RU2661088C1 - Method for purification of sustained-release drug based on recombinant interferon beta-1b analogue for the treatment of multiple sclerosis - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, genetic engineering and biotechnology, namely to method for preparing a recombinant analogue of interferon beta 1b with an oligosaccharide-like polymer. Present method comprises production of a recombinant analogue of interferon beta 1b, coupled to a thioredoxin stabilizing protein via an enterokinase cleavage site, chromatographic purification of the resulting analog, its conjugation with a polymer of oligosaccharide nature, hydrolysis with enterokinase enzyme, isolation of the resulting conjugate and its purification. This method differs in that the polymer of oligosaccharide nature is D3000-7(Sa-Sp)-(Sp-M), in which D3000 is a dialdehyde dextran, (Sa-Sp) – Neu5Ac-Lactose-N(CH)-CH(2)-CH(2)-NH(2), (Sp-M) – maleimide-CH(2)-NH(2), and the recombinant analog of interferon beta 1b is optimized for expression in E. coli cells.

EFFECT: present invention allows to obtain a conjugate of a recombinant analogue of interferon beta 1b with an oligosaccharide polymer in high yield.

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Description

The invention relates to biotechnology, in particular to a method for producing a hybrid protein, comprising conjugating a recombinant analog of interferon beta 1b, which can be used to treat multiple sclerosis.

Multiple sclerosis (PC) is the most common demyelinating disease that affects mainly people of working age. In recent years, there has been a steady trend towards an increase in the incidence of PC and rejuvenation of the patient population. One of the main characteristics of PC is the polymorphism of clinical manifestations, due to the characteristics of the course and localization of the demyelinating process. The combination of acute demyelination and pathological changes in axons underlies the progression of neurological deficit and disability of patients with PC.

PC is a disease with high-cost treatment regimens and methods of social rehabilitation of patients.

For the treatment of this disease, immunomodulators are used that change the course of the PC, reduce the severity of exacerbations, increase the length of remission and thereby slow down the progression of the disease. One of the first such drugs is interferon beta, and is still used as a first-line drug.

Interferon beta-1b has the highest activity against multiple sclerosis in various in vitro tests.

RU 2614264 describes a nucleotide sequence encoding a recombinant protein, an analogue of interferon beta, which is optimized for expression in E. coli cells. The invention allows to obtain a recombinant protein - an analogue of interferon beta with a biological activity of 1.8 * 10 ⁹ U / μmol in the A549 / VSV system. However, this recombinant protein, an analogue of interferon beta, does not have pharmacokinetic parameters that exceed native unmodified preparations based on interferon beta.

An advantage of the invention is the improved pharmacokinetics of the resulting fusion protein compared to native interferon beta 1b. The half-life is 16 hours, which is 3-5 times higher than the values for native drugs.

The currently available technologies for purification of recombinant beta interferons expressed in CHO cells consist of 3-5 purification procedures, including primary purification by affinity chromatography (US 4541952), metal chelate chromatography (US 4257938), size-controlled porous glass chromatography then CGP (US 5244655 and others) or chromatography on Konkavalin A (US 4658017) followed by cation exchange chromatography or reverse phase chromatography.

The use of metal-chelate chromatography for purification can lead to environmental pollution, since it uses heavy metals. Chromatography on porous glass or on Concavalin A has low specificity. Moreover, purification techniques using affinity chromatography include washing and eluting with ethylene glycol using monoclonal antibodies or dyes containing resins. However, affinity chromatography using monoclonal antibodies requires a separate step for removing the non-glycosylated form of interferon beta, which makes mass production difficult and the ethylene glycol used for washing is very toxic to the body, which limits the use of this purification method.

The aim of the invention is to obtain and purify a recombinant analogue of interferon beta 1b conjugated in the vicinity of the natural glycosylation site with an oligosaccharide represented by the sequence SEQ ID NO 1 with an increased yield of the target product.

Obtaining a hybrid protein with therapeutic activity against multiple sclerosis includes the following steps:

- obtaining a recombinant protein - an analogue of interferon beta, coupled with a protein stabilizer;

- chromatographic purification of recombinant protein - an analog of interferon beta, coupled with a protein stabilizer, on a metal-affinity sorbent;

- obtaining, purification and analysis of an oligosaccharide containing seven sialic acid residues per molecule of oligosaccharide;

- conjugation of the oligosaccharide with a recombinant protein - an analog of interferon beta 1b, coupled to a protein stabilizer;

- chromatographic purification of the conjugate of the recombinant analogue of interferon beta 1b with an oligosaccharide, which takes place in one step on an ion-exchange sorbent.

