WO2022178922A1 - Interleukin-29 mutant protein preparation - Google Patents

Abstract

Provided is an interleukin-29 mutant protein preparation, which is characterized by containing an interleukin-29 mutant protein, a buffer system, a metal ion chelating agent and an osmotic pressure regulator. The IL-29 mutant protein preparation greatly reduces the degradation and aggregation of the IL-29 mutant protein during storage; and the impurity growth rate of the IL-29 mutant protein preparation is slow, thus significantly improving the stability of the IL-29 mutant protein.

Description

A kind of interleukin 29 mutant protein preparation technical field

The present application belongs to the field of pharmaceutical preparations. Specifically, it relates to an interleukin 29 (IL-29) mutant protein preparation.

Background technique

Interferons are an important class of familial cytokines with broad-spectrum antiviral and immunomodulatory effects. To date, seven (α, β, ω, δ, τ, γ, λ) forms of interferons have been identified, which are divided into three broad groups: Type I, Type II, and Type III. So-called "type I" interferons include interferon alpha, interferon beta, interferon omega, interferon delta, and interferon tau. Currently, interferon gamma is the only type II interferon. Type III interferons are a recently discovered family of cytokines that include interferons λ1, λ2, and λ3, also known as IL-28A, IL-28B, and IL-29.

IL-28A, IL-28B and IL-29 have sequence homology with type I interferon and gene sequence homology with IL-10. Functionally, IL-28 and IL-29 are similar to type I interferons in that both induce antiviral effects in cells, and unlike type I interferons, they do not show antiproliferative effects on certain B cell lineages active.

The wild-type IL-29 (interferon λ1, abbreviated as IFN-λ1) gene encodes a protein of 200 amino acids, as shown in SEQ ID NO:3. Wherein 1-19 amino acids in this sequence are signal peptide sequences, and the mature amino acid sequence of this protein is 181 amino acids, as shown in SEQ ID NO: 2. The IL-29 molecule is composed of six protein helices A-F, in which helices A, C, D and F form a classical up-up-down-down four-helix bundle. IL-29 initiates downstream signaling pathways by interacting with its receptor complex, which consists of IFN-λR1 and IL-10R2. Notably, IFN-λR1 is unique to the IFN-λ signaling pathway. IFN-λ1 specifically binds to IFN-λR1 to form an IFN-λ1/IFN-λR1 complex, wherein the amino acid residues of the active center of IFN-λ1 and IFN-λR1 binding are Pro25, Leu28, lys32, Arg35, Asp36, Glu39 respectively , Trp47, Phe152, Phe155, Arg156, Arg160.

Whether it is type I interferon, type II interferon or type III interferon, as a protein drug, due to factors such as poor stability, low activity, short half-life in vivo, etc., it is greatly limited in clinical treatment. Therefore, it is expected to obtain more stable and higher specific activity interferon recombinant protein drugs through genetic engineering technology. Especially when using aerosol inhalation therapy to prevent and/or treat respiratory diseases, a nebulizer device is required to atomize the drug solution into tiny particles, and the drug is inhaled into the respiratory tract and lungs to deposit the drug in the respiratory tract and lungs, which is important for the stability of the drug. The requirements for sex and activity are higher, so people are more eager to obtain IL-29 mutant proteins with higher stability and better activity, and there is an urgent need for a stable IL-29 mutant that can be used for clinical use and suitable for storage protein preparations.

SUMMARY OF THE INVENTION

In order to solve the above problems, the purpose of the present application is to provide a stable and long-term storage IL-29 mutant protein preparation.

The specific technical solutions of this application are as follows:

1. An interleukin 29 mutant protein preparation, characterized in that it comprises: an interleukin 29 mutant protein, a buffer system, a metal ion chelating agent and an osmotic pressure regulator.

2. The preparation according to item 1, wherein the interleukin 29 mutant protein comprises a substitution mutation at the 161st or 162nd amino acid in the amino acid sequence shown in SEQ ID NO: 1, wherein the 161st Aspartic acid (D) at position 162 or glycine (G) at position 162 is substituted with other natural amino acids.

3. The preparation according to item 1 or 2, wherein the aspartic acid (D) at position 161 is substituted with glutamic acid, threonine or serine, or the glycine (G) at position 162 is substituted with glutamic acid, threonine or serine replaced by aliphatic amino acids.

4. The preparation according to any one of items 1 to 3, wherein the interleukin 29 mutant protein comprises the following amino acid sequence: SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO :8.

5. The preparation according to any one of items 1 to 4, wherein the interleukin 29 mutant protein further comprises the amino acid sequence shown in SEQ ID NO: 1 at position 165 from cysteine ( C) Substitution mutation to serine (S).

6. The preparation according to any one of items 1 to 5, wherein the interleukin 29 mutant protein comprises the following amino acid sequence: SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO :9.

7. The preparation according to any one of items 1 to 6, wherein the mass-volume concentration of the interleukin-29 mutant protein is 0.1-1 mg/mL.

8. The preparation according to any one of items 1 to 7, wherein the buffer system is a phosphate buffer, an acetate buffer or a histidine buffer, preferably a phosphate buffer.

9. The formulation according to any one of items 1 to 8, characterized in that the formulation has a pH of 4.0 to 5.0, preferably 4.0 to 4.5.

10. The preparation according to any one of items 1 to 9, wherein the substance concentration of the phosphate buffer is 20 to 80 mmol/L, preferably 20 to 40 mmol/L.

11. The preparation according to any one of items 1 to 10, wherein the metal ion chelating agent is disodium edetate.

12. The preparation according to any one of items 1 to 11, wherein the mass volume concentration of the disodium edetate is 0.01 to 0.1 mg/mL, preferably 0.01 to 0.05 mg/mL.

13. The preparation according to any one of items 1 to 12, wherein the osmotic pressure regulator is sodium chloride.

14. The preparation according to any one of items 1 to 13, wherein the mass volume concentration of the sodium chloride is 6 to 10 mg/mL.

15. The preparation according to any one of items 1 to 14, wherein the mass-volume concentration of the interleukin-29 mutant protein is 0.5-1 mg/mL, and the mass-volume concentration of the disodium edetate is 0.5-1 mg/mL. The concentration is 0.01 mg/mL.

effect of invention

The IL-29 mutant protein preparation of the present application greatly reduces the degradation and aggregation of the IL-29 mutant protein during storage, and the impurity growth rate in the IL-29 mutant protein preparation is slow, which significantly improves the IL-29 mutant protein. 29 Mutant protein stability.

Especially when the phosphate buffer system is used and the amount of metal chelating agent is controlled at a low level, on the one hand, it ensures no irritation, on the other hand, it is safer, more suitable for direct access to internal organs, and has high safety requirements. dosage forms, such as inhalation dosage forms.

