ -. ρ 3 a

BATH ORIGINAL

35 mg of pure trypsin were displaced from the column. They don't
specific adsorption was much smaller than that of one
Bed based on commercial unbound, desulfated, and reduced agars.

You could use the gel an unlimited number of times with no noticeable reduction in the value Apply adsorption capacity. Triggering of the trypsin inhibitor could not be established.

As in Examples 19-22, the bed had excellent flow properties.

109853/189 9

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Claims (22)

"" C f ™ Exploaterings Aktiebolaget T.B.F., Uppsala, Sweden. Claims Agart product for separation purposes, for example in the bioeinie, as well as Molckülabseiher, stabilizers in electrophoresis or as an inert starting material in production of well-defined ion exchangers or specific adsorbents of various types, such as gel matrices for fixing enzymes or other chemical products or for other, similar purposes, characterized in that agars or gel-forming fractions produced from agars (e.g. agarose), preferably in bead form practically complete insolubility in water and in strong alkali, for example 1 M NaOH, brought about by cross-linking and possibly had been made practically sulfate-free and / or by being completely or partially freed from carboxyl ions have been left with an extremely small ability to adsorb basic substances, as well as that they may have an enzyme or protein component has been coupled. 2. Product according to claim 1, characterized in that that epichlorohydrin or epibromohydrin is the cross-linking substance. 3. Product according to claim 1, characterized in that that a bisepoxide is the cross-linking substance. k. Product according to claim 1, characterized in that a divinyl sulfone or other cross-linking vinyl compound is the cross-linking substance. 5. Agar product according to claim 1- * f, characterized in that the carboxyl groups before or after Abachulfeln completely or partially removed, by conversion into a reaction-ready Form and then to act with strong reducing agents, such as hydrides from lithium, magnesium, aluminum, boron or their derivatives, which increases the adsorption capacity of the product further reduced. 109853/1899 6. Product according to claim 5> characterized in that a cross-linked, sulphurized agar product is used as the starting material was applied. 7. Product according to claim 5 or 6, characterized in that that the starting material is a cross-linked agar derivative which consists of the ester of a lower carboxylic acid, e.g. acetic acid, and that after reduction with aluminum or Borohydride, the remaining ethyl groups were removed by alkaline hydrolysis in the presence of sodium borohydride. 8. agar product according to claim 1, characterized in that that the insoluble, particulate matrix part of the agars or the agar product that contains epihalohydrin, bisepoxide, divinyl compounds how divinyl sulfone was cross-linked, or substances that cause such compounds, etc. and possibly desulfated and / or decarboxylated, chemically coupled with a protein component in a reversible molecular complex or protein-particle coraplesi · » was pelt. 9. agar product according to claim 8, characterized in that that the protein is an enzyme, chymotrypsin, ribonuclease ubw = or is an enzyme inhibitor. 10. agar product according to claim 8, characterized in that that the protein is an immunoglobulin or equivalent antigen. 11. Agar product according to claim 8, characterized in that that the protein is a phythemagglutinin or other carbohydrate binding protein. 12. A process for the production of agar products according to claim 1-11, characterized in that agar or agarose particles, preferably obtained the shape of homogeneously swollen pearls from a homogeneous water solution, in a water sludge with epichlorohydrin, epibromohydrin, bisepoxides or compounds that how these substances react, treated with the reagent 109853/1899 may possibly be dissolved in an organic solvent and the reaction in an alkaline medium in the absence of oxygen and / or possibly in the presence of strongly reducing substances, after which the sulphate content (sulfur content) of the product can be lowered by alkaline hydrolysis, likewise under oxygen-free or reducing conditions, until the adsorption capacity of the product of basic substances has fallen to or below the desired limit, and that the product may then is decarboxylated. 13. The method according to claim 12, characterized in that that the reagents have been dissolved in organic solvents such as ethanol, dioxane, etc., 14. The method according to claim 12 or 13, characterized in that the gel concentration of the mixture in the case of cross-connections can vary between e.g. 20 and 80 #. 15. Method according to claim 12-1 * +, characterized in that that the mixture with cross-linking agents takes place in an alkaline environment / elevated temperature. 16. The method according to claim 15 »characterized in that that the alkali concentration is between 0.2 and 1.0 M. 17 · Method according to claims 15 and 16, characterized in that the temperature is preferably between 50 and 70 C. 18. The method according to claim 15-17 »characterized in that that the reaction time for the cross-connection is more than 1 hour. 19. The method according to claim 12-17 »characterized in that that the sulfur content is lowered after the cross-connection by alkali extraction and the sulfate ester hydrolysis with washed, Querverbundecem agargel is carried out in 0.5-1.5 M NaOH solution by means of boiling without oxygen present or with high sulfur 109853/1899 content by repeated boiling or autoclaving in the presence of sodium borohydride. 20. The method according to claim 12, characterized in that a reduced adsorptive capacity is brought about by completely or most of the carboxyl groups being freed from the agar product before or after sulphurizing, by converting it into a reactive form and then using strong Roduzie- · rungsmittel as % .. B "hydrides of lithium, magnesium, aluminum, boron or their derivatives such as, for example, lithium aluminum hydride (LiAlH.) reduced. 21. The method according to claim 20, characterized in that that the willingness to react to reduction thereby makes it possible is brought by swelling the starting product first in water and then with a suitable organic solvent washes, e.g. n-propanol, and then with dioxane. 22. The method according to claim 20 or 21, characterized in that the organic solvent used is tetrahydrofuran Dibutyl ether or high-boiling alkyl ether is used. 23 · The method according to claim 20-22, characterized in that the water content in the system before the reduction is brought down to practically zero. Zh, the method according to claims 20-23, characterized in that the reduction at high temperature, preferably 80-120 C, is carried out. 109853/1899 ORIGINAL