RU2769201C2 - Method for producing a highly purified recombinant human c1-esterase inhibitor for application in medicine - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to a method for purifying a recombinant human C1-esterase inhibitor and to an application of the method for producing an active pharmaceutical substance of a recombinant human C1-esterase inhibitor. The proposed method for purification includes viral inactivation, metal chelate chromatography, affinity chromatography, and anion exchange chromatography. When the content of proteins of the producer strain in the product therein is less than 3,000 ng/mg and the level of DNA is less than 50 pg/mg, the product is purified by finishing hydrophobic chromatography using a sorbent containing phenyl groups. Also proposed is an application of the above method for producing an active pharmaceutical substance characterised by the content of a monomeric form of the recombinant human C1-esterase inhibitor of no less than 99.0%, the content of the main form of the recombinant human C1-esterase inhibitor based on the data of reverse-phase HPLC of at least 99.5%, the content of residual proteins of the producer strain of no more than 10 ng/mg, the content of DNA of the producer strain of no more than 5 pg/mg. The specific activity of the resulting active pharmaceutical substance is no less than 9.0 IU/mg.

EFFECT: ensured purification of a recombinant human C1-esterase inhibitor.

11 cl, 1 dwg, 6 tbl, 2 ex

Description

The invention relates to the field of biotechnology, and in particular to the problem of developing highly efficient production technologies that make it possible to obtain highly purified drugs for medical use. In particular, the invention relates to the development of a technology for the production of an active pharmaceutical substance of a recombinant human C1-esterase inhibitor. The invention can be used in the pharmaceutical industry, namely, when creating a drug for the treatment of hereditary angioedema (angioneurotic edema).

Currently, in the treatment of the above diseases, a drug based on a recombinant inhibitor of human C1-esterase obtained from the milk of genetically modified rabbits, Rukonest®, as well as a drug isolated from donor plasma, Berinert®, is used. The recommended dose of these drugs is 500 IU by slow intravenous injection or infusion. In order to improve the quality of life of patients, reduce the incidence of adverse events associated mainly with the degree of purity of the drug used, the inventors proposed a technology for obtaining a highly purified drug.

The following were used as critical quality parameters: the content of proteins of the producer strain, the content of DNA, the content of the monomeric form of the active substance, the content of the main form of the protein according to reverse-phase HPLC, specific activity.

Description of drawings.

Rice. 1. Scheme for the purification of a recombinant human C1-esterase inhibitor.

The following solutions are known in the art for the production technology of a human C1 esterase inhibitor.

Application EA201591278 discloses a method for treating or preventing a C1 esterase inhibitor deficiency disorder by administering a composition containing a C1 esterase inhibitor, wherein the inhibitor is present at a concentration of 400 units/ml or more. The compositions are administered subcutaneously, intravenously. The inhibitor may be plasma derived or recombinantly produced. The data given in the patent do not disclose the production technology and do not allow assessing the quality of the resulting drug.

Application WO 2015131154 discloses a method for the treatment of acute attacks of HAE by administering successive doses of a recombinant C1-esterase inhibitor at a dose of 50 units/kg of body weight, relates to the therapeutic use of a drug. This decision does not disclose the technological aspects of obtaining a drug based on a recombinant human C1-esterase inhibitor.

Application WO 1992022320 discloses a method for the treatment of systemic inflammatory diseases in mammals, incl. preeclampsia, by administering a therapeutically effective amount of C1-INH or variants thereof, without disclosing aspects of the manufacturing technology.

