A pegylated recombinant canine uricase injection preparation

Abstract

The application provides a polyethylene glycolized recombinant canine human uricase injection preparation and a preparation method thereof. The injection preparation is composed of polyethylene glycolized recombinant canine human uricase, a beta cyclodextrin derivative, a buffer and an osmotic pressure regulator. The preparation method comprises the steps of solution preparation, sterilization, canning, plug pressing and the like. The polyethylene glycolized recombinant canine human uricase injection of the application has high purity, few impurities and little PEG residue, avoids the problem of increased drug immunogenicity after PEG shedding, and has high stability; compared with a freeze-drying preparation, the production process is simple, the production cycle is short, the freeze-drying process is not needed, and the energy consumption is low.

Description

Technical Field

[0001] This invention belongs to the field of protein and polypeptide drug technology, specifically relating to a polyethylene glycol-modified recombinant canine human uricase injection preparation and its preparation method. Background Technology

[0002] Gout is a common inflammatory joint disease. High uric acid levels cause urate crystals to crystallize and precipitate, leading to gouty arthritis and uric acid urolithiasis. Polyethylene glycol-modified recombinant canine human uricase lowers serum uric acid levels by catalyzing the oxidation of uric acid to allantoin. Uricase can rapidly reduce blood urate levels in patients who cannot tolerate conventional treatments or for whom conventional therapies are ineffective. However, as an exogenous protein, it has high antigenicity, affecting uricase activity. Therefore, a water-soluble polymer needs to be bound to the surface of the uricase molecule to reduce its antigenicity and prolong its half-life in vivo. Protein PEGylation technology has developed rapidly over the past 20 years. Numerous studies have reported that PEG-modified protein drugs have reduced immunogenicity, improved stability, and prolonged half-life, and are used clinically to treat various diseases. A novel long-acting mammalian uricase product (code-named PEG-UHC) is constructed from polyethylene glycol-modified recombinant canine human uricase (UHC). The unmodified protein UHC consists of 297 amino acids and is active in a tetramer form, with the monomers being almost inactive. Polyethylene glycol-modified recombinant canine human uricase injection is a new generation of long-acting uricase injection prepared by saturating and modifying high-purity canine human uricase through recombinant, expressed, and purified Escherichia coli system with 5kDa mPEG-SPA. It can be used to treat hematologic malignancies chemotherapy and hyperuricemia and chronic gout caused by metabolic disorders.

[0003] Long-term research and clinical practice have shown that PEGylated recombinant canine human uricase injection, like other protein drugs, suffers from poor stability and short shelf life, affecting its therapeutic efficacy. Furthermore, the PEGylation in PEGylated recombinant canine human uricase is prone to detachment, reducing its solubility and causing drug precipitation. The immunogenicity of the drug after PEG detachment increases, seriously impacting the safety of clinical use. This invention optimizes the formulation by adding hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin, resulting in a PEGylated recombinant canine human uricase injection preparation with high stability, simple production process, and low cost, suitable for clinical use. Summary of the Invention

[0004] The purpose of this invention is to provide a PEGylated recombinant canine uricase injection formulation that is highly stable, has a simple manufacturing process, and is low in cost, making it suitable for clinical use. This formulation has a stable protein system, facilitating large-scale production, storage, and transportation, and is convenient for clinical administration. This invention improves the solubility and stability of the PEGylated recombinant canine uricase by adding hydroxypropyl β-cyclodextrin or sulfobutyl β-cyclodextrin to the formulation to form an inclusion complex with the PEGylated recombinant canine uricase, thereby reducing the residual PEG content and enhancing the overall stability of the formulation.

[0005] The technical solution adopted by this invention to solve its technical problem is as follows:

[0006] A polyethylene glycol-modified recombinant canine-human uricase injection, comprising polyethylene glycol-modified recombinant canine-human uricase, a β-cyclodextrin derivative, a buffer, and an osmotic pressure regulator.

