KR840001516B1 - Process for preparing human fibroblast interferon - Google Patents

Abstract

Affinity chromatog. and high-pressure liq. chromatog. (HPLC) were used in the purifn. of human fibroblast interferon and in the prodn. f a homogeneous protein having a specific activity ˜4x108 units/mg, an apparent mol. wt. ˜20,500 <= 3 glucosamine residues, and no galactosamine or mannosamine residues. Concanavalin A or Blue dextran-Sepharose colmns were used for affinity chromatog. HPLC was carried out with columns contg. a silica matrix bound to cyclohexyl groups; pyridine-HCO2H-iso-PrOH-H2O (8:8:20:64) and pyridine-HCO2H-iso-PrOH-BuOH-H2O (8:8:25:20:39 vol/vol) were used as the initial and the final eluents in HPLC. Parental formulations were prepd. contg. 1.5mg interferon and 25mL of serum albumins.

Description

Method of manufacturing human fibroblast interferon

The present invention relates to a method for producing human fibroblast interferon in homogeneous protein state using affinity chromatography and high pressure liquid chromatography (HPLC).

Since interferon was first discovered in 1957 by Isaac and Lindenmann, regardless of leukocytes or fibroblast types, the interferon is isolated into homogeneous peptides in amounts sufficient to confirm the biological and chemical properties of the interferon. In order to do so, scientists all over the world have failed to isolate interferon as a homologous peptide, despite attempts over 20 years. Several scholars have claimed that mouse or human interferon has been purified to homogeneity, but the homogeneity of the protein has not been demonstrated and, as claimed, the properties as pure compounds have not been described.

The use of high performance liquid chromatography for protein purification is a well known method in the prior art. The literature describes ion exchange and size exclusion type columns for protein purification (eg Regnier and Noel, J. Chromatog. Sci. 14, 316 [1976] and Chang et al., Anal. Biochem. 48, 1839 [1976]), and also in the case of reverse phase partition chromatography, Lichrosorb RP-18 (octadecyl silica fine particle column) was used to successfully obtain peptides such as β-endorphin. Purification is described. (Rubinstein et al., Proc. Natl. Acad. Sci. USA, 74, 4969 [1977]).

It has been described in the prior art that affinity chromatography can be used as a manipulation step to purify human fibroblast interferon. Davy et al. Describe the use of Concanavalin A-Sepharose 4B affinity chromatography to prepare purified human fibroblast interferon and described by Davey et al., J. Biol. chem. 251, 7620 (1976), Zankoski et al. Describe the use of Blue Dextran-Sepharose for this same purpose. [Jankowski et al., Biochemistry 15, 5182 (1976)] Finally, according to US Pat. No. 4,172,071 (de Maeyer et al.), Purification of leukocyte and fibroblast interferon crude solution was performed on a bluedextran-sepharose column. It is described that preparations having activities of 1 to 5 × 10 ⁸ interferon international units were prepared by performing by the affinity chromatography used. No other purification steps have been described, and the resulting interferon preparations contain little contaminated protein and contain high concentration products with interferonic activity.

The improved formulation of the present invention allows for efficient purification of human fibroblast interferon by combining affinity chromatography and high pressure liquid chromatography steps.

In particular, the inventive method combines the following steps:

A. Impure human fibroblast interferon aqueous solution is passed through an affinity chromatography column to adsorb the interferon to the column, and then the interferon is eluted from the column to obtain a specific fraction of the eluate containing the interferon of increased purity;

B. The obtained interferon fraction is a hydrocarbon-bonded siltica matrix column equilibrated with one or more buffers under high pressure liquid chromatography conditions, wherein the hydrocarbon is selected from cyclohexyl, phenyl, diphenyl, octyl, octadecyl and cyano propyl. Interferon is adsorbed on the column, and the interferon is eluted from the column using an acidic aqueous buffered mixture gradient of water-miscible solvent, and the homogeneous protein state as one specific peak in a specific fraction of the eluate obtained. To obtain interferon.

