CS237930B3 - A method for isolating serum albumin on spherical macroporous cellulose affinity sorbents - Google Patents

Abstract

The invention relates to the isolation of serum albumin on blue cellulose sorbents. The essence of the invention is a method of isolating serum albumin on spherical macroporous cellulose affinity sorbents with bound reactive blue 2 according to Czechoslovak Patent Application No. 237 620, characterized in that albumin is isolated from fractions obtained during blood serum fractionation by the ethanol precipitation method or ion exchange chromatography in such a way that albumin is captured from the protein solution during flow through the column or stirring of the suspension at pH 7.0 to 8.5 and an ionic strength corresponding to 0.01 to 0.05 M sodium chloride solution and is released from the binding complex by elution with a suitable solution of higher ionic strength, preferably 1 M to 6 M sodium chloride solution in acetate buffer pH up to 4.8. The method according to the invention is further characterized in that pure albumin is obtained from the intermediate fraction IV-V of the ethanol fractionation. Advantageously, albumin can also be isolated from other globulin fractions according to the invention.

Description

The invention relates to the isolation of serum albumin on blue cellulose sorbents.

A number of procedures have already been developed for fractionation of blood serum proteins (Allen P. C., Hill E. A., Stokes A. M., Plasma Protelns, Analytical and Preparative Techniques (1977), Curling J. M., Methods of Plasma Protein Fractionation (1980)). However, procedures based on ethanol fractionation by Cohn (Cohn E.J. et al., J. Am. Chem. Soc. 68, 459 (1946)) are still most commonly used to isolate proteins for therapeutic and prophylactic purposes on a production scale. Only recently have 1 in this field began to promote the chromatographic methods.

For example, ion exchange chromatography is used in the preparation of albumin and globulin as a major purification step or in improving the purity and stability of fractions obtained mostly by precipitation procedures (Curling JM: Methods of Plasma Protein Fractionation, 1980, pp. 77-158). Finding that, under appropriate conditions, serum albumin interacts selectively with reactive blue 2 (Cibachron blue 3G-A, Procion blue H, Ostazine blue H-3G) supported on the support (Travis J., Paneli R .: Clin Chim. Acta 49, 49 (1973)), has elicited interest in utilizing this behavior for albumin isolation (dye-ligand chromatography) (Saint-Blancard J, Kirzin JM, Riberon P., Petit F .: in Affinity Chromatography and Related Techniques, edited by Gribnau TCJ, Visser J., Nivard RJF-Elisevier 1982 - p.

The decisive role is played by the structure of the dye, its concentration and the method of binding. Also important from an application point of view is a carrier that determines the availability of functional groups during sorption and desorption, and its non-specific sorption. Until now, most commonly used dextran or agarose type polysaccharides and more recently also synthetic polymers of methacrylate type have been used as carriers for the preparation of blue affinity sorbents.

Traditional forms of cellulose yielded blue albumin-bound derivatives insufficient for practical purposes (Angal, S., Dean, P. D., Biochem, J. 167, 301 (1977)). This drawback is overcome by using spherically regenerated macroporous cellulose as a starting material. Compared to dextran or agarose, blue derivatives on this basis are more mechanically strong, have better flow properties even when working in a high column column, their volume practically unchanged due to changes in pH and ionic strength. This, together with better availability, also makes it possible to use on a production scale. They were used for separation and isolation of albumin from blood serum (Pommerening K., Jobsky D., Benes M., Stamberg J .: MS AO No. 237 620).

The essence of the method of isolation of serum albumin on spherical macroporous cellulosic affinity sorbents with reactive blue 2 bound according to U.S. Pat. AO No. 237,620 according to the invention is characterized in that albumin is isolated from the fractions obtained in the fractionation of blood serum by ethanol precipitation method or ion exchange chromatography, such that the albumin is collected from the protein solution during flowing through the column of said solution or agitating the suspension of the albumin solution and said sorbents wherein the albumin solution has a pH of 0.7 to 8.5 and an ionic strength corresponding to 0.01 to 0.05 M sodium chloride solution and the serum albumin is released from the binding complex by elution with a suitable solution of higher ionic strength, preferably 1 M to 6 M sodium chloride solution in acetate buffer pH 5.6 to 4.8.

The process according to the invention is further characterized in that pure albumin is obtained from the IV-V intermediate fractionation of ethanol fractionation. Preferably, according to the invention, albumin can be isolated from other globulin fractions.

An advantage of the method of isolating the serum albumin of the invention is to increase the yield in the production of pure albumin from donor blood plasma or surrogate raw materials using a combination of the classical fractionation process with (pseudo) affinity chromatography. Thus, pure albumin corresponding to pharmacopoeia requirements is obtained from the intermediate fraction IV-V ethanol fractionation according to Cohn, the globulin fraction or the waste liquid after separation of the immunoglobulin fraction. Removal of albumin from crude gamma globulin yields a product of higher electrophoretic purity.