Application WO 2017/087882 A1 (Recombinant human C1 esterase inhibitor and uses thereof) discloses a large number of aspects related to the medical application, pharmaceutical composition and production technology of a human C1 esterase inhibitor. This solution discloses the process of cultivating the producer strain both in perfusion mode and in fed-batch mode in a 5-liter bioreactor; 10 l; 200 l; 500 l; 500 l; 1000 l; 2000 l; 5000 l; 10000 l; 15,000 l or 20,000 l with final productivity in the culture liquid at the level of 5, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mg per day (perfused). The application also states that one or more methods based on the use of affinity chromatography, gel filtration, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic chromatography, as well as non-chromatographic methods (centrifugation, ethanol precipitation, etc.) .) to obtain a C1-esterase inhibitor preparation with a purity of at least 90% (purity assessment method is not specified). The description indicates general approaches to the removal of contaminants: proteins of the producer strain, DNA, viruses using chromatographic methods with different operating principles. Additionally, the application discloses pharmaceutical compositions containing as an active substance a human C1-esterase inhibitor and excipients from the following categories: salts, salts with buffering properties, alcohols, various sugars, amino acids, etc. These compositions can have different pH values, as well as different format: solution or lyophilisate. Application WO 2017/087882 is the closest to the claimed invention, affecting the technological aspects of the production of a medicinal product. The main disadvantages of the solutions disclosed in the application WO 2017/087882 are:

- only general manufacturing approaches are described that can be applied to the production of any protein-based drug, without being tied to a specific product;

- the cleaning technology described in the examples is not related to the production process, but is in the nature of a research work. At the same time, methods of elution on sorbents POROS XS, GigaCap Q with fractional analysis are used, which are low-tech and require a significant amount of resources to implement the strategy of in-process control;

- the authors of the decision conducted a large amount of research to reduce the level of proteins of the producer strain and DNA in the final product, however, the results obtained indicate an extremely high level of these contaminants in the final product. In particular, the stage of filtration through the Sartobind STIC membrane, declared by the authors as the main one for purification of the proteins of the producer strain, is not sufficiently effective, which is confirmed by the results given in the patent (Table 1). The level of protein reduction in the producer strain was 0.21 Log, which made it possible to obtain the protein content of the producer strain in the final preparation at a level of about 2000 ng/mg.

The purification method known from WO 2017/087882 may include various chromatography steps routinely used in protein purification, such as affinity, ion-exchange, anion-exchange, cation-exchange, multimodal, hydrophobic chromatography on a POROS XS sorbent, as well as the stages of viral inactivation and diafiltration are obligatory. Despite the variety of approaches used, the authors of the application failed to achieve the final purity of the drug required for the drug: the content of HCP after all stages of purification was about 1960 ng/mg. The yield of the monomeric form was about 90%. The content of residual DNA after all stages of purification ranged from 5 to 13 pg/mg.

The results obtained by the authors of the solution on the content of DNA are shown in Table 2.

Thus, the authors of WO 2017/087882 failed to create a highly efficient technology that allows obtaining a drug with a minimum amount of contaminants. For a more complete elimination of HCP, the authors of the claimed invention significantly changed the conditions for cleaning on a hydrophobic sorbent, using a sorbent with phenyl groups. This modification is an unexpected technical step, since, as far as it is known from the prior art (see APPLIED BIOCHEMISTRY AND MICROBIOLOGY, 2009, volume 45, No. 3, pp. 378-383), protein purification on phenylsepharose allows you to get only about 73% monomeric form of the protein. Such a low proportion of the monomeric form is theoretically an obstacle to the use of phenyl-containing sorbents in the purification of a therapeutic protein, since in the case of the C1 inhibitor, it is this form that exhibits biological activity. Nevertheless, the authors used these sorbents in the purification of the C1 inhibitor in one of the final stages of chromatography, where they unexpectedly showed high efficiency, while maintaining a high yield of the monomeric form. When the C1-esterase inhibitor was introduced into the purification procedure at the stage of chromatography on various hydrophobic sorbents containing a phenyl group, the authors obtained an unexpectedly high yield of the monomeric form - 99%, with a very high purity according to HCP (content less than 10 ng/mg) and DNA (less 1.0 pg/mg). The use of hydrophobic sorbents with a phenyl group to increase the purity of the C1-esterase inhibitor is unknown from the prior art, is not obvious to a specialist and directly affects the production of a new technical result - obtaining a C1-esterase inhibitor of a high degree of purification from host cell proteins and DNA with a high yield of the target protein in its biologically active monomeric form.

