FI106465B - A process for preparing virus-safe pharmaceutical compositions - Google Patents

Description

, 106465

METHOD FOR MANUFACTURING VIRUS-SAFE PHARMACEUTICAL COMPOSITIONS

This invention relates to the preparation of virus-safe pharmaceutical compositions from biologically active proteins. In particular, the invention relates to a process for the preparation of a virus-safe liquid composition of interferon α, preferably multicomponent α interferon, which has a long storage stability. The present invention also relates to the use of nonionic detergents as stabilizers for pharmaceutical compositions, and to non-viral multicomponent α-interferon solutions which can be used for injection for the treatment of diseases.

The pharmaceutical compositions of the biologically active proteins must be virus-safe, i.e., free from contaminating, potentially pathogenic viruses and other infectious agents. In addition, such pharmaceutical compositions should have a long shelf life which allows their long-term use. The following is an overview of the viral safety and shelf life problems of protein-containing pharmaceutical compositions, with particular reference to interferon compositions. However, the principles can be generally applied to physiologically active substances derived from human, animal, blood, urine or viscera and the corresponding recombinant proteins produced in cultured animal cells or transgenic animals.

• Human alpha interferons (IFN-α) are a family of closely related proteins with anti-proliferative, antiviral and immunomodulatory effects. Human leukocytes and lymphoblastoid cells are known to produce several IFN-α subtypes in culture induced by Sendai virus (Cantell et al., Methods Enzymol. 78, 29-38, 1981, Mizrani, Methods Enzymol. 78, 54-68, 1981). Purified multicomponent IFN-α drugs are used in the treatment of a variety of diseases including neoplastic and viral diseases. Multicomponent IFN-α drugs have been shown in the art to have therapeutic advantages over bacterial recombinant IFN-α drugs containing only one IFN-α subtype.

Commercial leukocyte or lymphoblastoid cells are cultured in the commercial production of human multicomponent IFN-α and are induced by Sendai virus. These products therefore carry a risk of viral contamination. Blood-derived viruses that may be present in 2,106,665 in leukocytes and serum or their fractions used in culture media include HIV, hepatitis C and B viruses, and small non-enveloped viruses such as parvovirus B19, which is resistant to many physico-chemical treatments . Lymphoblast food cell lines may contain, for example, retroviruses.

5

An effective method for the removal of viruses having various physico-chemical properties is filtration on membranes having a pore size of 10 to 40 nm; the procedure is also known as nanofiltration or viral filtration. A particular advantage of filtration is that it also removes viruses that are non-enveloped and other infectious agents 10 such as infectious spongiform encephalopathies ("prions") that are resistant to conventional heat and chemical-resistant treatments (physico-chemical antibodies).

Stabilizing agents are added to solutions containing purified IFN-α to prevent binding of IFN-α proteins to filters, end product bottles and other surfaces. In addition to the short-term stabilizing effect described above, the stabilizers also inhibit protein aggregation and thus provide extended storage stability. Albumin is the most commonly used stabilizer in multi-component IFN-α products and is used in many commercial formulations (Alfanative®, Alferon® N, Wellferon®).

20

However, the use of albumin as a stabilizer for IFN-α products can cause at least two problems. First, albumin has been reported to lead to albumin IFN aggregates in products and these may be antigenic and lead to IFN-. a (Braun et al., Pharm. Res. 14, 1472-1478, 1997). These problems apply to recombinant products. A working and important consideration with regard to the preparation of virus-safe compositions is that if the formulated IFN-α solution is filtered with a virus-removing filter, as is the case for multi-component leukocyte or multi-component lymphoblastoid IFN-α compositions, the use of albumin as a stabilizer decreases the ability of the filter to remove viruses, since it has been shown that the activity of the virus-removing filter is reduced as the concentration of protein present increases (Hirasaki et al., Mem brane 20,135-142,1995). This is apparently due to protein clogging of the filter, which is reflected in the reduced filtration rate when the pressure is kept constant. As shown in Example 2 below, the filtration level was reduced by about 80% after filtration of 20 l / m 2 of well purified IFN-α solution containing 1 g / l albumin.

