CN113116831A - Freeze-drying method of recombinant human prourokinase for injection - Google Patents
Freeze-drying method of recombinant human prourokinase for injection Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/49—Urokinase; Tissue plasminogen activator
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
Abstract
The invention relates to a freeze-drying method of recombinant human prourokinase for injection, which comprises the following steps: 1) pre-freezing and annealing products: mixing the raw materials and the auxiliary materials, subpackaging into penicillin bottles, then putting into a freeze-drying box, pre-freezing the product, wherein the temperature of a plate layer is set to be-38-42 ℃ in the pre-freezing process, the holding time is 50-70 minutes, and annealing at-22-28 ℃ after the temperature of the product is stable; 2) primary drying: the freeze-drying box body needs aeration, the aeration is controlled to be 20 +/-5 Pa, the temperature of the plate layer is increased to-20 ℃ to-10 ℃, and the plate layer is kept stable until a product waterline disappears; 3) secondary drying (analysis drying): after the primary drying is finished, the temperature of the plate layer is raised to 30-40 ℃, secondary drying is carried out, and the drying temperature is controlled to be 35 +/-5 ℃.
Description
Technical Field
The invention relates to a preparation method of a pharmaceutical preparation, in particular to a freeze-drying method of recombinant human prourokinase for injection.
Background
Prourokinase (pro-UK) is a serine protease which can activate plasminogen in vivo to convert into active plasmin, thereby dissolving fibrin in thrombus, and thus has wide application in the treatment of thrombolytic.
The peptide bond between Lys158-Ile159 in pro-UK is easily hydrolyzed by plasmin and other enzymes to generate Urokinase (UK). UK consists of two peptide chains, namely an A chain and a B chain, and is connected by an interchain disulfide bond. Compared with UK, pro-UK has higher affinity with fibrin, and the part for activating the fibrinolytic system is usually at the position of thrombus formation, so that the side effect of causing the tendency of systemic hemorrhage is less than that of urokinase, and the pro-UK is a more excellent plasminogen activator. The pro-UK also has the advantages of small dosage, safe use, good thrombolysis effect, low re-thrombolysis rate, no anaphylactic reaction and the like, is a safe, nontoxic and ideal biological thrombolysis preparation, and has wide market prospect.
Freeze drying (Lyophilization), also known as vacuum freeze drying, is a drying process in which a water-containing material to be dried is frozen into a solid and the material is dehydrated at a low temperature by utilizing the sublimation property of water under a low temperature and reduced pressure condition to achieve the purpose of drying. Improper formulation and lyophilization profile can cause various problems with the product, such as: poor product molding, surface hardening, collapse or bottle spraying, middle depression, poor re-solubility and even influence the color of the product.
CN1730098A (application No. 2004100580060, entitled composition containing active prourokinase, its freeze-drying method and freeze-dried preparation) discloses a pharmaceutical composition containing active prourokinase, its freeze-drying method and freeze-dried preparation. The composition contains 1.5-5.5mg/ml prourokinase, in specific embodiment, the excipient is mannitol, the buffer solution is phosphate buffer (adjusted to a concentration of 10-20mmol/L), and the protective agent is albumin. The freeze drying method of the composition comprises the following steps: 1) putting the composition into a freeze dryer which is pre-cooled to minus 35 ℃ to minus 45 ℃ for pre-freezing for 2 to 4 hours; 2) then, starting to vacuumize, and heating to-25 to-35 ℃; 3) And after the water in the container is basically sublimated, raising the temperature for the second time to 25-35 ℃, and keeping the temperature for 0.5-1.5 hours after the temperature is stabilized. The product prepared by the method is easy to have the phenomenon of atrophy delamination.
