INTERFERON PURIFICATION PROCESS

Abstract: A process for the purification of a crude human leukocyte interferon, comprising the steps: a) applying a solution of said crude interferon onto an immuno-affinity adsorption column; b) eluting the adsorbed interferon from said column using a buffer solution; c) concentrating the eluate resulting from step b) with a thiocyanate solution by either precipitation or an exchange chromatography; and d) recovering the precipitated interferon obtained in step c).

Description: Interferon purification process. The present invention relates to a process for the purification of a crude human leukocyte interferon. Intβrferons are a group of proteinβ naturally produce in the body in response to an exterior stimulus, for example virus causing an infection. Interferon may also be preβent i small quantities in the blood in connection with certain vi¬ rus and tumor diseases. Interferons are formed in the body as part of the defense against infection and is considered to constitute an immediate defense mechanism in the protection against a virus infection. Several types of interferons are known. As examples there may be mentioned α-Interferon produced by leukocytes, β-Interferon produced by binding tissue cells and gamma-in- terferon produced by immuno-competent cells. Native interferon produced from leukocytes consists of a native mixture of different α-interferon subtypes or ' inter feron components. The different interferon components are named Interferon (X-l, Interferon α-2, Interferon α-3 etc. Moreover, there is an allelic variation within the different α-interferon components named Interferon 0C-2a, α-2b, (X-2c etc . Interferon produced from leukocytes according to the "Cantell method", so called PIF-Interferon or Cantell-Inter- feron has been used since 1971 in the treatment of different virus and tumor diseases. The expectations on interferons as a drug against different tumor and virus diseases arising during the 1970: ies are exclusively based on clinical result obtained in the use of Cantell-Interferon. In the early 1980:iβs interferon was cloned and inter feron manufactured using recombinant-DNA-techniques is today a drug in some fifty countries. Native interferon manufactured from leukocytes con- sists of a mixture of some twenty different α-interferonβ . Recombinant interferon consists of a cloned α-interferon sub type . The present invention resides in new techniques for the purification of native leukocyte interferon. The origina Cantell-interferon originally manufactured in Finland in the early 1970: ies haβ a relatively low purity, of the order of IX. As such, it is useful but leaves much to be desired due to today's stringent requirements as to purity and reproduci bility of drugs. The present invention has for a main purpoβe to provi de a process for the manufacture of a highly purified leuko- cyte interferon for clinical use. Another object of the invention is to provide such purification process which is highly reproducible, relativel inexpensive and provides for relative ease of performance. For these and other objects which will be clear from the following description the present invention resides in a process for the purification of a crude human leukocyte in¬ terferon, said procesβ comprising the following steps: a) applying a solution of βaid crude interferon onto an immuno-affinity adsorption column; b) eluting the adsorbed interferon from said column usin a buffer solution; c) concentrating the eluate resulting from step b) with thiocyanate solution by either precipitation or an exchange chromatography; and d) recovering the precipitated interferon obtained in step c) . In such process it is preferred to use for the prepa¬ ration of an immunoaffinity adsorption column a recombinant interferon for the generation of the antiserum from which polyclonal antibodies are isolated and covalently attached t the column matrix. The process of the invention is preferably performed using a further step of concentrating the eluate from the immuno affinity adsorption column using an ion exchange co- l m . The interferon is suitably eluted from such ion ex¬ change column by increasing the pH. Such pH increase can be obtained by applying a buffer solution to the column. In addition to the precipitation step using for examp le a thiocyanate solution the process may involve another precipitation step, wherein the interferon resulting from such precipitation is again precipitated in ethanol before its recovery. After the single or double precipitation the interfe¬ ron obtained is preferably made subject to gel filtration, and the proper effluent fractions