Concanavalin A-Sepharose 4B or Bluedextrin-Sepharose can be used in the affinity chromatography process according to the present invention, providing a much higher yield and also a more stable product than the Concanabaltin A-Sepharose 4B process. More preferably using blue dextran-sepharose.

The Concanavalin A-Sepharose 4B process can be performed in the following manner:

(a) 20 to 30 times the volume of human crude fibroblast interferon (highly active about 10 ⁴ units / mg) of the column in 5% native calf serum containing Eagle's lowest essential medium Pumped into a Concanavalin A-Sephatose 4B column at a flow rate of from 60 cm / hour

(b) washed with phosphate buffered saline (PBS) at pH 7.2,

(c) washed with PBS-0.1M α-methyl mannoside (α-MM),

(d) elute with PBS-0.1M α-MM containing 50% ethylene glycol.

The specific activity for the purified interferon obtained from step (d) is about 10 ⁷ units / mg, and the yield is about 10-30%.

A suitable sequence for using bluedextran-sepharose in an affinity chromatography step is as follows.

(a) A saturated sodium chloride solution was added to a culture supernatant (1-3 × 10 ⁴ units / ml, 0.2-1 mg protein / ml) of 10 to 40 times the volume of the column, and adjusted to 1.25M sodium chloride solution. Pumping the column at a constant flow rate of from 40 cm / hour,

(b) 5 to 15 times the volume of the column, washed with 5 to 15 times the volume of 1 M NaCl / 0.02 to 0.05 M phosphate followed by 5 to 15 times the volume / volume of ethylene glycol (pH 7.2). Washed with 1M NaCl / 0.02 to 0.05M phosphate,

(c) Eluate the interferon with 1M NaCl / 0.02 to 0.05M phosphate containing 50% (volume / volume) ethylene glycol pH 7.2. The obtained interferon has an intrinsic activity of about 3 × 10 ⁶ units / mg and a yield of about 80%.

The bluedextran-sepharose used in the affinity chromatography described above can be conveniently prepared by combining blue-dextran [Cibacronblue F3GA combined with dextran] with cyanogenbromide-activated Sepharose 4B at pH9.5. Can be.

Washing and aging of the resin (Ageing), column load and elution as well as the above-mentioned binding method is also very important for obtaining the human fibroblast interferon formulation in maximum yield and maximum purity.

When separating the interferon sample from the serum-free medium, 30 to 50% (volume / volume) ethylene glycol / buffered aqueous solution (pH 7.2) was passed through the Blue Sepharose-4B column as an eluent once or twice in a homogeneous state. It can be purified.

In order to further purify the interferon produced in the affinity chromatography step, the separation and yield are high when the interferon is subjected to one or more high pressure liquid chromatography steps on a production scale. The liquid chromatography process uses a column containing a porous silica matrix bonded to cyclohexyl, octyl, octadecyl, phenyl, diphenyl or cyanopropyl groups. This column, which can be used continuously under various pH and organic solvent gradient conditions, is characterized by a single band on sodium dodecyl sulfate (NaDodSO ₄ ) polyacrylamide gel electrophoresis, constant intrinsic activity, a single peak on HPLC and matching activity and protein concentrations. As confirmed by the human fibroblast interferon can be seen to be purified to a homogeneous material.

A generally porous silica microparticle column (particle size about 10 microns, pore size about 100 mm 3) having an irregular shape combined with cyclohexyl, octyl, octadecyl, phenyl or cyanopropyl groups used in the present invention is commercially available.

High pressure liquid chromatography, which is convenient to use the aforementioned columns, is described in US Pat. No. 4,116,046 (Stanley Stein).