The preparation method is improved to find that: in the product freeze-drying process, the change of the vacuum degree of the freeze-drying box body can be controlled in an air entrainment mode, so that the temperature rise speed of the product and the sublimation speed of the water content of the product are improved. Therefore, a new invention is proposed:
201810149830.9 (lyophilization method of recombinant human prourokinase for injection, not yet disclosed) provides a lyophilization method of recombinant human prourokinase preparation for injection, which is an improvement of the method of the CN1730098A patent, the formula of the lyophilization method is the same as that of CN1730098A, and the lyophilization method comprises the following steps: step 1, pre-freezing a product: pre-freezing the subpackaged products for 1-3 hours at the temperature of-35 to-45 ℃ of the plate layer; step 2, primary drying: vacuumizing, aerating after the temperature of the plate layer is increased to-30 to-40 ℃, keeping the temperature and controlling the pressure of the box body to be 10 +/-3 Pa until a product waterline disappears; and 3, secondary drying: and after the primary drying is finished, gradually heating the plate layer to 25-35 ℃ for secondary drying, and finishing the freeze-drying until the product is stable and qualified.
However, the existing method still has the following disadvantages: 1. the marketed recombinant human prourokinase (Pro-UK)5mg preparation specification prescription for injection is applied to clinic, 10 pieces are needed for one-time medication, and the operation is very inconvenient. 2. In the freeze-drying process, the eutectic point of the preparation is low, 5mg of the preparation is slightly loose in frozen shape, and the freeze-dried powder is still easy to collapse. Accordingly, the present invention has been made.
Disclosure of Invention
The invention is an improvement on the prior art.
In order to improve the medication convenience, the application improves on the basis of the original prescription, carries out process development with larger preparation specification, develops a new preparation with 10mg per unit and improves the medicine concentration in the prescription by at least one time. The salt ion concentration in the prescription is reduced, so that the damage of local high salt concentration to protein caused by low-temperature concentration in the freeze-drying process can be reduced, the disintegration temperature of the product can be increased, the freeze-drying parameters are optimized, and the time is shortened.
The invention provides a prourokinase lyophilized preparation for injection, which is prepared from the following components in parts by weight:
preferably, the injection prourokinase lyophilized preparation of the present invention is prepared from the following components in parts by weight:
most preferably, the injection prourokinase lyophilized preparation of the present invention is prepared from the following components in parts by weight:
in the above lyophilized powder raw materials:
the buffer solution is 0.01mol/L phosphate buffer solution, has pH of 7.0 +/-0.1, and contains 0.4mol/L NaCl.
The acid or alkali used for adjusting the pH is hydrochloric acid or sodium hydroxide solution.
The second purpose of the invention is to provide a preparation method of the injection prourokinase lyophilized preparation, in particular a lyophilization method, which comprises the following steps:
1) pre-freezing and annealing products: mixing the raw materials and the auxiliary materials, subpackaging into penicillin bottles, then putting into a freeze-drying box, pre-freezing the product, wherein the temperature of a plate layer is set to be-38-42 ℃ in the pre-freezing process, the holding time is 50-70 minutes, and annealing at-22-28 ℃ after the temperature of the product is stable;
2) primary drying: the freeze-drying box body needs aeration, the aeration is controlled to be 20 +/-5 Pa, the temperature of the plate layer is increased to-20 ℃ to-10 ℃, and the plate layer is kept stable until a product waterline disappears;
3) secondary drying (analysis drying): after the primary drying is finished, the temperature of the plate layer is raised to 30-40 ℃, secondary drying is carried out, and the drying temperature is controlled to be 35 +/-5 ℃.
Preferably, the lyophilization method of the invention comprises the following steps:
1) pre-freezing and annealing products: mixing the raw materials and the auxiliary materials, subpackaging into penicillin bottles with the volume of 2-4 ml/bottle, then filling into a freeze-drying box, pre-freezing the product, wherein the plate layer is set at-40 ℃ in the pre-freezing process, the holding time is 60 minutes, and annealing at-25 ℃ after the product temperature is stable;
2) primary drying: the freeze-drying box body needs aeration, the aeration is controlled to be 20 +/-5 Pa, the temperature of the plate layer is increased to-20 ℃ to-10 ℃, and the plate layer is kept stable until a product waterline disappears;
3) secondary drying (analysis drying): and after the primary drying is finished, heating the plate layer to 30-40 ℃, performing secondary drying, controlling the drying temperature to be 35 +/-5 ℃, and if the pressure rise per minute for three times is not more than 0.01mbar/min and the pressure rise per minute for two times is not more than 0.003mbar/min, finishing the freeze-drying.