Example 1

Through a column of spherical cellulose (35 centimeters high, 1.35 cm diameter) with a bound reactive blue 2 - color index 61211 (Cibacron blue) (3.6 (umol dye / mol wet carrier) equilibrated with 0.05 M sodium chloride solution, an 8% protein solution was freely flowing containing 94 percent gamaglobulin, 1% alpha and beta globulin, and 5 o / o albumin, which was produced by ethanol serum fractionation according to the Cohn method, with an ionic strength equivalent to 0.08 M sodium chloride solution.

The ipH value was adjusted to 7.2 to 7.5. A ratio of 100 ml protein solution to 50 ml sorbents has proven to be successful. After running the entire amount of protein solution, the column was washed with a 0.05 molar sodium chloride solution if the eluate contained protein. The albumin-free gamma-globulin solution thus obtained was further processed in a known manner.

The sorbent was regenerated by washing with a 3.5 molar sodium chloride solution and equilibrated with a 0.05 M sodium chloride solution. After several of these cycles, the sorbent was recovered after washing with a 3.5 molar solution of sodium chloride according to the method known as ion exchange regeneration using 0.2 M hydrochloric acid and 0.1 M sodium hydroxide. The sorbent was then equilibrated with a 0.05 M sodium chloride solution.

Example 2

A 2% aqueous plasma protein solution containing 80% albumin formed freely through the sorbent column of Example 1, which was equilibrated with 0.05 M sodium chloride solution, was formed by dissolving the IV-V intermediate fraction according to the Cohn method or as a fraction in the mixture proteins on the ion exchanger. The ipH of the solution was adjusted to 8.0-8.5 and the ionic strength of the solution corresponded to 0.05 M sodium chloride solution.

A ratio of 100 ml solution to 130 to 150 ml sorbent has proven to be successful. After complete application of the protein, the column was washed with 0.05 M sodium chloride solution until the captured protein was flowing. This effluent did not contain albumin or contained a considerably reduced amount. The captured albumin was then desorbed by washing the column with a 3.5 M sodium chloride solution. The elution was continued as long as protein was released from the sorbent. The serum albumin solution obtained was further freed of excess salts, concentrated and further processed in a manner known per se.

Example 3

The Cohn method protein intermediate fractionation solution containing 19% ethanol and 2% protein was freely flowing through the sorbent column of Example 1, which was equilibrated with 0.05 M sodium chloride solution, after separation of fraction I-II-III. The pH of the solution was adjusted to 8.0 to 8.5. The ionic strength was between 0.05 and 0.1. Application of the ethanolic protein solution was carried out at 0 ^° C. Another procedure for washing and desorption of albumin was carried out as in Example 2.

Example 4

A waste liquid solution was passed through a column of the sorbent shown in Example 1, which was equilibrated with 0.05 M sodium chloride solution, after separating the immunoglobulin fraction containing 0.2 o / _o protein, the major part of which was albumin and 25% ethanol. The pH of the solution was adjusted to 7.8 to 8.0; the ionic strength was 0.08. Application of the solution was carried out at -1 ° C. Another procedure for washing and desorption of albumin was performed as in Example 2.

Example 5 ml of reactive blue 2 (Cibacron blue) affinity-bound spherical cellulose, washed with distilled water, was suspended with 100 ml of waste liquid resulting from the separation of the immunoglobulin fraction of the ethanol fractionation method according to Cohn containing about 0.2% proteins of which the major part was albumin and contains 25% ethanol.

The pH of this liquid was pre-adjusted to 7.8-8.5. The sorbent suspension with the protein solution was stirred until the protein content was substantially reduced. Then, the sorbent was separated by sedimentation or filtered, the liquid was discarded and the sorbent was washed with a 0.05 M sodium chloride solution. The captured albumin was released from the sorbent by means of a 3.5 M sodium chloride solution, either by suspending the sorbent and decanting, or by washing the column on the filter.

Example 6

The serum albumin captured on the sorbent of Examples 2 to 5 was eluted with an aqueous solution consisting of 6% sodium chloride

1.6 o / o sodium octanoate (sodium caprylate) and 10% ethanol.

Claims (5)

A process for isolating serum albumin on a spherical macroporous cellulose affinity sorbent with reactive blue 2 bound by co. AO No. 237,620, characterized in that albumin is isolated from the fractions obtained in the fractionation of blood serum by ethanol precipitation method or ion exchange chromatography by capturing during the flow of the albumin solution through a column of said sorbent or by agitating the suspension of the albumin solution and said sorbent. 0 to 8.5 and an ionic strength corresponding to 0.01 to 0.05 M sodium chloride solution and serum albumin are released from the binding complex by elution with a suitable solution of higher ionic strength, preferably 1 M to 6 M invented with sodium chloride solution in acetate buffer opH 5.6 to 4.8. 2. Process according to claim 1, characterized in that the pure albumin is obtained from the intermediate fraction IV to V of the ethanol fractionation. 3. The process of claim 1 wherein the albumin is removed from the globulin fraction of ethanol fractionation. 4. The method of claim 1, wherein the albumin is recovered from the waste liquid formed after separation of the immunoglobulin fraction of the ethanol fractionation. 5. The process of claim 1, wherein the albumin is isolated from fraction IV-1.4 ethanol fractionation.