The technical result achieved by the implementation of this invention is the improved pharmacokinetics of the obtained hybrid protein compared to native interferon beta 1b and an increase in the degree of purification of the recombinant analogue, which leads to uniformity and almost 100% chromatographic purity of the obtained product.

Brief Description of the Drawings

FIG. 1. Scheme of a recombinant protein - an interferon beta analog connected to a stabilizer protein: 1 - human interferon beta analog sequence; 2 - site cleavage of the enzyme enterokinase; 3 is a sequence of a protein from the chaperone family stabilizing the entire recombinant construct; 4 - polyhistidine sequence.

FIG. 2. Chromatographic profile of the elution of the recombinant protein - an analog of interferon beta, coupled with a protein stabilizer, with Ni-Sepharose.

FIG. 3. Scheme for the synthesis of activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M.

FIG. 4. Chromatographic elution profile of D3000-7 (Sa-Sp) -Sp-M from a mono-Q preparative column.

FIG. 5. Chromatographic elution profile of D3000-7 (Sa-Sp) -Sp-M from a mono-Q analytical column.

FIG. 6. Mass spectrum of MALD-TOF monoion of the activated oligosaccharide. The matrix is 2,5-dihydroxybenzoic acid, the laser is 336 nm.

FIG. 7. Chemical conjugation reaction of activated oligosaccharide and recombinant protein.

FIG. 8. Chromatographic profile of conjugate gel filtration through Sephacryl S400. The arrow indicates the yield of the conjugate.

FIG. 9. Hydrolysis of a conjugate of a recombinant protein, an analog of interferon beta, coupled to a protein stabilizer with an oligosaccharide enzyme enterokinase.

FIG. 10. Electrophoresis of a hydrolysis mixture of a recombinant protein - an analog of interferon beta, coupled with a protein stabilizer, and enterokinase at different time intervals. The effectiveness of prolonged hydrolysis (60 minutes) is shown. Track 1 - the reaction mixture half an hour after the start of the experiment (time T = 30 min); track 2 — reaction mixture one hour after the start of the experiment (time T = 60 min); track 3 — reaction mixture at the beginning of the experiment (time T = 1 min); Track 4 - molecular weight standards. The arrows indicate: the position in the gel of the conjugate connected to the protein stabilizer (a), pure conjugate (b).

11. Chromatographic profile of elution of a recombinant protein, an analogue of interferon beta with SP-Sepharose.

FIG. 12. Dynamics of changes in the concentration of the recombinant analogue of interferon beta 1b conjugated to an oligosaccharide in rat serum after intravenous administration at a dose of 107.5 μg / kg and pharmacokinetics parameters.

DETAILED DESCRIPTION OF THE INVENTION

Example 1. PRODUCTION OF RECOMBINED PROTEIN - ANALOGUE OF INTERFERON BETA

To obtain the recombinant protein represented by the sequence SEQ ID NO: 1, a genetic construct was created according to the standard procedure (Maniatis T., Fritsch E.F., 1982) based on the commercial plasmid pET32a. This plasmid transfected cells of E. coli strain BL21 (DE3). The resulting plasmid pET-32a / IFN beta transform competent bacterial cells of strain E. coli BL21 (DE3) to obtain a soluble fraction of the protein. Strain BL21 (DE3) (Invitrogen) is intended for expression of recombinant proteins. The designation DE3 means that the strain contains the lysogen of phage λ DE3, which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter. IPTG (isopropyl-β-D-1-thiogalactopyranoside) is required to induce the expression of T7 RNA polymerase. Strain E. coli BL21 (DE3) / pET-32a / IFN beta provides fusion protein thioredoxin-interferon beta. After transfection, the cells were subcultured into a culture flask with LB broth and kanamycin 25 μg / ml. The culture was grown on a shaker at 280 rpm at 37 ° C until the optical density reached OD585 = 0.8E. Protein expression was induced by adding IPTG to a concentration of 1 mM. The culture was additionally cultured for 4 hours, then the cells were precipitated by centrifugation at 5000 g. Cell pellet was lysed in 8 M urea and processed on an ultrasonic disintegrator. The cell lysate was centrifuged at 20,000 g for 2 hours. The pET-32a construct, carrying the gene encoding the recombinant IFN-beta protein in E. coli cells as a fusion protein with a protein stabilizer thioredoxin, increases the expression level and solubility of the fusion protein.