Most preferably, the pre-freezing time in the step 1) is divided into 4 stages, and parameters of each stage are set as follows:
| set temperature (. degree. C.) | Setting time (min) | Duration (min) | |
| Stage 1 | -2 | 1 | 30 |
| Stage 2 | -40 | 60 | 60 |
| Stage 3 | -25 | 10 | 120 |
| Stage 4 | -40 | 30 | 90 |
In the step 2), one-time sublimation is divided into 2 stages, and parameters of each stage are set as follows:
| set temperature (. degree. C.) | Setting time (min) | Duration (min) | Pressure (mbar) | |
| Stage 1 | -10±5 | 30 | 990 | 0.2 |
| Stage 2 | -10±5 | 1 | 240 | 0.2 |
The secondary drying in the step 3) is divided into 2 stages, and parameters of each stage are set as follows:
| set temperature (. degree. C.) | Setting time (min) | Duration (min) | Pressure (mbar) | |
| Stage 1 | 35±5 | 225 | 120 | 0.2 |
| Stage 2 | 35±5 | 1 | 300 | 0 |
The method comprises the following steps:
Δ P is the pressure rise.
The scheme of the invention is obtained by screening, and the specific screening process is as follows:
the existing preparation of prourokinase has the defects that the concentration of each product is too low, the clinical medication dosage is more, the salt ion concentration is higher, the frozen shape is slightly loose, and the product is easy to collapse, so that the inventor adopts the following auxiliary materials for improvement: increasing the concentration of the drug in the prescription; the salt ion concentration in the formulation is reduced.
Optimization of parameters of salt ion concentration
1. Data detection method
1.1DSC scanning, measuring the glass transition temperature by using Differential Scanning Calorimetry (DSC), wherein the temperature rise range is 10-110 ℃, the temperature rise rate is 200 ℃/h, the data acquisition frequency is 10sec, and the specific setting values are shown in Table 1.
Table 1: parameter settings for DSC scan
| Item | Set value |
| Start of sweep temperature | 10℃ |
| End of scan temperature | 110℃ |
| Scanning rate | 200℃/hr |
| Number of rescans per sample | 0 |
| Rate of cooling | Exp |
| Equilibrium time before scanning | 3mins |
| Wait time before next scan | 0 |
| Final instrument hold temperature | 25℃ |
| Frequency of data acquisition | 10sec |
| Thermal compensation mode | none |
| Capillary temperature before sample introduction | 30℃ |
1.2 appearance properties: each sample was taken, left to stand and allowed to return to room temperature, and then the color and clarity were checked.
1.3 content detection: HPLC method, specifically as follows:
phase A: containing 0.1% trifluoroacetic acid in water
Phase B: acetonitrile solution containing 0.1% trifluoroacetic acid
A chromatographic column: reverse direction C18Column: 4.6X 150mm, particle size 5 μm, pore size
Setting temperature of the column oven: 35 ℃, sample cell set temperature: 4 ℃, detection wavelength: 280nm, flow rate: 1 ml/min. Gradient elution method: a30 min gradient elution of 0-80% of phase B was performed.
1.4SEC-HPLC
An area normalization method in three parts (appendix III B) of the Chinese pharmacopoeia 2010 version is adopted. The chromatographic column is TSKgel G3000SWXL7.8X 300mm, 0.01mol/L phosphate buffer solution (pH6.8 contains 0.4mol/L sodium chloride), 25 ℃ column temperature, 4 ℃ sample pool temperature, 0.8ml/min flow rate, 20 mu g sample loading, and detection at 280nm wavelength.
1.5SDS-PAGE
Adopting a method of three parts (appendix IV C) of Chinese pharmacopoeia 2010 edition, using a non-reduction SDS-polyacrylamide gel electrophoresis method, dyeing by Coomassie brilliant blue R250, wherein the concentration of a separation gel is 15 percent, the concentration of a concentrated gel is 5 percent, mixing a sample with a sample loading buffer solution, and then keeping the temperature at 60 ℃ for 3min, wherein the sample loading amount is 10 mu g; and scanning by a scanner to analyze the purity of the protein.
1.6 biological Activity: the detection is carried out by adopting a bubble method (one of the detection of the prourokinase activity can be specifically referred to the literature, Mendelian and the like, the activity of urokinase in blood serum is measured by the bubble method, the modern application of pharmacy in China, No. 4, No. 17, 308-310 in 2000).