35

It is known in the art that certain proteins, in particular human growth hormone, can be prevented from adsorbing on a membrane filter by pretreating the filter with human serum albumin or polyvinylpyrrolidone, polyoxyethylene sorbitan mono laurate, gel polysorbate 80 or 11, polysorbate 80. In this known pretreatment, albumin or any of the above agents is adsorbed on the filter by filtration, impregnation or soaking in an aqueous solution.

Although said treatment may have some beneficial effect on the filtration level, it constitutes an additional, costly step. In addition, coating the filter with al-10 bumin does not reduce the adsorption of proteins on other surfaces that are in contact with the product, such as tubes, collecting containers, vials and caps.

The object of the present invention is to overcome the problems previously encountered in the art and to provide a novel method for the preparation of virally safe pharmaceutical compositions of biologically active proteins.

Another object of the present invention is to provide a novel liquid composition of multicomponent IFN-α that does not contain IFN-α polymers or albumin-IFN complexes, and which has the property of prolonged storage stability and can be used for injection.

A third object of the invention is to provide a new use of nonionic detergents as stabilizers in liquid compositions of IFN-α that can be filtered by virus removal filters, and provide improved yield and capacity, and can be used for injection.

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 and as set forth in claims T '' 30.

The present invention is based on the discovery that by using a nonionic detergent as a stabilizer for pharmaceutical compositions containing biologically active proteins, and adding said stabilizer to the composition prior to viral filtration, viral filtration yield and capacity can be greatly increased. This finding was surprising as it is known that nonionic surfactants such as polysorbate 80 have very low critical micelle concentrations (CMC). Thus, for example, the CMC of polysorbate 80 is about 0.013 g / L in aqueous solutions (Helenius and Simons, Biochim. Biophys. Acta 415.29-79, 1975). Above the CMC, nonionic surfactants form micelles of various sizes which penetrate very slowly e.g. into dialysis membranes.

In accordance with the present invention, nonionic detergents (surfactants) are added to pharmaceutical compositions at concentrations above the CMC prior to virus removal filtration to provide stabilized proteinaceous compositions, e.g., multicomponent IFN-α compositions substantially free of ingredients. (including viruses and prions) having a size greater than 10-40 nm, in particular 10-20 nm, which are normally retained by a viral filter of this class.

More specifically, the process for the preparation of virus-safe pharmaceutical compositions of biologically active proteins of the present invention is characterized in what is stated in the characterizing part of claim 1.

The multicomponent IFN-α compositions of this invention, in turn, are characterized by what is stated in the characterizing part of claim 11.

20

The invention provides considerable advantages. Thus, according to the present invention, the stability of a multicomponent IFN-α solution stabilized with a nonionic detergent is improved. In addition, multicomponent IFN-α compositions stabilized with a non-ionic detergent do not contain albumin IFN complexes formed in albumin-containing compositions and are believed to be harmful in combination products. By replacing albumin with a nonionic detergent as a stabilizer, the IFN-α solution can be filtered with a virus removal filter without clogging the filter. In other words, by replacing albumin with a nonionic detergent, it is possible to filter the IFN-α solution with a virus removal filter with improved yield and capacity. Compared to the US patent • <? In addition to the known process, the present invention not only increases filtrate yield, but also prevents losses caused by adsorption of protein from the filtrate to other surfaces in contact with the product, such as tubes, collecting containers, vials and caps. By incorporating a nonionic detergent into the composition prior to filtration, it is not necessary to pre-treat the filter. In fact, the test has shown that such pre-treatment does not improve the yield to any appreciable extent.

5, 106465

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

In the accompanying drawings, Figure 1 shows adsorption of IFN-α on a glass surface in the presence of various stabilizers; Figure 2 shows the presence of albumin-IFN complexes in solutions stabilized with albumin and the absence of aggregates in solutions stabilized with polysorbate 80;

Figure 3 shows the viral filtration flow rates of polysorbate 80 and purified IFN-α solutions stabilized with albumin, respectively.

According to the present invention, the nonionic detergent is added to a solution of purified, biologically active protein, which is then filtered through a virus removal filter having a pore size of about 10-40 nm and then optionally sterile filtered to provide a virus safe, sterile and stable protein solution.

