Method of producing anticoagulative protein, extracted from nematodes (nap)

FIELD: biology.

SUBSTANCE: present invention relates to genetic engineering, more specifically to obtaining anticoagulative protein extracted from nematodes (NAP) and can be used in medicine. To obtain a medicinal preparation based on NAP, methanotrophic yeast host cells are cultured, encoding rNAPc2 or rNAPc2/praline, until attaining the desired cell density. NAP is then extracted from the said yeast host cells through cation-exchange chromatography on an expanding layer. To purify the NAP medicinal preparation, hydrophobic-interaction chromatography is used. NAP is extracted and purified at pH levels below 4.

EFFECT: simple and more efficient method of obtaining anticoagulation proteins from nematodes.

25 cl, 8 dwg, 7 tbl, 6 ex

 

RELATED APPLICATIONS

This application claims priority to jointly pending application "Method of treatment of hemorrhagic disease with the use of an inhibitor of factor VIIa/tissue factor", filed may 6, 2003

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to a method for production of proteins that are anti-clotting substances of human plasma, and to proteins produced by this method. Specifically, the present invention relates to a method of production of purified, extracted from nematode anticoagulant protein (NAP), and refers to the purified NAP produced in this way. In particular, the present invention relates to medicinal substances NAP and medicinal products NAP, and methods for their production.

The LEVEL of TECHNOLOGY

The discovery and purification of therapeutic proteins that have potential value as pharmaceuticals, can be performed in a research laboratory using materials and methods that are not suitable for large-scale industrial production of pharmaceutical products. To produce pharmaceutical products in industrial scale biotech manufacturing operations must be reliable and scalable without jeopardizing the quality of the product Gottschalk, 2003, BioProcess Intl 1 (4):54-61). Production processes for pharmaceutical products should ensure cost-effective ways, an increased production of the product, sufficient to meet the demand, and, ideally, should provide a scalable way to respond to fluctuations in demand. Manufacturing processes for therapeutic proteins should improve the cost-effective production of large quantities of protein in a functional form, as well as methods of protein purification, to produce the pharmaceutical product of suitable purity for its intended use.

"Research" methods of protein purification, also known as "lab" or "poster" ways, often closely associated with the methods that were used to detect and characterize therapeutic protein. Often develop only in micrograms or milligrams of purified protein is sufficient for characterizing protein and determination of the sequence of its amino acids. Even after developed expression system for recombinant production of therapeutic protein, such expression systems are not necessarily suitable for the production of a protein on an industrial scale. In addition, in the research methods of cleaning can be used organic rest ritali, strong acids or other reagents that may be undesirable or impractical to use on an industrial scale, and sometimes they are not allowed to be used in the manufacture of pharmaceutical products. In addition, these cleaning methods can be used such methods of separation, as exclusion or high performance liquid chromatography (HPLC), which are powerful cleaning methods in the laboratory, but which are difficult to scale to the level of industrial production.

Methods pilot production, for example the volume of fermentation from 10 l to 100 l of host cells expressing therapeutic protein, suitable for further studies of the mode of production or for the production of therapeutic quantities of protein, sufficient for the early clinical trials, but even the ways of the pilot production is not always possible to scale up to production quantities required for later phases of clinical trials.

One approach to increase the biotechnological production includes an extension to the performance or efficiency of microbial expression systems. For the production of therapeutic proteins, there are many known biological "factories". However, as the product of a functioning protein is closely associated with the cellular mechanisms of the organization of the mA, producing a protein, each expression system has advantages and disadvantages for use in large-scale production of pharmaceutical products, depending on the protein. E. coli was the "factory" of choice for the expression of many proteins, because it is easy to apply, it grows rapidly, requires inexpensive nutrient medium and can secrete the protein into the growth medium, which facilitates the selection. However, many eukaryotic proteins produced in E. coli, produced in the defunct, unfinished form, are deglycosylation or have other posttranslational modifications, in addition, they do not form the corresponding disulfide bridges and do not form the corresponding three-dimensional conformation. In addition, a substance produced in E. coli, may be contaminated with endotoxin. These restrictions are often faced with the use of Bacillus species as expression systems. Culture of mammalian cells provide a small number of eukaryotic proteins with the proper glycosylation and conformation, but the culture of mammalian cells costly, their scale may be difficult to increase to the industrial level, they can be unstable and may require the use of animal serum. The expression system of the cells of the insect quickly, and is relatively easy to develop, and they offer good levels of protein expression mammals, but can be donostiako only moderately scalable, and can give improper glycosylation. Yeast expression systems are popular because they are easy to grow, they are fast and scalable; however, some yeast expression systems give unstable results, and it is sometimes difficult to achieve a high output.

One of the yeast expression systems, which seemed promising, is methanotrophic Pichia pastoris. Compared to other eukaryotic expression systems Pichia offers many advantages, because it is deprived as endotoxin problems associated with bacteria, and the problem of viral contamination of the proteins produced in cell cultures, animals (Cino, Am Biotech Lab, May 1999). In the absence of glucose Pichia as a carbon source uses methanol using induced by methanol promoter of alcoholiday (AOX1), which normally inhibits the expression of the enzyme catalyzing the first step in the metabolism of methanol, as induced by methanol promoter to control the expression of heterologous proteins. The proliferative rate of growth of Pichia makes it easily scalable to industrial levels of production, although difficulties when zoomed include panel is free pH the limited oxygen, limited nutrients, temperature and security considerations when using methanol (Gottschalk, 2003, BioProcess Intl 1(4):54-61; Cino Am Biotech Lab, May 1999). Production with Pichia pastoris, according to the current conditions of Good Manufacturing Practice (cGMP), possibly at the scale of 1000 l of fermentation (Gottschalk, 2003, BioProcess Intl 1 (4):54-61).

Another approach to increase the biotechnological production is to improve the separation of proteins and processing of fermentation products. In recycling processes should be adjustable to adapt to the changes and improvements titer fermentation, the composition of the nutrient medium and cell viability while maximizing performance in an existing volume (Gottschalk, 2003, BioProcess Intl 1 (4):54-61). Recent advances in chromatography and filtration provide a significant increase in selectivity, separation and offer large volumes and low cycle time, comparable with the large volume and high levels of expression in the modern periodic processes fermentation with water (Gottschalk, 2003, BioProcess Intl 1 (4):54-61).

Despite great strides in improving the biotechnological production, there are no comprehensive solutions suitable for any protein. Method of production of a particular therapeutic protein requires new and innovative solutions to problems that can be the ü specific for a given protein or protein family. Similarly, successful commercial applications often depend on a combination of specific properties of the protein or family of proteins and methods of production used in the production of a given protein or family of proteins as pharmaceutical products.

The INVENTION

In the present invention proposes a method of production of purified extracted from nematode anticoagulant protein (NAP) and the purified drug substance NAP and medicinal products NAP produced by this method. In the present invention proposes a method of production of large (industrial) scale quantities of medicinal substances NAP and NAP drug product. In particular, the present invention proposes a method of production of medicinal substances NAP, comprising the steps of: (a) the fermentation process, including the production of the NAP in a suitable host, where at least one sequence encoding a NAP, integrated into the host genome; (b) the allocation process, in which the NAP is separated from the cells and cell fragments; and (c) a cleaning process for cleaning medicinal substance NAP from contamination. A suitable host is Pichia pastoris. The method may additionally include the introduction of medicinal substances NAP in the final pharmaceutical composition. The method may additionally include the Zap process the log, includes filtering the flow of medicinal substance NAP in the final pharmaceutical composition, and the filling stage, which may include the division of medicinal substance NAP in the final pharmaceutical composition in dosage forms to produce a medicinal product NAP, and may additionally include lyophilization of the drug product NAP. This proposed method can be used for the production of purified drug substance NAP or NAP drug product of rNAPc2 (AcaNAPc2), rNAPc2/Proline (AcaNAPc2/Proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44 or AcaNAP46.

In the present invention is proposed drug substance NAP produced by the method disclosed here. In the present invention is proposed drug substance NAP produced using NAP, selected from, but not limited rNAPc2 (AcaNAPc2), rNAPc2/Proline (AcaNAPc2/Proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44 or AcaNAP46. In one embodiment, the medicinal substance NAP of the present invention can be produced using rNAPc2/Proline. The present invention additionally provides a medicinal product NAP produced by the method disclosed here. In the present invention is proposed drug product NAP, made using NA, selected from, but not limited rNAPc2 (AcaNAPc2), rNAPc2/Proline (AcaNAPc2/Proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44 or AcaNAP46. In one embodiment, the medicinal substance NAP of the present invention is manufactured using rNAPc2/Proline.

In accordance with another aspect of the present invention proposes a method of production of drugs rNAPc2/Proline and medicinal products rNAPc2/Proline. The present invention additionally offers the medicinal substance rNAPc2/Proline and medicinal products rNAPc2/Proline produced by the method disclosed here. In particular, the present invention proposes a method of production of medicinal substances rNAPc2/Proline, which includes the process of fermentation, separation and purification process. Here, the method includes the process of fermentation, where rNAPc2/Proline is produced in Pichia pastoris, having at least one sequence encoding a rNAPc2/Proline integrated into the genome, where the fermentation process involves the fermentation of seed to multiply the host cells to the desired cell density, and the process of industrial fermentation, including fermentation with periodic feeding of glycerol fermentation by feeding glycerin, fermented with adaptation to methanol and fermentation with induction with methanol p is abolitionniste approximately seven days. The method proposed here, additionally provides for the allocation process, including ion-exchange chromatography in the suspended layer to separate the rNAPc2/Proline from cells and cell fragments. The method proposed here, additionally offers the cleanup process, including hydrophobic chromatography using environment for hydrophobic chromatography, collecting fractions of rNAPc2/Proline at least one ultrafiltration/diafiltration (UF/DF) fractions rNAPc2/Proline, ion-exchange chromatography and collecting fractions of rNAPc2/Proline with ion-exchange chromatography, where the fraction of rNAPc2/Proline with ion exchange chromatography containing the medicinal substance rNAPc2/Proline. In accordance with one aspect of the method includes the temperature control of fermentation, in particular, maintaining the temperature of the fermentation adaptation to methanol approximately 28±2°C for approximately the first four hours and approximately 25±1°C in the remaining time of fermentation adaptation to methanol. In accordance with another aspect, supported pH approximately 2,9±0,1 during fermentation adaptation to methanol and fermentation induction with methanol. In one embodiment, the allocation process includes chromatography in the suspended layer on the ion-exchange resin Streamline SP XL at pH approximately 3,2±0,2, and the step of cleaning includes hydrophobic chromatograph is Yu Source 15PHE at a pH of approximately 3.0±0,1, and ion chromatography on a Source 15Q followed UF/DF fractions NAP with ion exchange chromatography.

Further provides a method of manufacturing a liquid medicinal product rNAPc2/Proline, including the production of medicinal substances rNAPc2/Proline according to the method described above, followed by the introduction of the drug rNAPc2/Proline in the final pharmaceutical composition, the process of filling, including the filter in the flow and the filling stage, including the distribution of rNAPc2/Proline in the final dosage form, such distribution into the container or bottle to get the liquid pharmaceutical product rNAPc2/Proline, and may additionally include lyophilization of the drug product rNAPc2/Proline. In the present invention proposes a liquid medicinal product rNAPc2/Proline produced by this method, and liofilizovannye medicinal product rNAPc2/Proline produced by this method.