1.7 Single Strand ratio: the detection is carried out by an S2444 chromogenic substrate reaction method.
1.8 moisture: karl fischer moisture assay.
2. The experimental results are as follows:
2.1 results of salt ion concentration screening
From the design interval view to make 0.095mol/L, 0.10mol/L and 0.105mol/L three NaCl concentration samples. The specific preparation volumes and ratios are as follows (calculated by taking 1000 samples as a standard preparation unit), and are respectively numbered as 10-1, 10-2 and 10-3, and the numbers are used for replacing samples with different salt ion concentrations in the following, and are specifically shown in Table 2.
Table 2: salt ion concentration screening
Table 3: thermodynamic data analysis table
And (3) analysis: under the condition of different salt ions (NaCl concentration is 0.095 mol/L-0.105 mol/L), the eutectic point is about 31 ℃, the disintegration temperature is about-40 ℃, and the glass transition temperature is not detected. The data show that the eutectic point is still lower under the salt ion concentration, the disintegration temperature is more about-40 ℃, and therefore, the product is shrunk and collapsed when the freeze-drying is carried out at the temperature of-30 ℃ according to the current production process, and the NaCl concentration of 0.095 mol/L-0.105 mol/L is selected as the final salt ion concentration of the product.
II, comparing the prescription: the prourokinase composition is improved on the basis of the prior art: the concentration of the drug in the prescription was doubled while the concentration of the salt ion was reduced, and the comparison of the original prescription and the modified prescription is shown in Table 4.
Table 4: comparison of the modified prescription with the original prescription
Note: c is the concentration of the protein in the stock solution
Among them, stock solution (Bulk) refers to a homogeneous substance used for manufacturing a Final Formulation (Final Formulation) or a semi-finished product (Final Bulk).
Table 4 the results show: the final drug concentration of the product is about 2.5mg.ml, the final salt ion concentration is about 0.2mol/L, and the final drug concentration of the invention is about 5mg.ml, the salt ion concentration is about 0.095mol/L to 0.105mol/L (optimally 0.1 mol/L).
And thirdly, improving the freeze-drying process:
1. screening by prefreezing
Determining the influence of different pre-freezing modes on the activity of prourokinase, putting the prepared sample into a penicillin bottle, pre-freezing in a freeze dryer by adopting quick pre-freezing, slow pre-freezing and pre-freezing annealing processes respectively, re-melting at room temperature, detecting the activity and the protein content (HPLC method), and comparing the activity and the protein content before and after freeze-drying.
1.1 Rapid Pre-freezing procedure: placing the sample on a clapboard of a freeze dryer, rapidly cooling the temperature of the clapboard to-40 ℃ (time: 30min), and maintaining for 90 min;
1.2 Slow Pre-freezing procedure: placing the sample on a clapboard of a freeze dryer, slowly cooling the temperature of the clapboard to-45 ℃ (time: 180min), and maintaining for 90 min;
1.3 prefreezing annealing procedure: placing the sample on a clapboard of a freeze dryer, slowly cooling the temperature of the clapboard to-45 ℃ (time: 180min), maintaining for 90min, then heating the clapboard to-20 ℃ for 30min, maintaining for 90min, then cooling the clapboard to-45 ℃ for 30min, and maintaining for 90 min;
the frozen samples obtained from the three procedures were tested for the relevant items and compared at 0 min.
In the prefreezing stage, the sample is mainly subjected to different prefreezing modes (fast freezing, slow freezing and prefreezing annealing) and then is subjected to thawing, so that the influence of the prefreezing mode on the prourokinase protein content (HPLC) and the activity (bubble method) is only considered, and the table is shown in tables 5-1 and 5-2.
Table 5-1: effect of prefreezing regimen on prourokinase protein Activity
Tables 5-2: effect of prefreezing on prourokinase protein content
Tables 5-1, 5-2 analysis of results: the operation of quick pre-freezing, slow pre-freezing and pre-freezing annealing is used, biological activity shows that the activity of the recombinant human prourokinase is reduced to the lowest extent by using a pre-freezing annealing mode, and the reduction level of protein content is lower, and theoretically, the pre-freezing mode using the pre-freezing annealing can strengthen crystallization (crystallization components are not enough to be completely crystallized), improve the glass transition temperature of amorphous phase frozen concentrated solution, change the shape and size distribution of ice crystals, improve drying efficiency and the like, so the pre-freezing annealing mode is selected as the pre-freezing mode of a new freeze-drying process.
