IE41485B1 - Process for the purification of protein conjugates - Google Patents

Abstract

Protein conjugates are obtained in highly pure form from crude conjugates which, besides the desired protein conjugate, contain unreacted conjugation partners as well as byproducts by preparative electrophoresis. The process is particularly applicable to the purification of protein-dyestuff conjugates which are used in medical diagnosis. The precondition for carrying out the process is the presence of different electrophoretic properties which result in differences in the rate of migration of the desired protein conjugate on the one hand and of the byproducts on the other hand.

Description

The present invention relates to the purification of protein conjugates, especially protein-dyestuff conjugates.

Protein conjugates are protein or protein mixtures linked by covalent bonds to organic compounds.

Protein-dyestuff conjugates, especially immune sera containing antibodies and conjugated with fluorescent dyes, are especially important in the so-called immunofluorescence technique reported by A. H. Coons in 1942, which permits a combined investigation of serologicimmunologic specificity and of the morphological characteristics of the material to be analysed. This method is based on the so-called antigen-antibody reaction, in which one of the two immunologica components—generally, the antibody—is conjugated with a fluorescent dyestuff.

The antigen-antibody reaction compound is made visible in the microscopic preparation by provoking the visible fluorescence by means of rays in the ultraviolet range.

The immunofluorescence technique has acquired great significance in diagnosis for identifying linked and free antigens and antibodies.

According to known methods, fractions with a high concentration of antibodies are reacted as so-called immune sera with fluorescent dyes, preferably with fluorescein isothiocyanate. The reaction product is then predominantly purified by gel filtration and by ion exchange chromatography on diethyl-amino-ethyl (DEAE) cellulose, whereby the fluorescein-conjugated immune globulins are isolated.

Those known methods have the drawback that the separation of the dyestuff, which is not linked by a covalent bond, from the fluorescein-conjugated protein composition by means of gel filtration or dialysis is incomplete; in particular, a dyestuff which is linked to protein by adsorption or by a weak covalent bond that can relatively easily be split up by hydrolysis, is not eliminated. Moreover, the elimination of dyestuffs of high molecular weight, owing to autopolymerisation or autocondensation, is unsatisfactory.

Compositions containing such free dyes lead to unspecific fluorescence colourations in the microscopic preparation and thus to false results.

The widely used prior art process for obtaining conjugated protein fractions containing antibodies by means of ion exchange chromatography on DEAE cellulose, generally provides only the antibody fraction belonging to the IgG class. Under the conditions applied for elution and separation, the antibodies of the IgM and IgA classes are separated only unsatisfactorily or are not separated at all, so that the preparation obtained partly shows a substantial loss of the desired specific antibodies. When modifying the elution conditions suitable for the recovery of fluorescein-conjugated IgM and IgA antibodies, so-called overlabelled γ-globulins as well as basophilic proteins are also eluted. Owing to their strongly acid nature, these overlabelled protein fractions lead, in the immunologic reaction, to an 4148S unspecific linkage to a number of basic proteins in the material to be examined and thus again to a misinterpretation of the fluorescence results.

The present invention provides a process for the purification of a protein conjugate of the type as hereinafter defined, which comprises subjecting the crude conjugate in its preparative mother liquor state to electrophoresis to separate it from undesired byproducts and unreacted conjugation components. By the term unreacted conjugation component there is meant the organic compound which has not reacted with the protein.

It will be understood that only protein conjugates which differ in electrophoretical properties from the organic compound used for the conjugation and from the unreacted protein are to be considered.

Since the protein conjugate to be purified differs from undesired conjugation products and from the organic compounds used for conjugation in its electrophoretical properties, preparative electrophoresis of a mixture of the above products produces a separation into a zone containing the desired conjugates from those zones containing undesired protein conjugates and free organic compounds.

This process is especially used for the purification of protein-dyestuff conjugates, preferably of antibody-containing immune sera or protein fractions that have been reacted by conjugation with dyes, especially with fluorescent dyes, for example fluorescein isothiocyanate, by means of preparative electrophoresis.