In the present invention proposes a method of production of large (industrial scale) quantities of medicinal substances NAP, in particular medicinal substance rNAPc2/Proline. From medicinal substances NAP produced by the proposed method, may be prepared in dosage form, which can be prepared in the form of a medicinal product NAP, including in the form of liquid is about NAP drug product or liofilizirovannogo medicinal product NAP. In addition, from medicinal substances rNAPc2/Proline produced here by the way, may be prepared in dosage form, which can be distributed in the form of a medicinal product rNAPc2/Proline, including in the form of a liquid pharmaceutical product rNAPc2/Proline or liofilizirovannogo medicinal product rNAPc2/Proline.

This method is suitable for efficient industrial production of medicinal substances NAP and medicinal products NAP desired levels of activity and purity. In contrast, the previously disclosed methods for cleaning the NAP research in ways that are impossible to scale for large-scale production of NAP, and used reagents and materials are not desirable in the manufacture of drug substances and drug products. For example, the previously disclosed method of allocation included centrifugation to remove cells. In the previous method supernatant was then purified by cation-exchange chromatography, gel-filtration chromatography (also known as size-exclusion chromatography), and, finally, by reversed-phase chromatography. However, as suggested here, the properties of the NAP, especially rNAPc2/Proline allow modifications to the research method in order to replace the stage centrifugation scalable and provide clearance pic is BOM, to resolve trudnorastvorimye stages gel-filtration chromatography and reverse phase high-performance liquid chromatography (RP-HPLC), which include the use of flammable organic solvent and specialized equipment, and to improve the purity of the final product. In this way the stage ion-exchange chromatography in the suspended layer, in particular, the phase chromatography in suspended layer of Streamline SP XL, eliminated the operation of many blocks, typically used to process industrial allocation (for example, a combination of microfiltration to ultrafiltration). As suggested here, the phase of Streamline SP XL was used to separate the rNAPc2/Proline from cell fragments and put the product in a buffer suitable for the first stage of the treatment. Although in the previously open method was used gel filtration and reversed-phase chromatography, the data column stages in the present invention substituted hydrophobic chromatography (HIC) and anion-exchange chromatography, in particular HIC using Source 15PHE and anion-exchange chromatography using Source 15Q, which resulted in a significant purification of rNAPc2/Proline by removing protein and non-protein contaminants. It seems that the relatively low isoelectric point rNAPc2/Proline (pH 4,1) and other NAP can participate in the building at the unexpected result higher binding matrix and a higher overall selection of the product from step HIC depends on the execution of this phase chromatography at low pH is around 3.2. In addition, the return process was the result of the execution stages at a pH of approximately 3, since the later stages of fermentation and finishing chromatography to Streamline and HIC that eliminated the replacement buffer between stages.

BRIEF DESCRIPTION of FIGURES

Figure 1 shows a vector map of the expression vector rNAPc2/Proline Pichia pastoris pYAM7sp8/rNAPc2/proline used in the production of rNAPc2/pro showing the reference point.

In Fig. 2A and 2B depicts a block diagram of fermentation, showing the materials and reagents used in the process and equipment, and adjustable and controllable conditions at each stage of the process of fermentation; fermentation begins with preparation bulb for inoculum and fermentation lasts from seed to industrial fermentation.

Figure 3 shows the block scheme showing the materials and reagents used in the process and equipment, and adjustable and controllable conditions at each stage of the allocation process.

In Fig. 4A and 4B depicts a block diagram cleanup, showing the materials and reagents used in the process and the equipment, and reguliruemyi controlled conditions at each stage of the purification process; treatment includes the steps of hydrophobic chromatography on Source 15PHE, stage ultrafiltration/diafiltration #1 (UF/DF#1), step ion-exchange chromatography on Source 15Q, the final UF/DF, filtering in the stream, filling and storage of the pure product.

Figure 5 shows a block diagram of a liquid medicinal product, showing the materials and reagents used in the process and equipment, and adjustable and controllable conditions at each stage of the manufacturing process of a liquid medicinal product. This process includes the step of mixing, stage filtration and filling, and the transfer of the vials of liquid medicinal product to the warehouse.

Figure 6 shows the block diagram of the preparation of liofilizirovannogo medicinal product, showing the materials and reagents used in the process and equipment, and adjustable and controllable conditions at each stage of the process of the preparation of liofilizirovannogo medicinal product. The process includes a stage UF/DF, phase mixing, filtration stage in the stream and filling, and transfer to unit lyophilization.

DETAILED description of the INVENTION

In the present invention proposes a method of production of purified extracted from nematode anticoagulant protein (NAP), such as disclosed in U.S. patent No. (hereinafter "USA") 5863894; 5864009; 5866542; 5866543; 5872098; and 5945275 (on the ing the contents of each is incorporated by reference), where NAP is characterized to date have anticoagulative action and/or have activity of serine proteases. The present invention offers peeled NAP produced by the claimed process, where such purified NAP is a medicinal substance NAP, from which may be prepared in dosage form as a drug product NAP. The present invention may be particularly suitable for the production of polypeptides comprising at least one NAP domain. The present invention offers medicinal substance NAP and medicinal products NAP produced by the method, opened here. In one embodiment, the present invention proposes a method of production of medicinal substances rNAPc2/Proline and medicinal product rNAPc2/Proline, and offers medicinal substance rNAPc2/Proline and medicinal product rNAPc2/Proline produced according to the method, opened here.

Extracted from nematode anticoagulant protein (NAP) are so named because the first initially selected NAP was extracted from nematodes, dog hookworms, Ancyclostoma caninum. The term "domain NAP" refers to a sequence, which, as I believe, has anti-clotting properties. Usually NAP domain is an amino acid sequence, with the holding is less than about 120 amino acid residues, and contains 10 cysteine residues, as disclosed in U.S. 5863894; 5864009; 5866542; 5866543; 5872098 and 5945275. Domain NAP" can also refer to nucleic acids or nucleotide sequences encoding one or more amino acid sequences or polypeptides having domains NAP. Illustrative NAP domains, amino acid sequence NAP, characteristics, broadly define this family of proteins and nucleic acid molecules that encode such proteins are disclosed in U.S. 5863894; 5864009; 5866542; 5866543; 5872098 and 5945275.

Medicinal substances NAP of the present invention include anticoagulation means, characterized by the inhibition of blood clotting, which includes coagulation of plasma. Medicinal substances NAP of the present invention include, among others, those that increase the clotting time of human plasma, as measured by prothrombin time (PT) and/or activated partial thromboplastin time (APTT), as disclosed in U.S. 5863894; 5864009; 5866542; 5866543; 5872098 and 5945275. The person skilled in the art may use other methods of analysis to determine the anticoagulant activity of medicinal substances NAP. The person skilled in the art can similarly use other methods of analysis to determine whether any other biological activity of medicinal substances NAP.

The terms "AcaNAPc2" or "rNAPc2" refers to a recombinant protein family NAP. Obtaining and sequence AcaNAPc2 described in U.S. 5866542.

The terms "AcaNAPc2/Proline", "AcaNAPc2P", "rNAPc2/Proline and rNAPc2/Pro" refers to a recombinant protein having the amino acid sequence of AcaNAPc2, which was amended in order to add the Proline residue to the C-end of the sequence AcaNAPc2.

"Medicinal substance or active pharmaceutical ingredient (API)" refers to a pharmaceutically active substance, which together with fillers can be prepared dosage form to produce a medicinal product. The medicinal substance can be in undivided dose form. "Medicinal product" refers to a finished dosage form (e.g. capsule, tablet, liquid product in the bottle, liofilizovannye powder in vial)containing the medicinal substance in the final buffer composition and usually contains the inactive ingredients. The medicinal product may be a dosage form prepared from medicinal substances. "Filler" refers to an inactive ingredient that is intentionally added to the medicinal product, where it is understood that inert fillers do not have pharmacological properties in quantities is H. "Pollution" refers to a component present in the drug substance, the composition of the API or drug product that is not the desired product associated with the product, substance or inert filler, where it is understood that the contamination may be associated with the product or associated with the process. "Degradation products" refers to variants, in particular to molecular variants that occur over time from changes in the drug substance or drug product due to light, pH, temperature, water or the reaction with an inert filler or system capacity/packaging/covers.

"USP" refers to the standards presented in the US Pharmacopoeia (USP) and the National pharmaceutical formulary (NF) (United States Pharmacopeial Convention, Inc., Rockville, Maryland (2002), "USP26-NF-21", the full content of which is hereby incorporated by reference), and USP Standards. Additional information can be found on http://www.usp.org or in USP-NF.

In the present invention proposes a method of production of medicinal substances NAP of high purity, in which at any stage of fermentation or purification is not used raw materials of animal origin. This method is scalable and is suitable for carrying on an industrial scale. In the present invention proposes a method of production of medicinal substances NAP and Le is artenova product NAP where the method includes the processes of fermentation, separation, purification, filtration and filling. Proposed method of fermentation, by which the NAP is produced in a suitable host, where encoding NAP sequences integrated into the host genome. Proposed allocation method, which improves the yield and purity of proteins secreted to the fermentation stage, where the allocation method allows you to more efficiently capture NAP compared with traditional methods, such as microfiltration and ultrafiltration. Suggested cleaning method, in which the medicinal substance NAP cleaned of contaminants, where the desired composition is obtained using a combination of methods, including but not limited to ultrafiltration, diafiltration, hydrophobic chromatography and ion exchange chromatography. Features optional filling process, where the medicinal product NAP is introduced into the packaging container and can be liofilizovane.

The methods of the present invention are suitable for the production of medicinal substances NAP and medicinal products NAP. The person skilled in the art can modify the methods as disclosed here, in order to improve the expression, isolation, purification, preparation of dosage forms or completing specific medicinal substance NAP. In a non-limiting example, the specialist in D. the authorized area may determine the isoelectric point (pI) of particular interest to NAP and can slightly adjust the conditions such as binding capacity or pH phase chromatography to achieve improved purification of the desired medicinal substance NAP. The present invention offers medicinal substance NAP, purified as disclosed here, from the NAP, including, but not limited to AcaNAPc2, AcaNAPc2/Proline, AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP46 and AcaNAP44. In particular, the present invention proposes rNAPc2/Proline. The person skilled in the art can identify other proteins NAP, suitable for use in the methods disclosed here, for the production of purified drug substance NAP.

Fermentation

In the present invention proposes a process of fermentation, in which the NAP is produced in a suitable host. As suggested, one or more sequences encoding NAP, integrated into the host genome, and the owner produces a NAP during the fermentation process. In one embodiment, rNAPc2/Proline is produced by Pichia pastoris in the form of a secreted protein in the fermentation process, as suggested here.

As suggested here, the fermentation process involves the fermentation inoculum, where the cells of the host to multiply the desired cell density, and the process of industrial fermentation, where the NAP is made to the desired title. The fermentation inoculum provide the supports, respectively, a dense inoculum for the process of industrial fermentation, producing high levels of NAP. The fermentation process, provided here, further includes industrial fermentation for the production of high levels of NAP. Industrial fermentation includes different phases: the periodic supply of glycerin; recharge of glycerol; adaptation to methanol and induction with methanol. In the phase of the periodic filing of glycerol is formed biomass. In the phase of feeding glycerol to the culture is fed enriched with glycerin solution, to increase the biomass and to suppress the expression. In the phase of adaptation to methanol supply of glycerin is terminated and replaced by a feed of methanol, which induces the production of the NAP master. In the phase of induction with methanol supported the terms of the end phase of adaptation to methanol to support the production of the NAP.