The annealing step has the following functions: (1) the annealing can change the shape and size distribution of the ice crystals, and the phase behavior and recrystallization in the annealing process can reduce the size difference of the ice crystals and the nonuniformity of the drying rate caused by the nucleation temperature difference, thereby improving the drying efficiency and the uniformity of the product. (2) Annealing can strengthen the crystallization. Pharmaceutical formulations generally contain the active substance, water, and various ingredients such as stabilizers, excipients, and fillers. (3) The annealing can increase the glass transition temperature t of the amorphous phase frozen concentrated solutiong. Removal of t from amorphous phasegLower crystalline component, and can increase t of amorphous phaseg. To prevent collapse of the drug, primary drying typically controls the drug temperature below tgIs carried out under conditions to increase the t of the amorphous phasegIt allows the shelf temperature to be increased during sublimation drying, speeding up the drying process without collapsing the drug. Impact on the final results: the product has better uniformity, better frozen shape and difficult collapse.
2. Annealing temperature optimization
In the pre-freezing stage, the pre-freezing at-40 ℃ and the annealing process at-25 ℃ are adopted to compare with the prior pre-freezing process (direct pre-freezing at-40 ℃, no annealing and falling into the range disclosed in CN 1730098A), the freeze-dried product is subjected to accelerated stability inspection for 1 month at 37 ℃, and the optimal annealing temperature is screened according to the stability of the product, which is shown in Table 6.
Table 6: optimization of annealing temperature
And (3) analysis: in the pre-freezing stage, the annealing process at-40 ℃ and the annealing process at-25 ℃ are compared with the current pre-freezing process, and accelerated stability data of protein content at 37 ℃ show that the prourokinase freeze-dried product with-25 ℃ as the annealing temperature is most stable.
3. Study of Primary drying (Primary sublimation) Process
Four groups of experimental conditions are set in a primary drying process research according to the challenging experimental result and the disintegration temperature detection result, the range of the freeze-drying process during primary sublimation is determined, and the four groups of experiments respectively set the primary drying temperature as follows: 0 ℃, 10 ℃, 20 ℃ and 33 ℃ and judges the end point of the first sublimation by a pressure rise test, the analysis and drying temperature adopts 30 ℃ of the prior art, and the pressure rise test result is taken as the freeze-drying end point.
The appearance, water content and protein activity of the freeze-dried product are detected, and accelerated stability examination is carried out for one month at the temperature of 37 ℃.
3.1 Freeze drying time and product appearance, see Table 7
Table 7: freeze-drying method and product appearance
Table 7 the results show: except that the freeze-dried product with the primary drying temperature of 0 ℃ has a certain bottom-removing phenomenon, the appearance and the freeze type are good under other conditions; in four experiments, the freeze-drying time is prolonged along with the reduction of the primary drying temperature, and 72 hours are needed under the condition of-33 ℃.
3.2 Freeze-dried product stability: see Table 8
Table 8: stability of
Table 8 the results show: the test is at 0.4m2The freeze dryer is operated on a large scale, the moisture can be controlled to be 2.3 percent by taking 0 ℃, 20 ℃ and 33 ℃ as the primary drying temperature, the quality requirement is met, and the test moisture of the-10 ℃ group is higher, which is probably related to the relatively higher out-of-box pressure rise result.
The RSD requirement of the activity detection methodology is 10%, and the activity data shows that the activity reduction of all the samples of the test groups after 1 month of accelerated stability test at 37 ℃ is within the RSD requirement range, so that the reduction of the activity of all the products of the test groups under accelerated conditions is small and relatively stable.
The RSD requirement of the methodological detection of the protein content is 3%, and the protein content of all test group samples is reduced within the RSD range after 1 month of accelerated stability examination at 37 ℃, so that the protein content of all test group products is reduced slightly and is relatively stable under the accelerated condition.