The present invention also provides a process for the preparation of a protein conjugate of the type as hereinbefore defined, which comprises reacting a protein and a conjugation component and purifying the resulting protein conjugate by the above-described purification process.

This invention further provides protein conjugates that have been purified according to the process of the present invention.

The present invention further provides a method of immunological diagnosis, especially by the ummunological fluorescence technique, wherein there is used a protein conjugate which has been prepared by a process of the present invention.

Due to the electrophoretical properties of the starting material and of the reaction products, the process of the invention permits the separation of the substantially pure protein conjugate having a specified degree of reaction from unreacted protein and from the organic compound used for conjugation, provided that the protein conjugate has a different electrophoretic mobility from the other two components.

The process of this invention generally avoids the above-mentioned drawbacks of the known processes. The electrophoretic purification of dyestuff-conjugated protein preparations, preferably fluorescein-conjugated protein preparations, carried out preferably in an inert carrier material after the conjugation reaction, offers the following advantages: 1. The process permits an optimum or near optimum recovery of accurate zones containing antibodies. 485 - 6 Undesired protein fractions that are, for example, free of antibodies can easily be eliminated together with the separation of the desired fraction. The yield of the dyestuff-conjugated protein preparations containing antibodies is optimum or near optimum. For example, with a fluorescein-labelled γ-globulin fraction, 70 to 90% of the amount used may be recovered. 2. The conjugation reaction of proteins with acidic organic compounds reduces the isoelectric points in all the proteins reacted with the organic molecules, so that under the electrophoresis conditions, the reaction products migrate toward the anode at a higher speed than non-conjugated proteins. There is no difficulty in eliminating proteins which have become too acidic owing to an intensive reaction with the compound used for conjugation, since these migrate toward the anode at a higher speed. This finding is especially significant with the proteins that are-conjugated with fluorescein isothiocyanate as the acidic fluorescent dyestuff (guinoid system with a free carboxylic group). 3. The process of the invention ensures the optimum or near optimum separation of the acidic organic compound, which is not linked (t>r linked by adsorption) and which, if present in the fluorescein-conjugated compositions, leads to undesired, unspecific fluorescent colourations. The reaction for this easy elimination of this dyestuff portion is that the acidic organic compound itself has a strong tendency to migrate toward the anode. The electric potential established under the conditions of the electrophoresis causes the conjugates which are relatively loosely bound to protein to be split off and enhances the quality of the protein conjugates recovered by electrophoresis. In the case of proteins conjugated with fluorescent dyes, the stability of such preparations, when stored in an aqueous solution, is increased in comparison with those obtained according to the state of the art. 4. When antibodies are used as proteins for the conjugation, the process of the invention allows all the dyestuff-conjugated antibody classes to be obtained, for example IgA, igG, IgM. This is of importance especially for the use of antibodies obtained from sera, for example of man, goat, sheep and horse, in which—besides the antibodies of the IgG class—also antibodies of other immunoglobulin classes account for a relatively high percentage of the total antibody amount. This factor is also of a substantial economic interest since antibody-containing sera are available to only a limited extent.

Used for the purification of antibodies conjugated with fluorescent dyes, the process of the invention brings about an increase in the relationship between specific antibodies (Ab) and total protein (P), the socalled Ab/P quotient, which again intensifies the desired specific fluorescent effect.

. The process, which can be operated without using expensive chromatographical adjuvants, results in a substantial cost reduction for the manufacture of substantially pure protein conjugates. The inert carrier material used may be employed several times.

The preferred electrophoretic method used is carrier electrophoresis, in particular electrophoresis using the 414 8 5 inert carrier material in a horizontal container. When, for example polyvinyl chloride granules are used as the carrier material, it is possible after the separation of the fluorescein-conjugated protein fractions containing the antibodies to cut out the desired preparation in accordance with the visible bands and then elute it-from the carrier by means of simple solvents.