In accordance with one aspect, to achieve the desired high titer NAP, control the pH range of fermentation. In one embodiment, the pH of the fermentation is kept in the pH range of around 2.9±0.1 units of pH during fermentation adaptation to methanol and fermentation induction with methanol. In accordance with another aspect control the temperature of fermentation. In one embodiment, a phase of adaptation to methanol is kept a temperature of approximately 28±2°C within the first four hours, in order to facilitate successful adaptation under the che methanol, and in the remaining time phases of adaptation to methanol is kept a temperature of approximately 25±1°C, in order to promote high titer NAP. In accordance with one aspect titer NAP continues to increase without detrimental effects on the product for about seven days, which results in high overall yield NAP.

In illustrative embodiments, the implementation proposed here, the fermentation process was carried out in 15 l, 100 l, 150 l and 1000 l fermentors and material of 15, 100 and 150 l fermentation was purified to produce a medicinal substance NAP. In one embodiment, the fermentation process is produced rNAPc2/Proline with high titer. In various embodiments, the implementation of the fermentation for the production of rNAPc2/Proline was carried out in 15 l, 100 l, 150 l and 1000 l fermentors, and the medicinal substance rNAPc2/Proline was cleared of 15, 100 and 150 l of fermentation.

Selection

In the present invention proposes a method of allocation that increases the yield and purity of proteins NAP with the fermentation stage. Here the allocation process allows you to capture the NAP and remove cells and cell fragments, where the suggested allocation method is more efficient than traditional methods, such as microfiltration/ultrafiltration. Not wanting to be limited by this theory, increased the effectiveness of the proposed allocation may witecka the ü from a combination of aspects of the system Pichia, namely that Pichia pastoris produces a dense biomass during fermentation. In addition, NAP proteins are relatively small proteins and, therefore, require membranes for ultrafiltration with small pores which have a small flow rate, which leads to long duration of the process. In accordance with one aspect of the allocation process uses ion exchange chromatography, including the use of ion exchange chromatography in a suspended layer to separate NAP from cells and cell fragments and put the product in a buffer suitable for use in the subsequent steps of purification. In one embodiment, to highlight the medicinal substance NAP, as described here, uses the block chromatography in the suspended layer on the ion-exchange resin Streamline SP XL (Amersham Biosciences). In another embodiment, rNAPc2/Proline is separated from the fragments of host cells, expressmusic rNAPc2, using chromatography in the suspended layer. In a particularly preferred embodiment, rNAPc2/Proline stands out using ion-exchange block Streamline XL.

Alternatively, the allocation method, effectively breathtaking NAP after fermentation stage may be performed using methods that differ from ion-exchange chromatography in the suspended layer. The person skilled in the art can in order to test and evaluate alternative ways of capturing NAP and removal of cells and cell fragments, including but not limited to affinity chromatography, centrifugation, filtration, differential precipitation and other methods that will be defined.

Clean

In the present invention proposes a process of purification by which the medicinal substance NAP cleaned of contaminants. As suggested here, the cleaning method includes hydrophobic chromatography, collecting fractions NAP, at least one ultrafiltration and diafiltration (UF/DF) fractions NAP, ion exchange chromatography, collecting fractions NAP with ion-exchange chromatography, another stage UF/DF and final filtration. It is clear that each phase proposed in the cleaning process, increases the purity of the drug substance NAP, so that the person skilled in the art can determine the degree of purity desired for the particular application, and choose the steps and conditions necessary to achieve the desired level of purity of drug substances NAP. The overall efficiency of the method was improved by maintaining a low pH (about 3) solution from a liquid medium for fermentation and including phase separation and the first stage of the treatment (hydrophobic chromatography on Source 15PHE). These stages have been specifically designed to run at the same pH, to remove the stages of change pH/buffer required in other ways, so smart is a large investment of time and labor, as well as reducing the potential loss of product. These steps are performed at a pH below about 5, preferably at a pH below about 4, more preferably at a pH of about 3. In one embodiment, in the hydrophobic chromatography is used environment for hydrophobic chromatography Source 15PHE at a pH of approximately 3.0±0,1. As suggested here, in the cleaning method used hydrophobic chromatography, to remove impurities, where the use of low pH allows hydrophobic environment to connect larger numbers of NAP and using a gradient elution allows you to separate closely related pollution. As suggested here, the fraction NAP, allerona with environment for hydrophobic chromatography, subjected to ultrafiltration and diafiltration (UF/DF), in order to concentrate the product and perform the change of buffer, and then NAP in a suitable buffer is applied to the ion exchange medium to remove most of the remaining protein and non-protein contaminants, including closely-related pollution. Finally, as suggested here, the fraction NAP collected from ion-exchange chromatography, containing highly refined medicinal substance NAP (API), is subjected to UF/DF, in order to concentrate the medicinal substance NAP and translate it into the final buffer composition.

In one embodiment, the implement is placed filtered, presented in good condition the eluate from step chromatography on Streamline SP XL used to allocate NAP, is introduced into the column with the environment for hydrophobic chromatography Source 15PHE (Amersham Biosciences) at low pH (approximately 3,0±0,1), where a significant amount of NAP is associated with the column, followed by gradient elution Source 15PHE, the addition of sodium hydroxide to raise the pH to about 5 or higher, and then UF/DF elyuirovaniya faction NAP, after which the solution NAP is introduced into the column with the liquid ion chromatography Source 15Q (Amersham Biosciences), and gradient elution is used to separate the medicinal substance NAP from closely related pollution. In one embodiment, the fraction NAP after ion-exchange chromatography containing the medicinal substance NAP. In another embodiment, the cleaning process is performed as described above to obtain highly refined medicinal substance rNAPc2/Proline.

As suggested here, the solutions containing NAP, may be subject to different stages of filtration, to obtain the medicinal substance NAP in the desired concentrations or in the desired compositions. Optionally may include additional stages of filtering. Accordingly, the fraction NAP, erwerbende with stages chromatography, can be filtered, concentrated, desalted or Powergen what are you replacing the buffer with only the ultrafiltration (UF) or its combination with diafiltration (DF), or a combination of ultrafiltration and diafiltration (UF/DF). As suggested here, UF/DF can be used to replace an eluting buffer hydrophobic chromatography on boot buffer ion-exchange chromatography or replace an eluting buffer ion chromatography on a final buffer composition or the principal amount of the medicinal composition, which is used for medicinal product NAP. UF/DF can be performed using one or more filters. In accordance with one aspect to UF/DF using one filter (or membrane) pore size for the selected molecular weight. Alternatively, multiple filters may be used as necessary to zoom in. In one embodiment, the fraction NAP with adjusted pH with phase hydrophobic chromatography is subjected to ultrafiltration using a filter with pores for substances with a molecular weight of 3 kDa to achieve the desired concentration, then the set of retentate containing NAP, subject to diafiltration against 5 or more volumes bootable ion-exchange buffer using the same filtration membrane with pores for substances with a molecular weight of 3 kDa, until it is determined that achieved the desired state of the buffer. In one embodiment, the coat is AI NAP with ion exchange chromatography are subjected to at least one final UF/DF. In another embodiment, the fraction NAP with stage ion-exchange chromatography are subjected to UF/DF, as described above, in order to transfer the medicinal substance NAP in the buffer of the final pharmaceutical composition. In another embodiment, ultrafiltration or diafiltration used filters for ultrafiltration of artificial cellulose fibers with pores for substances with a molecular weight of 3 kDa. The person skilled in the art can select and evaluate filters or filtration membranes that are suitable and compatible with the experimental conditions and the desired order.

Filtering in the thread

As suggested here, the medicinal substance NAP in the final buffer composition may be filtered and stored in stock or be subjected to further processing steps. In one embodiment, the medicinal substance NAP in the final composition is subjected to a process of filling. In one embodiment, the main volume of medicinal substance NAP is transferred into suitable sterile conditions, for example clean room class 100 in a manufacturing facility, and filtered into a sterile container. In one embodiment, the medicinal substance NAP is filtered, for example, using the 0.2-μm filter, autoclaved containers made of suitable material, for example E. the bone, made of fluorinated ethylene propylene (FEP), a copolymer of ethylenetetrafluoroethylene (EFTE) or other material that meets the requirements of the amendments on food additives of the Federal law the U.S. food, drug and cosmetic products, as well as the requirements of USP class VI. In one embodiment, the main volume of the medicinal product rNAPc2/Proline is transferred into a clean room of class 100 and filtered using the 0.2-μm Millipak filter in autoclaved 1-liter cast bottle series Nalgene Tefzel® FEP 1600 with unlined cast uncontaminated screw cap Tefzel®ETFE, after which the bottles are transferred to a freezer with a temperature of -20±10°C for storage.

The main volume of medicinal substance NAP can be re-filtered and bottled using the same method final filter, for example, the contents of the smaller bottles, filled as described above, may be transferred to a larger container. In one embodiment, the contents of the Teflon FEP bottles containing the medicinal substance rNAPc2/Proline as described above, is transferred to autoclaved wrapped the bottle in a clean room of class 100, re-filtered and bottled.

The filling stage

In accordance with one aspect of the present invention additionally offers the filling stage,where the medicinal substance NAP in the final pharmaceutical composition is packaged in aseptic vial, capacity or another package. The filling stage may include additional stages of filtering and may include the use of filler kit before filling in separate vials, containers or other packages. The filling stage can provide a bottle of medicinal product NAP, where the medicinal product NAP is in the final dosage form. Phase liquid fill can provide a bottle of medicinal product NAP in liquid form. Medicinal product NAP can be used in the form, entered during the filling stage, for example, a standard dose of a solution containing medicinal product NAP. Alternatively, the composition of the medicinal product NAP can additionally be controlled after the filling stage, for example, the medicinal product NAP in the vial after filling stage liquid can be liofilizovane. As suggested here, the medicinal substance NAP in the desired final concentration can be filtered under aseptic filling kit and then poured into a separate pre-sterilized bottles.

In one embodiment, the main volume of medicinal substance rNAPc2/Proline (with a concentration of 12±1.2 mg/ml) was diluted to 3 mg/ml solution of 0.2m alanine and 25 mm sodium dihydrophosphate, pH 7.0. Diluted rNAPc2/Proline then exposed diafil the radio vs. ≥5 volumes of a solution of alanine/phosphate. A solution of rNAPc2/Proline removed and the filters washed with a solution of alanine/phosphate. Subject diafiltration solution rNAPc2/Proline is then diluted to 2 mg/ml (measured UV breakdown, described below in the examples), the washing liquid with filters and a solution of alanine/phosphate. A solution of rNAPc2/Proline, 2 mg/ml, then diluted with an equal volume of 25 mm sodium phosphate, 8%sucrose, pH 7.0, in order to achieve concentrations of rNAPc2 of 1.0±0.1 mg/ml. Finally created a solution rNAPc2/Proline, 1 mg/ml, before filling stage is filtered using 0.2 μm Millipak filter (Millipore Corp.). Medicinal product rNAPc2/Proline, 1 mg/ml, filtered under aseptic filling kit in two passage of 0.2-μm Millipak filter. Then rNAPc2/Proline poured into pre-sterilized glass bottles 3 cm3and stoppered.

As an alternative implementation of the main volume of the medicinal substance rNAPc2/Proline may be prepared in dosage form for liquid medicinal product. Described in example 5.1.