In conclusion, the final product quality is relatively stable by taking 0 ℃, -10 ℃, -20 ℃, -33 ℃ as the temperature of one sublimation of the freeze-drying process and taking pressure rise as the qualified end point standard, but the requirements of appearance, quality and freeze-drying time can be met by combining the appearance result of the product and the freeze-drying time, wherein the temperature of one drying is in the range of-20 ℃ to-10 ℃.
The primary drying temperature disclosed in CN1730098A is very low, approaching to the disintegration temperature (-38 ℃) of the product at 0.20M NaCl concentration, and in the primary drying stage, the product is melted, thereby causing the problem that the frozen type has collapsed. In the application, the primary freeze-drying temperature is higher than the product disintegration temperature, and the frozen type is not easy to collapse.
4. Final lyophilization profile:
the above results are combined to obtain temperature parameters at each stage of the process, wherein the prefreezing temperature is set to-40 ℃, the annealing temperature is set to-25 ℃, the primary drying temperature is set to-15 +/-5 ℃, the secondary drying (desorption drying) temperature is set to 35 +/-5 ℃, and the specific time settings and pressure controls are shown in the following table 9:
table 9: freeze-drying key process parameter setting
The method provided by the invention has the following advantages:
1. the aimed formula is different: the existing preparation of prourokinase has the disadvantages of low concentration of each product and large clinical dosage, and the analysis reason is that the salt ion concentration is high, the frozen shape is slightly loose and easy to collapse, the salt concentration in the original process is 0.30-0.20mol/L sodium chloride, the modified formula contains 0.4mol/L sodium chloride, and the buffer system with lower salt ion concentration is used for replacing the phosphate buffer system in the original formula.
2. The freeze-drying process, because the raw material formula is slightly different, has the following differences compared with the prior art (especially the invention patent 201810149830.9, which is referred to as literature for short below):
1) pre-freezing part:
the temperature of the plate layer is set to be-40 ℃ in the literature, and the pre-freezing is finished after the temperature of the product is stabilized for 110-130min, but the annealing step is added in the invention, and the annealing at higher temperature not only keeps the advantages of the annealing, for example, the annealing can change the form and size distribution of ice crystals, but also can reduce the water content in the freeze-dried powder, the annealing can improve the glass transition temperature tg of the amorphous phase freezing concentrated solution, the product after filling is put into a freeze-drying box for pre-freezing, the temperature of the plate layer is set to be-40 ℃ in the pre-freezing process, and the annealing is carried out at-25 ℃ after the temperature of the product is stabilized.
In addition, the annealing temperature selected by the invention is also critical, and accelerated stability data of protein content at 37 ℃ show that the prourokinase freeze-dried product with-25 ℃ as the annealing temperature is most stable.
2) Primary drying:
the literature is that the vacuum pumping is firstly carried out to 15Pa, the aeration is started when the temperature of a slab layer rises to-33 ℃, the temperature is kept and the pressure of a box body is controlled to be 10 +/-3 Pa until a product waterline disappears, and the invention has two differences: firstly, the pressure is increased by aeration without vacuumizing. And secondly, the temperature is higher, and compared with the operation parameters of documents, the operation parameters of the invention are more beneficial to the volatilization of free moisture in the product.
3) And (3) secondary freeze-drying: after primary drying is finished, the temperature of the plate layer is gradually increased to 30 ℃ for secondary drying, a pressure rise test is carried out after the secondary drying is carried out for 9 hours, and the product is taken out of a box after the test is qualified; the total secondary drying time is maintained for 12 hours, the front box is filled with nitrogen until the pressure is-0.05 MPa to-0.02 MPa, the product is fully pressed and taken out of the box, and the temperature of the plate layer is raised to 35 ℃ for secondary drying. The secondary drying temperature is increased by 5 ℃, which is more beneficial to the volatilization of the crystal water in the product.
The summary is as follows: pre-freezing at-40 deg.C, annealing at-25 deg.C, drying at-10 deg.C, heating to 35 deg.C, drying again until the pressure is not higher than 0.01mbar/min, and taking out.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: urokinase zymogen freeze-dried powder raw material for injection
Is prepared from the following components in parts by weight (1000 for example): 10.7g of prourokinase, 500ml of phosphate buffer solution, 120g of mannitol and 6g of human serum albumin, wherein the volume of the mixture is 2000ml by water, and the pH value is adjusted to 6.9 +/-0.5 by acid and alkali.
