DE3932311A1 - Purifying equine interferon beta - using anion and cation-exchange columns, useful for antiviral or immuno:modulatory medicament - Google Patents

Abstract

A method for purifying equine interferon beta (Eq IFN-beta) comprises: (a) inactivating E. coli host cells with acid and extracting the EqIFN-beta with an alkaline medium. (b) precipitating the cell culture supernatant with polyethylamine and running this on an ion-exchange column and/or a molecular sieve. (c) eluting the EqIFN-beta with a phosphate buffer; and (d) purifying the eluted interferon further using an anion and/or cation exchanger. The extraction pH is 10 and in stage (b), adsorption on the column is achieved by the use of hydroxyapatite. The anion-exchanger is monoQ and the cation-exchanger is Mino S. Elution on the anion exchange column uses a linear gradient of sodium chloride. For the cation exchange column, a linear gradient of ammonium sulphate is used. USE/ADVANTAGE - The purified EqIFN-beta is useful as an antiviral medicament and also has immunomodulatory activity.

Description

The present invention relates to a method for the purification of beta interferons.

Three decades of research into the extraordinary antiviral properties of interferons (IFNs) resulted in the registration of natural and recombinant alpha and beta interferons as medicines for various viral diseases in humans. Unlike classic low molecular weight antiviral agents that directly interfere with viral replication, the interferons interact with protein receptors in the membrane of the target cells. Receptor structure differences in different species are therefore held responsible for the characteristic species preference of the interferons. The development of interferons into antiviral drugs for animals therefore requires homologous proteins; only in exceptional cases is the "cross-species activity" the use of heterologous interferons, e.g. B. human INF-alfa in cattle. Some working groups have therefore created various animal interferons using recombinant DNA technology. In addition to mouse and rat interferons, which have proven to be invaluable tools for biological and medical research in rodent models, recombinant bovine ( 1 , 2 ) and pig interferons ( 3 , 4 ) are now available.

In addition, Himmler et. al. ( 5 ) described the cloning of the genes for both the dog interferons alfa (CaIFN-α) and for the horse interferons alfa, beta and omega ( 6 ) (EqIFN-α, -β, -ω).

The interferon beta gene family comprises at least two genes in the horse; one of these genes was isolated from a liver DNA library and sequenced ( 6 ). The gene contains no introns and codes for a protein composed of 186 amino acids (horse interferon beta-1 = EpIFN-β1); the N-terminal 21 amino acids form a signal peptide. The mature protein consists of 165 amino acids: compared to other beta interferons (human, bovine, mouse), about 50% of the amino acids, including three characteristic cysteines, are conserved. The molecule contains two potential N-glycosylation sites, one of which is also conserved in all known beta interferons. EqIFN-β1 was expressed in E. coli HB101 using the S. marcescens trp promoter and a synthetic ribosome binding site ( 6 ), but can also be expressed in eukaryotes or in mammalian cells.

In order to use EqIFN-β as a medicine, it is essential that it be in a highly purified form. Methods for purifying HuIFN-β have already been described ( 15 ). As far as is known, processes for the purification of EqIFN-β have not yet been described.

The object of the present invention was a suitable one for the provision of pharmaceuticals to produce high-purity IFN-β, in particular EqIFN-β.

This task was solved by the in the characteristics cleaning process characterized by the claims.

The method according to the invention is exemplified based on the purification of EqIFN-β1. The same But other beta interferons can also be preferably the non-human beta interferons especially the horse's beta interferons.  

Acid-inactivated E.coli cells were homogenized in a 10-fold volume of 1% acetic acid. EqIFN-β1 was extracted at an alkaline pH, preferably at pH 10 (about 5% of the soluble protein). The extracts were neutralized before centrifugation. The EpIFN-4b1 concentrations of the crude extract varied between 0.1 and 0.15 mg / ml, which means 1-1.5 mg per gram of wet cell cake. The extraction at lower pH ( 3-7 ) surprisingly resulted in lower yields.