Description of drawings

Figure 1 and Figure 2 are the SDS-PAGE electropherograms of the IL29 mutants of Example 2 (from left to right, in Figure 1, lanes 1 to 4 are the IL29 control substance, IL29DE, IL29DS, and IL29GA in sequence; in Figure 2, lane 1 ~4 are IL29CS, IL29DE+CS, IL29DS+CS, IL29GA+CS in order).

Figures 3 to 10 are the purity profiles of the IL29 reference substance, IL29DE, IL29DS, IL29GA, IL29CS, IL29DE+CS, IL29DS+CS, and IL29GA+CS, respectively (the one with high main peak content is 0 Point determination curve, the main peak content is low on behalf of the 14-day curve).

Figure 11 is a diagram of the aerosol collection device in the nebulization stability experiment of IL29 mutants.

Figure 12 shows the in vivo efficacy results of ribavirin and IL29 mutants under different dosing regimens on RSV-infected mouse models.

Figure 13 shows the in vivo efficacy results of ribavirin and IL29 mutants under different dosing regimens on RSV-infected cotton rat models.

Detailed ways

Specific embodiments of the present application will be described in more detail below. It should be noted that, "comprising" or "including" mentioned in the entire specification and claims is an open-ended term, so it should be interpreted as "including but not limited to". Subsequent descriptions in the specification are preferred embodiments for implementing the present application, however, the descriptions are for the purpose of general principles of the specification and are not intended to limit the scope of the present application. The scope of protection of this application should be determined by the appended claims.

Application related terms

The "mutant protein" in this application refers to a protein obtained by altering the amino acid sequence of a wild-type protein, such as a protein obtained by mutating the amino acid sequence of the wild-type protein through genetic engineering methods. In this application, "mutant protein", "mutant protein" or "mutant" express the same meaning and can be used interchangeably.

In this application, "disodium edetate" is disodium edetate, which is a white or milky white crystalline or granular powder that is tasteless, odorless or slightly salty, odorless and tasteless. It is soluble in water and extremely insoluble in ethanol. It is an important chelating agent that can chelate metal ions in solution. It can prevent discoloration, deterioration, turbidity and oxidative loss of vitamin C caused by metals, and can also improve the oxidation resistance of oil (trace metals in oil, such as iron, copper, etc., can promote the oxidation of oil). The chemical formula is C ₁₀ H ₁₄ N ₂ Na ₂ O ₈ , it has six coordinating atoms, and the complex formed is called a chelate complex. Disodium edetate is often used in coordination titration, generally for the determination of metal ions. content. It has important uses in dyestuff, food, medicine and other industries. In this application, "edetate disodium dihydrate" is also called ethylenediaminetetraacetic acid disodium salt dihydrate, the molecular formula is C ₁₀ H ₁₄ N ₂ Na ₂ O ₈ ·2H ₂ O, which is a white crystalline powder , odorless, the application field covers many industries such as chemical industry, medicine, food, agriculture, etc., and has a good complexation effect.

The present application provides an interleukin 29 mutant protein preparation, which comprises: an interleukin 29 mutant protein, a buffer system, a metal ion chelator and an osmotic pressure regulator.

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein comprises a substitution mutation of the 161st or 162nd amino acid on the amino acid sequence shown in SEQ ID NO: 1, wherein the 161st amino acid Aspartic acid (D) or glycine (G) at position 162 is substituted by other natural amino acids, for example by amino acids selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, Methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamine amino acid, lysine, arginine or histidine.

It will be understood by those skilled in the art that positions 161 and 162 of SEQ ID NO: 1 are related to SEQ ID NO: 2 (wild-type interleukin 29 mature protein) and SEQ ID NO: 3 (wild-type leukocytes comprising a signal peptide interleukin 29 full-length protein), therefore, based on SEQ ID NO: 2 or SEQ ID NO: 3 (or amino acid sequences of different lengths derived from SEQ ID NO: 2 or SEQ ID NO: 3) Amino acid mutations at the corresponding positions are also covered within the protection scope of the present application. The protection scope of the present application also covers the interleukin 29 mutant proteins derived from SEQ ID NO: 2 or SEQ ID NO: 3, but having different lengths of the amino acid sequence at the corresponding positions of the above-mentioned positions 161 and 162 containing substitution mutations .

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein comprises a substitution mutation of the 161st or 162nd amino acid on the amino acid sequence shown in SEQ ID NO: 1, wherein the 161st amino acid Aspartic acid (D) or glycine (G) at position 162 was substituted with other natural amino acids, including the starting methionine (M). This is because when IL-29 is expressed in prokaryotic cells (eg E. coli), the expressed IL-29 protein has N-terminal or amino-terminal methionine.

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein comprises a substitution mutation at the 161st or 162nd amino acid in the amino acid sequence shown in SEQ ID NO: 1, that is, from SEQ ID NO: 1 Mutations at position 161 or 162, eg, aspartic acid (D) at position 161 or glycine (G) at position 162, are calculated from the N-terminus or amino-terminus of the indicated protein. Other natural amino acid substitutions. In a specific embodiment, the IL-29 mutant protein of the present application is a mutant protein expressed in prokaryotic cells (eg, E. coli), contained at position 162 or 163 (because of the addition of an N-terminal M, Substitution mutations on (counted from M), for example, the aspartic acid (D) at position 162 or the glycine (G) at position 163 is replaced by other natural amino acids. In a specific embodiment, the IL-29 mutant protein is shown in SEQ ID NOs: 4-9.

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein comprises a substitution mutation at the 161st or 162nd amino acid in the amino acid sequence shown in SEQ ID NO: 1, for example, wherein the 161st position The aspartic acid (D) of SEQ ID NO: 1 is replaced by glutamic acid, threonine or serine, or the glycine (G) at position 162 is replaced by an aliphatic amino acid; the gene sequence corresponding to the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO: 18. In a specific embodiment, the IL-29 mutant protein is a mutant protein expressed in prokaryotic cells (eg, E. coli), contained at position 162 or 163 (because of the addition of an N-terminal M, from Substitution mutations on M), for example wherein the aspartic acid (D) at position 162 is substituted with glutamic acid, threonine or serine, or the glycine (G) at position 163 is substituted with an aliphatic amino acid .

In a specific embodiment, in the formulation of the present application, the IL-29 mutant protein comprises or consists of the following amino acid sequence: SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein further comprises an amino acid sequence from cysteine (C) to serine (S) at position 165 on the amino acid sequence shown in SEQ ID NO: 1 substitution mutation. As described above, when the IL-29 mutant protein is expressed in prokaryotic cells (such as E. coli), the substitution mutation from cysteine (C) to serine (S) at position 165 will appear at the first position. 166, at this time, the IL-29 mutant protein sequence is shown in SEQ ID NO: 10.