The given technology of chromatographic purification makes it possible to obtain a preparation with a low content of DNA, however, during the production of a series under GMP conditions, the obtained DNA level was 13 pg/mg, which almost corresponds to the declared upper limit of the norm (14 pg/mg), and indicates the variability of the process with t.z. DNA removal.

The technical result to which the claimed invention is directed is to develop a method for producing a highly purified recombinant inhibitor of human C1-esterase for medical use, with a high yield of a biologically active monomeric form of the protein relative to the content of the target protein in the culture fluid.

To achieve these technical results, a highly efficient technology for obtaining the active pharmaceutical substance of a recombinant inhibitor of human C1-esterase was developed, scaled up and transferred to production, which makes it possible to obtain a drug containing no more than 10 ng/mg of proteins of the producer strain; no more than 5 pg/mg DNA; not less than 99.0% of the monomeric form of the protein; not less than 99.5% of the main form according to reverse-phase HPLC and a specific activity of not less than 9.0 IU/mg.

The claimed production technology makes it possible to obtain an active pharmaceutical substance suitable for use in clinical trials and commercial release.

The development of a highly efficient production technology was carried out taking into account the following criteria:

- ensuring acceptable key quality parameters of the final product;

- high yield of the final biologically active purified protein relative to the protein content in the culture fluid;

- low cost of production.

Implementation of the invention

Purification on a metal-chelate sorbent

The inventors have shown for the first time that for the purposes of the claimed invention, when capturing the target protein from the culture liquid, metal-chelate sorbents are best suited. When choosing a sorbent for capture, the following process parameters were evaluated:

- output of the target protein from the stage;

- the content of the monomeric form of the target protein in the eluate;

- the content of proteins of the strain-producer in the eluate;

- DNA content in the eluate;

- the degree of staining of the sorbent by the components of the culture liquid and the possibility of regeneration of the sorbent;

- capacity of the sorbent for the target protein.

The results obtained for the screening of sorbents for the capture stage are presented in Table 3.

The data obtained demonstrated that the best results can be achieved with metal chelate sorbents such as IMAC Sepharose or EMD Chelate. At the same time, it was experimentally found that when purifying the C1 esterase inhibitor, sorbents with cation-exchange and hydrophobic properties are not effective enough, since they have an extremely low capacity, and when using sorbents with anion-exchange properties, the level of contaminants in the eluate (proteins of the producer strain and DNA) is at a high level. . For this stage, an additional stage was developed for washing with a buffer solution containing from 5 to 50 mM imidazole, pH value from 5.5 to 8.5 units, while adding sodium chloride to the washing buffer solution at a concentration of 200 mM to 1 M. Also developed the composition of the eluent buffer solution containing from 100 mm to 1 M imidazole, pH value from 5.5 to 8.5 units.

The combination of the above conditions made it possible to obtain a protein solution with a protein content of the producer strain at the level of 5000 ng/mg; DNA at the level of 7000-8000 pg/mg; the content of the monomeric form of the protein at the level of 95.0%; the protein yield from the stage was about 95% of its content in the culture liquid.

Purification on an affinity sorbent

At the next stage, a purification stage was developed using the Heparin Sepharose FF affinity sorbent. This sorbent can significantly reduce the content of proteins of the producer strain and DNA. It has been established that at a pH value of 7.0 to 8.0 and a conductivity of 0.1 to 10.0 mS/cm (mS/cm) of the applied protein solution, as well as the elution of the target protein by increasing the sodium chloride content in the mobile phase to values of 0.2 - 0.4 M, the protein content of the producer strain is reduced to a level of about 3000 ng/mg, and the DNA content to a level of less than 50 pg/mg. Table 4 shows the screening data for elution conditions on Heparin Sepharose FF.

Thus, the purification stage was developed, which makes it possible to significantly reduce the amount of proteins of the producer strain in the eluate of the target protein - to a level of less than 3000 ng/mg, while bringing the DNA content to a level of less than 50 pg/mg.