[0007] Preferably, the β-cyclodextrin derivative is hydroxypropyl β-cyclodextrin or sulfobutyl β-cyclodextrin.

[0008] Preferably, the content of the polyethylene glycol-modified recombinant canine uricase is 1-15 mg / mL; more preferably, the content of the polyethylene glycol-modified recombinant canine uricase is 5-10 mg / mL.

[0009] Preferably, the weight ratio of the polyethylene glycol-modified recombinant canine uricase to the β-cyclodextrin derivative is 1:5 to 20; More preferably, the weight ratio of the polyethylene glycol-modified recombinant canine uricase to the β-cyclodextrin derivative is 1:8-12.

[0010] Preferably, the buffer is a phosphate buffer, a Tris / hydrochloric acid buffer, a glycine / sodium hydroxide buffer, or a sodium carbonate / sodium bicarbonate buffer.

[0011] Preferably, the buffer content in the polyethylene glycol-modified recombinant canine uricase injection is 3–30 mmol / L; more preferably, the buffer content in the injection is 8–15 mmol / L.

[0012] Preferably, the osmotic pressure regulator is sodium chloride, glucose, mannitol, sorbitol, sucrose, or trehalose.

[0013] Preferably, the content of the osmotic pressure regulator in the polyethylene glycol-modified recombinant canine uricase injection is 20-100 mg / mL; more preferably, the content of the osmotic pressure regulator in the injection is 40-60 mg / mL.

[0014] Preferably, the pH of the injection solution is 7.5 to 9.5; more preferably, the pH of the injection solution is 8.0 to 9.0.

[0015] Preferably, the polyethylene glycol recombinant canine-human uricase injection solution comprises:

[0016] (1) Polyethylene glycol-modified recombinant canine human uricase 1-15 mg / mL;

[0017] (2) β-cyclodextrin derivatives, wherein the weight ratio of polyethylene glycol-modified recombinant canine uricase to β-cyclodextrin derivatives is 1:5 to 20;

[0018] (3) Buffer 3-30 mmol / L;

[0019] (4) Osmotic pressure regulator 20-100 mg / mL;

[0020] The pH of the PEGylated recombinant canine uricase injection solution is 7.5–9.5.

[0021] More preferably, the polyethylene glycol-modified recombinant canine-human uricase injection comprises:

[0022] (1) Polyethylene glycol-modified recombinant canine human uricase 5-10 mg / mL;

[0023] (2) β-cyclodextrin derivatives, wherein the weight ratio of polyethylene glycol-modified recombinant canine uricase to β-cyclodextrin derivatives is 1:8-12;

[0024] (3) Buffer 8-15 mmol / L;

[0025] (4) Osmotic pressure regulator 40-60 mg / mL;

[0026] The pH of the PEGylated recombinant canine uricase injection solution is 8.0–9.0.

[0027] The preparation method of the polyethylene glycol-modified recombinant canine uricase injection of the present invention is as follows: The prescribed amount of β-cyclodextrin derivative and osmotic pressure regulator are added to a buffer solution and stirred to dissolve. Then, the prescribed amount of polyethylene glycol-modified recombinant canine uricase is added and stirred to dissolve. The pH is adjusted to 7.5–9.5 with hydrochloric acid solution or sodium hydroxide solution, and the volume is brought to a final volume with buffer solution. After passing through a 0.22 μm filter membrane, the solution is filled into vials, stoppered, and capped to obtain the finished formulation.

[0028] Compared with the prior art, the present invention has the following outstanding advantages:

[0029] 1. Polyethylene glycol-modified recombinant canine human uricase is a biological macromolecular drug with poor stability. PEG is prone to detachment, which reduces its solubility and leads to drug precipitation. The immunogenicity of the drug increases after PEG detachment, seriously affecting the safety of clinical use. The formulation prepared using this invention effectively solves the above problems and meets the requirements for clinical use.