In carrying out the process, a solution of human fibroblast interferon in an aqueous buffer of pH 4-8 purified by affinity chromatography is passed through a silica matrix column. Preferred buffers for the purposes of the present invention are pyridine / formic acid / water (8: 8: 84 vol / vol) solutions. Typically the process is carried out under pressure, preferably from about 3.4 to about 340 atmospheres. The interferon is adsorbed on the column and then eluted according to an optional method using a solution added to the aqueous buffer with varying amounts of water miscible solvent. Suitable water-miscible solvents for this purpose include alkanols such as n-propanol, iso-propanol, n-butanol, tert-butanol, ethanol, methanol and the like. Particularly preferred for the elution of human fibroblast interferon is a mixture of propanol and butanol.

Classification of the eluate is carried out using a fraction collector according to known methods while simultaneously tracking the protein content contained in each fraction by means of a peptide monitor operated with high sensitivity. Suitable systems for this purpose are described by Bolton et al. [Bohlen et al., Anal. Biochem. 67, 438 (1975). See also US Pat. No. 3,876,881.

The choice of the particular resinous form used in the high pressure liquid chromatography step depends on the source and purity of the human fibroblast interferon starting material used in this step. Thus, the material prepared by affinity chromatography on a concanavalin A-Sepharose column is conveniently homogenized by performing high pressure liquid chromatography on a cyclohexyl bound silica column followed by high pressure liquid chromatography on an octyl bound silica column. Purify with water. On the other hand, when using a bluedextran column in an affinity chromatography step, one or more octyl-bonded silica columns are passed through one or more times, or in some cases, through an octyl-bonded silica column, followed by cyanopropyl. After passing through the bound silica column, it can be purified to a homogeneous material by passing through the diphenyl bound silica column if necessary. In the case of using the Blue Sepharose-4B column and the serum-free material, a homogeneous preparation can be obtained by only passing the octyl-bonded silica column once.

Chromatography is performed at acidic pH due to the sensitivity of human fibroblast interferon activity at neutral pH in the presence of organic solvents such as propanol, butanol or 2-methoxyethanol. Human fibroblast interferon has a higher hydrophobicity than leukocyte interferon, and partially purified fibroblast interferon preparations contain other proteins with higher hydrophobicity. Therefore, it is recommended to use a mixture of propanol and butanol as the eluting solvent of the reversed phase column. More preferred is the use of n-propanol alone. The use of such mixtures / solvents limits the organic solvent concentrations required for complete elution of the absorbed protein, thereby reducing the generation of artificial structures under load due to the low solubility of the protein.

The homogeneous fibroblast interferon obtained according to the present invention is derived as one peak in the final high performance liquid chromatography column, and one narrow band on NaDodSO ₄ polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Form. The extract of the gel has one peak with antiviral activity consistent with the protein bands. Conventional antiviral activity assays for human fibroblast interferon activity can be used for the purposes of the present invention.

The molecular weight of the homogenous fibroblast interferon measured by the polyacrylamide gel method is about 20,500. The specific activity of this purified material is about 4x10 ⁸ units / mg. The N-terminal sequence included in the homogenous fibroblast interferon sample was Met ¹ -Ser-Tyr-Asn-Leu ⁵ -Leu-Gly-Phe-Leu-Gln ^10- Arg-Ser-Ser-Asn-Rhe ^15- Gln- … -Gln-Lys ¹⁹ -... to be.

Interferon has antiviral, anticancer, growth inhibitory and immunosuppressive activities. These activities were confirmed in clinical trials using relatively crude formulations containing less than 1% human interferon in 1-10 × 10 ⁶ units per day. The purified homogeneous human fibroblast interferon of the present invention can be used in the same manner as the preparations used so far by adjusting the dosage to provide the interferon at the required level.

Regarding the process and product of the present invention, it is explained in more detail by the following examples which are incorporated by reference. Titers of all interferons are expressed in standard units / ml or standard units / mg for the standard for human leukocyte interferon (GO 23-901-527) prepared by the US Department of Health.