The spectrum of biologically active proteins within the scope of this invention extends to all therapeutically used proteins, which may contain viruses and are filtered by a virus removal filter. Such proteins include coagulation factors and their activated forms (eg factor IX, factor VII), other proteinases, their activated forms and protein inhibitors (eg protein C), growth factors and colony stimulating factors (eg IGF-1, G). -CSF, GM-CSF), neurotrophic factors (eg NGF, GDNF, NT-3), hormones (eg erythropoietin, growth hormone) and other proteins that modify the cellular biological response (eg, interferons and interleukins). In addition to naturally occurring proteins, there are also recombinant proteins produced in cultured animal cells or transgenic animals.

The use of nonionic detergents in various pharmaceutical compositions is known per se. It is also known in the art that polysorbate 80 can be used as a stabilizer for recombinant IFN-α2a instead of albumin to prevent the formation of "albumin-IFN aggregates" (Hochuli, J. Interferon Cytocine Res.

Suppl. 1, SI5 - S21,1997). Liquid interferon alpha and y compositions containing nonionic detergents are also described in European Patent Application 0 736 303 A2 and WO 89/04177. However, all references to the inclusion of non-ionic detergent in the pharmaceutical composition prior to virus filtration are completely silent.

106465 In accordance with a preferred embodiment of the present invention, nonionic detergents are used as stabilizers in multicomponent IFN-α compositions that are subjected to viral filtration to remove any substances retained on filters having a pore size of 10 to 40 nm. These compositions include purified leukocyte and lymphoblastoid-5 interferons containing two or more of the following IFN-α subtypes: α1, α2, α4, α7, α8, α10, α4, α7 and α21. Human leukocyte interferon has been shown to contain at least nine IFN-α subtypes (Nyman et al., Biochem. J. 329,295-302, 1998), and lymphoblastoid interferon contains the same or similar subtypes (Zoon et al., J. Biol.) Chem. 267, 15210-15216, 1992). Some of the subtypes secreted by producer cells may be lost during purification, depending on the purification process used (U.S. Patent No. 5,503,828).

Methods for producing multicomponent IFN-α have been described in detail previously. Multicomponent IFN-α can be produced in leukocyte or lymphoblastoid isolates by Sendai virus induction. A process for producing a highly purified drug may include precipitation, filtration and chromatography steps. Methods of purifying multicomponent IFN-α using monoclonal or polyclonal antibodies have also been described. The production process may include additional viral inactivation steps such as low pH treatment and solvent / detergent treatment. IFN-α composition 20 and methods of producing it from human peripheral blood leukocytes are described, e.g., in U.S. Patents 5,503,828 and 5,391,713; these publications are incorporated herein by reference.

The purification process yielding all major IFN-α subtypes is described in Example 2. Generally, it comprises, e.g., the step of contacting a solvent / detergent-treated composition with at least two murine monoclonal IgG antibodies, with complementary subtype specificities. The α-interferon subtypes that bind to the monoclonal antibodies are eluted and the eluate is purified and filtered on a virus removal filter.

Other pharmaceutically useful proteins that can be subjected to virus removal filtration may be produced by methods known per se, for example, isolation from human or animal blood, or recombinant cells in cultured cells or transgenic animals.

The protein solution formulated in accordance with the present invention is prepared by diluting a calculated amount of purified, biologically active protein with formulation buffer containing 7,106,465 polysorbate 80 or other nonionic detergent in an amount to give a final concentration of 0.05 to 1 g / L, preferably ca. 0.1-0.5 g / l nonionic detergent. Preferably, the protein has a purity level of at least about 90%. The formulated solution may be pre-filtered with a 0.04 to 0.2 µm filter and then filtered with a virus removal filter having a pore-5 size preferably between 10 and 40 nm. The non-ionic detergent does not cause the filter to become clogged, and depending on the molecular size of the protein, filtration can be carried out at constant pressure without loss of filtration flow, and thus with higher capacity and constant virus removal. If desired, two virus filters can be used sequentially.

The recovered filtrate is filtered through a sterile filter and placed in vials, syringes or other containers suitable for parenteral injection. It is also possible to perform virus filtration and sterile filtration in reverse order.

Virus removal filters (nanofilters) include filters suitable for removing viruses from pharmaceutical protein solutions. The size of the filter pores or perforations should be small enough to effectively remove even small, non-enveloped viruses such as parvoviruses less than 25 nm in size. Pore sizes in the range of 10-40 nm, especially 10-20 nm, are preferred.