In FIG. 1 shows a diagram of a recombinant protein - an analog of interferon beta coupled to a protein stabilizer: 1 - a sequence of an analog of human interferon beta; 2 - site cleavage of the enzyme enterokinase; 3 is a sequence of a protein from the chaperone family stabilizing the entire recombinant construct; 4 - polyhistidine sequence.

Example 2. ISOLATION AND CLEANING OF RECOMBINANT PROTEIN - ANALOGUE OF INTERFERON BETA 1B, CONNECTED WITH A PROTEIN-STABILIZER

Chromatographic purification of a recombinant protein, an analogue of interferon beta, coupled to a stabilizing protein, takes place on a metal-affinity sorbent. The carrier used was Ni-Sepharose from GE Helthcare. These columns are prefilled, but they can be cascaded through a serial connection. In a chromatographic purification of a recombinant protein, an analogue of interferon beta, coupled to a stabilizing protein, a cascade of five columns of HisPrep FF 16/10 (17-5256-01, GE Helthcare) was used.

The entire volume of the cell lysate was run through a cascade of Ni-Sepharose columns pre-equilibrated with 10 mM acetic acid. Due to the polyhistidine sequence, the recombinant protein was adsorbed onto the carrier. All other cell components passed through the column. The presence of TRITON X-100 in the mixture avoids the non-specific sorption of a number of proteins having a negative charge, because the sorbent also has ion-exchange properties.

The target protein is eluted with a linear gradient. Starting buffer: 10 mM acetic acid, final buffer: 200 mM acetic acid. The volume of the gradient is 7500 ml. The eluate was fractionated. The presence of protein in the fraction was determined by electrophoresis in PAAG. In FIG. Figure 2 shows the chromatographic profile of elution of a recombinant protein, an analog of interferon beta, coupled to a protein stabilizer, with Ni-Sepharose. Numbers 1-7 indicate the fractions selected for analysis.

Example 3. SYNTHESIS, CLEANING AND ANALYSIS OF ACTIVATED OLIGOSACCHARIDE

The synthesis of activated oligosaccharide occurs in three stages:

a) the interaction of dialdehyde dextran (DBP) with Neu5Ac-Lactose-N (CH) -CH (2) -CH (2) -NH (2) (Sa-Sp),

b) the conversion of the COOH group of the terminal glucose residue of the dextran core to the aldehyde group of COH by treatment with ethanolamine,

c) the introduction of the maleide group in the dextran core of the oligosaccharide due to the interaction of Malemid-CH (2) -CH (2) -NH (2) (Sp-M) with the SON-aldehyde group of the terminal glucose residue.

The reaction proceeds by mixing and incubating solutions containing DBP, Neu5Ac-Lactose-N (CH) -CH (2) -CH (2) -NH (2) (Sa-Sp), Malemid-CH (2) -CH (2 ) -NH (2) (Sp-M) and ethanolamine.

The DBP and Sa-Sp solutions are mixed and incubated at 24 ° C for 2 hours, then the ethanolamine solution is added, and the mixture is incubated at the same temperature for 10 minutes with stirring, then the Sp-M solution is added, and after stirring after 30 minutes the synthesis reaction of activated oligosaccharide goes to the end. The synthesis scheme of the activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M is shown in FIG. 3.

The synthesis of D3000-7 (Sa-Sp) -Sp-M results in a mixture of oligosaccharides with various amounts of sialic acid residues on the dextran cortex. To isolate the product with the desired degree of substitution (seven sialic acids), ion-exchange HPLC chromatography was performed on a mono-Q column. Eluted with a linear gradient. Starting buffer: 5 mM NaCl, final buffer: 80 mM NaCl. The target product leaves within the range of NaCl concentrations from 38 mM to 50 mM. To improve the quality of the product, “shoulders” of the peak were not selected. In FIG. 4 shows the chromatographic elution profile of D3000-7 (Sa-Sp) -Sp-M from a mono-Q preparative column.

The activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M was analyzed by HPLC chromatography on a mono-Q analytical column. D3000-7 (Sa-Sp) -Sp-M was diluted 10 times with 5 mM NaCl, loaded onto a mono-Q assay column. Eluted with a linear gradient. Starting buffer: 5 mM NaCl, final buffer: 100 mM NaCl. The target product was obtained at a concentration of NaCl 45 mM. In FIG. 5 shows the chromatographic elution profile of D3000-7 (Sa-Sp) -Sp-M from a mono-Q analytical column.

The peak is symmetrical, separation on the columns of this type occurs according to the magnitude of the charge of the molecule, which proves the absence of molecules with other charges in the composition of the substance, i.e. molecules with more or less sialic groups.