From the supernatant of a polyethyleneimine precipitation was EpIFN-β1 on an adsorbent with ion exchanger and / or molecular sieve properties, preferably on Hydroxyapatite bound and with phosphate buffer eluted. It was then chromatographed over Anion and / or cation exchanger (e.g. Mono Q or Mono S) obtained a highly purified protein that in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Gel Permeation HPLC (GPC) and Reverse Phase HPLC (RP-HPLC) showed a purity of more than 98%. The Yields were about 0.3 to 0.5 mg per gram E.coli cells (about 25-30% relative to the crude extract).

Table 1 summarizes the results of a typical one Cleaning sequence together.

The molecular weight of EqIFN-β1 was 19,400 (SDS-PAGE) or 21 000 (GPC) determined (theory: 19 596). There were no impurities in this system noticeable. Analysis of the N-terminal Amino acid sequence showed that surprisingly at more than 95% of the molecules do not contain the initiator methionine was split off. The amino acid composition corresponded to that within the precision of the method theoretical value.  

A major peak that corresponded to at least 98% of the total peak area was observed in the reverse phase HPLC ( FIG. 3). A weak peak that eluted with a somewhat shorter retention time may correspond to a methionine sulfoxide derivative of EqIFN-β ( 14 ). A trace contamination (<1%), which elutes somewhat after the main peak, consists of dimeric EqIFN-β1 (identified by Western blot analysis according to SDS-PAGE of the associated eluted fraction). All detectable trace contaminants together made up less than 2% of the total protein. These trace contaminants can be further reduced, for example, by non-denaturing HPLC.

Examination of the N-terminus of EqIFN-β1 from different fermentations and purification cycles confirmed the assumed sequence ( FIG. 1).

The antiviral activity of EqIFN-β1 was determined using the CPE reduction assay. The absolute specific activity of the highly purified protein varied between 2 × 10 ⁸ and 2 × 10 ⁹ U / mg.

Specific activities of 2-3 × 10 ⁸ U / mg have been reported for natural HuIFN-β. One group reported recombinant HuIFN-β from E. coli with only a slightly lower specific activity, namely with 1-2 × 10 ⁸ U / mg, other authors, however, reported significantly lower activities, namely 3 × 10 ⁷ U / mg ( 15 , 16 ). These differences were explained by the different proportions of incorrect disulfide bridges in IFN-β produced in E. coli ( 16 ). In view of the high specific activity of the protein purified according to the invention, a high proportion of the protein molecules purified according to the invention surprisingly appears to be correctly folded.

The antiviral activity of EqIFN-β1 was also increased Cells of other species determined: human cells (A 549, Lung carcinoma); Mouse (L-M: connective tissue); Bovine (MDBK, kidneys and BT, turbinate); Sheep (SPC-choroid plexis) infected with VSV and / or EMCV. HuIFN-α2c or HuIFN-α / β were positive Controls used.

As expected, HuIFN-α2c showed strong antivirals Activity on bovine and ovine cells and was also in a little active on equine fibroblasts. in the In contrast, EpIFN-β1 surprisingly showed a remarkably high level of species specificity: It was even at the highest concentration that was tested (1 µg / ml), inactive on all non-epuine cells, which is equivalent to less than 0.001% of activity on homologous cells (Table 3).

These results contradict previous reports that IFN induced by poly (I: C) in equine fibroblasts shows comparable activities on epuine, bovine and ovine cells ( 17, 18 ). This obvious discrepancy can be explained by the fact that other forms of EqIFN-β are present in the "natural" epuinen "fibroblast" interferon. At least two genes coding for EqIFN-β had been identified in the equine genome ( 6 ). However, it seems more likely that superinduced epine fibroblasts produce not only IFN-β but also IFN-α. EqIFN-α (obtained from Newcastle disease virus-induced leukocytes) shows higher activity on bovine and ovine than on homologous cells ( 17 , 18 ). IFN-α as an admixture in the "natural" equine "fibroblast" interferon could therefore be responsible for the cross-species activity.