The buffer of the liquid composition is less essential and may be an inorganic buffer or an organic buffer. The pH of the buffer may be in the range of 4.5 to 7.5, and the buffer may contain other substances, e.g. inorganic salts, sugars, amino acids, polyols or cyclodextrins. Other stabilizers may be added to the IFN-α solution after the viral filtration step.

25

The activity of the IFN-α solution to be filtered by the virus removal filter may be close to the final product or may be significantly higher. In the latter case, the solution is diluted after virus filtration. The activity of IFN-α.η in the final product is selected based on a number of variables including the disease to be treated, the treatment plan, and the delivery system. In general, the activity of the IFN-α solution prior to virus filtration is in the range of 3 to 50 milliliters. IU / ml.

Examples of nonionic detergents that can be used as stabilizers include polyoxyethylene-based detergents such as polyoxyethylene sorbitan monooleate: 35 (polysorbate 80), polyoxyethylene sorbitan monolaurate (polysorbate 20), polyoxyethylene lauryl ethyl, 10-laoxyethylene,

O

(poloxamer 188). A polysorbate such as polysorbate 80 is most preferred. Polysorbate 80, as with other nonionic detergents, is used at concentrations above the critical micelle concentration, typically about 0.05 -1 g / L for polysorbate 80. A preferred range is from 0.1 to 0.5 g / L, and the most preferred concentration is about 0.2 g / L.

5

In a preferred embodiment, the nonionic detergent used has a low peroxide number to prevent any adverse oxidation reactions in the pharmaceutical compositions. Preferably, the peroxide number is less than 5.0 mEq / kg, tested at Ph. Eur. according to 1997. Optionally, an antioxidant may be added to the composition to prevent oxidation of IFN-α.

The following non-limiting examples illustrate the invention: IFN-α concentration IFN-α concentration was measured by time-resolved fluoro-immunoassay (FIA) on microtiter plates. An IgG fraction of bovine antiserum to human leukocyte IFN-α was used for capture and a mixture of two murine Eu-labeled monoclonal IgG antibodies to IFN-α was used for detection. The monoclonal antibodies were the same as those used for purification of IFN-α (Example 1). Details of the assay are described elsewhere (Rönnblom et al., APMIS 105, 531-536,1997). IFN-α concentration was expressed in IU / ml using a laboratory standard calibrated by viral plaque reduction assay against the International Reference Preparation of Interferon, Human Leukocyte 69/19 (NIBCS, United Kingdom).

25

Interferon Antiviral Activity The antiviral activity of IFN was determined by a plaque reduction assay in 35 mm petri dishes using human epithelial cells (Human Epithelial 2 (HEp2)) exposed to vesicular stomatitis virus (VSV). IFN-α samples, control and standard dilutions were diluted to 0.25 log increments of 0.3 to 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 for four dilutions and at least two series of assays. To the plates were added 1 ml of cell suspension (2 x 10 6 cells / ml) in EMEM and 1 ml of sample dilution. For each assay time: 35 virus control plates without IFN were included. After overnight incubation at 37 ° C in a 3-4% CO 2 atmosphere, the solutions were removed from the confluent cell layers and 150 to 200 PFU VSV in 1 ml 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. Viral plaques were counted after overnight incubation. 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 5 was expressed in International Units (IU) using a laboratory standard calibrated against the international reference preparation of interferon, Human Leukocyte 69/19 (NIBCS, United Kingdom).

10 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).

15 Western blots

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the Laemmli method using 15% gels. Proteins were electroblotted onto a nitrocellulose membrane, blocked with 0.5% Tween 20, and washed with 0.05% Tween 20 in 0.011 M sodium phosphate buffer, pH 7.0, 0.14 M NaCl (PBS). . The membrane was incubated with bovine polyclonal IgG against IFN-α (Wellcome Research Laboratories), 4 pg / ml in PBS containing 0.05% Tween 20 and 0.1% human albumin for 2 hours at room temperature. The membrane was washed and incubated with peroxidase-conjugated rabbit anti-bovine IgG (Jackson Immunoresearch Laboratories, PA, USA). After washing, the positive bands were visualized using 4-chloro-1-naphthol as a peroxidase substrate.