Example 1

[Concanavalin A Affinity Chromatography]

50 ml of large cannavaline A-Sepharose 4B equilibrated with PBS at pH 7.2 containing 0.1 M α-MM was charged into a 50 ml polyethylene column equipped with polyethylene disc frit. It was loaded with crude interferon (1.25 L of 2 × 10 ⁷ units of specific activity of 2 × 10 ⁴ units / mg) at a flow rate of 180 mL / hour (35.5 cm / hour). Wash with 600 mL PBS buffer containing 0.1 M α-MM buffer and finally interferon is eluted with the above-mentioned buffer containing 50% (volume / volume) ethylene glycol. The yield of the product at 1 × 10 ⁷ units / mg is 9% (1.8 × 10 ⁶ units) The yield of a wide range of fractional cuts including other fractions with an intrinsic activity of 2 to 4 × 10 ⁶ units / mg is 15% ( 3 × 10 ⁶ units).

[HPLC]

A total of 120 mL of eluate (2.5 × 10 ⁶ units: about 2-4 × 10 ⁶ units / mg) from the Concanavalin A-Sepharose 4B column was 0.4-0.8 mL while maintaining the maximum back pressure below 306 atm. It is applied directly to a chromegabond 10 μcyclohexyl column (4.6 × 300 mm) equilibrated with pyridine / formic acid / isopropanol / water (8: 8: 20: 64: volume / volume; buffer A) at a flow rate of / min. The apparatus used for the HPLC process is described by Bohlen et al. (Anal. Biochem. 67, 438 [1975]).

From the next gradient of equilibration buffer A, the final buffer B (pyridine / formic acid / isopropanol / n-butanol / water, 8: 8: 25: 20: 39, volume / volume) is drained at a rate of 0.4 ml / min. 0-25% B, 32 minutes; 25 to 60% B, 225 min; Combine 60-100% B, 63 minutes, 2.0 ml fractions. Take 26-29 (2 × 10 ⁶ units) fractions from the center of the activity peak and dilute with 4 ml of pyridine / formic acid. This solution is applied to a chromobond 10μ octyl column (4.6 x 300 mm) via a pump. Elution conditions are the same as in the cyclohexyl column described above. Activity The intrinsic activity at the center of the peak is about 4 × 10 ⁸ units / mg, and the total amount of purified human fibroblast interferon is 10 ⁶ units.

Note that when the interferon purified on Concanavalin A is HPLCed on a cyclohexyl column or octyl column, the interferon elution locations of these two columns are very close to the main central peak. If the initial HPLC step is carried out on an octyl column, the interferon elutes immediately before the main central peak, while under the same chromatographic conditions on the cyclohexyl column, immediately after this main peak. Human fibroblast interferon samples purified by HPLC were tested on 12.5 or 15% NadodSO ₄ polyacrylamide gels in Tris / Glycine buffer with experimental 1 μg protein. After electrophoresis, staining with Coomassie blue forms one band. The same sample material is applied to the same type of gel, cut and used for antiviral experiments. The maximum value of antiviral activity coincides with the center of the colored band. Bovine serum albumin, chymotrypsin, cytochrome C and ribonucleases were used as standards to determine the molecular weight of this sample about 20,500. The relative mobility of human fibroblast interferon bands is the same as whether mercaptoethanol is present in the sample.

Example 2

[Production of Bluedextran-Sepharose 4B Resin]

50 g of packed Sepharose 4B is washed with 1 L of water and resuspended in 50 ml of distilled water. 15 g of finely ground cyanogen bromide is added to the suspension with gentle stirring and the pH is adjusted to 11 ± 0.2 by the addition of 10N NaOH dropwise and maintained. Add a small amount of crushed ice to maintain the temperature at 20 ± 5 ℃. When the reaction is stopped (15-20 minutes) about 1x of ice water is added and the suspension is transferred to a Buchner funnel and washed with 5x 0.01N HCl.