The purification of EpIFN-β1 from E.coli extracts was done initially followed up by a bioassay. With the help of monoclonal antibodies was an enzyme immunoassay with which the cleaning steps are assessed were.

The EqIFN-β1 purified in this way showed in primary leukocytes of horses in vitro as well as ex vivo after parenteral Application (see c., I. M.) Antiviral and immunomodulating activity.

The present invention relates in particular to: Process for the purification of EqIFN-β, thereby characterized in that

• a) EqIFN-β from acid-inactivated cells of the Expression host organism preferably from E.coli cells at an alkaline pH is extracted
• b) EpIFN-β from the supernatant of a polyethyleneimine Precipitation on an adsorbent with Ion exchange and / or Molecular sieve properties is bound
• c) EqIFN-β is eluted with a phosphate buffer,
• d) and then the EqIFN-β eluted via Anion and / or cation exchanger is cleaned up.

Process, characterized in that the pH at the extraction is set to 10.  

Process, characterized in that as an adsorbent Hydroxyapatite is used.

Process, characterized in that as Anion exchanger Mono Q and as a cation exchanger Mono S is used.

Process, characterized in that a Linear gradient of sodium chloride for the elution of the anion exchanger and a linear gradient of Ammonium sulfate for the elution of that Cation exchanger is used.

EpIFN-β, producible according to one of the invention Method.

EpIFN-β according to one of the methods according to the invention cleaned, for use as a medicine.

EqIFN-β according to one of the methods according to the invention cleaned, for use as an antiviral Drug.

EqIFN-β according to one of the methods according to the invention cleaned for use as a medicine with immunomodulating activity.

The following examples are only intended to illustrate the invention explain without restricting them.  

Legend to the figures

Fig. 1 amino acid sequences of EqIFN-β1 and HuIFN-β. The conserved cysteines are boxed, and potential N-glycosylation sequences are underlined. An asterisk indicates a deletion in EqIFN-β1. 89 amino acids (59%) are identical in both proteins. The sequences are from Himmler et al. ( 6 ) and Taniguchi et al. ( 20 ).

Fig. 2 purification of EqIFN-β1. Left field: Anion exchange chromatography (Mono Q). 8 mg protein eluate from the hydroxyapatite column was applied. Right field: cation exchange chromatography (Mono S). 3 mg protein eluate from the Mono Q column was applied. The main peaks in both fields correspond to EqIFN-β1.

Figure 3 SDS-PAGE of purified EqIFN-β1. 1, 3 and 9 µg protein were reduced with 5% thioglycerol, developed on a 15% gel and stained with Coomassie Blue R250. A trace contamination (Mr. = 60,000) comes from the thioglycerol preparation (see sample buffer control, trace SB). Lane M: molecular weight marker (Mrx10 ³ ).

Fig. 4 Reverse phase HPLC of purified EqIFN-β1. RP-HPLC was carried out as described. 80 µg protein was applied to the column. The main peak corresponds to monomeric EqIFN-β1 (<98% of the integrated peak areas) and is off-scale to show trace impurities.

example 1 Purification of EpIFN-b

Acid-inactivated Escherichia coli cells were found in 10 Volume parts 1% acetic acid homogenized. The pH was adjusted to 10 by adding NaOH; the Homogenizate was extracted at 4 ° C for one hour and then neutralized with HCl. The cell debris were removed by centrifugation. Polyethyleneimine was up to a final concentration of 0.25% added and the solution for 1 hour at 4 ° C touched. The precipitate was centrifuged away; 150 ml of the supernatant was added to a Hydroxyapatite column (Bio-Rad HTP; 2.6 × 20 cm; Flow rate 3 ml / min). The pillar was washed with 150 mM sodium phosphate pH 6.8 and with 250 mM sodium phosphate pH 6.8 eluted. Peak fractions different runs were collected, concentrated and overnight against 20 mM Tris-HCL, pH 7.5 / 20 mM NaCl dialyzed. Solutions containing up to 25 mg of protein were used on a Mono Q anion exchanger Column (Pharmacia, 0.5 × 5 cm; flow rate 1 ml / min) upset. The column was filled with the same buffer washed and EqIFN-β-1 with a linear gradient (134 up to 248 mM in 15 ml). Peak fractions different runs were collected, concentrated and overnight against 100 mM ammonium acetate, pH 5.5 dialyzed. Solutions containing up to 10 mg of protein were included on a Mono S Cation Exchange Column (Pharmacia; 0.5 x 5 cm; Flow rate 1 ml / min). The pillar was washed with the same buffer and with one Linear gradients from ammonium acetate (370 to 500 mM in 12 ml) eluted. Peak fractions from different runs  were collected, concentrated and at -70 ° C kept. All chromatographic Process steps were below at room temperature Performed using a Pharmacia FPLC system. Protein concentrations were determined by Coomassie Blue Staining Binding Assays (Bio-Rad, Pierce) using bovine serum albumin as Standard determined.

