CS262555B1 - A method for isolating immunoglobulin by affinity chromatography - Google Patents

Abstract

The solution concerns the method of Isolation of plasma immunoglobulin by affinity chromatography on substituted porous spherical celluloses with immobilized ligand of histidine, lysine, reactive Sulti 13 (C.I. 18 990) or 2-(4-aminophenyl)sulfonylethyl groups and products of their diazotization and spontaneous transformation. The principle of the procedure, which is based on the non-specific immunoaffinity of the marked functional groups to immunoglobulin, is based on its sorption, separation of uncaptured proteins and subsequent desorption with a concentrated solution of salts, preferably 3.5M magnesium or calcium chloride. To increase the effect, it is recommended to add 1,4-dioxane, or possibly a stabilizing component such as sodium acetate, glucose or maltose. The obtained eluate is freed from excess salts by dialysis or diafiltration. By the above method, it is possible to prepare a fully native electrophoretically pure immunoglobulin fraction of the IgG class.

Description

The invention relates to the isolation of an IgG class of immunoglobulin by affinity chromatography from solutions containing said immunoglobulin fraction in a mixture of proteins, such as blood plasma, serum or fractionated solutions thereof.

A variety of methods are known for isolating the immunoglobulin fraction from blood plasma or serum. These procedures are described in detail in review monographs (E. E. Schultze, JF Heretnans: Molecular Biology of Human Proteins, Elsevier, Amsterdam 1966), (FW Putnam: The Plasma Proteins, vol. III, Acad Press, New York (1977), (P. Allen, EA Hill,

Stokes, A. M.: Plasma proteins, Blackwell Sci. The method of ethanol fractionation in the cold is most commonly used to isolate immunoglobulin from human blood plasma on a production scale (Cohn et al., J. Amer. Chem. Soc 72, 465 (1950)). All these processes, which can be described as classical, have a relatively low partitioning effect and therefore the separation processes need to be suitably combined or repeated to achieve the desired purity of the final product. The affinity chromatography method has proven to be highly efficient and advantageous in terms of selectivity in terms of maintaining the tertiary structure of the isolated proteins and simplicity of execution. The method is based on the principle of the interaction of a binding ligand immobilized on a solid support with an appropriate site on the isolated protein molecule. For example, the process is very useful in the isolation of enzymes. This relatively new method is discussed in a number of comprehensive publications (Ch. R. Lowe, PDG Dean: Affinity Chromatography, ed. J. Wiley Ltd. London 1974), (J. Turkova: Affinity Chromatography, Elsevier Sci. Publ. Co. Amsterdam 1978 SP Colovick, NO Kaplan, WB Jakoby: Methods in Enzymology-Affinity Chromatography vol. 104,

Part. C, Acad. Press INC 1984). The specificity of the method is that each separation example requires its own specific selection of optimal conditions, starting with the selection of the appropriate carrier and the appropriate binding ligand to the sorption and desorption conditions of the partition. A specific group of binding ligands are immobilized dyes (P. D. G. Dean. F. Qadri: Affinity Chromatography on Immobilized Dyes, Solid Phase Biochemistry, ed. W. H. Scouten,

J. Wiley 1983). A typical example of their use is isolation of serum albumin on a sorbent with immobilized reactive blue (Procion Blue HB) (J. Saint-Blacard, J. Kirzin, P. Riberan, F. Petit: Affinity Chromatography and Related Techniques, eds. Elsevier 1985 p. 305 ), (K. Pommerening, D. Jobsky, M. Benes, J. Stamberg: AO No. 237 620), (M. Pokorny, M. Benes,

Holinka M., Pommerening K., Ulrych S.: Cs. AO 257,930). Another example is the use of reactive HAC1 yellow immobilized on Sepharose to isolate immunoglobulin (PGH Bylield: Mol. Immunol. 21 (7), 647 (1984)), (G. Birkenmeier: FEBS Letters 174 (1), 162 (1984)). . Another example of a covalently linked affinity ligand is some amino acids. For example, Arginine-Sepharose 4B was used to isolate plasminogen (M. Wu, G. K. Azimura, A. A. Yunis: Biochemistry 16,

908 (1977) or Lysin-Sepharose 4B (D.G. Deutsch: Science 170, 1095 (1970)). Possibilities of using sorbent with bound histidine on Sepharose matrix or porous glass were discussed at the International Symposium on Bioaffinity Chromatography in Prague (L. Amourache, S. Kanoun,

S. Krishnan, M.A. Vijayalakshni: Abstracts of the 6th International Symposium on Bioaffinity Chromatography, September 1985 Prague p. 149). A special case are methods based on the principle of immunoaffinity chromatography. As an example, isolation of an immunoglobulin with a specifically increased content of anti-tetanus or anti-staphylococcal antibodies may be used. (M. Pokorny, S. Ulrych, M. Benes, M. Kyselova, N. Odehnalova, J. Fincova: AO No. 256,654).

For the practical application of any affinity chromatography procedure, optimal conditions must be selected throughout the separation process. It is primarily the choice of a suitable solid support, a binding ligand, a process for sorption of the desired substance, an elution medium, and a number of other conditions that allow the native, fully active product to be obtained in the desired purity and yield. only the interplay of all factors takes full advantage of the affinity chromatography procedure. Affinity chromatography methods can also be used to isolate an immunoglobulin from a mixture of other proteins.