Example 2: freeze-drying process
1. Pre-freezing and annealing products: filling the filled product into a freeze-drying box, pre-freezing the product, setting the temperature of a plate layer to be-40 ℃ in the pre-freezing process, setting parameters in the following table, and annealing at-25 ℃ after the temperature of the product is stable;
table 10: setting of prefreezing parameters
| Set temperature (. degree. C.) | Setting time (min) | Duration (min) | |
| Stage 1 | -2 | 1 | 30 |
| Stage 2 | -40 | 60 | 60 |
| Stage 3 | -25 | 10 | 120 |
| Stage 4 | -40 | 30 | 90 |
2. Primary drying: in the primary drying process, the freeze-drying box body needs to be aerated, the aeration is controlled to be 20 +/-5 Pa, the temperature of the plate layer is increased to minus 20 ℃ to minus 10 ℃, and all parameters are set in the following table and are kept stable until the product waterline disappears.
Table 11: setting parameters of primary drying
| Set temperature (. degree. C.) | Setting time (min) | Duration (min) | Pressure (mbar) | |
| Stage 1 | -10±5 | 30 | 990 | 0.2 |
| Stage 2 | -10±5 | 1 | 240 | 0.2 |
3. Secondary drying (analysis drying): after the primary drying is finished, the temperature of the plate layer is raised to 35 ℃, and secondary drying is carried out. If the pressure rise per minute does not exceed 0.01mbar/min and the pressure rise per minute does not exceed 0.003mbar/min for three consecutive times, all the parameters are set as shown in the following table, and the freeze-drying is finished.
Table 12: setting parameters of secondary drying
| Set temperature (. degree. C.) | Setting time (min) | Duration (min) | Pressure (mbar) | |
| Stage 1 | 35±5 | 225 | 120 | 0.2 |
| Stage 2 | 35±5 | 1 | 300 | 0 |
Experimental example 1: analytical drying screening process
And (4) performing secondary drying on the basis of the determination of the pre-freezing parameter, the annealing parameter and the primary drying parameter, namely analyzing the optimization of the drying parameter. Three groups of experiments TA, TB and TC are designed in the research process, the analytic temperature of the TA group is set to be 30 ℃, the analytic temperature of the TB group is set to be 35 ℃, and the analytic temperature of the TC group is set to be 40 ℃. The specific parameter control of each order is as follows in table 13:
table 13: analytical drying optimization of experimental parameters
Table 13 shows the parameters, in-40 ℃ after pre-freezing annealing at-25 ℃, in-10 ℃ under the conditions of primary drying, then temperature rise to 30 ℃, 35 ℃, 40 ℃ for analysis drying, until the pressure rise does not exceed 0.01mbar/min after the full pressure plug out of the box. According to the distribution of the freeze dryer, 10 samples at different positions are taken for water detection.
The results are shown in Table 14:
table 14: analytical drying optimized moisture detection results
The results in Table 14 show that the moisture for all three experimental temperatures is less than 2% and less than the standard of the prior art (less than 3%), so that all three temperatures are feasible as temperatures for analytical drying.
Claims (8)
1. A prourokinase lyophilized preparation for injection is prepared from the following components in parts by weight:
2. the lyophilized formulation according to claim 1, which is prepared from the following components in parts by weight:
3. the lyophilized formulation according to claim 1, which is prepared from the following components in parts by weight:
4. the lyophilized formulation according to claim 1, wherein the buffer is 0.01mol/L phosphate buffer solution, pH7.0 ± 0.1, containing 0.4mol/L NaCl.
5. The lyophilized formulation according to claim 1, wherein the acid or base used for adjusting the pH is hydrochloric acid or sodium hydroxide solution.