Lyophilization

The present invention offers an optional step of freeze-drying, which produces liofilizovannye medicinal product NAP. After the filling stage of the medicinal product NAP in bottles or other containers dried by sublimation and then sealed, for example cork in the bottle is pushed deeper and vial dress caps. Liofilizovannye composition retains a high purity and long-term stability, when the medicinal product NAP is subjected to severe thermal stress, for example 28 days at 50°C.

The present invention will be further explained through specific examples are presented below to demonstrate the characteristics of the production processes rNAPc2/Proline and characteristics of rNAPc2/Proline produced by these processes, including data and methods of the study of the pure product. In the following examples, the above-mentioned effects are explained by disclosing methods of production of medicinal substances rNAPc2/Proline suitable for the preparation of dosage forms in the form of a medicinal product for use in pharmaceutical compositions. This alternative implementation, however, is formulated to illustrate the invention and should not be construed as limitation, the invention will be defined by the claims.

EXAMPLES

Example 1. Creation of a Bank of cells for expression of the system of drug substances NAP

Example 1.1. Expression system rNAPc2/Proline

Gene rNAPc2 cloned in the expression vector Pichia pastoris, pYAM7sp8 (Laroche et al, 1994, Biotechnology 12:1119-1124), using the selection using PCR. Vector pYAM7sp8 (figure 1) is derived from the pHIL-D2 (Despreaux and Manning, 1993, Gene 106:35-1). It contains the promoter and signal termination of transcription of the gene AOX1 Pichia pastoris, signal peptide secretion (fusion of the signal sequence of acid phosphatase Pichia pastoris and proposedvalue hybrid α-factor of sexual reproduction in S. cerevisiae) and HIS4 marker gene for selection of transfectants.

The PCR primers used for gene selection rNAPc2/Proline from phage clone (Jespers et al., 1995, Biotechnology 13: 387-382), were:

A8:5'GCGTTT AAAGCA ACG ATG CAG TGT GGT G3'(SEQ ID NO: 1)

A9:5'C GCT CTA GAA GCT TCA TGG GTT TCG AGT TCC GGG ATA TAT AAA GTC3'(SEQ ID NO: 2)

These primers add lots Dral and Xbal to the 5'- and 3'-ends of the selected DNA fragment, respectively. The underlined nucleotides that hybridize with the matrix. Primer A9 (SEQ ID NO: 2) also inserts a Proline codon immediately before the termination codon, which converts the coding sequence of the sequence coding AcaNAPc2 (SEQ ID NO: 3), in sequence, encoding AcaNAPc2/Proline (SEQ ID NO: 4). The resulting PCR fragment was digested by Dral and XbaI and cloned in pYAM7sp8, split by Stul and Spel. Ligation of blunt ends pYAM7sp8 (Stul) and PCR fragment (Dral) led to the merger within the scope of the signal peptide for the secretion of P. pastoris and Mature part of rNAPc2/Proline. Ligation of XbaI ends and Spel PCR fragment and pYAM7sp8 led to the destruction of the site Spel pYAM7sp8.

Expression strain P. pastoris b the l created by the integration of expression cassettes into the genome of P. pastoris homologous recombination. Design pYAM7sp8/NAPc2 were digested in Notl. Cleaved plasmid was introduced by electroporation into cells of P. pastoris GS115 (his4-). Organized the screening of transfectants to phenotype using methanol (mut+) and a high level of expression of rNAPc2. The only isolate (designated as GS115/AcaNAPc2P-55), was selected to create a master cell Bank (MCB). Industrial strain was analyzed by the method of blotting by Southern using as probes of radioisotope-labeled genes rNAPc2 or HIS4. This blotting showed that multiple copies of the expression cassettes were integrated into the 3'-AOX1 gene.

Example 1.2. The master cell Bank (MCB)

The master cell Bank (MCB) was prepared using pregbancy isolate from a single colony (GS115/AcaNAPc2P-55). The flask containing the nutrient medium YEPD (bactopeptone, yeast extract, and dextrose) with 2% glucose sowed 0.5 ml of pregbancy and were grown to optical density (ANM) of 0.5-1.0. The culture was collected, diluted with glycerol as cryoconserved to a final concentration of 15% and froze in cryoplane, which was kept at a temperature below -60°C.

Example 1.3. The working cell Bank manufacturer (MWCB)

A new working cell Bank manufacturer (MWCB) prepared from a vial of the MCB. Vial of the MCB was used to sow the flask containing the yeast Pat the new medium (peptone and yeast extract) and 2% dextrose. The flask is incubated at 28±2°C and 250 rpm until the optical density (A600Nm) not was 17.0±5,0. The culture was collected and diluted with glycerol as cryoconserved to a final concentration of 9%. Aliquots of 1.1±0.1 ml were transferred into kriplani 2.0 ml, frozen and stored at -70±10°C.

Example 1.4. The methods used for analysis of the Central Bank of cells

Identification of the owner. Cell culture rNAPc2/Proline from the Bank was planted touch on Petri dishes with soy agar Trypticase (TSA), and the Petri dishes were incubated for growth. The isolate was prepared to identify using system identification Vitek®, which uses a camera with controlled temperature and block photometric sensor to monitor changes in the turbidity of the suspension of the isolate, which was planted on a test map for yeast Vitek®containing substrates for 26 conventional biochemical tests. To identify the owner of rNAPc2/Proline from the Bank of cells resulting Borisenok reactions were compared with Borisenko body reactions, which was the positive control (Pichia pastoris, ATCC No. 76273).

The concentration of viable cells. The concentration of viable cells from a cell Bank rNAPc2/Proline was measured by counting viable colony forming units (CFU) by making serial dilutions of the bottles from lankaclear (each from the beginning, mid and end). Breeding were sown in triplicate Petri-dish with TSA and incubated CFU were counted, and performed calculations to determine the cell concentration in CFU/ml

The definition of a structural gene sequence. Culture of cell Bank was prepared to determine the sequence of a gene by a gene amplification rNAPc2/Proline integrated into the host genome using polymerase chain reaction (PCR). The PCR product was purified and determined concentration. Then determined the sequence of the PCR product using the method of termination of ddeoxyribo (Sanger method). The resulting sequence of a gene from the cell Bank was compared with the known DNA sequence rNAPc2. The identity was confirmed 100%match.

Analysis of contamination by other microorganisms. Liquid nutrient medium for fermentation rNAPc2/Proline were checked for contamination by other microorganisms, sowing 100 ál of each of the nine Petri dishes with TSA. Three Petri dishes were incubated at three temperatures (20-25°C, 30-34°C and 35-39°C). Within seven days of incubation, the Petri dishes were examined for microbial colonies that differed from the typical owner, was particularly noted differences in colony morphology, color and/or size of the colony. The final Petri dish, which is th read result it was also examined by staining gram. The analysis incorporated an adequate negative controls.

Example 1.5. Methods of analysis of the working cell Bank manufacturer

Identification of the owner. Culture rNAPc2/Proline from the Bank cells were sown touch on Petri dishes with agar Saburo containing dextrose (SDA), the Petri dishes were incubated for growth at 20-25°C for 7 days. In parallel, the positive control (strain ATCC K. pastoris is the second name for P. pastoris) were sown touch on Petri dishes with SDA in the same way. Selected grown colonies were examined by staining gram.

After incubation for at least two morphologically similar colonies from each Petri dish filled with SDA was coming out of the Petri dishes with SDA, sowed the analyzed sample, and Petri dishes with SDA, under positive control. These colonies were perseval on separate Petri dishes with SDA and incubated at 20-25°C for 7 days. Growth on each Petri dish with persianas culture was subjected to the test API 20C AUX and staining gram. Test the API system (biomérieux basis SA; Marcy l'etoile, France) is a manual test for the identification of microbes, which contains 20 miniature biochemical tests. Test strip 20C AUX contains 20 biochemical tests that are specific for the identification of yeast. The results of the test API samples from the cell Bank rNAPc2/Proline compared the results obtained for the positive control to confirm the identification.

The concentration of viable cells. The concentration of viable cells from a cell Bank rNAPc2/Proline was measured by counting viable colony forming units (CFU) by making serial dilutions of the two vials from the Bank of cells, one vial was removed before freezing Bank and one vial was removed after the Bank was frozen. Aliquots of 100 µl of each dilution were sown on two Petri dishes with TSA and incubated for 5-7 days. Include all Petri dishes with countable colonies (30-300 CFU). To calculate the average of numbers obtained from the Petri dishes with the same dilution of the investigated samples was multiplied by their breeding and divide by the size of the aliquot 100 ál to report the results as CFU/ml

Determination of the DNA sequence. The newly created cell Bank (sample test) was isolated DNA entirely. Gene NAPc2/Proline amplified polymerase chain reaction (PCR), using primers homologous to the 5'- and 3'-sequences of the cloned gene NAPc2/Proline. The resulting DNA fragment (approximately 500 base pairs) was purified using standard methods and used as a matrix to determine the DNA sequence using the method of agaysex" primers, made using a kit Thermo Sequenase for cycle sequencing with the labeled terminator (Amersham Biosciences, Piscataway, NJ). Film sequences were read digitization, and sequence data were assembled and analyzed using the software SequencherTMversion 3.0 (Gene Codes Corp., Ann Arbor, Michigan). The consensus sequence obtained from the sample for testing, and then compared with theoretical sequence for gene NAPc2/Proline.

Contamination by other microorganisms. Before freezing the newly created cell Bank (sample testing), the vial was subjected to analysis of microorganisms, different from the owner. The sample liquid was diluted with saline in a thousand times. Two Petri dishes with nine different types of culture media were seeded with 100 µl of the diluted measured sample. In addition, the positive control (strain ATCC K. pastoris is another name for P. pastoris) was diluted and seeded on Petri dishes in the same way. Another set of Petri dishes was not sown and was appointed as a negative control. All Petri dishes, in addition to containing SDA were incubated at 30-35°C for 48-72 hours; Petri dishes with SDA were incubated at 20-25°C for 7 days. Petri dishes were examined for growth after 1-St and 2-nd or 3 rd day. the moreover, Petri dishes with SDA was studied on growth after 7 days. Any colony, deviating from the normal type, identified using the test API and gram staining.

After the 2nd or 3rd day at least two morphologically similar colonies from each Petri dish filled with TSA were selected from the Petri dishes with the TSA, under the investigated sample, and Petri dishes with TSA, under positive control. These colonies were perseval on separate Petri dishes with TSA and incubated at 30-35°C for 48-72 hours. Growth on each Petri dish with persianas culture was subjected to the test API 20C AUX and staining gram. Test the API system (biomérieux basis SA; Marcy l'etoile, France) was a manual test identification of microbes, which contained 20 miniature biochemical tests. Test strip 20C AUX contains 20 biochemical tests that are specific for the identification of yeast. The results of the test API sample for testing were compared with the results obtained for the positive control to confirm the identification.

Example 2. The production of medicinal substances rNAPc2/Proline

Industrial process for the production of medicinal substances rNAPc2/Proline consisted of fermentation, separation, purification, filtration, flow and filling. In the subsequent sections describe the operation of the individual blocks for each stage of the process. The block diagram for each of the operations of the unit shown in Fig. 2-4, where the flowchart summarizes the equipment, buffers, components, and input and output parameters. Replacement suppliers and equivalent materials may be as necessary as long as compliance with the Protocol requirements of good manufacturing (GMP) for active pharmaceutical ingredient (API), the International conference on harmonization of technical requirements for registration of pharmaceuticals for human use (ICH).