The activated resin is immediately suspended in 50 ml of 0.4 M sodium carbonate buffer (pH9.5) containing 1.00 g of dissolved bluedextran and placed in a round bottom flask at 4 ° C. for overnight shaking. The resin was then washed with 50% ethylene glycol (pH 7.2) containing 10 times of 1M NaCl, 2 times of 1M NaCl and 0.05M sodium phosphate, left overnight in 50% ethylene glycol, then 1x 80 Wash as the lowest essential medium of 5 times Gibco No. 11 containing% ethylene glycol and 5% native calf serum (MEM) and leave at 50 ° C. in this medium overnight. The resin is then further washed with three times 80% aqueous ethylene glycol containing 1M NaCl followed by two times MEM and then slurryed in MEM to fill the column for use.

Affinity Chromatography

1.5 L of crude fibroblast interferon (2 × 10 ⁴ units / ml: 1 mg protein / ml; intrinsic activity 2 × 10 ⁴ units / mg protein) is mixed with 0.27 times saturated saline (about 6.3 M). This solution is pumped at a flow rate of 100 ml / hr (20 cm / hr) through Bluedexran-Sepharose 4B (35 ml) filled in a 50 ml polypropylene column equipped with a porous polyethylene disc at the bottom. The filled resin was successively washed with 200 ml of 1 M NaCl containing 0.05 M sodium phosphate buffer (pH 7.2), 500 ml of 1 M NaCl and 75 ml ethylene glycol containing 0.05 M sodium phosphate buffer (pH 7.2). It is washed and finally eluted with 500 ml of 1 M NaCl and 250 ml of ethylene glycol containing 0.05 M sodium phosphate (pH 7.2). Elution with 50% ethylene glycol reveals about 90% interferon in the peak in the first half. The specific activity at the maximum peak at which 50% or more of the total interferon was eluted is 1 × 10 ⁷ units / mg.

[HPLC]

(A) A total of 125 ml of interferon, eluted from a Bluedextran-Sepharose 4B column (3 × 10 ⁷ units: 1 × 10 ⁷ units / mg), was previously equilibrated with 1M NaCl containing 50% ethylene glycol. Pump into a 4.6 x 300 mm chromobond 10μ octyl column. Immediately after the sample was added, buffer A (pyridine / formic acid / isopropanol / n-butanol / water, 8: 8: 20: 3.3: 60.7, volume / volume) was pumped into the column. The following gradient of eluent for Buffer B (pyridine / formic acid / isopropanol / n-butanol / water: 8: 8: 25: 20: 39, volume / volume) flows out at a flow rate of 0.45 mL / minute; 0-25% B, 30 minutes; 25 to 55% B, 190 minutes; 55-100% B, 20 minutes; 100% B, 60 minutes. Interferon activity is almost identical to one protein peak identified in 18-23 fractions, each fraction containing 1.5 ml of eluent.

(B) When about 1/4 load of interferon in (A) was applied to the same column and the elution conditions used for the cyclohexyl column in Example 2 were applied, the interferon activity was found in 24 to 26 fractions of protein peaks. Coincides with

(C) Approximately 1/9 of the load per column volume by using the same load, gradient and outflow rate as in (A) and the A and B buffers used in (B), and using a 9.6 × 500 mm chromagabond octyl column. And the separation is better.

(D) Using pyridine / formic acid / water (8: 8: 84, volume / volume) as the substance having the maximum specific activity (4 × 10 ⁸ units / mg) in (A), and the flow rate was 0.3 ml / The major peak was symmetrically produced by rechromatography on a cyanopropyl column with an n-propanol gradient of 0-40% (volume / volume) over 1 hour in minutes.

[NaDodSO ₄ Polyacrylamide Gel Electrophoresis]

When electrophoresis was performed on the samples obtained from the above-mentioned steps (A), (B) and (C) as described in Example 1, the main band appeared in the 20,500 MV band, and the gelled material was separated and analyzed. If activity is also coincident with peak center. The second strong band (about 20-50% of all material, estimated from 10,500 MW / staining intensity) varies greatly from sample to sample and has no antiviral activity. If the sample is not treated with mercapto ethanol, a 40,000 MW band can be observed, which has borderline activity. The results of amino acid analysis of the 20,000 MW band and the 40,000 MW band are not unusual, whereas the results of the 10,000 MW band are very unusual.

