FI105319B - A process for preparing multicomponent alpha interferon - Google Patents

Description

105319 1.

METHOD FOR MULTI-COMPONENT ALFA-INTERFERON

This invention relates to the preparation of an alpha interferon drug from a human leukocyte or lympho-5 blastoid cell culture. In particular, the invention relates to the preparation of a highly purified and virus-safe solution of interferon alpha, wherein the solution contains the major natural α-interferon subtypes.

Interferons α (IFN-α) are important drugs with antiproliferative, antiviral and immunomodulatory effects. Human leukocytes are known to produce several IFN-α subtypes in culture induced by Sendai virus (Nyman et al., Biochem. J. 329, 295-302, 1998). Similarly, human lymphoblastoid cells produce several IFN-α subtypes after induction with Sendai virus (Zoon et al., J. Biol. Chem. 267, 15210-152216, 1992). Both leukocyte and lymphoblastoid IFN-α (hereinafter referred to as "multicomponent α-interferon" or "multicomponent α-interferon") are used as drugs in the treatment of neoplastic and viral diseases.

IFN-α drugs are also produced by recombinant DNA technology in bacteria. In contrast to leukocyte and lymphoblastoid IFN-α, the recombinant products contain only one IFN-20 or subtype, which is structurally different from the corresponding natural subtypes. Recombinant IFN-α may induce antibodies against interferons in patients, which may result in loss of therapeutic response. Multicomponent IFN-α products have returned a therapeutic response to these patients, and these products may also have other therapeutic advantages over combination products (Farrell, Hepatology 26, 96S-100S, 1997).

An important goal in purifying IFN-α from a leukocyte or lymphoblastoid cell culture is to include all major natural subtypes in the finished product. This must be done in a reproducible manner so that the product subtype composition is constant, which is t. a prerequisite for maintaining the quality of the drug. In addition, it is equally important for purification of IFN-α from 30 leukocyte or lymphoblastoid cell cultures that viruses · in the starting materials are inactivated and removed. The leukocytes and serum, or fractions thereof, used to produce leukocyte and lymphoblastoid interferon may contain viruses with different physico-chemical properties that cannot be effectively inactivated by a single viral inactivation treatment. Blood-derived viruses that may be present in serum-35 fractions and leukocytes include enveloped viruses such as HIV, hepatitis C and B viruses, as well as non-enveloped viruses resistant to many physicochemical treatments such as parvovirus. B19. Lymphoblastoid cell lines may contain e.g. retroviruses.

Several preparation methods have been developed using human leukocyte cultures 5 as starting material to obtain the multicomponent IFN-α drug.

U.S. Patent No. 5,391,713 describes a process for purifying leukocyte interferon. This comprises an immunoaffinity chromatography step using a polyclonal goat antibody against IFN-α. The purification process comprises the precipitation steps with thiocyanate solution and aqueous ethanol, but no other specific steps for viral inactivation or removal. The use of polyclonal antibodies in purification can be problematic because the specificity of the antibodies obtained at different times of immunization may be different, leading to changes in the subtype composition and impurity profile of the drug purified with different batches of antibody.

US Patent 5,503,828 relates to a purification process of leukocyte interferon comprising immunoaffinity chromatography with NK2 monoclonal antibody. The use of this monoclonal antibody results in a composition comprising at least 50% of IFN-α subtypes a2 and a8, and further subtypes of <x4, <x7, α10, a16, a17 and oc21.

However, during the purification process, at least the subtypes IFN-? 1 and IFN-? 4 disappear. Of these subtypes, IFN-? 1 is the most abundant natural subtype (Nyman et al., Biochem. J. 329,295-302,1998). The purification process includes treatment at low pH for at least 2 days, but no other specific steps of virus inactivation or removal.

It may be possible to obtain all major IFN-α subtypes by immunoaffinity chromatography with a polyclonal antibody, but in practice it seems impossible to obtain polyclonal antibodies that bind only IFN-α and no other related cytokines. Furthermore, it is not possible to maintain a continuous supply of polyclonal antibodies with identical binding specificity. Therefore, the use of polyclonal antibodies in purification does not ensure a constant subtype composition and impurity profile for the product over a long period of time. The use of a monoclonal antibody in immunoaffinity chromatography enables constant binding specificity in long-term production. However, at least in the case of NK2 antibody, the use of one monoclonal antibody resulted in the disappearance of the major IFN-α subtypes from the product.