When the conjugate is uncoloured, the positions of protein, reaction component and protein conjugate may be established indirectly, for example by means of a paper dabbing which is then coloured in known manner. in addition to polyvinyl chloride, other compounds for example polyacrylamide, cellulose, starch, glass beads and sand, may be used as electrophoretic carrier materials.

The electrophoresis equipment used according to the invention may also be vertical arrangements with or without the use of carrier material. Very good purifications are also obtained using continuously operating equipment.

The process of this invention generally separates antibody preparations of any origin which have been linked by covalent bond to dyes of acidic nature, especially to fluorescein isothiocyanate.

However, it is also possible to react non-fluorescent dyes with proteins and to separate the proteins coloured by the conjugation reaction according to the process of the invention, provided the dyes used show different electrophoretic migration properties.

Instead of antibodies, other proteins of animal, vegetable and microbial origin may be used, if they exhibit as conjugates an electrophoretical property that differs from that of the starting protein and of the organic compound used for the conjugation. Organic compounds preferably used for the conjugation are those having their isoelectric points within the acid pH-range.

The essential feature, of this invention resides in applying preparative electrophoretic methods for the manufacture of purer protein conjugates, especially of gamma-globulin fractions reacted with fluorescent dyes. The reaction of γ-globulin fractions with conventional fluorescent dyes is not expected to yield an electrophoretically homogeneous product, since the structures of the immunoglobulin classes used for the reaction are known to be different and rather have a different number of amino groups available for the reaction. Although electrophoretical methods are known toprovide relatively uniformly migrating zones of unreacted γ-globulins, the uniform migration of immunoglobulins reacted with dyes and belonging to different classes has to be regarded as a surprise. Moreover, the dyes linked to protein molecules by adsorption could not be expected to be separated from the protein in an electrio field. Finally, owing to the known intense linkage of fluorescent dyes to ion exchange material as used in the state of the art, it could not be forseen that the free dyestuff used in an electrophoretic method, especially together with polyvinyl chloride as a carrier material, generally migrates over a particularly long distance as compared to that of the protein conjugates.

The known process of electrophoresis generally yields a product of substantially higher purity than chromatographical methods do. The product obtained generally exhibits, surprisingly, a higher specificity when used as an immunological reagent than the product obtained according to the state of the art.

The following Examples illustrate the invention.

EXAMPLE 1 A 3% antibody-containing serum protein solution (goat) was reacted in known manner with fluorescein isothiocyanate in an alkaline medium. In horizontal electro phoresis equipment (size: 65.0 cm in length, 76.0 cm in width, and 1.2 cm in height), which contained 3.6 1 of PVC powder (Geon X 427, trade mark of Messrs. Serva.

Heidelberg, W. Germany), the conjugated antiserum was separated at pH 8.1, at a field potential of 6 V per cm, and at an amperage of 0.6 A, over a period of 15 hours. The γ-globulin fraction containing the antibodies, which generally remains static at the point of application or even migrates toward the cathode unless conjugated to fluorescein isothiocyanate, migrated clearly toward the anode. The light yellow to greenish colouration of the γ-globulin fraction containing the antibodies stood clearly out against the rest of the protein fractions and free dyestuff portions with their dark yellow to reddish brown colour. The desired γ-globulin fraction containing the antibodies and having the optimum F/P quotient (molar ratio of the bound fluorescent dye (F) and protein (P)) -which can also be determined bandwise immediately after separation—was cut out as a PVC zone and washed clear of the carrier material by means of a 0.9% sodium chloride solution. The eluate was concentrated to about 1 9 %of protein by ultra-filtration.

This last operation was followed by the final standardisation and quality control of the product.

Prom 3.6 g of a fluorescein-conjugated protein solution containing antibodies (120 ml of antiserum), 0.5 g of fluorescein-conjugated γ-globulin fraction containing antibodies was obtained.