In a specific embodiment, in the formulation of the present application, the IL-29 mutant protein comprises or consists of the following amino acid sequence: SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein is the IL-29 mutant protein (IL-29 DE) shown in the following SEQ ID NO: 4:

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein is the IL-29 mutant protein (IL-29 DE+CS) shown in the following SEQ ID NO: 5:

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein is the IL-29 mutant protein (IL-29 DS) shown in the following SEQ ID NO: 6:

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein is the IL-29 mutant protein (IL-29 DS+CS) shown in the following SEQ ID NO: 7:

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein is the IL-29 mutant protein (IL-29 GA) shown in the following SEQ ID NO: 8:

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein is the IL-29 mutant protein (IL-29 GA+CS) shown in SEQ ID NO: 9:

In a specific embodiment, in the preparation of the present application, the IL-29 mutant protein further comprises a short sequence (such as a short sequence of 6 histidines) that facilitates protein purification, or a short amino acid sequence that prolongs half-life .

In a specific embodiment, the preparation of the present application is a liquid preparation, which can be made into dosage forms such as injections, tablets, capsules, inhalants, suppositories and the like. Preferably, the formulations of the present application can be formulated into inhalants, such as dry powder inhalers or liquid inhalers, atomized inhalers, aerosols, soft mists and sprays, etc., by inhalation devices such as nebulizer inhalers, metered doses Inhalation device, dry powder inhalation device for administration.

In a specific embodiment, in the preparation of the present application, the mass volume concentration of the interleukin 29 mutant protein is 0.1-1 mg/mL, for example, it can be 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL , 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, etc.

In a specific embodiment, in the preparation of the present application, the buffer system is phosphate buffer, acetate buffer or histidine buffer, preferably phosphate buffer. Wherein, the substance concentration of the buffer solution is 20-80 mmol/L, preferably 20-40 mmol/L. Because phosphate buffer is non-irritating, and is used in a variety of products on the market, it is safer, so phosphate buffer is preferred; the substance concentration of the phosphate buffer is 20-80mmol/L, for example Can be 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L, 55mmol/L, 60mmol/L, 65mmol/L, 70mmol/L, 75mmol/L, 80 mmol/L, etc., preferably 20 to 40 mmol/L.

In a specific embodiment, the pH of the formulation is 4.0-5.0, such as 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, etc., preferably 4.0-4.5.

In a specific embodiment, in the preparation of the present application, the metal ion chelating agent is disodium edetate dihydrate, and its mass volume concentration in the preparation is 0.01-0.1 mg/mL, for example, it can be 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.1mg/mL, etc. , preferably 0.01 to 0.05 mg/mL. The metal ion chelator can reduce protein degradation reactions caused by metal ions.

In a specific embodiment, in the formulation of the present application, the content of the osmotic pressure regulator keeps the osmotic pressure of the formulation at 280-320 mOsmol/kg. Wherein, the osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride and magnesium chloride. Preferably, the osmotic pressure regulator is sodium chloride, and the mass volume concentration of the sodium chloride is 6-10 mg/mL, such as 6 mg/mL, 6.5 mg/mL, 7 mg/mL, 7.5 mg/mL , 8mg/mL, 8.5mg/mL, 9mg/mL, 9.5mg/mL, 10mg/mL, etc. Osmotic pressure regulators can balance the osmotic pressure of the preparation and various liquids in the human body and maintain isotonicity.

In a specific embodiment, in the preparation of the present application, the mass volume concentration of the interleukin 29 mutant protein is 0.5-1 mg/mL, and the mass volume concentration of the disodium edetate is 0.01 mg/mL, The pH of the formulation is 4.0 to 5.0.

In a specific embodiment, in the preparation of the present application, the preparation is: 0.5 mg/mL interleukin 29 mutant protein, 0.01 mg/mL disodium edetate, 20 mmol/L phosphate buffer, 8 mg/mL mL of sodium chloride, the pH of the formulation was 4.5.

In a specific embodiment, in the preparation of the present application, the preparation is: 1.0 mg/mL interleukin 29 mutant protein, 0.01 mg/mL disodium edetate, 20 mmol/L phosphate buffer, 8 mg/mL mL of sodium chloride, the pH of the formulation was 4.5.

In a specific embodiment, in the preparation of the present application, the preparation is: 0.5 mg/mL interleukin 29 mutant protein, 0.05 mg/mL disodium edetate, 20 mmol/L phosphate buffer, 8 mg/mL mL of sodium chloride, the pH of the formulation was 4.5.

In a specific embodiment, in the preparation of the present application, the preparation is: 1.0 mg/mL interleukin 29 mutant protein, 0.05 mg/mL disodium edetate, 20 mmol/L phosphate buffer, 8 mg/mL mL of sodium chloride, the pH of the formulation was 4.5.

There are also many methods for testing the stability of interleukin 29 (IL-29) mutant protein and IL-29 mutant protein preparations, such as reversed-phase high performance liquid chromatography can be used to analyze hydrophobicity and polarity difference. Impurities; ion exchange high performance liquid chromatography can be used to separate impurities with large differences in charge; and molecular sieve exclusion chromatography is used to analyze dimers, polymers and monomers. Each assay has a different focus, but all can be used to characterize the purity of the protein to determine the stability of the protein preparation.

The present application will be described in detail below by using examples. It should be understood, however, that the application may be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present invention will be more thoroughly understood, and will fully convey the scope of the application to those skilled in the art.

Example

Example 1 Preparation of IL-29 mutant protein

1.1 Construction of mutant protein expression engineering bacteria

The IL-29 mutant protein gene fragment SEQ ID NO: 11-17 was obtained by chemical synthesis, and the above fragment was inserted into the prokaryotic expression plasmid pET-30a(+) (Novagen) through Node I and Xho I sites and sequenced verify. The resulting expression plasmids were used for transformation assays. The plasmid containing the target gene obtained above was transformed into Escherichia coli BL21 (DE3) competent cells (Invitrogen), 50 μL of BL21 competent cells were thawed on an ice bath, the plasmid was added, shaken gently, and placed in an ice bath 30 minutes. Then heat shock in a water bath at 42°C for 30 seconds, then quickly transfer the centrifuge tube to an ice bath for 2 minutes without shaking the centrifuge tube. 500 μL of sterile LB medium (without antibiotics) was added to the centrifuge tube, mixed well, and then placed at 37° C. and incubated at 180 rpm for 1 hour to recover the bacteria. Pipette 200 μL of transformed competent cells onto LB agar plates containing kanamycin resistance, and spread the cells evenly. Place the plate at 37°C until the liquid is absorbed, invert the plate, and incubate at 37°C overnight. The next day, the monoclonal colonies in the transformed plates were picked using an inoculation loop, and inoculated into 15 mL of sterile LB medium (containing kanamycin), and cultured at 30°C overnight.