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Current methods for purifying and producing leukocyte interferon do not include a number of specific steps of viral inactivation or removal with different modes of action and do not allow inactivation of viruses resistant to e.g. low pH or aqueous ethanol and thiocyanate.

The object of the present invention is to eliminate the problems associated with the prior art known in the art and to provide a method for producing a highly purified IFN-α solution from a leukocyte or lymphoblastoid cell culture. In particular, it is an object of the invention to provide a novel process for the production of a highly purified, virus-safe, multicomponent IFN-α-10 solution from crude leukocyte or lymphoblastoid interferon.

The foregoing and other objects of the invention, as well as its advantages over known methods, will become apparent from the following detailed description of the invention, and will be achieved by the invention, as described below, as set forth in claim 15.

According to the present invention there is provided a process for the preparation of multicomponent IFN-α comprising a plurality of virus removal steps. The invention is based on the finding that by performing immunoadsorption chromatography on at least two monoclonal antibodies having complementary subtype specificities to the protein 20 intermediate of the isolation process of IFN-α, it is possible not only to reproducibly discriminate all IFN-α major also effectively remove the most persistent infectious agents from IFN-α, including diapers. fungal viruses and physico-chemical resistant infectious agents such as infectious fungal encephalopathies ("prions").

More particularly, the invention is characterized by what is stated in the characterizing part of claim 1.

9 * 30 The invention provides considerable advantages. Thus, in a nutshell; By combining immunoadsorption chromatography with prior solvent / detergent treatment followed by low pH treatment and filtration with a virus removal filter, it is possible to obtain four effective virus removal steps, at least two of which eliminate all types of viruses and infectious agents. present in cell cultures. This treatment has the capacity to thoroughly purify all major, native IFN-α subtypes in good yield, and to continuously provide a product of constant subtype composition.

In the following, the invention will be examined in more detail by means of a detailed description and with reference to a few embodiments.

5

In the attached drawings,

Figure 1 shows, in the form of a flow chart, a preferred application for the preparation of a highly purified, virally safe, IFN-α drug containing all major IFN-α subtypes from this process, and Figure 2 shows a typical reverse phase HPLC profile of purified leukocyte from IFN-α.

The present invention provides a process for the preparation of highly purified multicomponent IFN-α. Starting with leukocytes, the process generally includes the steps of removing cells from the leukocyte culture, concentrating the crude interferon thus obtained, filtering the crude interferon concentrate, and subjecting it to a solvent / detergent treatment (also referred to as S / D treatment), IFN-α obtained by means of at least two monoclonal antibodies by immunoadsorption chromatography, eluted from the IFN-α column and maintained at low pH for at least 16 hours, neutralizing the eluate and separating the murine antibody residues by gel filtration, and finally making the purified drug browning and sterile filtration in a composition suitable for the finished product. Similar process steps are used to isolate interferon from human lymphoblastic cell culture.

25

The virus security of the product is ensured by using at least two effective virus removal steps for all viruses with different physico-chemical properties. First, S / D treatment effectively inactivates all enveloped viruses. Secondly, immunoadsorption chromatography and the accompanying washing steps remove all types of viruses. Third, low-pH incubation effectively inactivates most viruses except for some acid-resistant viruses. Ultimately, viral filtering removes all viruses, including even the smallest, non-enveloped viruses. In addition, other physico-chemical resistant infectious agents such as "prions" are also effectively removed by immunoadsorption chromatography and virus filtration steps. Thus, method 35 comprises at least two effective steps against all types of physicochemically resistant infectious agents, and up to four steps against the most important blood-borne viruses, HIV and hepatitis B and C viruses, which have a lipid envelope and which are effectively inactivated S / D treatment and low pH, and removed by immunoad-sorption chromatography and virus filtration.

A preferred embodiment of the production process is shown in Figure 1.

Referring to Figure 1, it can be seen that the first step of the preferred embodiment comprises the production of crude interferon in a leukocyte culture, in particular by Sendai virus induction (Cantell et al., Methods Enzymol. 78, 29-38, 1981). The starting material may also be crude interferon derived from human lymphoblastoid cell culture by Sendai virus induction (Mizrahi et al., Methods Enzymol. 78, 54-66, 1981).