Electrophoresis of the sample from step (D) described above yields one band having a molecular weight of about 20,500 and an intrinsic activity of 4x10 ⁸ units / mg. The result of amino acid analysis of the homogeneous peptide, which was hydrolyzed in 6N HCl for 24 hours, is as follows when the leucine is approximately 25.0.

Example 3

[Bluedextran-Sepharose Affinity Chromatography]

(a) 5 L of the culture supernatant containing 19,400 units / mL of fibroblast interferon is filtered through a 0.3 μ filter and then applied to a column containing 275 mL of blue dextran-sepharose with bad volume. To the supernatant was added 1350 mL saturated NaCl solution (6.3 M) to adjust to a NaCl concentration of 1.25 M and dumped into the column at a flow rate of 360 mL / hour. About 4-6% of interferon passes at a rate that does not remain in the column.

(b) The column is then washed with a flow rate of 420 ml / h with 1300 ml of an aqueous solution containing 15% (volume / volume) ethylene glycol, 1 M NaCl and 0.02 M sodium phosphate, pH 7.2. About 1% of the interferon added to the column is eluted.

(c) The interferon is then eluted at a flow rate of 420 ml / hour with an aqueous solution containing 50% (volume / volume) ethylene glycol, 1 M NaCl and 0.02 M sodium phosphate (pH 7.2). Few interferons are eluted in the first 200 ml eluting eluent, and most of the interferon added to the column is then eluted in 300 ml eluent as shown in the following table.

[HPLC]

Fractions 2 to 6 (250 mL; 13 × 10 ⁷ units; high activity 8.12 × 10 ⁶ units / mg) eluted from the Bluedextran-Sepharose affinity column were 9.5 × 500 mm octyl-bonded silica at a flow rate of 4 mL / min. Dump to the matrix column. Buffer A (pyridine / formic acid / 2-propanol / n-butanol / water, 8/8/20 / 2.5 / 61.5, volume / volume) is then dumped into the column for 12 minutes at a flow rate of 2 ml / min. Buffer B (pyridine / formic acid / 2-propanol / n-butanol / water, 8/8/25/25/34, volume / volume) is then pumped at a flow rate of 1.25 ml / min in the following gradient: 0 to 20% B, 18 minutes; 20 to 29% B, 57 minutes; 29% B, 27 minutes; 29 to 30% B, 3 minutes; 30 to 100% B, 36; 100% B, 54 minutes.

Interferon activity appears in the fraction eluted with a 29% B gradient and elutes with the peak measured as a fluoroescamine tracer. 45 × 10 ⁶ units of interferon were recovered (yield 35%), and the specific activity of the aggregates (50 mL) was 2 × 10 ⁸ units / mg. After two months of storage in a closed polypropylene vial at −20 ° C., the activity is reduced to 10% in the HPLC step and no protein is lost.

50 ml of the aggregate from the previous HPLC column (about 0.2 × 10 ⁸ units / mg high activity) are mixed with 1 ml thiodiglycol and 20 ml n-propanol. Analysis of this mixture yielded a total of 4.26 × 10 ⁶ units. To this was added 100 ml of 0.1% Triton × -100 / 0.3% thiodiglycle. This mixture is a total of 5.1 × 10 ⁶ units and 5μ / 80Å 4.6 × 250 previously equilibrated with a mixture of pyridine / formic acid / water (8: 8: 84, volume / volume, buffer A) at a flow rate of 1.5 ml / hour for 30 minutes Dump with a millimeter Sphersolve CN column. Buffer B, pyridine / formic acid / n-propanol / water (8: 8: 50: 34, volume / volume) at 0.5 mL / min flow rate with the following gradient: 0-25% B, 30 min; 25-50% B, 210 minutes. Interferon activity occurs with only one peak observed. 4.2 × 10 ⁶ units are recovered (82%) in three 1 ml fractions.