This principle is utilized by a method of isolating immunoglobulin class IgG from blood plasma, serum or solutions containing, in addition to the immunoglobulin fraction, a mixture of other proteins by an affinity chromatography method characterized in that porous spherical cellulose containing covalently immobilized histidine, lysine, reactive yellow 13 GGL or 2- (4- aminophenyl) sulfonylethyl groups or their diazotization and self-conversion products are contacted with a solution of the protein mixture, preferably in a cold of +4 ° C, then the solution containing the uncontained protein is separated, the sorbent is washed with a sufficient amount of low ionic strength solution. corresponds to a concentration of 0.1 to 0.01M sodium chloride solution or 0.05M borate buffer pH 8.0 to 8.5 and the captured immunoglobulin is desorbed with a 3.0 to 3.5M magnesium or calcium chloride solution, which may further contain 0 1 to 0.2 M sodium acetate, 1 to 5% maltose or glucose, and 1 to 5% maltose or glucose 5% 1,4-dioxane, after which the immunoglobulin-containing eluate thus obtained is freed from salts by dialysis or diafiltration.

Said method for isolating an immunoglobulin is based on the principle of non-specific immunoaffinity.

When comparing with other fractionation! The methods have advantages, which are characterized by the method of affinity chromatography, ie high selectivity, reliability, simplicity of implementation, energy efficiency, low labor intensity, maximum risk reduction when working with infectious material and possibility of automation of the whole process. However, the condition of the execution remains to be met.

The use of spherical cellulose as a carrier for immobilization of histidine, lysine, reactive yellow 13 GGL and 2- (4-aminophenyl) sulfonylethyl groups has an advantage over other similar sorbents in good flow properties, stability, slight change in volume due to pH and ionic fluctuations, harmlessness for use in the preparation of medicines and in their availability. The proposed procedure provides an immunoglobulin fraction of high electrophoretic purity corresponding to the IgG class. Human immunoglobulin prepared in this manner is fully native without aggregated moieties and exhibits low anti-complementary activity. The sorbent can be regenerated repeatedly.

Example 1

A column of 12 x 1 cm spherical cellulose with immobilized histidine (30 µmol / g) in a 0.05 M borate buffer medium was allowed to flow at 10 ml / h with 11 ml of human blood plasma at 4 ° C. The column is washed with 30 ml of 0.2m sodium chloride solution at a rate of 40 ml per hour, thereby separating the non-retained protein fraction. The desorption was carried out with 30 ml of a 3.5 M solution of magnesium chloride in 0.2 M sodium acetate, to which 5% 1,4-dioxane and 1% glucose were added. Elution is completed by washing with distilled water. The sorbent regeneration is performed by washing with 0.05 M sodium hydroxide in 1 M sodium chloride, distilled water and stabilizing in 0.05 M borate buffer pH 8.5. The eluate containing immunoglobulin is dialyzed against 0.2% sodium chloride if the solution contains magnesium ions and further concentrated by ultrafiltration to the desired protein concentration. In this way, 775 g of protein was obtained which corresponded to 98.8% of an immunoglobulin class G (IgG) electrophoretically.

Example 2

To 10 ml of immobilized lysine ligand (30 µmol / g) was added 30 ml of a 2% protein solution formed by melting the precipitate of fraction II-III of the Cohn ethanol fractionation process. The suspension was stirred cold for two hours. The liquid on a glass frit is then separated and the sorbent is washed with a 0.2 H sodium acetate solution to separate the non-retained portion of the proteins. The sorbent is transferred to a chromatography column and the bound protein is desorbed with a 3.5 M calcium chloride solution. The eluate obtained is dialyzed until it contains no calcium ions. Finally, the solution is freeze-dried. This gave 45 mg of protein which corresponded to 96% IgG electrophoretically.

Example 3

A column of 40 x 1.5 cm of spherical cellulose with reactive yellow 13 GGL (C.I. 18,990) is allowed to flow slowly at 20 ° C with 50 ml of filtered serum. The column is washed. 0.05M borate buffer pH 8.5. The first portion of the protein was released by elution with buffered saline pH 7.2. The captured immunoglobulin is desorbed with a 40% magnesium chloride solution. Elution is completed with water wash. The resulting solution containing the immunoglobulin fraction was freed of excess salts by diafiltration of 262555 cl and dried by lyophilization. This gave 575 mg of protein corresponding to an electrophoretic of 91% IgG.

Example 4

10 ml of blood plasma is applied to a 7.5 x 1 cm cellulose with immobilized 2- (4-aminophenyl) sulfonylethyl groups and their diazotization and spontaneous conversion products. The further separation procedure is the same as that described in Examples 1, 3, 4 or 5. This yielded 40 mg of protein which corresponded to 95% IgG electrophoretically.

Claims (1)

SUBJECT OF THE INVENTION Method for isolating an IgG class of immunoglobulin from blood plasma, serum or solutions containing, in addition to the immunoglobulin fraction, a mixture of other proteins by affinity chromatography, characterized in that the porous spherical cellulose containing covalently lobobyzed histidine, lysine, reactive yellow 13 GGL or 2- (4-aminophenyl) the sulfonylethyl groups or their diazotization and spontaneous conversion products are contacted with a solution of the protein mixture preferably at + 4 ° C, after which the solution containing the non-retained protein fraction is separated, the sorbent is washed with a low ionic strength solution of 0.1 up to 0,01 M sodium chloride solution or 0,05 M borate buffer pH 8,0 to 8,5 and the captured immunoglobulin is desorbed with 3,0 to 3,5 M magnesium or calcium chloride solution, which may further contain 0,1 to 0, 2M sodium acetate, 1 to 5% maltose or glucose and 1 to 5% 1,4-dioxane, then eluting The immunoglobulin containing salt is dialysed or diafiltrated.