The cells are removed from the culture by decantation, microfiltration and / or centrifugation. The filtrate or supernatant is concentrated by ultrafiltration to obtain a crude IFN-15 concentrate, filtered and treated with S / D reagents to degrade the lipid enveloped viruses. S / D treatments are described in the prior art, particularly in U.S. Patents 4,540,573,4,764,369 and 4,820,805, which are incorporated herein by reference. The organic solvent is preferably selected from dialkyl phosphates and trialkyl phosphates having from 1 to 10 carbon atoms in alkyl groups. Examples of trialkyl phosphates include tri- (n-butyl) -20 phosphate, tri- (t-butyl) phosphate, tri- (n-hexyl) phosphate and tri- (2-ethylhexyl) phosphate. The concentration of the solvent is in the range of 0.01 g / l to 100 g / l, preferably from about 0.1 g / l to about 10 g / l, a typical concentration of tri- (n-butyl) phosphate is about 0.3 %. The solvent may be used in combination with a non-toxic detergent which is capable of increasing the solvent te-ν 'potency. Examples of such agents are nonionic surfactants, e.g.

Oxyethylated alkylphenols, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alcohols and sodium deoxycholate. The amount of detergent is in the range of 0.001 to 10%, preferably about 0.01 to 1.5%. Typically, the detergent comprises 1% polysorbate 80 (Tween 80), 1% Triton X-100 or 0.2% sodium deoxycholate. S / D agents may be used,. even higher concentrations such as 2% tri (n-butyl) phosphate without detergent.

The treatment time may be from 4 to 50 hours and the temperature from 24 to 30 ° C.

Protein impurities, DNA, potential viruses, and S / D chemicals are removed by binding IFN-α to an immunoadsorption comprising monoclonal antibodies linked to a solid matrix. To obtain all major IFN-α subtypes, at least two monoclonal antibodies with complementary subtype specificities should be used.

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Monoclonal antibodies can be generated using purified leukocyte interferon as an immunogen. Antibody-producing cell clones are selected by testing the ability of the antibodies to bind leukocyte interferon by immunoadsorption (Example 1). Monoclonal antibodies should collectively bind at least 90% of the interferon activity in crude interferon, and the recovery of low pH eluted interferon activity should be at least 80% of the bound activity. Antibodies should have complementary binding specificities for IFN-α subtypes such that they bind together all major IFN-α subtypes. In particular, antibodies should bind at least IFN-α subtypes a1, a2, a8, a10, a14, a17, and <x21. It is preferred that at least one antibody binds subtype a1 and one binds subtype a2.

The immunoadsorption column is washed thoroughly and the bound IFN-α is eluted with a low pH buffer (pH 2 to 2.5). The elution buffer may contain albumin or other stabilizers, such as a nonionic detergent, to stabilize IFN-α.η. The eluate is incubated at low pH for at least 16 hours to inactivate any remaining viruses. The eluate is neutralized by the addition of basic buffer or sodium hydroxide. The neutralized eluate is concentrated by ultrafiltration. Alternatively, IFN-α may be bound to an ion exchange chromatography gel.

Mouse IgG and fragments thereof that leak from the immunoadsorbent, and possibly albumin as a stabilizer, are removed by gel filtration or ion exchange chromatography. Fractions containing IFN-α are pooled and the purified IFN-α drug thus obtained is stored frozen at -70 ° C.

The IFN-α drug is stabilized with a suitable stabilizer, such as albumin or deionized detergent, and filtered with a virus removal filter having a pore size of 10 to 20 nm to remove even the smallest non-enveloped viruses. The yield of IFN-α from virus filtration and filter capacity is significantly higher when a nonionic detergent such as polysorbate 80 is used as a stabilizer instead of albumin, as described in Example 4. If desired, two virus removal filters can be used sequentially. Virus filtration may also be performed after sterile filtration of the formulated bulk solution. Other stabilizers may be added to the bulk solution after virus filtration.

It is also possible to formulate the IFN-α drug in a suitable pharmaceutical composition prior to the viral filtration step, as shown in Figure 1.

105319 The method of the present invention efficiently removes all types of impurities in leukocyte and lymphoblastoid cell cultures, as evidenced by more than a million fold removal of DNA and about one million fold removal of ovalbumin, the major heterologous protein in crude interferon (Example 2). The level of murine IgG leaking from Immunoad-5 sorbent is typically less than 10 ng / 3 milliliters. IU of purified IFN-α.