having absorbance at 280 n are collected and recovered. In the precipitation step it iβ preferred to use a solution of potassium thiocyanate. Other precipitants are trichloroacetic acid and ammonium sulphate. Elution of the interferon from the ion exchange column to concentrate the eluate from the preceding step is suitably performed by app- lying a buffer solution to increase the pH. Such pH increase is preferably carried to a pH above neutral, such as to abou 8 or higher. The invention also covers a purified human leukocyte interferon whenever prepared by the process described herein The present invention will now be further illustrated by specific examples, which, however, are not to be construe as limiting the scope of the invention otherwise than as de¬ fined in the appended claims. EXAMPLE 1 Preparation of antiserum. In the production of antiserum healthy goats not pre¬ viously immunized are used. The antigen used in the immuniza tion is a recombinant interferon, α-88, manufactured at the University of Umea and described in detail in published Euro pean Patent Application No. 86903650.9 , publication No. 0 263 102. This recombinant interferon is dissolved in physio¬ logic phosphate buffer, the specific activity of the solutio being about 2xl0 8 IU/ g. This solution is administered to th goats together with "Freund's complete adjuvant" at the ini¬ tial immunization. Then immunization using "Freund's imcom- plete adjuvant" is performed once per week or every fortnigh in volumes of from 0.2 ml to 1.0 ml. The goats are bled via the jugular vein every fort¬ night after the third immunization. The drained blood is in¬ vestigated with regard to contents of anti-interferon antibo- dies and unspecific binding. The contents of antibodies is analyzed using an assay measuring neutrilazing titre, i.e. the titre measuring the amount of interferon which is inacti¬ vated by the antibodies in an inter eron-sensitive biologic system. Specific and unspecific binding is measured by con- ventional "Western blot". 150-200 ml blood is normally bled each time. The fresh blood is kept for 24 hours in the refrigerator and coagulates during this period. The blood is then centrifugated and the serum is recovered. This serum containing antibodies against interferon is made subject to further purification as descri¬ bed below. EXAMPLE 2 Preparation of immuno-a finity adsorption column. The antiserum obtained in Example i is precipitated with 25 g ammoniumsul ate/100 g antiserum with slow agitatio in centrifuge bottles which are left over * night. The follow¬ ing day the antiserum is centrifuged at 4000 g for 30 min. The supernatant is discarded and the precipitate is washed twice with ammonium sulfate 1.75 mol/1 to decrease the amoun of hemoglobin and albumin. The precipitate is transferred to dialysis tubing and dialysis is performed alternately agains water and sodium acetate buffer 0.05 M NaAc, 0.021 M HAc, pH 5.0. During dialysis a precipitate forms consisting of li- poproteins, the precipitate being removed by centri ugation at 4000 g for 30 min. The supernatant containing gammaglobu- ® lins is applied to a column containing DEAE Sepharose FF (2 ml packed gel/100 ml crude antiserum) equilibrated with ace- tate buffer, pH 5.0. The dialysed antiserum is passed throug the column and eluted with about one column volume of acetat buf fer . Volume: 445 ml Absorbance 280 nm: 35.2 IgG cone: 25.3 g/ml Total IgG: 11.3 g Recovery: 16 mg IgG/ l serum The immunoglobulin containing eluate is dialysed against coupling buffer, sodium bicarbonate 0.1 mol/1 and sodium chloride 0.5 mol/1. Br-CN-activation of Sepharose 4B Sepharose is washed with distilled water on a glass funnel and sucked dry. About 40-60 g gel/g IgG is weighed out and suspended in potassium phosphate buffer 2 mol/1 pH 11.0. The gel slurry is cooled on an ice-bath. BrCN is dissolved in water 0.05-0.1g BrCN/ml. 3.3 g BrCN/100 g dry sucked gel is added and the sepharose is activated for 10 min under gentle stirring on a magnetic stirrer. The gel slurry is transferred to a glass funnel and washed with cold distilled water until neutral reaction of eluate. The activated gel is mixed with the immunoglobulin solution and incubated at room temperature over night under gentle agitation. The gel is transferred to a glass funnel and the filt rate is analyzed for protein content by measurement of A28O ' The gel is again suspended in coupling buffer and glycin 0.1 mol/1 is added to block remaining coupling sites and the gel is incubated for at least 1 hour at room temperature. After the blocking procedure the gel is repeatedly washed with phosphate buffer 0.02 mol/1 