The present invention is concerned with the field of electro- phoretic separation techniques and relates more particularly to compositions which are stable in storage and are to be used for the production of acrylamide gels with immobilized pH gradients for electrofocusing.

In electrofocusing procedures, sample components are caused under the action of an electric field to migrate in a medium having a pH gradient. Components reaching zero net charge at any of the pH values in the gradient will at this point not be influenced by the electric field but will there be focused into narrow bands; this is why the process is called "isoelectric focusing". Several methods are known for generating the pH gradient required for electrofocusing. The most important methods are

(i) using a mixture of so-called carrier ampholytes with different pi values distributed across the pH range chosen (see for example the U.S. patent 4,334,972 which describes a product suitable for this technique) ;

(ii) producing immobilized pH gradients, by copolymerizing the matrix material with a material containing charged of chargeable groups.

In actual practice when this latter technique is to be employed for focusing in polyacrylamide gels a plurality of acrylamide derivatives, each with either acid or base function(s) thus representing different pK values, are required for enabling the desired pH gradient to be generated,

A series of such buffering acrylamide derivatives of the general structure

O H n I CH ₂ =CH-C-N-R

where R contains a charged or chargeable group consisting of one or more of the elements C, 0, S, P, B or N, for example a carboxylic acid, phosphonic acid, sulfonic acid group or a tertiary or quaternary amino group, has been described by Gasparic et al. in the U.S. patent 4,130,470 and is marketed by Pharmacia LKB Biotechnology AB (Bromma, Sweden) under the commercial name of Immobiline . With the aid of acrylamide derivatives having the above formula it is possible to create gradients of varying widths within the range of from pH 3.5 to 10.5, in that a suitable amount of at least two acrylamide derivatives (Immobiline ) is mixed into the polymerization mix prior to polymerization. An elaborate description as to how these gradients are created is given in for instance "High Resolution Analytical Electrofocusing in Polyacrylamide Gels with Immobiline ^® pH Gradients", Application Note 324 from LKB (Bromma, Sweden) .

The said method (ii) avoids some of the problems inherent in the use of carrier ampholytes, for example in cases where focusing is to be effected within very narrow pH ranges. However, it has been reported that the chemicals employed have stability problems which in some cases may impair -experimental results. Such stability problems are discussed by e.g. Pietta et al. -(Electrophoresis 6 (1985) 162-170).

Stability of Immobiline solutions has been found to vary with temperature and pH; as far as acidic Iirimobilines (pK 3.6 and 4.6) are concerned, the problems have been most conspicuous at neutral pH. Alkaline Immobilines (especially pK 8.5 and 9.3) on the other hand are subject to degradation most quickly in alkaline solution. All Immobilines have been found to have their optimum storage stability in an acidic milieu (pH 3-4) .

In an article of 1986 Astrua-Testori et al. (Electrophoresis 7, pp. 527-529) report results obtained from studies regarding the stability of Immobiline solutions in a frozen state; an about 20 % per year hydrolysis rate has been found when Immobiline pK 9.3 was subjected to measurements during a period of two years.

The instability problems of the acrylamide derivatives in question have been found to be due in the first place to two reactions: hydrolysis and polymerization.

Hydrolysis of especially the alkaline derivatives has been discussed in detail by Pietta et al.. Even when stored at +4 °C these derivatives in the form of 0.2 aqueous solutions will produce acrylic acid in amounts such that a visible shift of the pH gradient is observable already after a short time. According to work implemented by Astrua-Testori et al. the method employed by some users comprising storage of the acrylamide derivatives in a frozen state, with or without addition of a buffer, is inadequate for providing a satis¬ factory solution to the problem.

The other major storage-associated problem resides in autopolymerization of acrylamide derivatives stored as the pure substance in the form of a powder, liquid or aqueous solution. Attempts have been made to add small amounts of an inhibitor to Immobiline pK 8.5 and 9.3; but note here that even if this may thwart formation of polymerization products then still hydrolysis problems will remain to be dealt with in cases where the products are stored in the form of aqueous solutions.

In an experiment where Immobiline ® pK 6.2, 8.5 and 9.3 were stored in an 0.1M Tris-phosphate buffer for varying periods of time at 60 C, with subsequent evaluation by casting of IPG 4.28 - 4.90 (according to the method described in Application Note 324 from LKB, Bromma, which is included in this specification) , followed by focusing, the pH gradients

obtained were highly acidic and the protein bands (ovalbumin) were very much displaced towards the cathode, or alternatively no protein bands could be observed on the gel (for pK 6.2 after storage for 3 days, 60 °C; for pK 8,5 after 1 hr, 60 °C; for pK 9.3 after 5 days, 60 °C) .

According to an article recently published (J. Chrom. 402

(1987) pp. 105-113) Rabilloud et al. have studied various methods for removing undesirable polymerisation products.

As far as methodology is concerned, demands as to purity and stability of this type of' acrylamide derivatives are very high, this- being so because even small concentrations of impurities will give rise to either a shift of the pH gradient or impaired resolution in protein separation procedures, or both.

Thus the problems due to hydrolysis and autopolymerization have been amply documented and have in fact occasioned restrictions in the use of this type of acrylamide derivatives, so that just that very technique which is believed to have an eminently large potential within the art of electrofocusing has failed, so far, to become accepted for a commensurate extensive use within this field.

We have now found that both autopolγmeri-_ation and hydrolysis of the type of acrylamide derivatives present in Immobiline can be eliminated or reduced to an acceptably low level if the derivatives are stored in the form of a composition consisting of one or more of the below-specified organic solvents plus the derivative in question.