This method is economical because it involves only two chromatographic steps and gives a good yield of the purified drug with an average of about 60% (calculated from the immunochemical concentration of IFN-α) as shown in Example 2.

By this method it is possible to obtain a constant subtype composition of IFN-α as demonstrated in Example 3 by comparing the subtype composition of 13 different batches of drugs. The use of monoclonal antibodies from different batches of antibodies results in a very similar IFN-α subtype composition, which illustrates the capacity of the method to provide a product of consistent quality over long term production.

The following examples illustrate the invention without limiting it: Analytical Methods Used in the Examples IFN-α Concentration IFN-α concentration was measured by time-resolved fluoroimmunoassay (FIA) on microtitre plates. An IgG fraction of bovine antiserum to human leukocyte IFN-? Was used to bind IFN-? And a mixture of two mouse Eu-labeled monoclonal IgG antibodies to IFN- ?. The monoclonal antibodies were the same as those used for purification of IFN-α. Details of the assay are described elsewhere (Rönn-Blom et al., APMIS 105, 531-536,1997). The concentration of IFN-α was expressed as IU / ml. . a laboratory standard calibrated by viral plaque reduction assay against? 30 international reference product of interferon (feron, Human Leukocyte 69/19 (NIBCS, U.K.)).

Interferon Antiviral Activity The antiviral activity of IFN was determined by a reduction of viral plaques in 35 mm 35 petri dishes using human epithelial cells (Human Epithelial 2 (HEp2)) exposed to vesicular stomatitis virus (VSV). IFN-α samples, control and standard 8 105319 were diluted at 0.25 log increments to 0.3-3 IU / ml in Eagle's Minimum Essential Medium (EMEM) supplemented with Calf Fetal Serum (FCS), 7%, and aureomycin, 0.004%. Samples were analyzed in triplicate at four dilutions in at least two sets of assays. Plates were supplemented with 1 ml of cell suspension (2 x 10 6 cells / ml) in EMEM and 1 ml of sample dilution. Virus control plates without IFN were included in each set of assays. After overnight incubation at 37 ° C in a 3-4% CO 2 atmosphere, solutions were removed from the confluent cell layers and 150-200 PFU of VSI in 1 ml of EMEM was added. After incubation for 40-45 minutes, the virus was removed and the cells were covered with 2 ml of 0.8% agar in EMEM. After overnight incubation, 10 viral plaques were counted. One unit of IFN activity is the highest dilution of the sample that inhibits 50% of the viral plaques compared to the viral control. Interferon activity was expressed in International Units (IU) using a laboratory standard calibrated against the Interferon International Reference Preparation (Interferon, Human Leukocyte 69/19 (NIBCS, United Kingdom)).

15

total protein

Total protein concentration was determined by the Lowry method using human albumin as a standard (Total Protein Standard, Finnish Red Cross, Blood Service, Helsinki, Finland).

20

Mouse IEG

Mouse IgG concentration was determined by enzyme-linked immunoassay (EIA) on microtiter plates. Polyclonal goat anti-mouse IgG, (Fc) F (ab ') 2 fragment (Jackson Immuno: Research Laboratories, USA) was used for IgG binding and polyclonal peroxidase-conjugated goat anti-mouse IgG (H + L) (Jackson Immuno Research Laboratories, USA). Mouse Myeloma IgG1 (Cappel, Belgium) was used as a standard.

ovalbumin

Ovalbumin concentration was measured by time-resolved fluoro-immunoassay (FIA) on microplates of rotary plates. Polyclonal rabbit IgG against egg albumin (Organon Teknika Co., USA) was used to bind albumin and polyclonal Eu-labeled goat IgG to egg albumin (Organon Teknika Co., USA). Egg albumin (Sigma, USA) was used as a standard. Fluorescence was measured with a time-resolved fluorometer (1234 Delfia Research Fluorometer, Wallac, Finland).

: 35 105319 9 '

Total DNA

Samples were treated with sodium dodecyl sulfate and proteinase K at pH 11 and the DNA concentration was determined with the Treshold Total DNA Kit (Molecular Devices, USA).