When serum proteins of rabbits containing antibodies were used in an amount of 8 g of a fluorescein-conjugated antiserum containing antibodies (120 ml), 1.0 g of a fluoroescein-conjugated γ-globulin fraction containing antibodies was obtained.

Analogous yields were obtained by recovering antibodies from human sera. Considering that the proportion of the γ-globulin fraction in an antiserum accounts for about 15 to 18%, these yields, calculated on this γ-globulin fraction, can be regarded as optimum.

EXAMPLE 2 A 3% human transferrin solution was reacted in known manner with fluorescein isothiocyanate in an alkaline medium. In horizontal electrophoresis equipment (size: 65.Ό cm long, 76.0 cm wide, and 1.2 cm high) containing 3.6 1 of a PVC powder (Geon X 427, trade mark of Messrs. Serva, Heidelberg, W. Germany), a conjugated protein was separated at pH 8.1, at a field potential of 6 V per cm and at an amperage of 0.6 A, over a period of 15 hours. The transferrin conjugate clearly migrated toward the anode. The yellow-brown colouration of the transferrin-containing zone stood clearly out against the different colours of the free dyestuff portions which had migrated still further toward the anode. The transferrin conjugate having the optimum F/P quotient—which can be determined bandwise immediately after separation— was cut out as a PVC zone and washed clear of the carrier material by means of a 0.9% sodium chloride solution. The eluate was concentrated to about lg% of protein by ultra-filtration.

This operation was followed by the final 5 standardisation and quality control of the product.

From 3.6 g of human transferrin, about 3.0 g of the corresponding fluorescein-conjugated protein were obtained.

Claims (20)

1. CLAIMS:1. A process for the purification of a protein conjugate of the type as hereinbefore defined, which comprises subjecting the crude conjugate in its preparative mother liquor state to electrophoresis to separate it from undesired by-products and unreacted conjugation components.

2. A process as claimed in claim 1, wherein the conjugation component for the protein is an organic compound having an isoelectric point within the acid pH range.

3. A process as claimed in claim 2, wherein the conjugation component is a dye.

4. A process as claimed in claim 3, wherein the dye is a fluorescent dye.

5. A process as claimed in claim 4, wherein the dye is fluorescein isothiocyanate.

6. A process as claimed in any one of claims 1 to 5, wherein the protein is an antibody-containing immune serum.

7. A process as claimed in claim 6, wherein the protein conjugate is an antibody-containing gammaglobulin fraction conjugated with a dye.

8. A process as claimed in claim 7, wherein the protein conjugate is an antibody-containing gamma-globulin fraction conjugated with a fluorescent dye.

9. A process as claimed in any one of claims 1 to 8, wherein the electrophoresis uses an inert carrier material.

10. A process as claimed in claim 9, wherein the carrier material is polyvinyl chloride.

11. A process as claimed in claim 9 or claim 10, wherein the electrophoresis is effected in a horizontal 5 electrophoresis apparatus.

12. A process as claimed in claim 1, carried out substantially as described in Example 1 or Example 2 herein.

13. A protein conjugate which has been purified 10 by a process as claimed in any one of claims 1 to 12.

14. A protein-dyestuff conjugate which has been purified by a process as claimed in any one of claims 3 to 12.

15. A process for the preparation of a protein 15 conjugate of the type as hereinbefore defined, which comprises reacting a protein and a conjugation component and purifying the resulting protein conjugate by a process as claimed in any one of claims 1 to 12.

16. A protein conjugate which has been prepared 20 by a process as claimed in claim 15.

17. A protein-dyestuff conjugate which has been prepared by a process as claimed in claim 15.

18. A method of immunological diagnosis wherein there is used a protein conjugate as claimed in claim 25 13 or claim 16.

19. A method of immunological diagnosis wherein there is used a protein conjugate as claimed in claim 14 or claim 17.

20. A method as claimed in claim 19, which is carried out 30 according to the immunofluoresence technique.