Polysorbate 80

The polysorbate 80 concentration was determined by the colorimetric method (Milwidsky, Analyst 94, 377-386, 1969).

: *. 30

Example 1

Preparation of Purified Leukocyte IFN-α This example describes the preparation of a highly purified leukocyte IFN-α drug. This • 35 IFN-α was used in the stabilization and filtration examples (Examples 2-5).

10 106465

Preparation of crude interferon was performed in leukocyte cultures induced with Sendai virus substantially as previously described (Cantell et al., Methods Enzymol.

78.29 to 38.1981). Residual cells from the culture supernatant were removed by microfiltration and the filtrate was concentrated 20-fold by ultrafiltration. The crude IFN concentrate was filtered through 1.2 µm and 0.22 µm filters and treated with 0.3% tri (n-butyl) phosphate and 1% polysorbate 80 at 16126 ° C (solvent / detergent treatment). ). The solution was applied to an immuno-adsorbent column containing two monoclonal antibodies against IFN-α coupled to a CNBr-Sepharose 4FF gel. Monoclonal antibodies have complementary binding specificities, and together they bind all major IFN-α subtypes. The immunoadsorption column was thoroughly washed and bound IFN-α was eluted with buffer adjusted to pH 2.0. The eluate was neutralized and concentrated to about 30 fold by ultrafiltration. The concentrated eluate was applied to a Superdex 75 gel filtration column equilibrated with PBS and eluted with it. The fractions containing IFN-α were combined and the purified drug thus obtained was stored frozen at -70 ° C.

15

The purified drug was analyzed for IFN-α subtype composition using procedures detailed elsewhere (Nyman et al., Biochem. J. 329, 295-302, 1998). It was found to contain subtypes α1, α2, α4, α7, α8, α10, α4, α7 and α21.

20

Example 2

Short-term adsorption of purified multicomponent IFN-α on glass of various compositions The stabilizing effect of various stabilizers over a short period of time was determined by analyzing the adsorption of IFN-α on glass.

Purified leukocyte IFN-α bulk solution was diluted in polypropylene flasks to a final concentration of 3 milliliters. IU / ml (0.02 g / l) in PBS containing one of the following stabilizing agents: * - 30 diluents: 1. polyoxyethylene lauryl ether (laureth 4, Brij® 35, CAS-9002-92-0) 2. polyoxyethylene sorbitan monooleate ( polysorbate 80, Tween® 80, CAS-9005-65-6) • 35 3. polyoxyethylene, polyoxypropylene block copolymer (poloxamer 188, Pluronic® F-68, CAS-9003-11-6) 11 106465 4. human serum albumin

Laureth 4, polysorbate 80 and poloxamer 188 were used at final concentrations of 0.1,0,2,0,5 and 1,0 g / l. Albumin was added at a final concentration of 0.5, 1.0, 1.5, 5 and 2.0 g / L. The IFN-α bulk solution used as a control was diluted in PBS. Formulated solutions were sampled immediately after mixing for determination of IFN-α concentration, and 100 μΐ of the formulated solutions were transferred to glass vials. The flasks were kept at 201 room temperature (23 ° C). Samples were taken for determination of IFN-α concentration. The results are shown in Figure 1. Adsorption was determined as the difference between the initial 10 and the final concentration of IFN-α in the flasks.

About 30% of IFN-α was adsorbed to glass vials in the absence of a stabilizer (Figure 1). The stabilizers studied inhibited the adsorption of IFN-α to varying degrees. Polysorbate 80 was most effective, followed by laureth 4, albumin and poloxamer 188.

The formation of IFN-containing aggregates was examined by western blot under non-reducing conditions. Purified leukocyte IFN-α was incubated in glass flasks in PBS containing polysorbate 80 or albumin for 20 hours at 23 ° C. Figure 2 shows western blots of samples containing 0.1 g / l (lane 3), 0.2 g / l (lane 4), and 0.5 20 g / l (lane 5) of polysorbate 80, and 0.5 g / l (lane 6), 1.0 g / l (lane 7) and 1.5 g / l (lane 8) albumin. Lanes 1 and 2 show a negative and a positive IFN-α aggregate control, respectively. In solutions containing polysorbate 80, only the bands corresponding to the monomers and dimer of IFN-α are visible. The intensity of the dimer band was weaker at 0.2 g / l and 0.5 g / l polysorbate 80 than at 0.1 g / l.