The activated oligosaccharide D3000-7 (Sa-Sp) -Sp-M was analyzed by MALDI-TOF mass spectrometry in monoion mode. The activated oligosaccharide at a concentration of 0.1 mg / ml and a volume of 5 μl was mixed with the matrix and allowed to dry. Then subjected to mass spectrometric analysis and received a mass spectrum of a monion of activated oligosaccharide.

In FIG. 6 shows the mass spectrum of the MALD-TOF monoion of the activated oligosaccharide. The matrix is 2,5-dihydroxybenzoic acid, the laser is 336 nm. Four major peaks and four minor peaks are visible in the mass spectrum. The minimum mass of the major peak is 7916 Da, the maximum - 8453 Da. The minimum mass of the minor peak is 7558 Da, the maximum is 8811 Da. From peak to peak, the mass changes by 179 Da, which corresponds to the mass of a glucose molecule lacking a proton (hydrogen atom). The presence of many peaks is due to the fact that the Dextran3000 preparation is not a homogeneous preparation and is a mixture of oligosaccharides.

By analyzing the obtained peaks, it is possible to confirm the absence of additional groups carrying sialic acid (mass change by 729 Da (spacer + Lactose-Neu5Ac), as well as additional maleide groups (mass change by 232 Da) in the activated oligosaccharide D3000-7 (Sa-Sp ) -Sp-M.

Example 4. CONJUGATION OF OLIGOSACCHARIDE WITH RECOMBINANT PROTEIN - ANALOGUE OF INTERFERON BETA 1B, CONNECTED WITH PROTEIN-STABILIZER

In the process of conjugation, a covalent bond is formed between the maleimide group of the activated oligosaccharide and the -SH group of the cysteine molecule of the recombinant analog of interferon beta. Conjugation takes place in the presence of 25 mM NaCl. In FIG. 7 shows a chemical conjugation reaction of an activated oligosaccharide and a recombinant protein.

The activated oligosaccharide is taken in excess. The reaction mixture is incubated for 8 hours, the reaction proceeds with a yield of more than 94%. Next, a dithiotriitol solution is added to the reaction mixture to reduce imines in the activated oligosaccharide molecule. After incubation for 10 minutes, the reaction mixture was applied to an XK 50 * 100 gel filtration column (18-87-68 GE Healthcare) with Sephacryl S400 (17-0506-01 GE Helthcare), previously equilibrated with water. In FIG. Figure 8 shows the chromatographic profile of conjugate gel filtration through Sephacryl S400. The arrow indicates the yield of the conjugate.

Example 5. Cleavage of a recombinant protein - an analog of the beta 1B interferon connected to a protein stabilizer, and isolation of a recombinant beta 1B interferon - conjugated olis

Cleavage of a recombinant protein, an analogue of interferon beta 1b, coupled to a stabilizing protein, as part of a conjugate with a specific protease, and isolation of a recombinant analogue of interferon beta 1b, conjugated to an oligosaccharide.

The conjugate of the activated oligosaccharide with a recombinant protein, an analogue of interferon beta 1b, coupled to a stabilizing protein, incorporates the enzyme cleavage site enterokinase. This enzyme allows you to split the protein fragment "flush". Those. after the Asp-Asp-Asp-Asp-Asp-Lys sequence, the enzyme breaks down the protein molecule, which avoids the appearance of additional amino acids in the target protein product, in our case, in the molecule of the recombinant protein - an analog of interferon beta. In FIG. Figure 9 shows the hydrolysis of a conjugate of a recombinant protein, an analog of interferon beta, coupled to a stabilizing protein with an oligosaccharide enzyme enterokinase.

Commercial kits containing the enterokinase enzyme allow 20 micrograms (μg) of protein to be cleaved with one unit of activity (1 unit). Thus, the amount of enterokinase enzyme consumed will be proportional to the molecular weight of the protein molecule. Because it is not possible to reduce the molecular weight of a recombinant protein, an analogue of interferon beta, coupled to a stabilizing protein, then a long time of hydrolysis with enterokinase due to the speed of the enzyme can reduce its amount.

A preliminary experiment was conducted showing the effectiveness of hydrolysis depending on the incubation time of the reaction mixture. In FIG. 10 shows electrophoresis of a hydrolysis mixture of a recombinant protein, an analog of interferon beta, coupled to a protein stabilizer, and enterokinase at different time intervals. The effectiveness of prolonged hydrolysis (60 minutes) is shown. Track 1 - the reaction mixture half an hour after the start of the experiment (time T = 30 min); track 2 — reaction mixture one hour after the start of the experiment (time T = 60 min); track 3 — reaction mixture at the beginning of the experiment (time T = 1 min); Track 4 - molecular weight standards. The arrows indicate: the position in the gel of the conjugate connected to the protein stabilizer (a), pure conjugate (b).