Development of the final stage of purification from the DNA of the producer strain To remove the DNA of the producer strain, a standard approach was used using an anion-exchange sorbent. It is shown that carrying out the process on the Q Sepharose FF sorbent under conditions of low conductivity of the eluting buffer solution and pH values from 6.0 to 8.5 units. allows to reduce the content of residual DNA to a level below

Development of the final stage of purification from proteins of the producer strain

To remove the proteins of the producer strain, an approach was chosen based on the high hydrophilicity of the target molecule, which makes it possible to purify the target protein in the filtration mode through a column with a sorbent. An experimental test was made of the possibility of using hydrophobic sorbents under various conditions to reduce the content of proteins of the producer strain in the final product to a level below 10 ng/mg. . When the content of sodium chloride in the applied protein solution was less than 3 mol/liter, the total yield of the target protein was at least 80%. The results of screening conditions for hydrophobic chromatography are presented in table 6.

Thus, a stage of the chromatographic purification process was developed, which makes it possible to reduce the protein content of the producer strain to a level of less than 10 ng/mg.

Additionally, the stages of viral inactivation were introduced into the purification scheme after the stage of purification on a metal-chelate sorbent, as well as nanofiltration and diafiltration for transfer to the final buffer solution. The finalized purification scheme is shown in Figure 1. This purification scheme makes it possible to obtain an active pharmaceutical substance with the following characteristics:

- monomer content not less than 99%;

- the content of the main form of the target protein according to reverse-phase HPLC is not less than 99.5%;

- the content of proteins of the producer strain is not more than 10 ng/mg;

- the content of the DNA of the producer strain is not more than 5 pg/mg;

- specific specific activity not less than 9.0 IU/mg.

The yield of the target protein relative to its content in the culture fluid was about 80% (500 mg of the target protein per 1 liter of the culture fluid).

As a suitable hydrophobic sorbent containing phenyl groups, Phenyl Sepharose FF, Tosoh Butyl 650M, Capto Phenyl, as well as other commercially available sorbents with a similar principle of action can be used.

Example 1

Carrying out chromatographic purification.

A 1 L culture fluid was applied to an IMAC Sepharose sorbent column (column volume (CV) 0.2 L) preactivated with 2 CV of 0.1 M zinc chloride solution and equilibrated with 20 mM Tris buffer, pH 7.2. After application, the sorbent was washed with 3 CV of the same buffer solution. The column was washed with 3 CV of 20 mM Tris buffer, 15 mM imidazole, pH 7.5. The target protein bound to the sorbent was eluted with a buffer solution of 20 mM Tris, 150 mM imidazole, pH 7.2.

Virus inactivation was carried out by the solvent-detergent method. A 20% solution of polysorbate-80 was added to the protein fraction with stirring to 1% (1/20 of the volume of the fraction) and tributyl phosphate to 0.3% (3/1000 of the volume of the eluate). Incubated with constant stirring at room temperature for 1 hour.

The resulting protein fraction was diluted with water 2 times and applied to a column with Heparin Sepharose (CV 0.1 L), pre-equilibrated with a buffer solution of 20 mm Tris, pH 7.2. After application, the sorbent was washed with 3 CV of the same buffer solution. The target protein bound to the sorbent was eluted with a buffer solution of 20 mM Tris, 200 mM sodium chloride, pH 7.2.

The next stage of purification was carried out on a column with a Q Sepharose FF sorbent (CV 0.1 l), which was preliminarily balanced with a buffer solution of 20 mM Tris, 50 mM sodium chloride, pH 7.0. Next, a protein solution was applied in a 50% gradient of purified water to a balanced column. After application, the sorbent with adsorbed protein was washed with a buffer solution of 20 mM Tris, 50 mM sodium chloride, pH 7.2. The target protein was eluted with a gradient of 20 mM HEPES, 200 mM sodium chloride, pH 7.5 buffer solution in 20 mM Tris, 50 mM sodium chloride, pH 7.5 buffer solution (from 0 to 100% over 10 column volumes).