[0030] 2. The polyethylene glycol-modified recombinant canine uricase injection prepared by the present invention has high purity, few impurities, less PEG residue, and high stability. After being stored for 2 years, the properties, activity, and impurities of the injection have changed little.

[0031] 3. Compared with freeze-dried preparations, polyethylene glycol-modified recombinant canine human uricase injection has a simpler production process, a shorter production cycle, does not require a freeze-drying process, and has lower energy consumption, thus greatly saving costs. Detailed Implementation

[0032] The present invention will be further illustrated by the following embodiments, but these embodiments do not constitute a limitation of the present invention. Therefore, any simple improvements to the present invention under the premise of the method of the present invention are within the scope of protection of the present invention.

[0033] Obtaining polyethylene glycol-modified recombinant canine human uricase: Following Novagen's pET system operation manual (10th edition), the pET-3c-UHC plasmid was constructed using conventional molecular cloning techniques. This plasmid was then transformed into the *E. coli* BL21 STARTM(DE3)plysS strain to obtain the engineered strain of recombinant canine human uricase protein. Fermentation yielded recombinant canine human uricase expressing the protein. At a fermentation volume of 100 L, the OD600 reached 120, and the target protein expression rate reached 38%. The resulting fermentation broth was purified to obtain recombinant canine human uricase. This recombinant canine human uricase was then saturated with 5 kDa mPEG-SPA to obtain polyethylene glycol-modified recombinant canine human uricase.

[0034] Example 1

[0035] (1) Prescription:

[0036] (2) Preparation method:

[0037] Add the prescribed amount of hydroxypropyl β-cyclodextrin and trehalose to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 8.5 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0038] Example 2

[0039] (1) Prescription:

[0040] Add the prescribed amounts of sulfobutyl β-cyclodextrin and mannitol to a buffer solution, stir to dissolve, then add the prescribed amounts of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 8.0 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0041] Example 3

[0042] (1) Prescription:

[0043] (2) Preparation method:

[0044] Add the prescribed amount of hydroxypropyl β-cyclodextrin and sucrose to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 9.0 with hydrochloric acid solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0045] Example 4

[0046] (1) Prescription:

[0047] (2) Preparation method:

[0048] Add the prescribed amount of hydroxypropyl β-cyclodextrin and sodium chloride to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 8.0 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0049] Example 5

[0050] (1) Prescription:

[0051] (2) Preparation method:

[0052] Add the prescribed amount of sulfobutyl β-cyclodextrin and glucose to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 7.5 with hydrochloric acid solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0053] Example 6

[0054] (1) Prescription:

[0055] (2) Preparation method:

[0056] Add the prescribed amount of hydroxypropyl β-cyclodextrin and trehalose to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 9.5 with hydrochloric acid solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0057] Example 7

[0058] (1) Prescription:

[0059] (2) Preparation method:

[0060] Add the prescribed amounts of glucosyl β-cyclodextrin and sorbitol to a buffer solution, stir to dissolve, then add the prescribed amounts of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 8.0 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0061] Example 8

[0062] (1) Prescription:

[0063] (2) Preparation method:

[0064] Add the prescribed amount of sulfobutyl β-cyclodextrin and sucrose to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 8.0 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0065] Example 9

[0066] (1) Prescription:

[0067] (2) Preparation method:

[0068] Add the prescribed amount of hydroxypropyl β-cyclodextrin and mannose to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 10.0 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0069] Example 10

[0070] (1) Prescription:

[0071] (2) Preparation method:

[0072] Add the prescribed amounts of hydroxypropyl β-cyclodextrin and trehalose to a buffer solution, stir to dissolve, then add the prescribed amounts of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 7.0 with sodium hydroxide solution, bring the volume to full with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0073] Comparative Example 1

[0074] (1) Prescription:

[0075] (2) Preparation method:

[0076] The prescribed amount of polyethylene glycol-modified recombinant canine uricase and sodium chloride were added to a buffer solution, stirred and dissolved, the pH was adjusted to 8.5 with sodium hydroxide solution, and the volume was brought to a final volume with buffer solution. The solution was then filtered through a 0.22 μm filter membrane and filled into vials, stoppered and capped to obtain the finished formulation.