Example 6. CHROMATOGRAPHIC CLEANING OF RECOMBINANT ANALOGUE OF INTERFERON BETA 1B CONJUGATED WITH OLIGOSACCHARIDE

Chromatographic purification of the conjugate of the recombinant analog of interferon beta 1b with an oligosaccharide takes place in one step on an ion-exchange sorbent. The carrier used was SP-Sepharose from GE Helthcare. HiTrap columns were used to standardize the process and its reproducibility. A HiPrep16 / 10 Q FF chromatographic column was used in the work, the capacity of which was sufficient for the entire volume of the secreted protein.

After hydrolysis and heating, the reaction mixture is filtered through a 0.22 μm filter from aggregates of denatured proteins. The filtered mixture is applied to a HiPrep16 / 10 Q FF column, pre-equilibrated with 25 mM NaCl.

The conjugate was eluted with a linear gradient. Starting buffer: 25 mM NaCl, final buffer: 450 mM NaCl. The eluate was fractionated. The presence of the conjugate in the fraction was determined by electrophoresis in SDS page. The chromatographic elution profile of the recombinant protein, an analogue of interferon beta with SP-Sepharose, is shown in FIG. eleven.

Example 7. STUDY OF THE PHARMACOKINETICS OF THE RECEIVED RECOMBINANT ANALOGUE OF INTERFERON BETA 1B CONJUGATED WITH OLIGOSACCHARID

To study the pharmacokinetics, the obtained recombinant analog of interferon beta 1b conjugated to an oligosaccharide with one ³ N substituted hydrogen atom was administered intravenously to sexually mature rats at a dose of 107.5 μg / kg at intervals of 2 minutes, 30 minutes, 60 minutes, 4 hours, 12 hours, 24 h, 36 h, 96 h. 1 ml of peripheral blood was taken from rats and at least 300 μl of serum was prepared. After sample preparation by adding one and a half times the volume of acetonitrile, the amount of ³ N of the substituted preparation was determined using a scintillation detector of nuclear radiation.

In FIG. 12 shows the dynamics of changes in the concentration of the recombinant analogue of interferon beta 1b conjugated to an oligosaccharide in rat serum after intravenous administration at a dose of 107.5 μg / kg and the pharmacokinetics parameters. The maximum concentration of the components of the recombinant analogue of interferon beta 1b conjugated to the oligosaccharide in the blood was detected immediately after administration of the drug (2 minutes), and 96 hours after administration, the sample was not detected in blood serum. The half-life for the recombinant analogue of interferon beta 1b conjugated to the oligosaccharide was about 16 hours, which is 3-5 times higher than the values for native unmodified preparations based on interferon beta (Jonasch E., Haluska FG Interferon in Oncological Practice: Review of Interferon Biology , Clinical Applications, and Toxicities. The Oncologist February - 2001, Vol. 6, No. 1 - P. 34-55 doi: 10.1634 / theoncologist. 6-1-34).

Claims (1)

A method for producing a conjugate of a recombinant analog of interferon beta 1b with an oligosaccharide polymer of nature, comprising preparing a recombinant analog of interferon beta 1b optimized for expression in E. coli cells represented by the sequence SEQ ID NO: 1 and coupled to the stabilizing protein thioredoxin via an enterokinase chromatographic cleavage site purification of the recombinant analogue of interferon beta 1b, coupled with a protein stabilizer, on a metal-affinity sorbent using Ni-Sepharose, conjugation of recombin of the analogue of interferon beta 1b, coupled to a protein stabilizer, with an oligosaccharide polymer of the nature D3000-7 (Sa-Sp) - (Sp-M), in which D3000 is dialdehyde dextran, (Sa-Sp) - Neu5Ac-Lactose-N ( CH) -CH (2) -CH (2) -NH (2), (Sp-M) - maleimide-CH (2) -NH (2), hydrolysis of the recombinant analog of interferon beta 1b, coupled to a protein-stabilizer, enzyme enterokinase, isolation of the conjugate of the recombinant analog of interferon beta 1b with an oligosaccharide polymer and purification of the conjugate of the recombinant analog of interferon beta 1b with an oligosaccharide polymer of ion exchange sorbent SP-Sepharose.

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