1.2 Expression and purification of IL-29 mutant protein

To 50 mL of LB medium, add 50 μL of the bacterial solution of the above-mentioned bacteria, and at the same time, add 50 μL of kanamycin, mix well, place it in a constant temperature shaker at 30°C, and inoculate overnight. Take 10 mL of the overnight inoculated bacterial solution and add it to 1000 mL of LB medium, and at the same time add 1000 μL of kanamycin. Shake well and place in a shaker at 37°C, 200 rpm, cultivate until the OD600 of the bacterial solution is 0.4-0.6 hours, add the final concentration of 0.5 mM IPTG to induce, continue to cultivate for 4 hours and collect the bacterial cells. The expressed IL-29 mutant accounted for about 30-50% of the total bacterial protein, mainly in the form of inclusion bodies.

The fermented thalline was washed 3 times with TE (10mmol/L Tris-HCl, 1mmol/L EDTA, pH 6.5) solution (m:V=1:10), then 60Mpa high pressure homogenization was broken, and after homogenization, the Bacterial breakage rate. When the bacterial cell fragmentation rate is about 95% (about 2-3 times of bacterial breaking), centrifuge at 8000 rpm for 15 min, and collect the fragmented bacterial cell pellet. Take the broken cell pellet and put it in a beaker, add inclusion body washing solution (10mM Tris-HCl+1mM EDTA+0.5%Triton-X100, pH 6.5, m:V=1:10), stir on a magnetic stirrer for 30min, wash 3 to 5 times. Inclusion bodies were lysed with inclusion body lysis buffer (7M guanidine hydrochloride+50mM Tris-HCl+10mM DTT, pH 6.5, m:V=1:10), stirred at room temperature, overnight. The cleaved protein was slowly added to the renaturation solution (100mM Tris-HCl, 0.5M arginine, 0.5% PEG3350 (m:V), 2mM GSH:0.5mM GSSG, pH 8.5) to make the final protein concentration 0.2mg/ mL, stirred at room temperature overnight.

The renaturation solution was centrifuged at 8000 rpm for 5 min, and the supernatant was collected. Using an ultrafiltration membrane pack with a pore size of 10 kDa, the ultrafiltration membrane was equilibrated with 20 mM phosphate buffer pH 7.0, and then 1 L of the supernatant was concentrated 10-fold. The concentrated solution was diluted with 5 times the volume of water for injection to be loaded, and the column was packed with Sepharose FF packing, 50 mmol/L Tris-HCl, pH 8.5, 0.1 mol/L NaCl equilibrated, and loaded; pH8.5, 0.15mol/L NaCl for elution, collect elution peak components and finally collect 600mL sample solution; Sepharose FF packing column, 20mmol/L phosphate, pH7.4, 0.05mol/L NaCl balance, load the sample , until the detector baseline stabilizes. Rinse with 20 mmol/L phosphate, pH 7.4, 0.2 mol/L NaCl, and collect the elution peak components.

Seven kinds of IL-29 mutant proteins (referred to as IL-29 mutants) were obtained, corresponding to the protein amino acid sequences SEQ ID NOs: 4-10, respectively named IL-29DE, IL-29DE+CS, IL-29DS, IL -29 DS+CS, IL-29GA, IL-29GA+CS, IL-29CS.

Wherein, the gene sequence of IL29 DE+CS is shown in SEQ ID NO: 11, the gene sequence of L29 DS+CS is shown in SEQ ID NO: 12, and the gene sequence of IL29 GA+CS is shown in SEQ ID NO: 13, The gene sequence of IL29 DE is shown in SEQ ID NO: 14, the gene sequence of IL29 DS is shown in SEQ ID NO: 15, the gene sequence of IL29 GA is shown in SEQ ID NO: 16, and the gene sequence of IL29CS is shown in SEQ ID NO: 16 : 17 shown.

Meanwhile, the IL-29 wild-type protein (abbreviated as IL-29 reference substance) was also synthesized by the same method as above, which corresponds to the protein amino acid sequence SEQ ID NO: 1 and has an additional M amino acid at the N-terminus.

Example 2 Detection of various indicators of the obtained IL-29 mutants

2.1 Molecular weight and purity of IL-29 mutants obtained by SDS-PAGE electrophoresis

Using SDS-PAGE electrophoresis loading buffer, in the case of adding mercaptoethanol, Marker and 10 μg of the above-obtained protein were loaded respectively, and electrophoresis was carried out. The electrophoresis conditions were 200V constant voltage for 45 minutes. The protein molecular weight and purity were detected by staining with Coomassie brilliant blue G-25. The results are shown in Figures 1 and 2.

It can be seen from Figures 1 and 2 that the molecular weights of the IL29 mutant protein and the reference substance are 20kDa respectively, indicating that the obtained target protein is correct, with only one band without other heterobands, and the purity can reach 100%.

2.2 In vitro RP-HPLC purity of IL-29 mutants detected by chromatography

2.2.1 Reversed-phase high performance liquid chromatography

Prepare mobile phase A: acetonitrile:water:trifluoroacetic acid volume ratio of 20:80:0.1, mobile phase B: acetonitrile:water:isopropanol:trifluoroacetic acid volume ratio of 70:20:10:0.1, use a chromatographic column (XBridge BEH C18 Column, 130A, 5μm, 4.6mm*100mm), analyze each protein test sample. The analysis conditions are: flow rate 1.0mL/min, acquisition time: 65min, acquisition wavelength 214nm, column temperature 45℃, elution gradient as follows:

Table 1

time (min)	0	5	5.01	45	55	55.01	65
B%	27	27	27	49	70	27	27

Two needles of the original solution of the test sample were injected in parallel, and the injection volume was set according to the sample concentration, and the injection volume was 15 μg to 20 μg; finally, the main peak purity of the test sample was calculated by integrating according to the area normalization method.

2.2.2 Ion exchange chromatography

Prepare mobile phase A: 25mmol/L phosphate buffer pH7.0, mobile phase B: 25mmol/L phosphate buffer, 0.5mol/L sodium chloride pH6.7), use a chromatographic column (Thermo ProPac WCX-10 4.0 *250mm), analyze each protein test sample. The analysis conditions are flow rate 0.8mL/min, acquisition time: 55min, acquisition wavelength 214nm, column temperature 25℃, elution gradient:

Table 2

time (min)	0	30	31	40	41	55
A%	80	70	10	10	80	80
B%	200	30	90	90	20	20

The injection volume was 20 μg, and the integration was performed according to the area normalization method to calculate the purity of the main peak of the two parallel needles of the test sample.