The next stage of purification was carried out on the Capto Phenyl High Sub sorbent. The volume of the column was 10 ml. Target protein loading is not more than 50 g of target protein per 1 liter of sorbent.

The Capto Phenyl High Sub column was pre-equilibrated with a buffer solution of 20 mM HEPES, 2 M sodium chloride, pH 7.0. The target protein for application was prepared by adding a 5 M sodium chloride solution until a conductivity of 145-155 mS/cm. Next, the protein solution was applied to the balanced column, collecting the filtrate containing the target protein. After loading, the column was washed with a buffer solution of 20 mm HEPES, 2 M sodium chloride, pH 7.0, while continuing to collect the filtrate.

Antiviral nanofiltration of the obtained protein fraction was carried out on a Planova 15N filter, then the filtrate was concentrated on a VIVAFLOW 200 tangential filtration membrane with a pore diameter of 30 kDa and dialyzed against a buffer solution of 9.4 g/l glycine, 2.9 g/l sodium citrate, 8 g /l sodium chloride, pH 7.0.

The resulting protein solution was filtered through a sterilizing filter with a pore size of 0.22 μm into a sterile container under aseptic conditions.

The total yield of purified protein was about 500 mg per liter of culture liquid.

The obtained active pharmaceutical substance has the following qualitative characteristics: 99.7% of the monomeric form of the target protein; 99.8% main form by reverse phase HPLC; 1.9 pg/mg DNA of the producer strain; 2.9 ng/mg of proteins of the producer strain; specific activity 9.3 IU/mg.

Example 2

Carrying out chromatographic purification in an industrial volume.

In the first purification step, a 250 L culture liquid was loaded onto an IMAC Sepharose column (column volume (CV) 25.0 L).

The preparation of the column was carried out by washing with 25.0 l of 0.5 M zinc chloride solution, followed by washing with a starting buffer solution: 20 mm Tris, pH 7.2.

The culture liquid was not subjected to preliminary preparation.

The culture liquid was applied onto the prepared sorbent, after which the sorbent was successively washed with a starting buffer solution and a buffer solution of 20 mM Tris, 5 mM imidazole, pH 7.2.

The target protein was eluted from the column with a buffer solution of 20 mm Tris, 150 mm imidazole, pH 7.2.

The next step was carried out by viral inactivation of the resulting solution by adding detergents: polysorbate 80 to 1%; tributyl phosphate to a concentration of 0.3%. The incubation time was at least 1 hour at room temperature.

At the next stage, the target protein was purified on the Heparin Sepharose FF affinity sorbent.

A Heparin Sepharose FF column (column volume (CV) 12.0 L) was pre-equilibrated with a buffer solution of 20 mM Tris, pH 7.5. Next, a protein solution was applied in a 50% gradient of purified water to a balanced column.

After application, the sorbent with adsorbed protein was washed with a buffer solution of 50 mM Tris, pH 7.5. The target protein was eluted with a buffer solution of 50 mM Tris, 200 mM sodium chloride, pH 7.5

The next stage of chromatographic purification was carried out on a Q Sepharose FF sorbent. The volume of the column was 2 liters. Target protein loading is not more than 70 g of target protein per 1 liter of sorbent.

The Q Sepharose FF column was pre-equilibrated with a buffer solution of 20 mM Tris, 50 mM sodium chloride, pH 7.5. Next, a protein solution was applied in a 50% gradient of purified water to a balanced column. After application, the sorbent with adsorbed protein was washed with a buffer solution of 20 mM Tris, 50 mM sodium chloride, pH 7.2. The target protein was eluted with a gradient of 20 mM HEPES, 200 mM sodium chloride, pH 7.0 buffer solution in 20 mM Tris, 50 mM sodium chloride, pH 7.5 buffer solution (from 0 to 100% over 10 column volumes).

The next stage of purification was carried out on the Capto Phenyl High Sub sorbent. The volume of the column was 2 liters. Target protein loading is not more than 50 g of target protein per 1 liter of sorbent.