Example 2.1 Fermentation

This section describes the procedures fermentation for the production of rNAPc2/Proline. Protein rNAPc2/Proline was produced in the form of a secreted protein Pichia pastoris. The fermentation process for rNAPc2/Proline consisted of flasks seeds, fermentation inoculum and industrial fermentation (figure 2, block diagram, fermentation). All components of the nutrient medium used purified water, USP.

Bulb seeds for fermentation inoculum. The purpose of the operation unit bulb seeds was to ensure that the inoculate the appropriate density for fermentation inoculum. Three bottles of MWCB thawed and one milliliter was used to aseptically to sow each of the three two-liter shake flasks with septum containing 250 ml of autoclaved nutrient medium with a pH of 6.0±0,1 (the figure 1). The vials were closed and transferred to thermostat with the shaker at 250±5 rpm and 28±2°C. the Flasks were incubated for a total of 27.5±2.0 hours until the cell density measured by wet weight of cells (WCW), was ≥30 g/L. once these two parameters have been reached, the contents of the two flasks were aseptically transferred into autoclaved inoculation bottle.

Table 1
Nutrient medium in a flask with seed
ComponentsConcentration
Potassium hydrogen phosphate2,30 g/l
Potassium dihydrophosphate11,8 g/l
Glycerin10 ml/l
Yeast nitrogen base without amino acidsa 13.4 g/l
Biotin0.4 g/l

Fermentation inocula

The purpose of the fermentation inoculum was to ensure that the inoculate the appropriate density for industrial fermentation. The nutrient medium for fermentation inocula (table 2),including trace salt PTM4 (table 3), transferred into the fermenter seed. Nutrient medium was steam sterilized, allowed to cool and brought the pH to 5,0±0,2 sterilized by filtration 28-30% ammonium hydroxide. Then through the septum was added to sterilized filtration 5% (vol./about.) the solution of antifoam KFO880 in 50%methanol to a concentration of 0.5 ml/L. When the temperature stabilized at 28.0±1.0°C, culture medium were seeded content inoculations bottles for bulb seeds in the ratio of 2.5%. the pH of the culture in the fermenter was maintained at 5.0±0,2 28-30% ammonium hydroxide. The growth of the fermentation was controlled by measuring the wet weight of the cells (WCW).

Fermentation was carried out for 15±2 hours and up to the final wet weight of cells ≥20 g/l of the culture fermentation inoculum was transferred sterilized by steam transmission lines in autoclaved inoculation capacity. A sample of the final fermentation inoculum was checked for contamination by other microorganisms.

Table 2
The nutrient medium for fermentation inocula
ComponentsConcentration
Phosphoramidate, 85%8.8 g/l
Calcium sulfate dihydrate0,93 g/l
Magnesium sulfate heptahydrate14.3 g/l
Potassium hydroxideto 4.2 g/l
Ammonium sulfate5.0 g/l
Potassium sulfateof 18.2 g/l
Glycerin, 100%of 7.9 ml/l
PTM4 trace of salt (see table 3)3.0 ml/l

Table 3
PTM4 trace salts
ComponentsConcentration
Copper sulfate, pentahydrate2.0 g/l
Sodium iodide0.08 g/l
Sodium molybdate, dihydrate0.2 g/l
Zinc chloride7,0 g/l
Iron(II) sulfate, heptahydrate2.0 g/l
Boric acid0.02 g/l
Cobalt chloride, uranyl0.5 g/l
Magnesium sulfate monohydrate3.0 g/l
d-Biotin0.2 g/l
Sulfuric acid1.0 ml/l

Industrial fermentation

The purpose of an industrial fermentation was to produce high levels of the protein rNAPc2/Proline. To achieve this, the culture was grown to high cell density before induction of gene rNAPc2/Proline. Nutrient media for industrial fermentation (table 4) prepared in an industrial fermenter. These components of the nutrient medium was dissolved and mixed with purified water USP, and then sterilized by steam. The vessel was cooled to the initial operating temperature of 28.0±1,0°C. Then was added to sterilized filtration 5% (vol./about.) the solution of antifoam KFO880 in 50%methanol. pH led to the initial operating range of 5.0±0,3 sterilized by filtration 28-30% ammonium hydroxide. When was achieved initial operating conditions, the nutrient medium were seeded content inoculations capacity fermentation inoculum against the AI 1 kg of inoculum per 10 kg initial dose of the nutrient medium (mass before making inoculum).

Table 4
Nutrient medium for industrial fermentation
ComponentsConcentration
Phosphoric acid, 85%of 8.8 ml/l
Calcium sulfate dihydrate0,93 g/l
Magnesium sulfate heptahydrate14.3 g/l
Potassium hydroxideof 4.13 g/l
Potassium sulfateof 18.2 g/l
Ammonium sulfate5.0 g/l
Glycerin, 100%to 23.8 ml/l
Salt PTM4 (see table 3)3.0 ml/l

Industrial fermentation consisted of four different phases: the periodic filing of glycerol feeding glycerin, adaptation to methanol and induction with methanol. Throughout the fermentation was maintained the level of dissolved oxygen of approximately 35% by adding air at a constant speed and use of back pressure, and AC is spent mixing. If required supplemental oxygen as soon as achieve maximum mixing, air flow, added oxygen. the pH of the culture in the fermenter was maintained 28-30% ammonium hydroxide. Periodically solution was added defoamer for controlling foaming.

In the first phase of fermentation, the phase of the periodic submission of glycerol formed biomass. The fermenter operated at 28±2°C until the exhaustion of glycerol in the medium, which is detected by peak oxygen caused by the cessation of metabolism of glycerol.

It was followed by phase feeding glycerin, in which 50% wt./wt. the glycerin solution was supplied to the culture with a speed of 18.0±1.0 ml/kg of body weight prior to the introduction of inoculum per hour in total over 8.5 hours, in order to increase the biomass and to suppress the expression. Within the first 4.5 hours of this phase feeding glycerin specified pH of the culture was diluted with 5.0 ą 0.3 to 2.9±0.1 at a rate of 0.5 pH unit per hour and maintained such a pH in the remaining time of fermentation, i.e. during induced by methanol phase induction of the gene. Throughout this phase maintained the temperature at 28±2°C. Before the end of phase feeding glycerin WCW was ≥225 g/l

In the phase of adaptation to methanol feed of glycerol was discontinued and replaced by the supply of methanol, which stimulated the body to produce rNAPc2. The flow of methanol (sod who rasego of 6.0 ml/l antifoam KFO880) started with 3.0 ml/kg before the introduction of the inoculum. The culture was tested for adaptation to methanol, starting 2 hours after the start of addition of methanol. Test adaptation to methanol consisted of short-term cessation and confirm peak of dissolved oxygen. After the first four hours of the addition of methanol, the temperature was lowered to 25±1°C for 2 hours. After the first four hours of the addition of methanol and after confirming that the culture used the methanol feed rate of methanol was increased to 1.0 ml/kg of body weight prior to the introduction of inoculum per hour. The methanol consumption was measured every hour, to ensure that the methanol is completely exhausted at this point, the feed rate of methanol was increased to 1.0 ml/kg of body weight prior to the introduction of inoculum per hour until the final feed rate of 6.0 ml/kg of body weight prior to the introduction of inoculum per hour.

During the phase of induction with methanol process conditions corresponding to the end of the phase of adaptation to methanol, maintained throughout the remainder of the fermentation. Starting in approximately 48 hours the total time of fermentation products rNAPc2/Proline controlled, determining the concentration in the supernatant liquid nutrient medium, measured by reversed-phase C8. The production fermenter was collected through 144-168 hours in an industrial fermenter and after concentration of rNAPc2/Proline measured by reversed-phase C8 was ≥0.55 g/l Sample final fer the orientation was checked for contamination by other microorganisms.

During the phase of adaptation to methanol and phase induction with methanol supported pH 2,9±0,1. Liquid medium for fermentation had a pH of 2.9±0,1.

Example 2.2 Selection

This section describes the selection operation for the production of rNAPc2/Proline. The allocation process rNAPc2/Proline consisted of the operation unit chromatography in the suspended layer, as shown in the block diagram in figure 3.

Ion-exchange chromatography on Streamline SP XL

The purpose of stage ion-exchange chromatography on Streamline SP XL was to separate the rNAPc2/Proline from cell fragments and put the product in a buffer suitable for the first phase chromatographic purification. The environment that was used to achieve separation, was a column for ion exchange chromatography in a suspended layer of resin Streamline SP XL (Amersham Biosciences).

The liquid fermentation medium (pH 2,9±0,1) was diluted with purified water until the conductivity reached ≤9 MSM/see Solution was brought to a concentration of 150 mm acetate, and the pH was brought to pH 3,1±0,2, using 17, 4M acetic acid. Boot solution was applied to the spreading layer of resin, which was balanced by 500 mm sodium acetate, pH of 3.2, followed by 50 mm sodium acetate, pH of 3.2. The column was washed upward flow of 50 mm sodium acetate, pH 3.2, and then 50 mm sodium acetate/150 mm NaCl, pH of 3.2. Layer of the resin allowed to settle and additionally is washed downflow 50 mm sodium acetate/150 mm NaCl, pH of 3.2. rNAPc2/Proline was suirable by injection of 50 mm sodium acetate/350 mm NaCl, pH of 3.2, and the concentration of rNAPc2/Proline was measured by the method of reversed-phase C8.

In preparation for the stage purification by chromatography on Source ONE solid sodium sulfate was added to the eluate to Streamline final concentration of 0.85 M pH resulted 3,1±0,2 using 2.4 M citric acid, and it was confirmed that the conductivity is 100±10 MSM/see the condition of the eluate Streamline was filtered through 0.45 µm filters.

Example 2.3. Clean

This section describes the cleaning procedures for the production of rNAPc2/Proline. Industrial purification method for rNAPc2/Proline consisted of phase, hydrophobic chromatography, stage ultrafiltration and diafiltration, stage ion-exchange chromatography, followed by stage ultrafiltration/diafiltration, and final filtration and filling the drug rNAPc2/Proline, also known as active pharmaceutical ingredient (API), as shown in figure 4.

Hydrophobic chromatography on Source 15PHE

The initial stage of purification of partially purified product by removing some protein and non-protein contaminants of rNAPc2/Proline, using a column with the environment Source 15PHE for hydrophobic chromatography (Amersham Biosciences).

Filtered contained in a proper state of the eluate Streamline was introduced the column with the Source 15PHE, pre-equilibrated with 50 mm sodium citrate/1,1M sodium sulfate, pH 3.0. After loading the column was washed equilibrating buffer. Protein rNAPc2/Proline was elyuirovaniya from the column with 15 volumes of the column with a gradient from 1,1M to 0,3M sodium sulfate in 50 mm sodium citrate, pH 3.0, followed by a gradient was maintained 0,3M sodium sulfate until the UV absorption ability did not return to the zero line. Faction climbed across the width of the peak elution of rNAPc2/Proline and then analyzed by reversed-phase C18. The fractions containing rNAPc2/Proline high purity were combined and examined the concentration of UV breakdown. the pH of the combined fractions Source 15PHE was brought to pH 5,3±0,1 addition of 5N. NaOH.

Stage ultrafiltration/diafiltration #1 (UF/DF #1)

The purpose UF/DF #1 was to concentrate the product and translate rNAPc2/Proline in the buffer used for chromatography on Source 15Q. Used filters for ultrafiltration of cellulose fibers with pores for substances with a molecular mass of 3 KD.