2.3 Determination of in vitro cell biological activity of IL-29 mutants by reporter gene method

HEK293-ISRE-Luc cells (purchased from China National Institute for Food and Drug Control) were grown adherently in complete culture medium. Passage at 1:4, 2-3 times a week, and grow in complete culture medium. The cultured cells were removed from the culture medium, washed once with PBS, digested and collected, and prepared into a cell suspension containing 3.5×10 ⁵ to 4.5×10 ⁵ cells per 1 mL with assay medium (BIBCO). The prepared IL-29 mutant protein and IL-29 control substance were transferred into a 96-well plate that can be used for cell culture and chemiluminescence microplate reader (Molecular Devices) reading, and 100 μL was added to each well, and then the above cell suspension was inoculated in In the same 96-well plate, 100 μL per well. Incubate at 37°C and 5% carbon dioxide for 19-23 hours. Carefully aspirate the supernatant in the 96-well plate, add the cell lysate and luciferase substrate according to the instructions of the luciferase detection kit (Bright-GloTM Luciferase Assay System, Promega), and use a chemiluminescence microplate reader to measure, _EC50 values for IL-29 mutants and IL-29 controls were recorded separately. Its relative biological activity was calculated as follows: with the IL-29 control as the standard, its 0-point activity was defined as "100%", and the relative biological activity = IL-29 control EC ₅₀ (0 point)/IL-29 mutant _EC50 .

The EC ₅₀ of each test substance of the IL-29 mutant was determined, and the IL-29 reference substance was used as the reference substance to calculate the relative biological activity of each mutant. The specific results are shown in Table 3 below:

Table 3 In vitro cell biological activity data of IL-29 mutants

protein type	EC50 (ng/mL)	Relative biological activity (%)
IL-29 Control	4.783	100%
IL-29DE	1.697	282%
IL-29DS	4.316	90%
IL-29GA	5.264	91%
IL-29CS	4.639	103%
IL-29DE+CS	1.428	335%
IL-29DS+CS	4.982	96%
IL-29GA+CS	3.985	120%

As can be seen from Table 3 above, although the D at position 162 of IL-29 is not located in the active center that binds to the IL-29 receptor, it was unexpectedly found through experiments that the EC ₅₀ of single-mutated IL-29DE was only 1.697, far Much lower than the 4.783 of the IL-29 control substance, the biological activity has increased nearly three times, while the other mutants IL-29DS, IL-29GA, IL-29CS with a single mutation site are compared with the IL-29 control substance. The biological activity of EC ₅₀ was basically unchanged. The double-mutated IL-29DE+CS is based on the D mutation at position 162 of IL-29 and the C mutation at position 166 is S, and its biological activity is further based on the single-mutated IL-29DE. Compared with the IL-29 control substance, the biological activities of the double mutant IL-29DS+CS and IL-29GA+CS mutants were not significantly improved. The present results indicate that the mutation of D at position 162 of the active center that does not bind to the IL-29 receptor to E has an unexpected effect of improving the biological activity of the mutated protein.

Example 3 Result of 50°C stability test of IL-29 mutant

The stability of the proteins in this application is primarily characterized by RP-HPLC purity.

Under the conditions of 50°C±2°C/75% relative humidity±5% relative humidity, take samples according to Table 4, and carry out the reverse phase of each test sample of IL-29 mutant in the same way as 2.2.1 in Example 2 The high-performance liquid phase purity determination was performed in the same manner as in 2.3 in Example 2 to determine the biological activity of each test substance of the IL-29 mutant. The results are shown in Table 5. At the same time, the purity values of day 0 and day 14 in Table 5 are compared with chromatograms, as shown in Figures 3 to 10.

Table 4 Stability verification scheme of IL-29 mutants at 50°C

Table 5 Determination results of reversed-phase high performance liquid phase purity of IL-29 mutants at 50°C

From the results in Table 5 and Figures 3 to 10, it can be seen that the two test substances with the greatest decrease in purity are IL-29 reference substance and IL-29CS, in which the purity of IL-29 reference substance dropped sharply from 85.82% to 34.46% within 14 days. %, the decrease was as high as 51.36%, the purity of IL-29CS dropped sharply from 97.03% to 46.20% within 14 days, and the decrease was as high as 50.83%; the two samples with the smallest decrease were IL-29DE and IL-29DE+CS, among which IL The purity of -29DE decreased from 92.68% to 77.39% within 14 days, a decrease of only 15.29%, and the purity of IL-29DE+CS decreased from 97.22% to 86.66% within 14 days, with a lower decrease of only 10.56%. The purities of other single-point mutants IL-29DS and IL-29GA decreased by 24.22% and 19.65% within 14 days, respectively, and the purities of other double-point mutants IL-29DS+CS and IL-29GA+CS within 14 days were decreased by 18.25% and 14.97, respectively. %.

From the above results, it can be inferred that the single-point mutant with the mutation from D to E at position 162 has the best stability and the smallest decrease in purity within 14 days. Moreover, because the stability of IL-29CS is much smaller than that of IL-29DE, according to conventional inferences, the stability of the double point mutant IL-29DE+CS may be lower than that of IL-29DE, while the stability of IL-29DE+CS in the present invention may be lower than that of IL-29DE+CS. The stability was also much higher than that of IL-29DE, indicating that the double-point simultaneous mutation also has a synergistic effect in improving the stability of the mutant. At the same time, the biological activities of the mutants were detected, and it was found that the biological activities of the mutants IL-29DE and IL-29DE+CS basically did not change within 14 days.

Example 4 Result of 25°C light stability test of IL-29 mutant

Under the conditions of 25°C ± 2°C/60% relative humidity ± 5% relative humidity/5000 ± 500 lux, the samples were taken as shown in Table 6, and the stability of the IL-29 mutant was determined by the same method as in Example 2. , the results are shown in Table 7, in which IL-29CS was used as the control.

Table 6 25℃ light stability scheme of IL-29 mutant

Table 7 The results of the determination of the stability of IL-29 mutants under light at 25°C

From the results in Table 7 above, it can be seen that the stability of the IL-29 mutant IL-29DE+CS is better than that of IL-29CS under the light condition of 25°C.

Example 5 Nebulization stability experiment of each mutant of IL29

The atomization experiment was carried out using a German PARI LCD type jet atomizer and a TurboBOY N atomizing pump, a total of 2 mL of samples were atomized, and the aerosol was collected in the manner shown in Figure 11 below. The same method was used to carry out the RP-HPLC purity determination of each test substance of IL29 mutants to verify the nebulization stability of each mutant. Wherein IL29 CS is used as reference substance, and the results are shown in Table 8 below.