5 IFN-a Subtype Composition?

The IFN-α subtypes were separated by reverse phase high performance liquid chromatography (RP-HPLC). A sample of IFN-α (about 1 mL IU in 400 μΐ) was applied to a Delta-Pak ™ HP1 C4 column (Waters, USA) and eluted with a linear gradient of 40-55% acetonitrile in 0.1% v / v ) in trifluoroacetic acid for 57 min at a flow rate of 0.1 ml / min. The absorbance was measured at 10,214 nm. IFN-α samples containing human albumin were first run on Superdex 75 HR

10-30 column (Pharmacia Biotech, Uppsala, Sweden) in 50 mM Tris pH 7.5 containing 0.15 mol / L NaCl to remove albumin from IFN-α.

To identify IFN-α subtypes from RP-HPLC peaks, the fractions were subjected to N-15 terminal end sequencing and peptide mass mapping, either directly or after further separation by SDS-PAGE, according to procedures detailed elsewhere (Nyman et al., Biochem. J. 329, 295-302, 1998).

Examples 20

Example 1

Production of Monoclonal Antibodies Against IFN-α This example describes the criteria for the preparation and selection of the monoclonal antibodies used in the immunoadsorption step of method o. IFN-α used for immunization was partially purified leukocyte interferon (Finnferon-alpha, Finnish Red Cross, Blood Service). It was further purified by immunoadsorption using bovine polyclonal anti-IFN-? Antibodies coupled to CN-Br-Sepharose (Pharmacia Biotech, Sweden). Bound IFN-α was eluted at low pH and stabilized with sodium dodecylisulfate (SDS). For immunization, female Balb / c mice were given a single intraperitoneal injection of 5 milliliters. IU IFN-α in 0.25 ml complete Freund's adjuvant, followed by 4 injections of 5-10 ml IU without adjuvant approximately every 14 days. Spleen cells from immunized mice were prepared for fusion on day 4 after the last injection. The fusion partner cell line was the non-secreting murine myeloma cell line Sp2 / 0-Agl4; 35 (ATCC CRL 1581), and thymocytes prepared from Lewis rats were used as feeder cells. The fuser was polyethylene glycol 1500 and the medium was DMEM supplemented with HT 10 and 10% FCS.

Following fusion, cells were subcloned and tested for the production of murine monoclonal antibodies against human IFN-α by enzyme immunoassay using purified human leukocyte IFN-α to bind IFN-α. In the next screening step, monoclonal antibodies which were strongly positive were selected for further testing. In the primary screening for binding, monoclonal antibodies were incubated with human leukocyte interferon. Rabbit anti-mouse IgG antibodies coupled to Sepharose 4B gel (Pharmacia, Biotech) were added, and after incubation, the gel containing the murine monoclonal antibodies and IFN-α was separated by centrifugation. The supernatant was assayed for interferon activity. Antibodies that bound at least 70% of the interferon activity were selected for further testing in a combination of two different antibodies, and antibodies that bound at least 90% of the activity as a mixture were considered suitable candidates for the immunoadsorption chromatography step. Further testing was performed by coupling the monoclonal antibodies to CNBr-Sepharose 6B (Pharmacia, Biotech) and testing their binding properties by immunoadsorption. The selection criterion was that the two monoclonal antibodies together should bind at least 90% of the interferon activity present in the crude leukocyte interferon and that the recovery of low pH eluted interferon activity should be at least 80% of the bound activity. The two murine monoclonal IgG antibodies selected for the purification process bind together all major leukocyte IFN-? Subtypes (IFN-α1, IFN-α2, IFN-α8, IFN-α10, IFN-α14, IFN). -17, and IFN-a21). Neither of them binds interferon beta or omega. Figure 2 shows a typical RP-HPLC chromatogram of a product purified using these two monoclonal antibodies for immunoadsorption.

Example 2 Purification of IFN-α drug from crude interferon This example was carried out by following the steps from crude interferon to purified drug according to the formula shown in Figure 1. The cells were first removed from the leukocyte culture by decanting and microfiltration using 0.3 µm membranes. The filtrate was concentrated 20 times by ultrafiltration using 10 kD membranes. If not used immediately, the concentrate was stored frozen at -40 ° C. After thawing,? 35 concentrate was filtered through 1.2 µm and 0.22 µm filters, and the clarified concentrate was treated with 0.3% tri (n-butyl) phosphate and 1% polysorbate 80 for 16 hours at 26 ° C.