In albumin solutions, the dimer bands were more intense, and in addition, bands with a higher molecular weight and corresponding to albumin-IFN complexes were visible. Polysorbate 80 compositions could not detect bands corresponding to higher molecular weight complexes.

Example 30

Comparison of polysorbate 80 and albumin in the production of virus-filtered and sterile-filtered multicomponent en-IFN-α-Liu

Purified leukocyte IFN-α was diluted to 5 ml activity. IU / ml (40 pg / ml) • 35 in PBS containing either 0.2 g / l polysorbate 80 or 1 g / l albumin. The formulated solutions were pre-filtered with a 0.1 µm filter and subjected to viral filtration using Pia-12 106465 nova 15N filters (Asahi Chemical Industry Co., Japan). Filtrations were carried out at room temperature under pressure at a constant pressure of 0.8 bar. The system was pressurized with nitrogen gas. At the end of filtration, the viral filter was washed with the formulation solution to recover all product from the filter system. Pressure, temperature and mass of 5 filtrate were monitored during filtration. Formulated solutions were sampled after pre-filtration, after virus filtration, and after sterile filtration for IFN-α concentration, polysorbate 80 and total protein, and for Western blot analysis.

The results are summarized in Table 1 below and Figure 3. Table 1 shows the yield of IFN-α in the preparation of a virus-filtered, finished product using polysorbate 80 (0.2 g / L) or albumin (1 g / L) as a stabilizer.

Table 1. Yield of IFN-α in the Preparation of Virus-Filed, Finished Product 15 Calculated from the FIA Results of IFN-α Cumulative Yield of IFN-α (%)

Production step polysorbate solution albumin solution (n = 3) __ (n ^ 3) __ formulated IFN-α bulk solution 100 '100 pre-filtered solution 99 97

Planova 15 filtered solution 102 88 sterile filtered solution 101 89

As shown in Table 1, the yield of IFN-α from the virus-filtered and sterile-filtered solution was consistently better in the presence of 0.2 g / l polysorbate than 1 * *: g / l albumin. Most of the IFN-α loss in albumin solutions occurred during virus filtration, whereas no significant IFN-α loss occurred in the polysorbate solution at the corresponding step. Notably, the recovery of polysorbate 80 was 99% to the filtrate in virus filtration, indicating that the polysorbate had no tendency to retire during virus filtration. The recovery of albumin in the filtrate was 87%, suggesting that the albumin was retained in the filter.

13 106465

Figure 3 shows the Planova 15N filtration flow levels of polysorbate 80 or albumin-stabilized purified IFN-α solutions. Purified leukocyte IFN-α (40 pg / ml) in PBS containing 0.2 g / l polysorbate 80 (open circles) or 1.0 g / l albinin 5 (filled circles) was filtered with a Planova 15N filter at a constant pressure of 0.8 bar in a ternary flow mode. The filtration level remained constant in the presence of polysorbate 80 at least during filtration at 2001 / m 2, whereas it decreased by about 80% in the presence of 1 g / l albumin already after filtration at 201 / m 2. This suggests that the filter was clogged when the albumin-containing solution was filtered, whereas there was no clogging tendency when the polysorbate-containing solutions were filtered. The same results were confirmed by filtration of pure albumin and polysorbate solutions (results not shown). Filtration of the polysorbate-containing solution could also be performed in the end mode without reducing the filtrate flow rate. Virus filtration did not cause any change in the molecular weight distribution of IFN-α, which was analyzed by Western blot.

15

Example 4

Preparation of Polysorbate 80-Stabilized, Viral-Filtered Ready IFN-α

The formulated IFN-α bulk solution was prepared by adding PBS and polysor-20 80 to a suitable vessel, mixing and adding purified multicomponent IFN-α to obtain the desired IFN-α activity in the calculated final volume of PBS. containing 0.2 g / l polysorbate 80. The formulated IFN-α solution was thoroughly mixed and pre-filtered with a 0.1 μιη: η filter. The pre-filtered IFN-α solution was filtered through a viral filter (Planova 15N, Asahi) at a constant pressure of 0.9 bar. The filtrate was collected and filtered through a sterile filter of 0.1 or 0.22 μιη: η and aseptically placed in the final product flasks.