[0077] Comparative Example 2

[0078] (1) Prescription:

[0079] (2) Preparation method:

[0080] Add the prescribed amount of L-cysteine ​​and sodium chloride to a buffer solution, stir to dissolve, then add the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stir to dissolve, adjust the pH to 8.5 with sodium hydroxide solution, bring the volume to the final volume with buffer solution, filter through a 0.22 μm filter membrane, fill into vials, stopper, and cap to obtain the finished formulation.

[0081] Verification example:

[0082] In accordance with the relevant research requirements, the stability study mainly examined appearance, specific enzyme activity, SEC purity, reversed-phase HPLC purity, and PEG residue. The specific test methods are as follows:

[0083] enzyme specific activity

[0084] The specific activity of PEGylated recombinant canine uricase injection was determined using a uric acid spectrophotometer. The sample was diluted 10-fold to obtain the test sample. Uric acid standard solution was diluted to 50 μM with activity assay buffer, mixed thoroughly, and equilibrated in a 37°C water bath to prepare the substrate reaction solution. The UV-Vis spectrophotometer was turned on, the wavelength was adjusted to 312 nm, and the water bath circulation system was activated to maintain the temperature at 37°C. Using the activity assay buffer as a blank control, 4 mL of the substrate solution was placed in a quartz cuvette, and then 10 μL of the test sample was added and quickly mixed. The absorbance was measured at 312 nm. Absorbance was measured every 30 seconds for a total of 2 minutes, with readings automatically obtained via software.

[0085] SEC purity

[0086] The active PEGylated recombinant canine human uricase protein is a tetramer, and as a strongly hydrophobic protein, the tetramers readily aggregate to form higher molecular weight polymers, which is one of the main factors causing immunotoxicity. SEC-HPLC analysis of the PEGylated recombinant canine human uricase injection was performed according to the "size exclusion chromatography" method in Chapter 0514 of the 2015 edition of the Pharmacopoeia of the People's Republic of China, Part III.

[0087] Reversed-phase purity (RP-HPLC)

[0088] The active pharmaceutical ingredient (API) content of PEG-UHC injection was determined by HPLC. The RP-HPLC method for PEGylated recombinant canine uricase injection was performed according to General Chapter 0512 of the 2015 edition of the Pharmacopoeia of the People's Republic of China.

[0089] PEG residue

[0090] The residual amount of PEG in PEGylated recombinant canine-human uricase injection was determined by HPLC using an ELSD detector and a phenomenex Jupiter C5 10μm, 150mm × 4.6mm column packed with alkyl-bonded silica gel. The determination of the residual PEG in PEGylated recombinant canine-human uricase injection was performed according to Section 0512 "High Performance Liquid Chromatography" in the 2015 edition of the Pharmacopoeia of the People's Republic of China, Part III.

[0091] The above-described embodiments and comparative embodiments were subjected to long-term stability studies at 2–8°C. Samples were taken at the end of 0, 3, 6, 12, and 24 months, and tested according to the stability study items. The results are shown in Table 1. Accelerated tests were conducted at 25°C ± 2°C and RH 60 ± 10%. Samples were taken at the end of 0, 3, 6, and 12 months, and tested according to the stability study items. The results are shown in Table 2.