Combined fractions Source 15PHE with adjusted pH was concentrated to 2.0±0.5 g/l (measured concentration by UF) membranes UF/DF #1, which had been pre-equilibrated with 50 mm sodium acetate, pH of 5.3. Then the combined fractions were diafiltration with ≥5 volumes of 50 mm sodium acetate, pH of 5.3, and while pH was 5.3±0,1, electraprobe the activity was < 6,0 MSM/see Subject diafiltration combined fractions UF/DF#1 was filtered through the 0.2-μm filter to prepare for loading the column with Source 15Q.

Ion-exchange chromatography on Source 15Q

The operation of the final block chromatography removed most of the remaining protein and non-protein contaminants of rNAPc2/Proline using columns with Source 15Q environment for ion-exchange chromatography (Amersham Biosciences).

The filtered combined fractions UF/DF #1 was injected into the chromatographic column with Source 15Q, pre-equilibrated with 500 mm sodium acetate, pH of 5.3, and then 50 mm sodium acetate, pH of 5.3. After loading the column was washed balanced buffer with 50 mm sodium acetate, pH of 5.3. In the column was introduced linear gradient from 0 to 400 mm NaCl in 50 mm sodium acetate, pH 5,3, volume 20 column volumes. Fractions were climbed during the peak elution and analyzed by reversed-phase C18. The fractions containing rNAPc2/Proline high purity were combined and examined their concentration UV breakdown.

The final stage of ultrafiltration/diafiltration (final UF/DF)

The purpose of the final UF/DF was to concentrate the product and translate rNAPc2/Proline in the final buffer composition. Used filters for ultrafiltration of cellulose fibers with pores for substances with a molecular weight of 3 kDa.

United Source15Q fractions were concentrated to 12.0±0.5 g/l (measured concentration by UV-sample) on the membranes of the final UF/DF, pre-equilibrated with buffer a final composition, 65 mm sodium phosphate/80 mm sodium chloride, pH 7.0. Combined fractions were then subjected to diafiltration with ≥6 volumes of buffer composition up until the pH was equal to 7.0±0,1.

Example 2.4. Filtering in the stream and filling

Purified API rNAPc2/Proline moved into the room class 100 and filtered using the 0.2-μm Millipak filter, autoclaved 1-liter cast bottle Nalgene Tefzel® FEP (fluorinated propylene-ethylene) series 1600 with cast uncontaminated Tefzel®ETFE (copolymer of tetrafluoroethylene and ethylene) screw cover without gasket. The bottle was moved to a freezer with a temperature of -20±10°C for storage.

The main volume of the API can be re-filtered and bottled using the same method for the final filtration. The contents of the Teflon FEP bottles was transferred into autoclaved wrapped the bottle in a clean room of class 100, re-filtered and bottled. Regarding storage tanks and covers: FEP, and ETFE meet the requirements of the amendments on food additives of the Federal law the U.S. food, drug and cosmetic tools. The material meets the requirements specified for class VI USP.

Example 3. The control process; research methods rNAPc2/Proline in the process

Conditions, which controls who were ovalis, including eligibility criteria of the process are listed in the flowcharts of the process (Fig. 2-4). Brief descriptions of the methods of the study process are listed below.

Research methods rNAPc2 in the process

pH. The sample was monitored using the pH meter, which was calibrated with NIST traceable standards pH immediately before the examination. the pH of the samples was read at 25±2°C.

The conductivity. Electrolytic components of the solution was measured using a conductivity meter that has been standardized by the standards of electrical conductivity in the range measurements. Electrical conductivity of the samples was read at ~25°C.

Wet weight of cells. Approximately 1.5 ml of sample was added to the fermentation balanced microcentrifuge tubes and centrifuged at 10,000 rpm for approximately 5 minutes. The supernatant in each tube was decanted from the sediment and tubes containing solid substance was weighed. Wet weight of cells was equal to the weight divided by the initial volume of the sample.

Analysis of contamination by other microorganisms. The final sample of liquid medium for fermentation inoculum and industrial fermentation was tested for contamination by other microorganisms, sowing 100 ál of each of the nine Petri dishes with TSA. Three Petri dishes were incubated p and three temperatures (20-25°C, 30-34°C and 35-39°C). Within seven days of incubation, the Petri dishes were examined for microbial colonies that differed from the typical owner, in particular, it was noted differences in colony morphology, color and/or size of the colony. The final reading data also were subjected to staining by gram stain. The analysis incorporated an adequate negative controls.

Analysis by reversed-phase C8 (concentration and purity). Supernatant samples of industrial fermentation samples Streamline SP XL was filtered through a 0,22 µm filter and then introduced into Kromasil CS, a 4.6×250 mm reverse-phase column. The column was equilibrated 22%acetonitrile, 0.1% of triperoxonane acid (TFA) prior to sample introduction. Then was running a linear gradient from 22-28% acetonitrile in 0.1% TFA for twenty minutes at 1 ml/min to the eluted substance rNAPc2/Proline. Standard cultivation rNAPc2/Proline with known concentrations were used to create a calibration curve based on linear regression mg/ml rNAPc2/Proline against peak area. The number of rNAPc2/Proline in any sample is extrapolated from the calibration curve and divide by the volume of the introduced sample to determine the concentration of rNAPc2/Proline in the samples. The purity of rNAPc2/Proline was calculated as a percentage of the total peak area.

Concentration by UV. The concentration of each of the combined purified fractions, starting with the Source 15PHE and d is the final UF/DF, inclusive, was determined using absorption capacity at 280 nm respectively on a calibrated spectrophotometer. Prior to entering the investigated samples, the instrument was zeroed using a suitable buffer solution. The investigated samples were prepared by diluting within the linear range (from 0.13 to 1.62 AU). The average absorption capacity at 280 nm divided by the absorbance (0,59 AU/cm-1(mg/ml)-1) and was multiplied by the dilution factor to obtain the concentration in mg/ml

Reversed-phase C18 (purity). The purity of fractions and common fractions Source 15PHE, United fractions UF/DF #1, fractions and common fractions Source 15Q and the combined fractions of the final UF/DF was in each case analyzed by reversed-phase C18. rNAPc2/Proline was separated from the other components of the sample by reversed-phase chromatography with a linear gradient. If necessary, samples were diluted to approximately 1 mg/ml in cPBS and 30 μl was injected into the reverse-phase column Waters Symmetry C18 (particle of 5 μm, inner diameter 4.6 mm, length 250 mm, Waters Corp., Bedford, Massachusetts), balanced 78% mobile phase A (0.1% OF TFU in water) and 22%mobile phase B (0.1% OF TFU in acetonitrile). The percentage of mobile phase B was then linearly increased to 26% in twenty minutes using a flow rate of 1 ml/minute. Peaks were verified UV detector is ω at 210 nm. The purity of rNAPc2/Proline was calculated by dividing the peak area rNAPc2/Proline on the total peak area on the chromatogram and expressing this ratio in percent.

Example 4. Research tools API rNAPc2/Proline

Appearance, pH and concentration: an aliquot of the sample for testing examined visually for color, opacity, and any visible impurities. The sample was read using a pH meter, calibrated NIST traceable standards pH immediately prior to the study. the pH of the samples was read at 25±2°C. the Concentration of samples was determined by its absorption capacity at 280 nm respectively on a calibrated spectrophotometer. The device was reset to zero using thinner samples to the input of the investigated samples. For studies made on three samples, diluting within the linear range (from 0.13 to 1.62 AU), established during the validation of the method. The average absorption capacity at 280 nm divided by 0.59 AU/cm-1(mg/ml)-1(attenuation coefficient) and multiplied by the dilution factor to obtain the concentration in mg/ml

Peptide map: before enzymatic splitting the sample rNAPc2/Proline for test and reference rNAPc2/Proline restored and alkilirovanie. rNAPc2/Proline was denaturiruet processing a high concentration of guanidine hydrochloride, and then restored ditio what raitala. Restored cysteine then alkilirovanie iodoacetamide. Drugs recovered and alkylated rNAPc2/Proline was cut to 2% wt./wt. trypsin for approximately 16 hours at 37±2°C. Trypsinogen peptides from each of the split samples protein rNAPc2/Proline were then separated by reversed-phase chromatography, to create a schema fragments in the form of a chromatogram or "fingerprint". The elution profile of the sample was visually compared with the standard elution profile using peak retention time. Profiles should be comparable, without new or missing peaks.

Electrophoresis in LTO-page with Kumasi (identity/purity): samples for testing, the standard of rNAPc2/Proline and a marker of the intensity of rNAPc2/Proline was diluted with the addition of reducing agent and without it, using Novex NuPAGE® LDS buffer for sample preparation (pH 8,4), to a final concentration of 0.5, 0.5 and 0.005 mg/ml, respectively. The mixture of protein standards (Novex Mark 12®) was diluted according to the manufacturer's instructions. The recovered samples were heated for five minutes to 95±2°C. the Restored and unrestored samples were separated on a single gel. Desyatikilogrammovye trial download of standard and samples for testing, 0,1-microgramma download token intensity (1% sample load) and adequate weight standard Mark 12 were analyzed what electrophoreses on the finished 4-12% acrylamide Bis-Tris gel NuPAGE Novex® with a pH of 6.4. Gels were stained colloid, Kumasi blue Novex. To confirm the identity, the main strip was visually compared with the standard and with a mixture of protein standards. The intensity of all bands contamination was visually compared with the marker strip 1%intensity. Noted any stripe contamination in excess of the marker. If the strip pollution, superior marker was absent, it was noted that the purity comparable to the reference standard. It is noted that the strip of rNAPc2/Proline did not appear in the expected size of the molecules. Due to its non-spherical shape rNAPc2/Proline moved as a molecule more apparent molecular weight. In unrecovered gels band rNAPc2/Proline moved between standards 21.5 and 31 kDa, while in the recovered gels band rNAPc2/Proline moved together with the standard of 21.5 kDa. (Products Novex from Invitrogen Corp., Carlsbad, California).

Analysis on a reversed-phase C18: rNAPc2/Proline was separated from the sample components by reversed-phase HPLC with a linear gradient. About the purity of rNAPc2/Proline reported as the ratio of the peak area rNAPc2/Proline to the total peak area on the chromatogram, expressed in percent. Tridtsatilitrovyj volumes of dilutions of the sample to approximately 1 mg/ml in cPBS was introduced in reversed-phase column Waters Symmetry C18 (particle of 5 μm, 4.6 mm internal diameter × 250 mm length, Bedford, mA) equilibrated 78% of mobile phase A (0.1% OF TFU in water) and 22% of mobile phase B (0.1% OF TFU in acetonitrile). The percentage of mobile phase B was then linearly increased to 26% in twenty minutes using a flow rate of 1 ml/minute. Peaks were monitored by UV detector at 210 nm.

Endotoxin: measuring the endotoxin was performed according to USP method.