The aggregates corresponding to 69% of the total protein initially added to the cyanocolum were mixed with 2 ml of 0.1% Triton × -100 and dumped into the same cyanocolumn as used previously. At the time corresponding to half of the total gradient time, the same gradient is performed. All active interferon (11.75 × 10 ⁶ units, 280% recovery) elute with only one peak.

The interferon active aggregates from the second cyanocolumn (4 ml) were pre-circulated from buffer A to buffer B (same buffer as cyano column) in a 2 hour gradient to equilibrate buffer A at 1.5 ml / min. Dump into a 4.6 × 250 mm diphenyl bonded silica matrix column.

Elution is carried out with a flow rate of 0.5 ml / min and the following gradient; 0-25% B, 22 minutes; 25-50%, 98 minutes. The second peak eluted when the gradient system indicates 33% buffer B is in agreement with the interferon activity plot. A total of 2.6 × 10 ⁶ units (22%) and 40 μg of protein elute at this peak.

Diphenyl-bonded silica matrix columns are prepared as follows.

The silica support (10 μm, 10 nm pore size) is immersed in 6N HCl (10: 1 volume / weight) for 24 hours with occasional stirring. The support is washed with water until the wash is neutral on a calcined glass filter and then with acetone and methanol to remove water. After overnight drying in vacuo, the support (10 g) was refluxed for 6 hours in anhydrous toluene (100 mL) containing 10 mL of dichlorodiphenyl silane. The bonded support is separated from the toluene solution using a sintered glass filter and washed with 200 ml of toluene. Subsequently, this bonded support was successively treated with 250 ml of toluene, acetone and methanol for 8 hours in a Soxhlet extractor followed by trimethylchlorosilane in the same manner as described above.

The bound support is filled into a 4.6 × 250 mm stainless steel column at 340 atm using a mixture of chloroform (10.7 mL) and n-butanol (2.3 mL) to suspend 3 g of the support.

The amino acid composition of the resulting interferon peak was analyzed under various hydrolysis conditions, and the results were summarized as follows (all hydrolysis was performed in 5.7 N HCl in a vacuum glass tube washed with acid.) And HCl blanks were corrected).

Fibroblasts from the hexamine assay after hydrolysis of the interferon at 110 ° C. for 6, 14 and 24 hours with 5.7 N HCl containing 0.02% thioglycolic acid using an eluate obtained from the central peak fraction of the diphenyl column. Interferon has been shown to contain about three residues of glucosamine. Galactosamine or mannozamine was not found.

The end group measurement was performed with 90 picoballs of a central fraction sample of diphenyl column fibroblast interferon peak, and Met was detected as the N-terminus.

Fibroblast Interferon and Leukocyte Interferon Samples Each of the 150 picomoles of trypsin immersed and chromatographically immersed, no peptide fragments common to both interferons were found.

The sequence of fibroblast interferon 1.25 nanomole is Met-identified at the N-terminus, and SEQ ID NOs: 3 to 10 by Hunkapillar and Hood indicate Tyr ³ -Asn-Leu-Leu-Gly-Phe-Leu-Gln ¹⁰ -has been confirmed. [Reference Biochemistry 17, 2124 (1978)] Further analysis of the sequence of 5.9 nanomole fibroblast interferon showed that the NH ₂ -terminal sequence was composed of the following: H ₂ N-Met-Ser-Tyr- Asn-Leu ⁵ -Leu-Gly-Phe-Leu-Gln ¹⁰ -Arg-Ser-Ser-Asn-Phe ¹⁵ -Gln-... -Gln-Lys ¹⁹ -...