EXAMPLE 5 Stability of virus-filtered IFN-α solution containing polysorbate 80

The stability of the finished virus-filtered IFN-α product prepared according to Example 4 was tested at 6 ° C and 25 ° C for six months. The results are shown in Table 2.

14 106465

Table 2. Stability of polysorbate 80, 0.2 g / L, stabilized virus-filtered IFN-α solution Time limit IFN-α concentration IFN-α antiviral act.

(months) mean ± SD (mill. IU / ml) mean ± SD (mill. IU / ml)

6 ° C 25 6 ° C 25 ° C

° C

0 4.5 ± 0.1 4.5 ± 0.1 4.0 ± 0.1 4.0 ± 0.1 1.5__4.6 ± 0.2 4.0 ± 0.1 4.5 ± 1.0 3.8 ± 0.0 3 4.5 ± 0.1 3.2 ± 0.0 4.3 ± 1.3 2.9 ± 0.5 6 4.3 ± 0.1 2.0 ± 0.0 4.4 ± 0.0 1.8 ± 0.2 5

As can be seen in Table 2, there is no decrease in the immunochemical concentration and biological activity of IFN-α within 6 months at 6 ° C. Some reduction (5-10%) occurs at room temperature after 1.5 months of storage, and approximately 30% reduction is observed at room temperature after 3 months of storage. From the results, it can be concluded that the stability of the polysorbate-stabilized IFN-α solution, which has been stored at 2-8 ° C, remains good for a long time.

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Claims (12)

1. A process for preparing a virus-safe pharmaceutical composition of biologically active protein consisting of interferon, characterized by 5. In an protein solution, an effective amount of ion-free detergent is added which gives the pharmaceutical composition an extended storage shelf life, - the solution containing the ion-free filter cleaner with filters for removing viruses whose pore size is 10 - 40 nm and the filtrate is used. 10 2. A process according to claim 1, characterized by ion-free cleaning agent, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaureate or polyoxyethylene lauryl ether are used. Process according to claim 2, characterized in that the ion-free detergent comprises polyoxyethylene sorbitan monooleate (polysorbate 80) which is added in an amount which exceeds the critical concentration of micelles. Process according to claim 3, characterized in that polysorbate is added in an amount of 0.05 - lg / l. Process according to any of claims 1-4, characterized in that the pharmaceutical composition comprises a purified α-interferon solution. A method according to any of claims 1-5, characterized in that the activity of the α-interferon solution is in the range of 3 to 50 mil. IU / ml. before the virus filtration. Process according to claim 5 or 6, characterized in that the pharmaceutical composition comprises an α-interferon solution containing at least two subtypes of α-interferon selected from a group wherein a1, a2, a4, a7, a8, alO , al4, al7 and a21. inglr. Process according to any of the preceding claims, characterized in that in the process a pharmaceutical composition is prepared comprising purified leukocyte or lymphoblastoid-α-interferon, where polymers and albumin interferon complexes of α-interferon are substantially absent. • «• ·« 106465 A method according to any of the preceding claims, characterized in that the proteinaceous solution of the process is pre-filtered with a filter of 0.04-0.2 µm, then filtered with a filter for removing virus with a pore size of 10 - 40 nm. and lastly, the filtrate is sterile and recovered. 5 Method according to any of claims 1-8, characterized in that the protein solution in the process is sterile filtered and the filtrate obtained by sterile filtration is subjected to filtration to remove virus with a filter having a pore size of 10 - 40 nm, and the filtrate is used. 10 A composition of multicomponent α-interferon, characterized in that it comprises ion-free detergent in an amount of stabilizer which exceeds the critical concentration of the micelle detergent and substantially lacks substances remaining in the virus filter, the pore size of which is in the range 10-40 nm. 15 Composition according to claim 11, characterized in that it contains a solution of α-interferon which contains at least two subtypes of α-interferon selected from a group containing ai, a2, a4, a7, a8, alO, ai4, al7 and a21, and which contains polysorbate as a stabilizer in an amount of 0.05 - lg / l. 20 The use of polysorbate as a stabilizing agent in pharmaceutical compositions of purified leukocyte-α-interferon which are subjected to filtration with a virus removal filter. 4 "9 Λ