DD215319A1 - PROCESS FOR ENRICHING INTERFERONS - Google Patents

Abstract

The invention has for its object to achieve a possible protease-free accumulation of interferons under industrial conditions of manufacture. According to the invention pretreated fumed silica is added to an interferon solution pretreated. After an exposure time of the protein to the pretreated silica from 1-20 h, this is separated from the supernatant. Subsequently, the fumed silica is washed with various buffer solutions, wherein contaminating macromolecules are removed, followed by the elution of the interferon in the presence of potassium thiocyanate or glycerol or Aethylengykol or Tetraaethylammoniumchlorid in the quoted Tris buffer followed.

Description

schadow

Steinborn

Eichmann

Process for the enrichment of interferons Field of application of the invention

The invention relates to a process for the enrichment of interferons.

Field of application is biochemistry, especially in the preparation of preparations of high specific activity.

Characteristics of the known technical solutions

For the purification of proteins; methods are known which are based on the separation according to the molecular weight of the molecule size or the hydrophobic interactions between protein and matrix. For this purpose, in most cases molecular sieves (Sephadexes), which have the property in the case of the <L- interferon, are used such macromolecules to separate according to their size * (N. Fuchsberger: Acta biol. raed. germ. 38, (1979), 795-800)

Furthermore, methods are known which rely on hydrophobic interaction of the proteins with substituted agarose molecules (eg octyl- or phenyl-Sepharose) in order to separate the strongly hydrophobic proteins, such as, for example, the 4-interferon, from the less strongly hydrophobic proteins (S. Yonehava, 3rd Biol. Chem. 256, 8 (1981) 3770-3775) This also includes the enrichment of interferon with the help

CPG glass column chromatography. (3 · Ε · Whitman: Dm Interferon Res · 1,2 (1981) 305-313; KC Chadka: Preparative Biochein 11 (4), 1981 467-482; M. Wiranowska-Stewart; Mol. Immunol. 17 (5 (1980) 625-633; M. Rosenbergova: Acta virgl. 25 (1981), 31-35).

Other mechanisms of action are based on affinity chromatographies in which polyribonucleic acid residues or dextran molecules are used as ligands. The method of high pressure liquid chromatography (HPLC ₁ ) can also be used for the separation of proteins, but only a few types of columns are suitable for the enrichment of hydrophobic proteins, such as the tlL interferon. (DS Hobbs: 0 Biol, Chera 257, 8 (1982), 4071-4076, M. Rabenstein: Methods in Enzymol 78, 464)

Disadvantages of these methods are, on the one hand, the complicated production of the carrier materials, the low yield of the purified proteins and the very limited applicability of these methods to certain macromolecular substances.

On the other hand, methods are known which rely on the different mobility of the macromolecules in acrylamide gels under the influence of the electric field. The limiting factor here is also the amount of proteins that can be purified by such methods.

Object of the invention

The object of the invention is to develop a process for the enrichment of interferons. The method should make it possible to enrich under industrial production conditions interferons, which are present in low concentrations.

Explanation of the subject of the invention

According to the invention, an interferon, eg. T <L-interferon-containing solution (2-lOOinl) with a protein concentration.

-15 -3 tion of IO -10 mol / 1 in the batch or column chromatography with 0.5-10 g pretreated fumed silica (0.2-10 m / g) added · The pretreatment of the silica is carried out with 2-50% hot nitric acid and subsequent neutral washing

After an exposure time of the interferon solution to the silica for about 1-20 hours under circulating conditions, the silica is centrifuged off and the protein content of the supernatant is determined. After further washing with a buffer solution in the pH range 6-8, the enriched interferon is passed through 0, 6-3.5 M KCN solution or "20-50% glycerol or polyethylene glycol or 0.2-0.4 M tetraethylammonium chloride eluted in a buffer pH 6-9. According to the method of the invention, the interferon is enriched 10-100 times ·

A great advantage of the method is that amn also reaches high yields in a batch process. Subsequently, the invention will be explained in more detail by way of example

Exemplary embodiment 2 ml of an interferoh-containing solution with an extinction

^Ε 280 ^β1 "" ^{2 unc} * of a mass of pt interferon from lQpg to 10 #ug, V is added to 0.5-0.7 g of pretreated silica. Very finely divided silica is used by the VEB Chemiewerk Bad Köstritz. The pretreatment of the silicic acid is carried out by:

1. Boil in 5% nitric acid, lh

2. Neutral wash with distilled water

3. Wash with 0.1 M phosphate buffer, pH 7.2.

The solution is stirred for 3-12 h under medium-circu-

lierenden conditions of fumed silica exposed. After centrifugation, the supernatant is discarded and the silica pellet washed under tnedium-circulating conditions with o · g · phosphate buffer until the absorbance E ₂₈₀ ^ O, 05 «Thereafter, the silica with 0.1 M Tris buffer pH 8, 0 washed 2-5 times. The elution of the bound hydrophobic protein 4 ' interferon is carried out by 2-4 times the action of 0.25 M tetraethylammonium chloride in 0.1 M Tris-HCl buffer pH 3.0 for 3 to 60 minutes under medium circulating conditions. The detection of the interferon-specific activity and the determination of protein with fluram of the dialyzed Elustes yielded a 10-1GOfsehe enrichment of the hydrophobic protein JL-interferon sit a yield of 60-80%.

2 »embodiment

2 ml of a proteinaceous solution having an extinction ^E 280 ^{= 1} ^ ^{2 and a mass of} anhydrophobic proteins, for example oC-interferon from 10 pg to 10, ug is added to 0.5-0.7 g. Of pretreated highly dispersed silicic acid Filler Suprasil from VEB Chemical Factory Fährbrücke Langenbach / Sa.

Pre-treatment of the silica is carried out by: 1 · boiling in 5% nitric acid, lh 2. neutral washing with distilled water 3 · washing with 0.1 M phosphate buffer pH 7.2 The protein-containing solution is 3-12 h under medium-circulating conditions After centrifugation, the supernatant is discarded and the silica pellet is washed under medium-circulating conditions with phosphate buffer mentioned above until the extinction E ₂ SO ^ ° * £ '§. Thereafter, the silica is washed with 0.1 M Tris buffer pH 8.0 2 -5 times. The elution of the bound hydrophobic protein o (interferon is effected by 2-4 times the action of 0.25 M tetraethylammonium chloride in 0.1 M for 3 to 60 minutes Tris-HCl buffer pH 8.0 under medium-circulating conditions · Detection of the interferon-specific activity and protein determination by means of amino acid analysis of the eluate yielded a 100-fold accumulation of the hydrophobic protein cC-interferon with a yield of ~ 90 %.

Claims (1)

invention claim A process for the enrichment of interferons, characterized λ ι that in the batch or column chromatography an interferon-containing solution of pH 6-8 with fumed silica «previously washed with hot nitric acid and neutralized, 1-2Oh under circulating conditions added» then the silica centrifuged, the enriched interferon is washed with a buffer solution and eluted with the aid of potassium thiocyanate or glycerol or ethylene glycol or tetraethylammonium chloride.