6. A method for preparing the lyophilized formulation of claim 1, comprising the steps of:
1) pre-freezing and annealing products: mixing the raw materials and the auxiliary materials, subpackaging into penicillin bottles, then putting into a freeze-drying box, pre-freezing the product, wherein the temperature of a plate layer is set to be-38-42 ℃ in the pre-freezing process, the holding time is 50-70 minutes, and annealing at-22-28 ℃ after the temperature of the product is stable;
2) primary drying: the freeze-drying box body needs aeration, the aeration is controlled to be 20 +/-5 Pa, the temperature of the plate layer is increased to-20 ℃ to-10 ℃, and the plate layer is kept stable until a product waterline disappears;
3) secondary drying (analysis drying): after the primary drying is finished, the temperature of the plate layer is raised to 30-40 ℃, secondary drying is carried out, and the drying temperature is controlled to be 35 +/-5 ℃.
7. The method of claim 6, comprising the steps of:
1) pre-freezing and annealing products: mixing the raw materials and the auxiliary materials, subpackaging the mixture into penicillin bottles with the volume of 2-4 ml/bottle, then filling the mixture into a freeze-drying box, pre-freezing the product, keeping the temperature of a plate layer in the pre-freezing process at-40 ℃ for 60 minutes, and annealing the product at-25 ℃ after the temperature of the product is stable;
2) primary drying: the freeze-drying box body needs aeration, the aeration is controlled to be 20 +/-5 Pa, the temperature of the plate layer is increased to-20 ℃ to-10 ℃, and the plate layer is kept stable until a product waterline disappears;
3) secondary drying (analysis drying): and after the primary drying is finished, heating the plate layer to 30-40 ℃, performing secondary drying, controlling the drying temperature to be 35 +/-5 ℃, and if the pressure rise per minute for three times is not more than 0.01mbar/min and the pressure rise per minute for two times is not more than 0.003mbar/min, finishing the freeze-drying.
8. The production process according to claim 7, wherein,
wherein, the pre-freezing time in the step 1) is divided into 4 stages, and parameters of each stage are set as follows:
in the step 2), one-time sublimation is divided into 2 stages, and parameters of each stage are set as follows:
the secondary drying in the step 3) is divided into 2 stages, and parameters of each stage are set as follows:
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730098A (en) * | 2004-08-06 | 2006-02-08 | 上海天士力药业有限公司 | Composition containing active prourokinase, freeze-drying process and freeze-dried preparation thereof |
| US20140199286A1 (en) * | 2013-01-15 | 2014-07-17 | Teva Pharmaceutical Industries, Ltd. | Lyophilization process |
| CN105078906A (en) * | 2015-08-11 | 2015-11-25 | 蚌埠丰原涂山制药有限公司 | Urokinase-containing pharmaceutical lyophilized preparation and preparation method thereof |
| US20180099049A1 (en) * | 2016-10-07 | 2018-04-12 | Regeneron Pharmaceuticals, Inc. | Room Temperature Stable Lyophilized Protein |
| CN110151705A (en) * | 2018-02-13 | 2019-08-23 | 天士力生物医药股份有限公司 | The freeze drying process of injection recombinant human urokinase zymogen |
-
2019
- 2019-12-26 CN CN201911364012.1A patent/CN113116831A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730098A (en) * | 2004-08-06 | 2006-02-08 | 上海天士力药业有限公司 | Composition containing active prourokinase, freeze-drying process and freeze-dried preparation thereof |
| US20140199286A1 (en) * | 2013-01-15 | 2014-07-17 | Teva Pharmaceutical Industries, Ltd. | Lyophilization process |
| CN105078906A (en) * | 2015-08-11 | 2015-11-25 | 蚌埠丰原涂山制药有限公司 | Urokinase-containing pharmaceutical lyophilized preparation and preparation method thereof |
| US20180099049A1 (en) * | 2016-10-07 | 2018-04-12 | Regeneron Pharmaceuticals, Inc. | Room Temperature Stable Lyophilized Protein |
| CN110151705A (en) * | 2018-02-13 | 2019-08-23 | 天士力生物医药股份有限公司 | The freeze drying process of injection recombinant human urokinase zymogen |
Non-Patent Citations (2)
| Title |
|---|
| 丁有学;李响;韩春梅;饶春明;王军志;: "重组人尿激酶原质控方法和质量标准的研究", 中国生物制品学杂志, no. 07 * |
| 陈光明: "蛋白质药品冷冻干燥技术研究进展", 《制冷空调与电力机械》, vol. 2, pages 4 * |
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