Bioactivity: rNAPc2/Proline depending on the concentrations were prolonged the clotting time of human plasma initiated by the addition of thromboplastin. The anticoagulant effect of rNAPc2/Proline on the coagulation of human plasma was measured directly in the automated analysis of prothrombin time (PT)using thromboplastin rabbit brain (tissue factor, Simplastin Excel)to initiate clotting. And the standard of rNAPc2/Proline, sample and rNAPc2/Proline was diluted to 1035 nm in analytical buffer. The measuring device (Coag-A-Mate® MAX, Organon Teknika, now belonging to biomérieux basis, Durham, North Carolina), then did a series of dilutions of human plasma starting mixture and measured the resulting clotting time (SU) in seconds. Graphs were constructed by fitting the linear regression of the logarithm of the sun rNAPc2/Proline against concentration dilution. Then calculate the bioactivity of the sample for testing as the ratio of the slope of a plot of sample for testing to the slope of a plot of the time pattern of activity pattern.

Binaratka: the total number of anaerobes (TAS) and total yeast/mold areas rich in mushrooms is (TYMC) in the sample was determined by filtration of two 10-ml aliquot through a separate 0.45 µm membrane filters from cellulose ethers. Membrane filters were processed and incubated in a Petri dish with agar TSA at 30-35°C for 48-72 h, and the other in a Petri dish with agar SDA at 20-25°C for 5-7 days. After an incubation period were counting colony-forming units (CFU) on both types of agar. Reported the combined number of CFU per 10 ml sample.

Residual DNA: Analysis on the threshold total DNA (Molecular Devices Corp., Sunnyvale, California) was specific for single-stranded DNA. It was held in three stages. In the reaction stage single-stranded DNA reacted with two binding proteins in the labeled reagent. One binding protein possessed high affinity, binding protein single-stranded DNA (SSB) from E. coli, conjugated to Biotin. Streptavidin, also present, tightly contacted with Biotin in the conjugate SSB. Another binding protein was a monoclonal antibody against single-stranded DNA, kongugirovanne to the enzyme urease. Data binding proteins form a complex compound with DNA in solution at 37°C.

Phase separation was carried out on the Threshold Workstation. Compound DNA was filtered through nitrocellulose membrane coated with Biotin. The Biotin on the membrane reacted with streptavidin in complex DNA connection, capturing the complex compound. In the stage of rapid washing of the membrane were removed especificas the th enzyme. During the detection phase coli (containing nitrocellulose membrane coated with Biotin) were placed in Threshold Reader, which contained the substrate urea. The enzyme reaction was change the local pH of the substrate solution. The silicon sensor recorded the change in surface potential, which was proportional to the change of the pH value. In each plot to measure changes in surface potential was controlled using the Threshold computer Workstation and Software Threshold. The computer analyzed the data kinetic measurements and quantify the results using the previously established standard schedule. Using Threshold Software to calculate the concentration of each sample in pilgrammage DNA.

Size-exclusion chromatography: rNAPc2/Proline was separated from the other components of the sample exclusion chromatography on the basis of differences in the size of the molecules. The identity of rNAPc2/Proline was confirmed by comparing the average retention time (RT) of the three repetitions of the sample with the five standards of fitness system. % RT should be 97,0 to 103,0%. The purity of rNAPc2/Proline was calculated by dividing the peak area rNAPc2/Proline on the total peak area of the chromatogram and expressing it in percentage. Prepared dilution in cPBS to a nominal concentration of approximately 1 mg/ml and was introduced to their exclusion column (Superdex 75 10/30, Amersham Biosciences). Maintained a flow rate of 0.5 ml/min Peaks to what was trenirovali UV detection at 210 nm.

Molecular weight according to mass spectrometry: molecular mass was determined by electrospray mass spectrometry using a VG Bio-Q (Quattro II Upgrade) quadrupole mass spectrometer (Micromass, Danvers, Massachusetts, currently owned by Water Corp., Bedford, Massachusetts). The sample was diluted to approximately 1 mg/ml in water with 0.1%solution triperoxonane acid and introduced into a pre-washed Trap Cartridge to remove salts. Then the cartridge was washed through the port for input and washout were introduced into the spectrometer.

Determination of N-terminal sequence: determined the sequence of the 15 N-terminal residues of the sample for testing using a Procise N-Terminal Sequencing System (Applied Biosystems, foster city, California). Before and after the test was determined sequence of 15 residues of the calibration standard β-lactoglobulin. Cys (cysteine) residues were observed on the Procise system. The obtained sequence was compared with theoretical sequence of sample for testing.

Example 5. The production of a medicinal product rNAPc2/Proline

Example 5.1. Production of liquid medicinal product

The production process of a liquid medicinal product rNAPc2/Proline displayed in figure 5, a block diagram of a medicinal product. Frozen API rNAPc2/Proline was extracted from -20°C storage and Tivoli at 2-8°C. After thawing API transferred to the mixing zone for combining and mixing. To produce a medicinal product, the API was diluted to 65 mm sodium phosphate/80 mm NaCl, pH 7.0, to a final concentration of 1.0±0.1 mg/ml, as measured by the concentration of the UV probe (described above). This dilution was performed stepwise with concentration measurements in the process to achieve the specified concentration. Diluted API then filtered through a 0.2 μm filter Millipore Millipak and placed in storage for short-term storage at 2-8°C.

For the filling stage diluted API was filtered under aseptic filling a set of two walk-through filter Millipore Millipak. To determine the sterility of the main volume was sampled. Diluted API then poured into sterilized vials of 2 cm3that was immediately closed with corks and caps. The final volume in the vials was 0.6 ml

To store 100% of the vials were visually examined under controlled conditions, using lights and backgrounds, designed to illuminate the bottles and the product is so defective vials or bottles containing visible particles could be easily detected and removed from the party. The vials were then loaded in labeled trays for storage and kept in storage for short-term storage at 2-8°C and storage for long-term storage the AI at -20±10°C. Table 5 shows the composition of the liquid pharmaceutical product rNAPc2/Proline on the bottle.

how much will it take to bring the pH to 7,0±0,1
Table 5
Liquid pharmaceutical composition rNAPc2/Proline
The name of the substanceQualityQuantity per vial
rNAPc2/Prolineno data0,6±0,06 mg
Sodium hydrogen phosphate heptahydrateUSP6.4 mg
Sodium dihydrophosphate monohydrateUSP2.1 mg
Sodium chlorideUSP2.8 mg
Water for injectionUSPhow much will it take to 0.6 ml
Phosphoric acidNFhow much will it take to bring the pH to 7,0±0,1
1 N. sodium hydroxide in water for injectionprepared from granules, corresponding NF

Example 5.2. Production of liofilizirovannogo medicinal product

Solution the main mass of the medicinal substance rNAPc2/Proline (with a concentration of 12±1.2 mg/ml in 65 mm sodium phosphate/80 mm sodium chloride with a pH of 7.0±0,1) was diluted to 3 mg/ml solution of 0.2m alanine and 25 mm sodium dihydrophosphate, pH 7.0. Diluted rNAPc2/Proline was then subjected to replace the buffer in the solution of alanine/phosphate. A solution of rNAPc2/Proline was then diluted to 2 mg/ml (the concentration was measured by UV breakdown) solution of alanine/phosphate. A solution of 2 mg/ml rNAPc2/Proline was then diluted with an equal volume of 25 mm sodium phosphate, 4%sucrose, pH 7.0, in order to achieve concentrations of rNAPc2 of 1.0±0.1 mg/ml. Finally created a solution of 1 mg/ml rNAPc2/Proline was filtered using the 0.2-μm filter.

To fill the solution rNAPc2/Proline, 1 mg/ml, filtered through the 0.2-μm filter. Then rNAPc2/Proline poured in separate sterilized glass bottles 3 cm3and covered tubes. Then the vials were subjected to freeze-drying in liofilizadora. After lyophilization stoppers pushed deeper and vial wore caps. Liofilizovannye drugs remains high purity and long-term stability if the medicinal product NAP subjected to heavy thermal load, for example 28 days at 50°C. In table 6 are listed with the becoming of liofilizirovannogo medicinal product rNAPc2/Proline on the bottle.

Table 6
Liofilizovannye pharmaceutical composition rNAPc2/Proline
The name of the substanceQualityQuantity per vial
rNAPc2/Prolineno data0,83 mg
AlanineUSP7.5 mg
Sodium dihydrophosphate monohydrateUSP2,9 mg
SucroseUSP3,3 mg
Water for injectionUSPhow much would it take to bring the volume to 0.83 ml
1 N. sodium hydroxide in water for injectionprepared from granules, corresponding NFhow much will it take to bring the pH to 7,0±0,1

Example 6. Forecast isoelectric point of the protein NAP

Isoelectric point (pI) of different NAP was determined to confirm that the method disclosed here are suitable for the manufacture of other drugs NAP and medicinal substances NAP. Sequences of proteins NAP disclosed in 5866542, were processed by programs predict pI. Table 7 presents pI NAP proteins disclosed in the U.S., as it was calculated ProtParam and Atalier BioInformatique. ProtParam, using ExPASy (ExpertProteinAnalysisSystem), developed by the Swiss Institute of Bioinformatics (SIB), found athttp://us.expasy.org/tools/protparam.htmlmaster server is the North Carolina Supercomputing Center (NCSS). Atalier BioInformatique (aBi) found athttp://www.up.univ-mrs.fr/~wabim/d_abim/compo-p.html,the master server is the University of Aix-Marseille I.

Table 7
Forecast isoelectric points (pI) of proteins NAP
Name sequencepI calculated ProtParampI calculated Atalier BioInformatique
AcaNAP54,324,10
AcaNAP64,254,03
AcaNAPc2or 4.314,10
AcaNAPc2/Prolineor 4.314,10
AcaNAP234,30
AcaNAP244,72of 4.45
AcaNAP254,724,48
AcaNAP31,of 42.464,284,07
AcaNAP444,744,48
AcaNAP484,344,13
AceNAP54,494,25
AceNAP7to 4.624,37
AduNAP44,554,33
HpoNAP5a 7.627,50

1. Method for the production of pharmaceutically acceptable anticoagulant protein (NAP) nematodes, including
(a) providing methanotrophic yeast host cells encoding NAP, selected from the group consisting of rNAPc2 and rNAPc2/Proline;
(b) the fermentation of the above host cells, where the fermentation process includes fermentation inoculum for the growth of these cells in host to the desired glue the face-to-face density, the productive process of fermentation, which involves the fermentation with periodic feeding of glycerol fermentation by feeding glycerin, fermented with adaptation to methanol and fermentation with induction with methanol, where this fermentation is carried out at pH values below 4;
(c) the allocation specified in the NAP of the above formatiruem host cells, where the process of selection includes cation exchange chromatography in the suspended layer to separate NAP from the said yeast host cells and cell remnants, where the specified allocation process is carried out at pH values below 4; and
(d) purging the specified NAP, where the cleaning process includes
i) the conduct of hydrophobic interaction chromatography, where
specified hydrophobic interaction chromatography is carried out at pH values below 4,
ii) collecting fractions NAP after a specified hydrophobic interaction chromatography,
(iii) conducting at least one ultrafiltration/diafiltration (UF/DF) fractions NAP
(iv) carrying out anion exchange chromatography of a solution NAP, and
(v) collecting fractions NAP after anion-exchange chromatography.

2. The method according to claim 1, where the NAP is a rNAPc2.

3. The method according to claim 1, where the NAP is a rNAPc2/Proline.

4. The method according to claim 1, where methanotrophic yeast cell host is Pichia pastoris.

5. The method according to claim 1, where the fermentation process which engages the fermentation inoculum, where the host cell grows to the desired cell density, and the productive process of fermentation, where the NAP is produced to the desired title.

6. The method according to claim 5, where the productive process of fermentation involves the following sequential order: fermentation with periodic feeding of glycerol fermentation by feeding glycerin, fermented with adaptation to methanol and fermentation with induction with methanol.