[0092] Table 1. Results of long-term stability test at 2–8℃

[0093]

[0094]

[0095] Table 2 Results of accelerated stability test at 25℃

[0096]

[0097] The stability data in Tables 1 and 2 show that the samples obtained in Examples 1-6 showed no significant changes compared to the samples from day 0 after 24 months of long-term stability testing and 12 months of accelerated stability testing, indicating that the samples prepared using this invention are of stable quality and meet the requirements for clinical drug use. In Examples 7 and 8, the inclusion effect was affected by factors such as the type and amount of β-cyclodextrin derivative used, resulting in increased PEG residue, but their storage stability was still better than that of Comparative Example 1. In Examples 9 and 10, although enzyme specific activity, reversed-phase purity, and SEC purity decreased significantly and the high molecular weight protein content increased due to the influence of factors such as the type and amount of buffer and osmotic pressure regulator, and pH, the appearance remained unchanged due to the inclusion effect of the β-cyclodextrin derivative, and the PEG residue was significantly lower than that of Comparative Example 1. The injectable formulations prepared in Comparative Examples 1 and 2 have poor storage stability, are prone to changes in properties, have high PEG residue and are easy to detach, which will reduce the solubility of polyethylene glycol-modified recombinant canine uricase, leading to drug precipitation. The immunogenicity of the drug after PEG detachment increases, which has an adverse effect on the safety of clinical use.

Claims

1. A polyethylene glycol-modified recombinant canine-human uricase injection, characterized in that, The solution comprises polyethylene glycol-modified recombinant canine uricase, a β-cyclodextrin derivative, a buffer, and an osmotic pressure regulator; the β-cyclodextrin derivative is hydroxypropyl β-cyclodextrin or sulfonyl β-cyclodextrin, the weight ratio of polyethylene glycol-modified recombinant canine uricase to the β-cyclodextrin derivative is 1:5~20, and the content of polyethylene glycol-modified recombinant canine uricase is 1~15 mg / mL; the buffer is phosphate buffer, Tris / hydrochloric acid buffer, glycine / sodium hydroxide buffer, or sodium carbonate / sodium bicarbonate buffer; the osmotic pressure regulator is sodium chloride, glucose, mannitol, sorbitol, sucrose, or trehalose; the pH of the injection solution is 7.5~9.

5.

2. The polyethylene glycol-modified recombinant canine human uricase injection solution according to claim 1, characterized in that, The content of the PEGylated recombinant canine uricase is 5-10 mg / mL.

3. The polyethylene glycol-modified recombinant canine-human uricase injection solution according to claim 1, characterized in that, The weight ratio of the polyethylene glycol-modified recombinant canine uricase to the β-cyclodextrin derivative is 1:8~12.

4. The polyethylene glycol-modified recombinant canine-human uricase injection solution according to claim 1, characterized in that, The buffer concentration is 3-30 mmol / L.

5. The polyethylene glycol-modified recombinant canine-human uricase injection solution according to claim 1, characterized in that, The buffer concentration is 8-15 mmol / L.

6. The polyethylene glycol-modified recombinant canine-human uricase injection solution according to claim 1, characterized in that, The content of the osmotic pressure regulator is 20~100 mg / mL.

7. The polyethylene glycol-modified recombinant canine human uricase injection solution according to claim 1, characterized in that, The content of the osmotic pressure regulator is 40~60 mg / mL.

8. The polyethylene glycol-modified recombinant canine-human uricase injection solution according to claim 1, characterized in that, The pH of the injection solution is 8.0~9.

0.

9. A method for preparing a polyethylene glycol-modified recombinant canine human uricase injection solution according to any one of claims 1 to 8, characterized in that, The process includes the following steps: adding the prescribed amount of β-cyclodextrin derivative and osmotic pressure regulator to a buffer, stirring to dissolve, then adding the prescribed amount of polyethylene glycol-modified recombinant canine uricase, stirring to dissolve, adjusting the pH to 7.5-9.5 with hydrochloric acid solution or sodium hydroxide solution, bringing the volume to full with buffer solution, filtering through a 0.22μm filter membrane, filling into vials, capping, and sealing to obtain the finished formulation.

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