Example 4

Crude interferon is prepared from human fibroblasts (cell line GM 2504A). This crude interferon is made into 1M sodium chloride solution by the addition of saturated sodium chloride. This material is passed through a Blue Sepharose-4B column (bed volume 20-25 mL, 2.5 cm, diameter 2.5 mL / min). Crude material is stored in ice while under load. After loading, the column is washed with 250 ml of the following solution (2.5 ml / min): 30% ethylene glycol, 1 M NaCl, 50 mM Na ₂ HPO ₄ (pH 7.2).

Finally, the interferon is eluted with 250 ml of the following solution at a flow rate of 2.5 ml / min: 50% ethylene glycol, 1 M NaCl, 50 mM Na ₂ HPO ₄ , pH 7.2.

The peak fractions are collected from the original Blue Sepharose column and diluted with 10% ethylene glycol as the following solution: 2M NaCl, 50 mM Na ₂ HPO ₄ (pH 7.2).

250 ml of 2M NaCl, 50 mM Na ₂ HPO ₄ (pH 7.2) and 30% ethylene glycol were suspended on a Blue Sepharose column (20-25 mL bed volume, 2.5 cm diameter, 2.5 mL / min flow rate). Washed with a solution of 2M NaCl, 50 mM Na ₂ HPO ₄ (pH 7.2) and eluted with a solution containing 50% ethylene glycol.

[HPLC]

Sample: The first sample used was a 50% ethylene glycol cut from Blue Sepharose. Subsequent samples were run twice through Blue Sepharose, in which case interferon in 30% or 50% ethylene glycol cleavage may be used.

Use a 25 × 0.46 cm Linkrosov RP-8 column. The column is first washed with water and then the blue-sepharose eluate is pumped. Use 5 ml mixing chamber before pumping. The HPLC column is added stepwise to the next gradient of n-propanol at constant pH4.2 maintained at 1 M formic acid / 0.8 M pyridine and flowed out at a rate of 22 ml / hour.

The column is then washed with 60% n-propanol and then rebalanced to 0% for operation.

Interferon elutes at the end of 32% steps (80-90 minutes). This material is homogeneous, as measured by NaomadSO ₄ acrylamide gel electrophoresis, either stained as Coomassie Blue or pre-labeled with Fluram.

Amino acid analysis (24 hour hydrolysis, 0.2% TGA, 5.7N HCl) was performed on nine separate samples prepared by four separate preparations. When leucine is set at approximately 22.0, the amino acid average composition as a relative value is shown in the following table.

Homogeneous fibroblast interferon by hydrolysis in 6N HCl containing 0.2% thioglycolic acid based on several samples analyzed at different times and manipulated according to different manipulations as shown in this and previous examples The analysis value of the representative amino acids analyzed (± 15%) is as follows.

Example 5

[Formula for parenteral administration of homogeneous human fibroblast interferon]

A total of 1.5 mg of homogeneous fibroblast interferon with an intrinsic activity of 4 × 10 ⁸ units / mg is dissolved in 25 ml of 5% normal human serum albumin. The solution is passed through a bacterial filter and then dispensed into 100 vials aseptically. Each vial contains 6 × 10 ⁶ units of pure interferon suitable for parenteral administration. The vial is preferably stored in a cold (-20 ° C.) prior to use.

Claims (1)

The aqueous solution of human fibroblast interferon in an impure state was passed through an affinity chromatography column to adsorb interferon to the column, and then the interferon was eluted from the column to obtain a specific fraction of the eluate containing the interferon in the state of increased purity. The fraction is passed through a hydrocarbon-bonded silica matrix column equilibrated with one or more buffers under high pressure liquid chromatography conditions, wherein the hydrocarbon is selected from cyclohexyl, phenyl, diphenyl, octyl, octadecyl and cyano propyl. Was adsorbed onto the column, and then the interferon was eluted from the column using an acidic aqueous buffered mixture gradient of water-miscible solvent, to obtain a homogeneous protein state of interferon as one specific peak in a specific fraction of the eluate obtained. Homogeneous Protein Status Method for producing human fibroblast interferon.