7. The method according to claim 6, where productive fermentation performed for up to about seven days, in this period, the titer NAP continuously increases.

8. The method according to claim 1, further comprising maintaining the temperature of fermentation.

9. The method of claim 8, comprising maintaining the temperature of the fermentation process of adaptation to methanol approximately 28±2°C for approximately the first four hours and approximately 25±1°C in the remaining time of the execution process of adaptation to methanol.

10. The method according to claim 1, further comprising maintaining the pH of the fermentation within around 2.9±0.1 pH units during fermentation adaptation to methanol and fermentation induction with methanol.

11. The method according to claim 1, where the ion-exchange chromatography in the suspended layer includes ion-exchange chromatography in a suspended layer of ion-exchange resin Streamline SP XL when the pH value is approximately equal to 2.9±0,1 common is C pH.

12. The method according to claim 1, where the chromatography hydrophobic interaction includes conducting hydrophobic chromatography on Source 15PHE when the pH value is approximately equal to 3.0±0.1 pH units.

13. The method according to claim 1, where anion-exchange chromatography involves the use environment Source 15Q for ion chromatography.

14. The method according to claim 1, additionally including at least one final UF/DF for fractions after anion-exchange chromatography.

15. Method for the production of pharmaceutically acceptable rNAPc2/Proline, including
(a) providing host cells Pichia pastoris, coding rNAPc2/Proline;
(b) fermenting the specified host cells in the fermentation process, where the fermentation process involves the fermentation inoculum, where the host cells multiply until the desired cell density, and the productive process of fermentation, which comprises the following sequential order: fermentation with periodic feeding of glycerol fermentation by feeding glycerin, fermented with adaptation to methanol and fermentation with induction with methanol, lasting approximately seven days, where this fermentation is carried out at pH below 4;
(c) allocation of rNAPc2/Proline of these formatiruem host cells, where the process of selection includes cation exchange
chromatography in the suspended layer on the I branch of the rNAPc2/Proline from cells and cell remnants, where the specified selection is carried out at pH below 4; and
(d) purification of rNAPc2/Proline of the selected rNAPc2/Proline using a purification process involving hydrophobic interaction chromatography using a medium for hydrophobic interaction chromatography, where this hydrophobic interaction chromatography is carried out at pH values below 4, collecting fractions of rNAPc2/Proline after the specified hydrophobic interaction chromatography, performing at least one ultrafiltration/diafiltration (UF/DF) fractions rNAPc2/Proline with obtaining the solution of rNAPc2/Proline, performance anion-exchange chromatography of a solution of rNAPc2/Proline and collecting fractions of rNAPc2/Proline containing pharmaceutically acceptable rNAPc2/Proline after anion-exchange chromatography.

16. The method according to clause 15, further comprising maintaining the temperature of fermentation.

17. The method according to clause 16, which includes maintaining the temperature of the fermentation adaptation to methanol approximately 28±2°C for approximately the first four hours and approximately 25±1°C in the remaining time of fermentation adaptation to methanol.

18. The method according to clause 15, which includes maintaining a pH of approximately 2,9±0,1 during fermentation adaptation to methanol and fermentation induction with methanol.

19. The method according to clause 15, where cation exchange chromatography include ion exchange chromium is ografia in suspended layer of ion exchange resin Streamline SP XL at pH approximately 3,2±0,2.

20. The method according to clause 15, where the hydrophobic interaction chromatography includes performing hydrophobic interaction chromatography using Source 15PHE at a pH of approximately 3.0±0,1.

21. The method according to clause 15, where anion-exchange chromatography include ion-exchange chromatography on Source 15Q.

22. The method according to clause 15, further comprising performing at least one final UF/DF fractions after ion exchange chromatography.

23. A method of obtaining a pharmaceutical composition comprising purified protein NAP, obtained by the method according to claims 1 to 22, including:
(a) the introduction of purified protein NAP in the final pharmaceutical composition comprising pharmaceutically acceptable excipients;
(b) implementation of the seeding process, including the filter in the flow of purified protein NAP in the final pharmaceutical composition, and
(c) implementation stage of filling, including the measurement of purified protein NAP in a unit dosage form in a container, to obtain a liquid medicinal product protein NAP.

24. The method according to item 23, further comprising a liquid pharmaceutical lyophilization of the product protein NAP in the tank.

25. The method according to item 23, where the NAP protein selected from the group consisting of rNAPc2 and rNAPc2/Proline.



 

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18 cl, 1 dwg, 4 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: adhesive protein containing protein and peptide repeated at least once, which is combined to carboxy- and/or amino-ends of the protein, is described. Polynucleotide containing nucleotide sequence, which encodes the adhesive protein, is represented. Expression vector containing functionally incorporated nucleotide sequence, which encodes the adhesive protein, is described. Transformant, which is transformed by the vector selected from the group, which contains prokaryote, eukaryote and cells, derived from eukaryotes, is represented. The methods of producing and purification of the adhesive protein are represented. Adhesive containing the adhesive protein as an active component in efficient number is offered. The method for regulation of adhesive strength of the present adhesive, including treatment with the substance selected from the group, which contains oxidising agent, filler and surfactant, or regulation of concentration of the adhesive protein, which is the surfactant of the adhesive, is described. Covering agent containing the adhesive protein as an active component in efficient number is offered.

EFFECT: present invention allows producing adhesive protein, which can be efficiently produced in volume to use them instead of chemical adhesive.

42 cl, 33 dwg, 1 tbl, 17 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to area of gene engineering and biotechnology and can be used for labeling biological objects. The nucleic acid molecule was extracted that encoded the fluorescing protein selected from fluorescing proteins represented by the Copepoda Crustacea biological kinds and fluorescing mutants of the aforesaid proteins. The aforesaid nucleic acid was functionally bonded to appropriate elements of expression regulation to be used in the method of producing aforementioned fluorescing protein. On the basis of the aforesaid extracted nucleic acid cloning and expressing vectors are produced as well as the expressing cassette. The cell and a stable cellular lineage, containing the aforesaid expressing cassette produce the fluorescing protein. Note that the said fluorescing protein, the nucleic acid encoding the protein above and expressive genetic structures containing the said nucleic acid, are used incorporated with the set designed to label a biological molecule. The fluorescing protein is also used in the methods of labeling a biological molecule, a cell or a cellular organella.

EFFECT: expanded biological objects labeling means.

12 cl, 9 dwg, 1 tbl, 7 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to gene engineering and biotechnology and can be used in a pharmaceutical industry. Cleared and extracted polynucleotide encodes protein with functional activity of a glucose carrier and the GLUT4 protein amino acid lineage wherein valine in position 85 is replaced by methionine. The protein under consideration features biological activity in yeast strains S. cerevisiae deprived of functional carriers of glucose and Erg4 protein.

EFFECT: production of protein with functional activity of glucose carrier in yeasty expressing system.

5 cl, 3 tbl, 1 ex

FIELD: medicine; pharmacology.

SUBSTANCE: it is obtained a chimerous photo protein (photin), presented by amino-acid sequence of obeline protein, which part (from the rest in position 50 to the rest in position 94) is replaced by a homologous site of amino-acid sequence of clitine protein (the rests 53-97). The method of obtaining new chimerous photo protein by the method of recombinant DNA and vectors applied to it and cells is described. It is offered to use photo protein under the invention as the calcium indicator in various test systems in vitro and in vivo.

EFFECT: increased level of bioluminescence in comparison with natural protein.

11 cl, 6 dwg, 2 tbl, 3 ex

FIELD: biology, gene engineering.

SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.

EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.

13 cl, 12 dwg,12 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and biotechnology and can be used for labelling of proteins, cells and organisms. Substitutes added to nucleic acid Aequorea coerulescens, coding colourless GFP-like protein acGFP is considered as means of production of nucleic acid coding fluorescent protein. Expression cassette containing this nucleic acid under regulatory elements control provides in-cell biosynthesis of fluorescent protein. This genetic structure is used for production of transgenic organism and cell producing fluorescent protein, coded by specified nucleic acid. This fluorescent protein is applied in methods for labelling of cells, intracellular structure and protein, as well as for promotor transcriptional activity registration.

EFFECT: invention application ensures widen range of labelling means used in biochemistry, molecular biology and medical diagnostics.

11 cl, 34 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: polynucleotide is obtained, coding chromo- or fluorescen mutant wild type DsRed (SEQ ID N0:2), where chromo- or fluorescent mutant contains a substitute in the amino acid position 42 SEQ ID N0:2, and optionally one or more substitutes in the amino acid positions, chosen from a group, consisting of 4, 2, 5, 6, 21, 41, 44, 117, 217, 145. Using the polynucleotide in the vector, the host cells which express chromo- or fluorescent mutant DsRed are transformed. The invention allows for obtaining chromo- or fluorescent polypeptide DsRed, which matures faster than wild type DsRed.

EFFECT: increased effectiveness.

26 cl, 10 dwg, 2 tbl, 4 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to methods of self-specific T-cell vaccine production. Self-specific T-cell vaccine for disseminated sclerosis treatment includes attenuated T-cells which are reactive relative to one or several epitopes, yet SEQ ID NOS: 1-6 contain these epitopes. Invention is intended for treatment of autoimmune diseases, such as disseminated sclerosis or rheumatoid arthritis using self-specific T-cell vaccines. Besides, invention provides diagnostics of diseases associated with T-cells. Advantage of this invention implies that it can be applied for production of T-call vaccines with heterogenous gene VR-Dp-JR to take into consideration clonal shift if self-reactive T-cells.

EFFECT: method has improved efficiency.

10 cl, 7 ex, 2 dwg

FIELD: technological processes.

SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.

EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.

7 cl, 20 dwg, 1 tbl

FIELD: technological processes.

SUBSTANCE: protein variants are suggested that possess lysozyme activity. Protein-based antibacterial substance is described. DNA molecule is discovered that codes the specified protein, and also procariotic cell is discovered that contained the specified DNA molecule. Method is described to prepare protein with the help of procariotic cell.

EFFECT: simplifies preparation of antibacterial substance in commercially significant volumes.

13 cl, 3 ex

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology. Description is given of a single-strand T-cell receptor (scTCR), containing an α segment, formed by a sequence of a variable region in a TCR chain, joined with the N end of the extracellular sequence with constant region in the TCR chain, a β segment, formed by a sequence of the variable region of the α TCR chain, joined with the N end of the extracellular sequence with constant region of the β TCR chain, and a linker sequence, joining the C end of the α segment with the N end of the β segment, or vice versa. Extracellular sequences of constant regions of α and β segments are joined by a disulphide bond. Extracellular sequences of constant regions can correspond to constant regions of α and β chains of native TCR, cut-off at their C ends such that, cysteic residues, which form the inter-chain native disulphide bond of the TCR, are excluded, or extracellular sequences of constant regions which are in the α and β segments, can correspond to constant regions of α and β chains of native TCR, in which cysteic residues, which form the native inter-chain disulphide bond, are replaced by another amino acid residue, or there is no uncoupled cysteic residue, which is in the β chain of the native TCR. This invention makes available a new class of alpha/beta analogues of scTCR, in which there is a disulphide bond between residues of a single amino acid, contributing to stability of the bond between the alpha and beta regions of the molecule.

EFFECT: such TCR are suitable for screening or for therapeutic purposes.

3 cl, 14 dwg, 3 ex

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