After 105319 S / D treatment, the concentrate was loaded onto an immunoadsorption column containing two monoclonal antibodies (4 mg / tnl each) coupled to a CNBr-Sepharose 4FF gel (Pharmacia Biotech). The load was 30-40 mill. IU IFN-a per ml gel 5. The immunoadsorption column was washed with 40 yellow volumes of 0.011 mol / L sodium phosphate buffer, pH 7, 0.14 mol / L NaCl (PBS) containing 0.1% polysorbate 80, followed by 10 volumes of PBS without polysorbate 80 . Bound IFN-α was eluted with pH 2 buffer. To stabilize IFN-α, 0.2 g / L human albumin was added to the pH 2 buffer (Finnish Red Cross, Blood Service). After incubation for 18-20 hours at pH 2, the eluate 10 was neutralized by the slow addition of 0.5 M NaOH and concentrated approximately 30-fold by ultrafiltration using 10 kD membranes.

The concentrated eluate was applied to a Superdex 75 column (Pharmacia Biotech) equilibrated in PBS. Fractions containing IFN-α were pooled and stored frozen at -70 to 15 ° C. The yield of IFN-α from 11 purification batches is shown in Table 1.

Table 1. Recovery of IFN-α from the purification process; presented are the mean values calculated from FIA results for IFN-α 11 purification batches 20

Process Step Yield Cumulative.

per step yield _% _% _ 25 crude interferon 100 100

Interferon Crude Concentrate 95 95 S / D Treated Concentrate 98 93

Immunoadsorption eluate 80 74

Neutralized and Concentrated eluate 89 66 30 Purified drug_91_60_ »No other protein constituents other than IFN-α subtypes can be detected in the purified" 35 drug with silver staining of SDS-PAGE gels. sensitive immunochemical assays for the determination of heterologous protein impurities, such as ovalbumin and mouse IgG, have been performed with batches of Oval-Bumin at a standard dose of 3 mg IU IFN-α from 0.1 to 0.3 ng and 40 g of mouse IgG. The clearance of 1-9 ng of IFN-α has been about one million times that of ovalbumin 12 (Table 2).

The amount of impurity from the main process, tri (t-butyl) phosphate, has been less than 0.2 pg per 3 mill. IU IFN-α in the three batches of drugs analyzed. The amount of DNA was measured from two batches of drug and was found to be less than 60 pg per 3 milliliter. IU IFN-aa. The clearance of IFN-α was more than a million-fold relative to DNA (Table 2).

10 Table 2 Purification of IFN - <x relative to ovalbumin and DNA in the purification process

Method Step Ovalbumin IFN-a DNA Purity IFN-a / 3 milliliters. IU purification pg / 3 mill. IU secretion of IFN-α relative to DNA in relation to ovalbumin

Crude IFN 1.8 x 105 1.1 x 10® Concentrate

Purified Drug 0.2 0.9 x 10 6 <60> 2x106 Substance Example 3 IFN-α Subtype Composition in Different Batches: Figure 2 shows a typical RP-HPLC chromatogram of the purified drug obtained using the procedure described in Example 2. . The RP-HPLC chromatogram was divided into seven peak groups, as indicated by the vertical lines in Figure 2. Comparison of the 11 batches of the drug shows that the relative areas of the peaks, and hence the subtype composition, remained very similar across batches (Table 3).

t », 3,105,319

Table 3. IFN-α subtype composition in 11 aliquots. The proportion of the different subtypes analyzed by RP-HPLC is shown RP-HPLC IFN-α subtype Mean RSD Range Peak (%) (%) (%) Group 1 IFN-? 4 7.2 15.8 6- 10 2 IFN-? cc2 19.7 4.7 18-22 3 IFN-a21, with IFN-a4 9.9 12.4 8- 11 4 IFN-aO 8.7 7.7 7- 10 5 IFN-a17, with IFN- a7 15.6 10.9 13-18 6 IFN-a 8 6.7 13.1 5-8 7 IFN-a 1 32.0 7.8 28 - 35 5

When different batches of two monoclonal antibodies were used to prepare the immunoadsorbent, both immunoadsorbents produced a very similar IFN-α subtype composition (Table 4).