Example 2 Biological interferon assay

Equine dermal fibroblasts (e.g. cell line E.derm = NBL-6, ATCC CCL 57) were found in Eagle's Minimum Essential Medium, which contains non-essential amino acids, 10% fetal calf serum, penicillin (100 IU / ml) and streptomycin (50 IU / ml), propagated. The cells were placed in the wells of microtiter plates (3 = × = 10 ⁴ cells in 0.1 ml) and incubated overnight. Samples were made with medium. containing 5% fetal calf serum, diluted and then serially diluted directly in the assay plates. After approximately 24 hours of incubation, the medium was removed and 0.1 ml of a vesicular stomatitis virus suspension (Indiana) was added to each well. Cell and virus control wells were placed on each plate. The plates were incubated until the virus controls showed complete cytopathic effect (20-24 hours); they were then examined under a microscope and finally stained with 0.3% methyl violet solution. The plates were air-dried and the optical density of the cell layers was determined in a microtiter plate photometer at 540 nm.

Dose-response curves were semilogarithmic Paper applied. A dilution that the cytopathic effect reduced to 50%, was considered 1 Containing unit / ml (U / ml) defined.

Antiviral assays with other cell lines (human A549, murine L-M; bovin MDBK and BT, ovin SCP; Available at the ATCC) were carried out in the same way, apart from the fact that in some cases the murine Encephalomyocarditis virus was used. Recombinant human IFN-α2C was positive Control used. All incubations were done at 37 ° C carried out.

Example 3 Interferon neutralization assay

A solution of EpIFN-β1 in cell culture medium was mixed with equal volumes of serially twice diluted antisera or monoclonal antibodies and 90 min. incubated at 37 ° C. The solution was then transferred to prepared monolayers from NBL-6 assay cells; the antiviral activity of the IFN was determined as described above. The titer of a solution was defined as the reciprocal of a solution that reduced the antiviral activity to 1 unit / ml ( 7 ). Neutralization bioassays for human IFN-β (partially purified from poly (I: C) -induced human skin fibroblasts) were carried out with human A549 cells. Antiserum against human IFN-ß was obtained from the National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA (Research Ref. Reagent G-028-501-568).

Example 4 SDS polyacrylamide gel electrophoresis

SDS-PAGE analyzes were carried out analogously to Laemmli ( 8 ), using a 15% polyacrylamide separating gel. The proteins were stained with Coomassie blue. The molecular weight was determined using the "Pharmacia Low Molecular Weight Marker Set".

Example 5 HPLC analysis

Gel permeation HPLC was performed using a Waters Protein Pak J 125 column (7.8 × 600 mm, particle size 10 µm) and a buffer consisting of 0.02 M sodium phosphate pH 7.0, 0.5 M sodium sulfate, 0.04% Tween 20 and 25% propylene glycol existed at a flow rate of 0.5 ml / min. carried out. The protein was detected by its absorption at 214 nm. The molecular weight was determined by using lysozyme (Mr = 14,400), trypsinogen (24,000), ovalbumin (44,000) and bovine serum albumin (66,000) as reference proteins. These proteins were also used as standards to determine the protein concentration of the peak areas. Reverse phase HPLC was carried out with a Bakerbond WP C ₁₈ column (4.6 × 250 mm; particle size 5 μm, pore diameter 300 A) at room temperature, using the following solvents: Solvent A: 0.1% trifluoroacetic acid in water Solvent B : 0.1% trifluoroacetic acid in acetonitrile The following program was used:
0-2 min: 20% B;
2-26 min: 20-68% B (linear gradient);
26-36 min: 68% B (flow rate 1 ml / min).