The Capto Phenyl High Sub column was pre-equilibrated with a buffer solution of 20 mM HEPES, 2 M sodium chloride, pH 7.0. The target protein for application was prepared by adding a 5 M sodium chloride solution until a conductivity of 145-155 µS/cm. Next, the protein solution was applied to the balanced column, collecting the filtrate containing the target protein. After application, the target protein was washed with a buffer solution of 20 mM HEPES, 2 M sodium chloride, pH 7.0.

To convert the resulting protein into the final buffer solution, diafiltration was performed using Sartorius diafiltration cassettes, PES material, with a cut-off size of 50 kDa. The cassettes were preliminarily washed with a buffer solution of 125 mM glycine, 11.2 mM sodium citrate, 136.8 mM sodium chloride, pH 7.0. The diafiltration process was carried out at a protein concentration of 50 mg/ml. After completion of the diafiltration process, the protein concentration was adjusted to a value of 15-18 mg/ml.

Next, antiviral nanofiltration of the resulting protein solution was carried out on a Millipore Virosolve filter. The filter was pre-washed with a buffer solution of 125 mM glycine, 11.2 mM sodium citrate, 136.8 mM sodium chloride, pH 7.0. Next, the protein solution was subjected to the prepared antiviral filter.

The resulting protein solution was filtered through a sterilizing filter with a pore size of 0.22 μm into a sterile container under aseptic conditions.

The total yield of purified protein was about 500 mg per liter of culture liquid.

The obtained active pharmaceutical substance has the following qualitative characteristics: 99.8% of the monomeric form of the target protein; 99.9% base form by reverse phase HPLC; 1.0 pg/mg DNA of the producer strain; 5.0 ng/mg of proteins of the producer strain; specific activity 9.5 IU/mg.

Claims (11)

1. A method for purifying a recombinant inhibitor of human C1-esterase, including viral inactivation, metal chelate chromatography, affinity chromatography and anion exchange chromatography, characterized in that when the protein content of the producing strain in the product reaches less than 3000 ng/mg and the DNA level less than 50 pg/mg, the product is purified by final hydrophobic chromatography using a sorbent containing phenyl groups. 2. The method according to claim 1, where affinity chromatography is used to pre-purify the product from host cell proteins. 3. The method according to claim 1, where Phenyl Sepharose FF, Tosoh Butyl 650M, Capto Phenyl or another sorbent with a similar principle of action is used as a hydrophobic sorbent. 4. The method of claim 1, wherein the sodium chloride content of the protein solution to be purified applied to the sorbent containing phenyl groups is in the range of about 2 to 3 mol/L. 5. The method according to p. 1, where the preferred load of the target protein on the sorbent containing phenyl groups is in the range from 50 to 100 mg/ml. 6. The method according to claim 1, where IMAC Sepharose FF, EMD Chelate or another sorbent with a similar principle of action is used as a metal-chelate sorbent when capturing the target protein from the culture fluid. 7. The method according to p. 1, where Heparin Sepharose, POROS Heparin, Heparin Hyper D or another sorbent with a similar principle of action is used as an affinity sorbent. 8. The method of claim 1 wherein the solvent-detergent or low pH inactivation method is used for viral inactivation. 9. The method according to claim 1, where when performing the final hydrophobic chromatography, the conductivity level of the target protein in the application sample is 140-160 mS/cm, preferably 145-155 mS/cm. 10. The method of claim 1 wherein the purified protein is transferred to the final buffer solution using diafiltration. 11. Application of the method according to p. 1 to obtain an active pharmaceutical substance with the following characteristics: the content of the monomeric form of the recombinant human C1-esterase inhibitor is not less than 99.0%; the content of the main form of the recombinant inhibitor of human C1-esterase according to reverse-phase HPLC is not less than 99.5%; the content of residual proteins of the producer strain is not more than 10 ng/mg; the content of the DNA of the producer strain is not more than 5 pg/mg; specific activity not less than 9.0 IU/mg.

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