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Table 4. IFN-α Subtype Composition in Batches Purified with Immunoadsorbents with Monoclonal Antibodies from Two Batches of Antibody,. RP-HPLC IFN-α lots (n = 2), purified IFN-α lots (n = 11), purified; peak group with antibodies from first batches of second batches Relative peak area (%)

Peak Relative Area (%) 1 2 3 4 5 6 7: 15 7.9 7.2 2 20.6 19.8 3 10.7 9.9 • l 4 8.2 8.7 5 14.2 15, 7 6 6.1 6.7 7 32.4 32.0 h 105319

Example 4

Preparation of Purified Ready IFN-α This example was carried out by formulating the purified drug and subjecting it to virus-5 filtration and sterile filtration according to the formula shown in Figure 1. Purified IFN-α drug was diluted to a concentration of about 4 milliliters. IU / ml in PBS containing 1 mg / ml albumin. The formulated solution was pre-filtered with a 0.1 µm particle filter followed by a Planova 15N filter (Asahi Chemical Industry Co., Japan). Planova filtration was carried out at room temperature using a constant pressure (0.9 bar). The IFN-α yield from Planova-10 filtration was about 90%.

The improved yield (about 100%) of IFN-α and the multiple filtration capacity was achieved in the viral filtration step when albumin was replaced by a nonionic detergent such as polysorbate 80, 0.2 g / L in a purified drug containing composition.

15

The virus-filtered IFN-α solution was filtered through a 0.1 or 0.22 µm sterile filter and aseptically dispensed into the final product vials.

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Claims (14)

A process for preparing a purified and virus-safe α-interferon product of raw leucocyte or lymphoblastoid interferon, characterized by (a) the pure interferon product is treated with solvent or detergent, (b) the composition treated with solvent or detergent is brought into contact with monoclonal Ig antibodies from a mouse in an immunoadsorption phase for cleaning substantially all of the major, natural subtypes of α-interferon, c) the α-interferon's bound to the monoclonal antibody-bound subtypes, d) the rest of the mouse IgG, and the other contaminants are removed from the eluent. preparing a purified α-interferon solution; e) the solution is stabilized and filtered through a virus removal filter; and f) the final composition of the product is controlled to produce an aseptic filling. Process according to claim 1, characterized in that the solvent reagents are di- or tri- (alkyl) phosphate and that it is used alternatively in combination with ion-free detergent. 20 3. A method according to claim 1 or 2, characterized in that the pure interferon product subjected to solvent or detergent treatment comprises a supernatant obtained from 10 ... 40-fold concentrated culture of leucocyte or lymphoblastoid. 25 Method according to any one of claims 1-3, characterized in that IFN-α in the b phase is bound to an immunoadsorbent containing at least two monoclonal antibodies with complementary subtype specifications bound to a solid matrix. 30 Process according to claim 4, characterized in that the immunoadsorbent binds at least 90% of the activity of the relay leukocyte interferon and that the yield of interferon activity eluted with low pH is at least 80% of the bound activity. 6. The method of claim 5, characterized by deprotecting the monoclonal antibodies form at least the subtypes a1, a2, a8, alO, al4, al7 and a21 of IFN-α. 5 Process according to any of the preceding claims, characterized in that the immunoadsorbent pillar is thoroughly cleaned after the b phase and that the bound α-interferon is eluted with a buffer with a pH less than 3.0. 10 Method according to claim 7, characterized in that the elution buffer used contains a α-interferon stabilizer. 9. A method according to any of the preceding claims, characterized by the eluate being incubated at low pH for 1-70 hours. 15 The process according to claim 7, characterized by the eluate being neutralized and concentrated 20 times 40 times. Process according to any of the preceding claims, characterized in that the d-phase is carried out by gel filtration or ion exchange chromatography. Process according to claim 11, characterized in that the α-interferon-containing fractions are combined and the obtained pure α-interferon drug is stored at a temperature of -70 ° C. 25 Method according to any of the preceding claims, characterized in that the α-9: interferon drug is formulated into a suitable pharmaceutical composition and filtered through a filter for removing viruses with a pore size of 10-20 nm, to remove small uncoated viruses and other infectious agents which have not been inactivated during the treatment with solvent or detergent and at a low pH, or which have not been removed by immunoadsorption chromatography. 105319 A method according to any of the preceding claims, characterized in that the process from and with the leukocytes comprises the following steps: - the cells from a leukocyte culture are removed; - The α-interferon is adsorbed by immunoadsorption chromatography with at least two monoclonal antibodies with complementary subgroup specifications. The eluate obtained from the immunoadsorption chromatography is subjected to treatment at a pH of 2- 2.5, the eluate is neutralized and concentrated, the final composition of the product is controlled and sterile filtered and filled aseptically.