βodium chloride 0.2 mol/1 pH 7.0 and citric acid 0.1 mol/1 sodium chloride 0.15 mol/1. The gel is stored in phosphate buffered saline with 0.04X sodium azi- de as preservative. Coupling procedure: 750 ml Sepharoεe 4B is washed with 2.5 1 diβtilled water on glass funnel and sucked dry, the weight of the "dry" gel is 520 g. The gel is resuspended in 500 ml potassium phosphate buffer 2.0 mol/1 pH 11.0 and cooled on an ice-bath. 17.3 g BrCN is dissolved in 200 ml water and added to the gel slurry and the gel is activated for 10 min. The gel is washed with 7 1 cold distilled water. The reaction of the eluate is neut¬ ral . The gel is mixed with the immunoglobulin fraction dia- lyzed against coupling buffer, sodium bicarbonate 0.1 mol/1 sodium chloride 0.5 mol/1 pH 8.5 and incubated over night at room temperature. The following day the gel is filtred and washed with one volume of coupling buffer and the protein concentration in filtrate and wash is determined by means of A 280- A 280 filtrate 184 mg protein A 280 wash 165 mg protein totally 349 mg protein Coupling efficiency: 96.9X The gel is resuspended in coupling buffer with glycin 0.1 mol/1 to block remaining activated sites for 1 hour. The gel is washed with phosphate buffer pH 7.0 and citric acid. The gel is stored in phosphate buffer saline with 0.04 sodium azide as preservative. EXAMPLE 3 Production of crude Interferon In this production buffy coats are used to prepare crude interferon. Buffy coats is a waste product at the ordi¬ nary blood banking in hospitals. The buffy coats used are no older than 48 hours and most of the buffycoats are not older than 24 hours . To prepare leukocytes from buffy coats the red blood cells thereof are lyzed by addition of ammonium chloride to a final concentration of 0.83%. After 10 min at room temperatu- re the leukocytes are centrifuged in a basket centrifuge with a flow rate of 300 ml/mm. The leukocytes are suspended in PBS. The process is repeated once and the leukocytes obtaine are then added to the culturing media. The purified leukocytes are suspended in Eagelβ mem with the following additives: Neomycin 25 μg/ml Tricine 3 mg/ml 4% human plasma precipitated with 6% Polyethylene glycol 6000 (Inglot A et al, Acta Virol. 19; 250-254, 1975) The use of PEG-fractionated plasma instead of α-gamma serum is the only major difference from the original Cantell method . The cells are used at a concentration of about 10? cells per ml in 4 1 round bottles containing approximately 2.0 1 each. Magnetic stirring is performed on the water bath at 37°C. The cell culture is primed through addition of crud leukocyte interferon 100 IU/ml. After 2 hours of priming Sen dai virus, about 150 haemaglutinating units per ml (about 40 ml Sendai virus per liter) is added. After 20 hours at 37°C the leukocytes are removed by centrifugation or filtration. The crude interferon will be stored maximum 7 days before purification. EXAMPLE 4 Concentration of crude Interferon The crude interferon solution obtained according to Example 3 is concentrated by ultrafiltration using a Filtron system, MW 10.000 filter. Before the ultrafiltration takes place, the crude interferon is filtered through a 0.45 μro membrane (Millipore, Prostak) to remove cells and cell deb- ris. The interferon solution is concentrated about 5 times. EXAMPLE 5 Affinity chromatographv of crude Interferon The affinity column ( BP 113/15, Pharmacia, gel volume: about 1 1) obtained as described in Example 2 is equilibrate with 0.02 M Tris-HCl 0.2 M NaCl 0.001 M EDTA pH 8.0 (Trie- -buffer) . The interferon solution obtained from Example 4 iβ then allowed to pass the column at a flow rate of about 4 1 per hour. After the interferon solution has passed the co¬ lumn, the column is washed using Tris-buffer until the abβor- bance of the eluate is zero or nearly zero. The affinity column is desorbed by passing 0.1 M cit¬ ric acid 0.15 M NaCl pH 2.0. The citric acid buffer is passed through the column at a flow rate of about 3 1 per hour. This acidification of the column results in desorption of the in- terferon taken up via the column. The desorption is continued until absorbance at 280 nm ceases and the collected interfe¬ ron solution is recovered. The volume is about 5 1. EXAMPLE 6 Concentration of desorbed Interferon The interferon resulting from the desorption according to Example 5 is passed through an ion exchange column (S- Φ Sepharose ) equilibrated with citric buffer, pH 2.0. The ion exchanger is then washed using citric buffer, and the inter- feron is eluted by increasing pH through the addition of Tris-buffer resulting in an pH increase to about pH 8. EXAMPLE 7 Precipitation