Table 8

From the results in Table 8, it can be seen that the purity of IL29 mutants IL29 DE+CS and IL29 GA+CS decreased by only about 3% after nebulization, and the purity of IL29 DS+CS decreased by 8.1% after nebulization, while the purity of IL29CS after nebulization was greatly reduced. A drop, by as much as nearly 11%. It can be seen that the nebulization stability of IL29 mutants is significantly enhanced compared with IL29CS, especially the IL29 mutants IL29 DE+CS and IL29 GA+CS have particularly good stability after nebulization, especially suitable for the preparation of aerosol inhalation preparation.

Example 6 In vitro pharmacodynamic assay of IL29 mutants on RSV infection of human bronchial epithelial cells (HBECs)

After the cell differentiation (Cell Application) of HBEC (human bronchial epithelial cell) was completed, the doubling-diluted IL29 mutant (IL29 DE+CS) and the positive control drug (BMS-433771, provided by Shanghai WuXi AppTec) For Respiratory Syncytial Virus Fusion Protein Inhibitor), differentiated HBEC cells were added and incubated at 37 °C in a 5% _CO incubator. Respiratory syncytial virus (respiratory syncytial virus, provided by Shanghai WuXi AppTec) was inoculated 24h before infection, and 1h and 24h after infection, and RSV with a titer of 100 TCID50 (half the tissue culture infectious dose) was added to each well of the activity test well, and the cells were placed in After 3 days of incubation in a 37°C and 5% CO ₂ incubator, RSV RNA was extracted from the cells using an RNA extraction kit (Cat. No. 74181, Qiagen) and quantified by RT-qPCR. Use GraphPad Prism software to analyze the compound dose-response curve and calculate the EC ₅₀ value and the average bacteriostatic rate. The results are shown in Table 9 and Table 10.

Table 9 Inhibition of RSV infection by positive control drug BMS-433771 in HBEC cell model

Table 10 Inhibition of RSV infection by IL29 mutants in HBEC cell model

The results showed that IL29 DE+CS showed a good anti-RSV efficacy in an in vitro model of human bronchial epithelial cells, with an EC ₅₀ of 0.13ng/ml (6.5pM), which was 1/100000 of the control drug and much lower than the control. medicine.

Example 7 Determination of in vivo efficacy of IL29 mutants on RSV infection in mouse models

On day 0, all mice (female, 6-7 weeks old, 16-18 g, specific pathogen-free (SPF) grade BALB/c mice (provided by Shanghai WuXi AppTec)) were injected intraperitoneally with sodium pentobarbital ( 75mg/kg) after anesthesia, inoculated with RSV (human respiratory syncytial virus A2 (RSV-A2;, purchased from BEI Resources, NIAID, NIHBethesda, MD) by intranasal method, the inoculation amount was 1.1×10 ⁵ PFU per animal , the inoculation volume was 50 μL.

From the 0th day to the 3rd day, the animals were administered according to the protocol in Table 11. The administration method was to spray the mice into the lungs after anesthesia, and then spray the liquid atomizing device (purchased from Shanghai Yuyan Instrument Co., Ltd.) with the pre-filled drug solution. The micro-spray cannula of the company) was gently inserted into the appropriate position of the mouse trachea, and the piston of the high-pressure push device of the nebulizer was quickly pressed to nebulize a quantitative volume of the drug into the mouse lungs (references Joseph D. Brain, Dwyn E. Knudson, Sergei P. Sorokin, Michael A. Davis, Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation, Environmental research 11, Volume 11, Issue 1, 1976, Pages 13-33 dosing regimen) with a frequency of daily once. The fifth day after infection was the end point of the in vivo experiment. All animals were euthanized and lung tissues were harvested to detect the virus titer (plaque assay). The results are shown in Table 12 and Figure 12.

Table 11 Dosing regimen in mice

Note: The IL29 mutant used in this example is IL29 DE+CS.

Table 12 In vivo efficacy results of different drugs on RSV infection mouse models

Note: Compared with the vehicle (normal saline) group, ***P<0.001, **P<0.01

The data showed that RSV could replicate in large numbers in mice after inoculation, and the positive control drug ribavirin significantly inhibited RSV replication in mice, showing the expected anti-RSV activity in vivo, proving the effectiveness of this model system. The administration time point of ribavirin was 1 h before virus infection (prevention model), while the test drug IL29 mutant was administered under set assay conditions (1 hour before virus infection, 1 hour after virus infection and after virus infection) 24 hours), can significantly inhibit the replication of RSV virus in mice, and the virus titers in the lung tissue of mice in the first administration group 1 hour before inoculation (10 μg) and 1 hour after inoculation (10 μg) are at the lower limit of detection Hereinafter, excellent in vivo anti-RSV efficacy was shown. The group administered 24 hours after infection also showed a good anti-RSV effect. The antiviral effect of IL29 mutants administered 1 hour after infection was comparable to the results of the prophylactic model in which ribavirin was administered 1 hour before infection. This also fully shows that the IL29 mutant has a very good antiviral therapeutic potential.

Example 8 Determination of in vivo efficacy of IL29 mutants on RSV-infected cotton rat model

On day 0, all mice (Sigmodon hispidus cotton rat, half male and female, 5 weeks old, SPF grade (purchased from Envigo)) were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg), and then intranasally administered RSV (human respiratory syncytial virus A2 (RSV-A2), purchased from BEI Resources, NIAID, NIHBethesda, MD), was inoculated with 1.1×10 ⁵ PFU per animal, and the inoculation volume was 50 μL.

From day 0 to day 3, animals were dosed according to the assay protocol in Table 13 by pulmonary nebulization (refs Joseph D. Brain, Dwyn E. Knudson, Sergei P. Sorokin, Michael A. Davis, Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation, dosing regimen of Environmental research 11, Volume 11, Issue 1, 1976, Pages 13-33) with a frequency of once daily. A 3ml syringe with a 22G needle was inserted into the trachea, 2mL of 0.9% saline was injected into the lungs, and bronchoalveolar lavage fluid (BALF) was collected. The collected BALF was divided into sterile 1.5mL EP tubes, frozen on dry ice, and stored at -80°C until plaque test analysis. The results are shown in Table 14 and Figure 13.

Table 13 Dosing regimen in cotton rat

Note: The IL29 mutant used in this example is IL29 DE+CS.

