KR920000051B1 - Method for purification of recombinant & interferon - Google Patents

Abstract

A purification method of recombinant alpha-interferon comprises (a) purifying alpha-interferon by monoclonal antibody IgG, (b) concentrating it by ultrafilter, and (c) passing it through a gel filtration column selected from sephadex G-50, sephadex G-75 or sephacryl S-200. The above procedure is repeated in order to elute pure monomeric alpha-interferon. The applying concn. of alpha-interferon to the gel filtration column is pref. 1- 10 mg/ml. The elution solvent is pref. 1-100 mM tetraalkyl ammonium solution.

Description

Method for Purifying Recombinant α-Interferon

1 is a result obtained by SDS-PAGE the fraction obtained by passing the α-interferon purified by Staehelin et al. Through the Sephacryl 5-200 column,

2 shows the result of SDS-PAGE of the fraction obtained by passing the monomer fraction passed through the Sephacryl S-100 column through the Sephadex G-50 column.

The present invention relates to a method for purifying recombinant α-interferon obtained by culturing genetically engineered Escherichia coli with high purity using gel filtration chromatography.

Interferon is known to be a group of proteins that induce antiviral status in target cells and have immunomodulatory and antitumor properties. Recently, the antiviral and antitumor effects of interferon prevent cancer treatment and prevent viral diseases. It is a small unit high-priced, high-titer medicine that has been actively used for the treatment and has been used as an ointment and injection for the treatment of hair loss and hairy cell leukemia.

Currently, interferon can be produced in large quantities by microbiological methods by recombinant DNA technology, but there is a significant amount of loss in the economic process, and it is difficult to purify only biologically active monomeric interferon with high purity. there was.

In general, as a method for purifying recombinant α-interferon, it is known that immunosorbents, that is, methods of coomatography on anti-interferon antibodies and methods of metal chelate deposition are very useful.

Recombinant interferon has high specificity by immunosorbent, so cultured microorganism black is separated from culture medium with high purity, and metal chelate resin can be used to separate biologically active monomer interferon with purity of 95% or more. Is known from Staehelin et al. J., Biol., Chem., 256 (1981), 9750-9754 and Korean Patent Publication No. 86-1150, and the description of U.S. Patent No. 4,172,071 to white blood cells and fibroblast interferon It is also known that a solution having a activity of 1 to 5 × 10 ⁸ IU can be prepared by affinity chromatography on a blue dextran sepharose column using a monoclonal antibody IgG and a metal chelate resin. Has oligomers such as dimers, and when used as an injection, pyrogen removal such as endotoxin is the biggest problem There was.

Therefore, the present inventors have studied and studied to improve the shortcomings of the conventional method as described above, and as a result, the interferon can be eluted in pure form by purifying the interferon by monoclonal antibody IgG and repeatedly using gel filtration chromatography. The present invention has been completed.

That is, according to the present invention, the monoclonal antibody IgG is attached to activated Sepharose 4B to perform affinity chromatography to selectively separate only -interferon containing a lot of oligomers (Staekelin et al., J. Biol. Chem. 256). (1981), 9750-9754), concentrated to an concentration of 1.0 to 10.0 mg / ml, preferably 4 mg / ml using an ultra filter, and then using a gel filtration column, preferably a Sephadex G-50 column. To separate only the monomer. As the buffer solution at this time, tetraalkylammonium buffer solution, preferably ammonium acetate solution is used as 1-100mM, pH 2-8.

Next, the eluted monomeric alpha interferon fractions are collected again, and the gel filtration chromatography is performed again, and when the purity is not 99.99%, it is repeated once more.

Thus obtained interferon not only has a purity of 99.99% or more, but also obtains almost no biological activity.

The intrinsic activity of the obtained interferon was 5 × 10 ⁸ IU / mg-protein or more, and the amount of endotoxin was 1 × 10 ⁻² EU / 1 × 10 ⁶ IU or less.

On the other hand, the purification method according to the present invention has the advantage that the same results can be obtained, as well as the operation can be simplified even if the order is changed appropriately.

Protein content was measured according to the method of Lowry et al. (J. Biol. Chem 193 (1961), 265-275) and quantitative determination of fragments and oligomers was described in the method of laemuli et al. As described by SDS-PAGE, staining was carried out by the Silver Staining method.

The present invention is explained in more detail in the following examples.

Example 1

770 ml of 0.2 mg / ml protein (containing 14.3% oligomer) solution according to Staehelin et al. Was made to 10 ml on 25 mM ammonium acetate solution using an ultrafilter. The pH of the solution at this time is 4.1.

It was loaded onto a Sephadex G-50 or G-75 column at 4 ° C. and continued to load 900 ml of 25 mM ammonium acetate (pH 4.1). Only eluted monomeric α-interferon was collected and eluted twice in the same manner.

Purity was measured by SDS-PAGE and Silver Staining. The purity at this time was 99.99% or more.

Intrinsic activity was above 5 × 10 ⁸ IU / mg-protein and endotoxin was below 1 × 10 ⁻² EU / 10 ⁶ IU.

Example 2

170 ml of a 0.5 mg / ml protein (containing 13.7% oligomer) solution obtained according to the method of Staehelin et al. Was concentrated to 10 ml in 25 mM ammonium acetate, pH 4.1 solution using an ultrafilter, and this was Sephacryl 5-200 at 4 ° C. After loading on the column, 900 ml of 25 mM ammonium acetate, pH 4.1 solution was continuously loaded and only the eluted monomeric α-interferon was collected and eluted in the same manner as described above using a Sephadex G-50 or G-75 column. I was.

Purity was measured by SDS-PAGE and Silver Staining. Purity at this time was 99.99% or more, specific activity was 5 × 10 ⁸ IU / mg-protein or more and endotoxin was 1 × 10 ⁻² EU / 10 ⁶ IU or less (see FIGS. 1 and 2).

Claims (5)

Α-interferon was purified using monoclonal antibody IgG, concentrated using ultrafilter, and then subjected to one gel filtration column selected from Sephadex G-50, Sephadex G-75 or Sephacryl S-200, respectively. A process for purifying recombinant α-interferon, characterized in that the pure monomeric α-interferon is eluted by repeating the passage. The method according to claim 1, wherein the concentration of the gel filtration column is 1-10 mg / ml. The method of claim 1 wherein the eluate is a 1 to 100 mM tetraalkylammonium solution. The method of claim 3 wherein the eluate is an ammonium acetate solution. The method of claim 1 wherein the eluate is between pH 2-8.

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