The proteins were isolated by absorption at 214 and 280 nm detected.

Example 6 Amino acid analysis

Purified EpIFN-β1 was desalted and reverse HPLC phase denatured. The peak fraction was collected and three alipuots of about 40 µg each (2 nmoles) were in a "Speed Vac Concentrator" dried. The protein was in the gas phase hydrolyzed. where 200 ul 6M HCl, the 1% phenol contained were used at 110 ° C for 24 hours. The samples were dried and in 100 ul Na-S Buffer (Beckmann Instruments) dissolved again.

50% of each sample was automated Amino acid analysis in a Beckman 6300 device using an anion exchange column and one downstream ninhydrin detection was connected, subject. The one recommended by the manufacturer Elution program was used. The peak areas of Leucine and alanine were used to get the results for the other amino acids to nMol protein normalize.  

The total deviation from the expected composition was obtained by summing the absolute values of the Deviations from all amino acids determined.

Example 7 Protein segregation

Purified EpIFN-β1 was desalted and denatured by reverse phase HPLC as described above; the peak fractions were collected and dried in a "Speed Vac Concentrator". The protein was redissolved in 75 μl of 70% formic acid and introduced directly into the cartridge of a gas phase sequencer (Applied Biosystems Model 470A; program 02 rpth). The phenylhydantoin derivative of the amino acids of each cycle of Edman degradation was analyzed off-line by reverse phase HPLC, using a Merck Supershere C ₈ column (4.6 × 250 mm; particle size 4 µm, 62 ° C) and an isocratic mobile phase from 31 % Acetonitrile and 1.5% dichloroethane in 0.01 M sodium acetate pH 5.2 ( 9 ) was used at a flow rate of 1 ml / min. The substances were detected by UV absorption at 254 nm. The retention times of the substances were compared with a standard set (Pierce).

Table 1

Purification of EqIFN-β1

EqIFN-β1 was extracted from 10 g of cell cake and purified as described. Total protein was determined by staining binding assay, EqIFN-β1 by ELISA certainly.  

Table 2

Amino acid composition of EqIFN-β1

Table 3

Antiviral activity of EqIFN-β1

bibliography

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Claims (9)

1. A process for the purification of EqIFN-β, characterized in that

• a) EqIFN-β is extracted from acid-inactivated cells of the expression host organism, preferably from E. coli cells at an alkaline pH,
• b) EqIFN-β from the supernatant of a polyethyleneimine precipitation is bound to an adsorbent with ion exchange and / or molecular sieve properties,
• c) EqIFN-β is eluted with a phosphate buffer,
• d) and then the EqIFN-β eluted in this way is highly purified via anion and / or cation exchangers.

2. The method according to claim 1, characterized in that that the pH during extraction to 10 is set. 3. The method according to any one of claims 1 or 2, characterized in that as adsorbent at b) Hydroxyapatite is used. 4. The method according to any one of claims 1 to 3, characterized in that as an anion exchanger Mono Q and used as cation exchanger Mono S. becomes.   5. The method according to any one of claims 1 to 4, characterized in that in stage d) Linear gradient of sodium chloride for the elution from the anion exchanger and a linear gradient of ammonium sulfate for the elution of that Cation exchanger is used. 6. EqIFN-β, producible according to one of the claims 1 to 5. 7. EqIFN-β according to claim 6 for use as Drug. 8. EqIFN-β according to claim 6 for use as antiviral drug. 9. EqIFN-β according to claim 6 for use as Medicinal products with immunomodulating activity.