Table 14 In vivo efficacy results of different drugs on RSV-infected cotton rat models

The data in Table 14 and Figure 13 show that RSV can replicate in a large amount in cotton rats after inoculation, and the test drug IL29 mutant can significantly inhibit RSV virus in mice under set conditions (a therapeutic model administered 1 hour after inoculation). In vivo replication, the virus titers in the cotton rat bronchial lavage fluid of the first administration group (groups 2, 3, and 4) at 1 hour after inoculation (1 μg) were statistically different from those in the control group, and the three doses were significantly different. Groups (Groups 2-4) showed a dose-related relationship, showing excellent in vivo anti-RSV efficacy. After cotton rats are infected with RSV, the virus will replicate in the bronchi of their lungs, and the viral load in the bronchial lavage fluid is a very good reflection of the virus replication in the lungs. Therefore, the IL29 mutants show a very good therapeutic potential in addition to respiratory syncytial virus.

Example 9 Buffer pH Screening

Weigh 3.4g of solid sodium acetate, dissolve it in 800mL of ultrapure water, add dilute hydrochloric acid/NaOH to adjust the pH to 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 respectively, and set the volume to 1L to obtain 25mmol/L Acetate buffer. Then, the IL-29 mutant protein IL-29DE+CS in Example 1 was dialyzed with the prepared dilution buffer. After the buffer was replaced by dialysis, the sample was diluted with the buffer to a concentration of 100 μg/mL, and the samples were packed in aliquots. 0.5mL/sample. The sample information is as follows in Table 15, using the same method as 2.2.1 in Example 2 for reversed-phase high-efficiency liquid phase purity determination, using the same method as 2.2.2 in Example 2 for ion exchange purity determination, and using reversed-phase high-efficiency Protein quantification by liquid chromatography.

Table 15

The buffer pH screening accelerated stability conditions and sampling design are shown in Table 16 below:

Table 16

The results of the RP-HPLC purity test of the accelerated experiment at 50°C are shown in Table 17 below:

Table 17

The ion exchange purity test results of the 50°C accelerated experiment are shown in Table 18 below:

Table 18

The 50℃ accelerated experiment reversed-phase high performance liquid chromatography and ion exchange purity test data show that the pH value of the buffer is relatively stable at 4.0～5.0; at the same time, the 50℃ accelerated experiment protein quantitative detection results show that the buffer pH value is between 3.5～5.0 At 6.0, there was no obvious content change in the quantitative detection of protein. Preferably, the pH of the buffer is between 4.0 and 5.0, and more preferably, the pH of the buffer is between 4.0 and 4.5. Combining the numerical results for the pH range, the most preferred pH for this formulation was determined to be 4.5. In this way, in the large-scale production process, the formulation can be controlled within a relatively optimal pH value range.

Example 10 Screening of buffer types

Acetate buffer, citrate buffer, histidine buffer and phosphate buffer were prepared as shown in Table 19 below.

Table 19

The IL-29DE+CS samples of the IL-29 mutant protein in Example 1 were dialyzed respectively with the prepared four buffer solutions, and after the dialysis was replaced with the buffer solution, the sample was diluted with the buffer solution to a concentration of 200 μg/mL. , aliquot 0.5mL/piece. The sample information is as follows in Table 20, using the same method as 2.2.1 in Example 2 for reversed-phase high-efficiency liquid phase purity determination, using the same method as 2.2.2 in Example 2 for ion exchange purity determination, and using reversed-phase high-efficiency Protein quantification by liquid chromatography.

Table 20

The buffer type screening accelerated stability conditions and sampling design are shown in Table 21 below:

Table 21

The 40℃ accelerated experiment reversed-phase high-performance liquid phase purity detection results are shown in Table 22 below:

Table 22

The ion exchange purity test results of the accelerated experiment at 40°C are shown in Table 23 below:

Table 23

The results of the RP-HPLC purity test of the accelerated experiment at 25°C are shown in Table 24 below:

Table 24

The ion exchange purity test results of the accelerated experiment at 25°C are shown in Table 25 below:

Table 25

The 40℃ accelerated experiment reversed-phase high-performance liquid phase purity detection data showed that the samples treated with citrate buffer showed poor stability, and the samples treated with the other three buffers showed good stability and no significant difference; The ion exchange purity test data of the accelerated experiment at 40 °C showed that the samples treated with citrate buffer showed poor stability, while the samples treated with acetate and histidine buffer showed good stability and no significant difference. , the stability of samples treated with phosphate buffer is better than that of samples treated with citrate buffer and slightly worse than that of samples treated with acetate and histidine buffer; accelerated at 40°C In the experimental protein quantitative detection experiment, the protein content of the samples treated with the four buffers did not change significantly.

The data of 25℃ accelerated experiment reversed-phase high performance liquid chromatography and ion exchange purity test showed that the samples treated with citric acid buffer showed poor stability, and the samples treated with the other three buffers showed good stability And no obvious difference; in the 25 ℃ accelerated protein quantitative detection experiment, the protein content of the samples treated with the four buffers did not change significantly.

It can be seen from the above that when acetate buffer, histidine buffer and phosphate buffer are used as buffer systems, the sample stability is good, and they can all be used as buffer systems in the preparation of the present application. Because phosphate buffer is non-irritating and used in a variety of marketed products, especially when the preparation of the present application is preferably made into an inhalant, the safety requirements are higher, so the preparation of the present application preferably uses phosphate buffer.

Example 11 Buffer concentration screening

Routinely weigh sodium dihydrogen phosphate monohydrate and dissolve it in ultrapure water, adjust the pH value with NaOH, and prepare phosphate buffer solutions of 10 mmol/L, 20 mmol/L, 40 mmol/L, 80 mmol/L, and pH 4.5, respectively. The IL-29 mutant protein IL-29DE+CS samples were dialyzed with the prepared phosphate buffers of different amounts and concentrations, respectively. After the target buffer was replaced by dialysis, the samples were diluted to a concentration of 300 μg/ mL, aliquot 0.5mL/piece. The sample information is as follows in Table 26, using the same method as 2.2.1 in Example 2 for reversed-phase high-efficiency liquid phase purity determination, using the same method as 2.2.2 in Example 2 for ion exchange purity determination, and using reversed-phase high-efficiency Protein quantification by liquid chromatography.

Table 26

The buffer concentration screening accelerated stability conditions and sampling design are shown in Table 27 below:

Table 27

The results of the RP-HPLC purity test of the accelerated experiment at 40°C are shown in Table 28 below:

Table 28

The ion exchange purity test results of the accelerated experiment at 40°C are shown in Table 29 below:

Table 29

The high-performance liquid phase purity test data of the accelerated experiment at 40 °C showed that the samples treated with phosphate buffer of 20-80 mmol/L showed good stability; The samples treated with phosphate buffer showed good stability; in the accelerated protein quantitative detection experiment at 40 °C, the protein content of the samples treated with the four concentrations of buffer did not change significantly, indicating that the phosphate buffer has When the concentration of the substance is in the range of 10-80 mmol/L, the effect of the concentration of phosphate buffer on the stability of the sample is not very different. In conclusion, when the concentration of the phosphate substance is 20-40 mmol/L, the stability of the IL-29 mutant IL-29DE+CS of the present application is the best, more preferably 20 mmol/L.