Since the composition is to be mixed with aqueous solutions of acrylamide and bisacrylamide the organic solvent has to be miscible with water up to at least about 5 % by weight, preferably 10 % by weight. Preferably the solvent is selected from the following groups: alcohols, especially lower alcohols of 1-5 carbon atoms such as e.g. methanol, ethanol,

n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, t-butanol, 2- or 3-pentanol and t-amyl alcohol; furthermore ethylene glycol, diethylene glycol and triethylene glycol; and mono- or dialkyl ethers of ethylene or diethylene glycol such as for instance ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether and diethylene glycol dimethyl ether; and glycerol; lower dialkyl ketones of -5 carbon atoms such as e.g. acetone and methyl ethyl ketone; esters, especially lower alkyl esters of acetic acid such as e.g. methyl acetate and ethyl acetate; ethers such as e.g. diethyl ether, THF and dioxane; nitrogen- or sulfur-containing solvents such as for example formamide, D F, acetonitrile, N-methyl pyrrolidone, nitromethane and N-methyl formamide, dimethyl sulfoxide and sulfolane.

In addition to the water miscibility of the solvent a further requirement is the following: Via the solvent no components may be entrained into the system that would affect the gel as formed in a manner adverse to the focusing operation, e.g. by the introduction of groups which will degrade biologically active molecules. Of course it is also important to avoid an inhibition of the polymerization procedure itself such as would necessitate unduly large additions of initiator.

The acrylamide derivatives present in the aforesaid composition have the general formula

0 H

I. I

CH ₂ =CH-C-N-R (1)

wherein R is a side chain containing a charged or chargeable group consisting of one or more of the elements C, 0, S, P, B or N, for example a carboxylic acid, phosphonic acid or sulfonic acid group or a primary, secondary, tertiary or quaternary amino group.

If these derivatives are mixed with one of the aforesaid solvents to a concentration of 0.01 - 2 M a composition is obtained which is stable in storage and is suitable for being directly mixed wit the monomers employed according to known techniques in the preparation of acrylamide gels for isoelectric focusing.

We have found that autopolymeri'.ation as well as hydrolysis are efficiently prevented during storage periods of up to 3 years - this being the longest period, so far, during which the system has been tested.

In addition to the aforementioned charged or chargeable derivatives the composition may of course contain neutral acrylamide derivatives in varying amounts.

From the literature it has been known that e.g. alcohols may be employed for effectively terminating a polymeri'.ation reaction, see for example US 2,921,933 according to which an aliphatic alcohol is used for arresting the reaction in a polyethylene manufacturing procedure.

In the present case liquid compositions are prepared which are stable in storage and form part of the reaction mixture employed for making electrophoretic gels for electrofocusing. We have found that the aforesaid solvent(s) of the composition will not affect the electrophoretic gel to any substantial degree.

The invention is thus concerned with a liquid composition which is stable in storage and contains at least one acrylamide derivative of formula (1) in a concentration of 0.01 - 2 M plus an organic solvent or mixture of organic solvents selected from the above-specified group in an amount of at least 75 % by weight, preferably at least 90 %. Among the acrylamide derivatives examined the most hydrolysis-sensi¬ tive specimens have been found to be those of pK 8.5,

so in their case the water content should be less than about 10 %. In case of the other derivatives a somewhat higher content is permissible, e.g. up to about 25 %, without the negative effects becoming too strongly pronounced.

Furthermore the invention is concerned with a method of producing electrophoretic gels for isoelectric focusing with immobilized gradients wherein at least one composition of the aforesaid kind is mixed with aqueous solutions of acrylamide monomers including crosslinking reagent such as e.g. bisacrylamide and initiator, whereupon the reaction mixture is caused to polymerize. In this method, a volume of the composition of this invention is added to monomer solutions known per se for the production of electrophoretic gels, the said volume being such that up to a maximum of about 15 % of the composition will be present in the reaction mixture.

The invention is illustrated in the following non-limitative examples.

For studying the degree of autopolymerization on storages, comparative tests were carried out as follows:

Table 1

Content of polymer, Immobiline pK 8.5 % by weight

Storage for 3 years

1. Sample stored at room temperature in the form of the substance as such 15.93

2. Sample stored at room temperature in the form of an 0.2M n-propanol solution <0»1

3. Sample stored at +4 C in the form of an 0.2M n-propanol solution <0.1

Table 2

Content of polymer, Immobiline pK 8.5 % of weight

Storage from February 1984 to August 1987

1. Sample stored at +4 C in the form of the substance as such 10.1

2. Sample stored at +4 C in the form of an 0.2M solution in n-propanol <0.1

3. Sample stored at room temperature in the. form of an -0.2M solution in n-propanol <0.1

Immobiline pK 6.2 and Immobiline pK 8.5 were stored at 60 C for 5 days at a concentration of 0.2M in the following solvents

methanol ethylene glycol

ethanol ethylene glycol monomethyl ether

n-propanol ethylene glycol monoethyl ether

n-butanol ethylene glycol dimethyl ether

isobutanol diethylene glycol

t-amyl alcohol diethylene glycol monomethyl ether

glycerol diethylene glycol monoethyl ether

diethylene glycol dimethyl ether

formamide acetone

DMF methyl ethyl ketone

acetonitrile

methyl acetate nitromethane ethyl acetate N-methyl formamide

dimethyl sulfoxide

sulfolane

dioxane

without the formation of any detectable amounts of polymer (content of polymer <0.02 %) .

In a series of experiments compositions according to the invention, i.e. acrylamide derivatives defined as above which had been dissolved in an organic solvent, were employed in the preparation of solutions for producing electrophoretic gels by polymerization. The gels were produced in a manner analogous to the method described in Application Note 324 (LKB Bromma, Sweden) . The gels then contained 6.7 % (by volume) of each respective solvent employed. The results showed that with the solvents defined above gel products are obtained which are suitable for electrophoretic separation.