of Interferon using potassium thiocyanate The interferon solution obtained from Example 6 is carefully added with 5M KSCN and human serum albumin (HSA) under stirring to a final concentration of 0.5M KSCN (weight ratio Ifn:HSA about 1:5-10). Hydrochloric acid (1M) is then added until the pH has decreased from about 8 to about 2.0. The cloudy precipitation is centrifuged at 5000 r/min for 30 min (Beckman J-21). The βupernatant is carefully recovered. EXAMPLE 8 Precipitation with Ethanol The interferon precipitate from the sodium thiocyanat precipitation in Example 7 is slurried in 95% acid ethanol together with HSA (weight ratio Ifn to HSN about 1:5-10) (a bout 300 ml) having a temperature of -20°C. The slurry is centrifuged for 30 min at 5000 r/min. The supernatant conta ning the interferon is recovered and the pH thereof is gent increased to pH 8.0 by adding 0.1M NaOH. The precipitate is then centrifuged for 30 min at 5000 r/min (Beckman J-21). T supernatant is decanted and the precipitate is dissolved in 10 ml 0.15M sodium phosphate buffer + 0.5M KSCN, pH 8.0 EXAMPLE 9 Gel filtration of precipitate The interferon solution obtained from Example 8 is centrifuged and made subject to gel filtration on a column (AcA-54, IBF, France, 2.5 x 90 cm). The interferon peak is then recovered, detected with UV-light, 280 nm. The purity the interferon obtained is about 80% and when assayed (Wes¬ tern blot) it is found to contain all interferon components present in the Cantell-type crude interferon starting mate¬ rial. The process of this invention offers many advantages, among. which there may be mentioned: efficient elimination of the riks for virus infectio high yield, simple and reliable performance. The examples given above are not to be construed as limiting the scope of the invention. They have been present merely for purposes of illustration and description of the invention. Obviously, many modifications and variations are possible to the skilled artisan in the light of the inventi as described. It is intended that the scope of the inventio shall be defined by the appended claims.

Claims: CLAIMS 1. A procesβ for the purification of a crude human leukocyte interferon, comprising the steps: a) applying a solution of said crude interferon onto an immuno-affinity adsorption column; b) eluting the adsorbed interferon from said column usin a buffer solution; c) concentrating the eluate resulting from step b) with thiocyanate solution by either precipitation or an exchange chromatography; and d) recovering the precipitated interferon obtained in step c) . 2. A process according to claim 1, wherein the eluate from step b) is concentrated using an ion exchange column, the interferon being -eluted from said column by increasing the pH by applying a buffer solution to the column. 3. A process according to claim 1, wherein the eluate from step b) is concentrated by precipitation using thiocya¬ nate, trichloroacetic acid or ammonium sulphate. 4. A process according to claim 1, 2 or 3, wherein th precipitated interferon from step c) is again precipitated i ethanol before recovery thereof. 5. A process according to any preceding claim, wherei the interferon resulting from the precipitation thereof is subjected to gel filtration, the effluent showing absorbance at 280 nm being recovered. 6. A process according to any preceding claim, wherei the column used in step a) contains polyclonal antibodies obtained by immunization using a recombinant interferon. 7. A process according to any preceding claim, wherei the precipitation in step c) is performed with a solution of potassium thiocyanate. 8. A process for the purification of a crude human leukocyte interferon comprising the steps: a) applying a solution of said crude interferon onto an immuno-affinity adsorption column; b) eluting the adsorbed interferon from said column usi a buffer solution; c) concentrating the eluate from step b) by passing same through an ion exchange column and eluting the interferon by applying a buffer solution increasing the pH; d) precipitating the interferon present in the eluate resulting from step b) with a thiocyanate solution; e) reprecipitating the interferon resulting from step d) in aqueous ethanol; and f) recovering the interferon obtained in step e). 9. A process according to any preceding claim, wherein any precipitation is performed in the presence of albumin. 10. A purified human leukocyte interferon whenever prepared by the procesβ of any preceding claim.

Inventors: BORG HAAKAN, BORG, HAAKAN
Applicants: BIONATIVE AB
Application year: 1990
Main classification: C07K3
IPC codes: A61K38, C07K1, C07K14, C12P21, C12R1