Example 12 IL-29 mutant protein preparation

Groups of formulations were prepared according to the formulations in Table 30 below. Firstly, after dissolving the excipients sodium dihydrogen phosphate monohydrate, sodium chloride and disodium edetate dihydrate in water for injection, use dilute hydrochloric acid to adjust the pH value of the solution to 4.5, and then use the prepared solution to separate each IL The -29 mutant protein IL-29DE+CS sample was dialyzed, and after dialysis replacement, the sample was diluted to the formula concentration for use. Use the same method as 2.2.1 in Example 2 for reversed-phase high-performance liquid phase purity determination, and use the same method as 2.2.2 in Example 2 for ion-exchange purity determination, while using reversed-phase high-performance liquid chromatography for protein determination. Quantitative.

Table 30

The accelerated stability conditions and sampling design are shown in Table 31 below:

Table 31

The 40°C accelerated experiment reversed-phase high-performance liquid phase purity detection results are shown in Table 32 below:

Table 32

The ion exchange purity test results of the accelerated experiment at 40°C are shown in Table 33 below:

Table 33

The results of the RP-HPLC purity test of the accelerated experiment at 25°C are shown in Table 34 below:

Table 34

The ion exchange purity test results of the 25°C light acceleration experiment are shown in Table 35 below:

Table 35

The 40℃ accelerated experiment reversed-phase high-performance liquid phase purity test data showed that under the same IL-29 mutant protein mass and volume concentration, the protein preparation samples with low edetate disodium mass and volume concentration of 0.01-0.05 mg/mL showed better performance The stability of protein preparation has reached a relatively good level, especially when the mass volume concentration of edetate disodium is a lower concentration of 0.01 mg/mL. 25 ℃ light acceleration experiment reversed-phase high performance liquid detection data showed that the volume concentration of low edetate disodium is 0.01mg/mL ~ 0.05mg/mL, and the volume concentration of high IL-29 mutant protein is 0.5 ~ 1mg/mL The stability of the protein preparation in mL is better.

Since the ion exchange chromatography method mainly controls the impurities to be oxidized impurities, the inhibition effect of disodium edetate on the oxidized impurities can be better reflected. The ion exchange purity test data of the accelerated experiment at 40°C showed that the protein preparation samples with a low mass volume concentration of edetate disodium 0.01-0.05 mg/mL showed good stability, especially the mass volume concentration of edetate disodium was higher. The protein preparation with a low concentration of 0.01 mg/mL and an IL-29 mutant protein concentration of 0.5-1 mg/mL had better stability, and the 21-day acceleration of impurity growth was controlled at a level below 3.962%. The ion exchange purity test data of 25 ℃ light acceleration experiment showed that the increase of oxidized impurities in each group of samples with disodium edetate was controlled within 1.0-2.5%, while the increase of impurities in the group without disodium edetate was 4.5%. %, showing that disodium edetate has a significant inhibitory effect on oxidative impurities, and the stability of protein preparation samples with disodium edetate is significantly better than that without disodium edetate. At the same time, the lower the concentration of disodium edetate in the protein preparation sample added with disodium edetate, the slower the growth rate of impurities; the ion exchange purity detection data of the 25°C light acceleration experiment showed that the low mass and volume concentration of disodium edetate (0.01 ～0.05mg/mL) and protein preparation samples with high IL-29 mutant protein volume concentration (0.5～1mg/mL) showed better stability. In particular, the protein preparation with disodium edetate concentration of 0.01 mg/mL and IL-29 mutant protein concentration of 0.5 to 1 mg/mL has better stability, and the increase in impurities in the 14-day and light-accelerated experiments Controlled below the level of 1.101%.

In the accelerated protein quantitative detection experiments at 40°C and 25°C, the protein content of each group of protein preparation samples did not change significantly.

Based on the above results, it is preferred that the impurity growth rate is slower and the stability is higher. ) protein preparation, especially the protein preparation whose mass volume concentration of edetate disodium is a lower concentration of 0.01 mg/mL, and the mass volume concentration of IL-29 mutant protein is (0.5-1 mg/mL). In the preferred formulation, the addition amount of the adjuvant disodium edetate can be kept at a low level, which further improves the safety index of the formulation.

The above are only preferred embodiments of the present application, and are not intended to limit the present application in other forms. Any person skilled in the art may use the technical content disclosed above to change or remodel to equivalent implementations of equivalent changes. example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present application without departing from the content of the technical solutions of the present application still fall within the protection scope of the technical solutions of the present application.

Sequence Listing:

Claims (10)

1. An interleukin 29 mutant protein preparation, characterized in that it comprises: an interleukin 29 mutant protein, a buffer system, a metal ion chelating agent and an osmotic pressure regulator.
2. The preparation according to claim 1, wherein the interleukin 29 mutant protein comprises a substitution mutation of the 161st or 162nd amino acid in the amino acid sequence shown in SEQ ID NO: 1, wherein the 161st position The aspartic acid (D) or the glycine (G) at position 162 is substituted by other natural amino acids.
3. The preparation according to claim 1 or 2, wherein the aspartic acid (D) at position 161 is substituted with glutamic acid, threonine or serine, or the glycine (G) at position 162 is substituted with Aliphatic amino acid substitutions.
4. The preparation according to any one of claims 1 to 3, wherein the interleukin 29 mutant protein comprises the following amino acid sequence: SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
5. The preparation according to any one of claims 1 to 4, wherein the interleukin 29 mutant protein further comprises the amino acid sequence shown in SEQ ID NO: 1 at position 165 from cysteine (C ) to serine (S) substitution mutation.
6. The preparation according to any one of claims 1 to 5, wherein the interleukin 29 mutant protein comprises the following amino acid sequence: SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
7. The preparation according to any one of claims 1 to 6, wherein the mass volume concentration of the interleukin 29 mutant protein is 0.1 to 1 mg/mL.
8. The preparation according to any one of claims 1 to 7, wherein the buffer system is a phosphate buffer, an acetate buffer or a histidine buffer, preferably a phosphate buffer.
9. The formulation according to any one of claims 1 to 8, wherein the pH of the formulation is 4.0 to 5.0, preferably 4.0 to 4.5.
10. The preparation according to any one of claims 1 to 9, characterized in that the substance concentration of the phosphate buffer is 20-80 mmol/L, preferably 20-40 mmol/L.

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