CN117338730A - Chymotrypsin oral freeze-dried powder and preparation method thereof - Google Patents
Chymotrypsin oral freeze-dried powder and preparation method thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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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
-
- 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
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- 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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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses chymotrypsin oral freeze-dried powder, which comprises chymotrypsin, mannitol, sucrose and dextran 20. Preferably, the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 units: (167 to 222) mg: (167 to 222) mg: (83-111) mg. The invention also provides a preparation method of the chymotrypsin oral freeze-dried powder. The chymotrypsin oral freeze-dried powder has the advantage of stable samples under high temperature conditions, the chymotrypsin sample purity is not obviously changed after being placed for 3 months at a high temperature of 40 ℃, and the activity recovery is more than 90%.
Description
Technical Field
The invention belongs to the field of biological preparations, and in particular relates to chymotrypsin oral freeze-dried powder and a preparation method thereof.
Background
Chymotrypsin (Chymotrypsin), also known as Chymotrypsin, is a proteolytic enzyme extracted from bovine or porcine pancreas, has endopeptidase effect, and specifically hydrolyzes carboxyl-terminal aromatic amino acid (tyrosine, tryptophan, leucine) or side bond bulky hydrophobic residue methionine by cutting off carboxyl-terminal peptide chain of tyrosine, tryptophan and phenylalanine in protein peptide chain. Chymotrypsin also has a lipase action, hydrolyzing certain lipids. Chymase, as a proteolytic enzyme, can be used to promote the digestive clearance of blood clots, purulent secretions, necrotic tissue, etc., for ophthalmic surgery to relax ligaments, alleviating traumatic iridocyclitis; it can also be used for wounds or local inflammation to reduce local secretion and oedema.
According to the statistics of the international cancer research institution, the new occurrence of gastric cancer is about 108.9 ten thousand cases worldwide in 2020, and the 5 th place of the number of malignant tumor patients is occupied. About 76.9 ten thousand cases of gastric cancer death occur worldwide in 2020, and the 4 th deaths caused by malignant tumors are located. Early gastric cancer is a curable disease, the survival rate of the gastric cancer after excision is more than 90% in 5 years, and the survival rate of the gastric cancer in the late stage is only 10% to 20% in 5 years; thus, early treatment was found to be very important for the cure of gastric cancer. Endoscopy is the most commonly used screening method for the discovery of early gastric cancer. Gastroscopy is a common diagnosis and treatment method for upper gastrointestinal diseases, and can find pathological changes, determine pathological change positions, morphology and properties, and perform endoscopic treatment. However, in a normal state, more mucus and foam exist in the upper digestive tract, which greatly affects the visual field under the endoscope and the detection of early lesions, repeated flushing increases the time of endoscopic examination, and increases the pain of the subject. Pronase acts as a viscosity-removing agent to eliminate the protein-induced mucus on the mucosal surfaces of the digestive tract. However, due to the high price of pronase, it has not been popular, and low-priced chymotrypsin and N-acetylcysteine are often used instead.
At present, chymotrypsin preparations in the market are chymotrypsin for injection, the packaging form is a 2mL penicillin bottle, the chymotrypsin preparations are required to be matched with a syringe for use, the chymotrypsin preparations are inconvenient to use and take orally in gastroscopy, and the chymotrypsin preparations are light in content and are not suitable for bagged split charging. The invention provides a novel chymotrypsin oral preparation for the first time, can be used for weighing, realizes bagging and split charging, has the advantages of high stability, convenient storage, simpler and more convenient use and long acting time, can effectively remove mucus in the stomach and improve the visual field definition, and is a modification and innovation of the traditional process. The preparation is reported as a biological medicine at present, and the chymotrypsin oral preparation has great innovation significance for improving biological medicine formulations.
Disclosure of Invention
The invention aims to provide a novel chymotrypsin preparation, and the preparation formulation of the chymotrypsin preparation is oral freeze-dried powder.
The chymotrypsin oral freeze-dried powder comprises chymotrypsin, mannitol, sucrose and dextran 20, wherein the proportion of the chymotrypsin, the mannitol, the sucrose and the dextran 20 is 4000 units: 167-222mg:167-222mg:83-111mg.
Preferably, the chymotrypsin is a human chymotrypsin.
More preferably, the chymotrypsin is a recombinant human chymotrypsin.
Preferably, the chymotrypsin, mannitol, sucrose and dextran 20 in the chymotrypsin oral lyophilized powder is 4000 units: 200mg:200mg:100mg.
The invention also aims to provide a preparation method of chymotrypsin oral freeze-dried powder, which comprises the following steps:
(1) Pretreatment: concentrating and dialyzing chymotrypsinogen liquid until the electric conductivity of the filtered liquid is lower than 0.5mS/cm to obtain chymotrypsin concentrated liquid;
(2) Preparing a mixed solution: adding 5% mannitol, 5% sucrose and 2.5% dextran 20 to the chymotrypsin concentrate according to the volume ratio, and stirring and uniformly mixing to obtain a mixed solution;
(3) And (3) sterilizing and filtering: sterilizing and filtering the mixed solution by adopting a filter membrane;
(4) And (3) freeze drying: freeze-drying the filtered filtrate in the step (3) to obtain freeze-dried powder;
(5) And (3) crushing the freeze-dried powder, and sub-packaging to obtain the chymotrypsin oral freeze-dried powder.
Preferably, step (1) uses a 5kD membrane package and water for injection at a temperature below 25 ℃ to concentrate and dialyze the chymotrypsinogen solution.
Preferably, the chymotrypsin content in the chymotrypsin concentration dialysis process of the step (1) is 1mg/mL-4mg/mL, and the activity is 2000U/mL-9600U/mL.
Preferably, the chymotrypsin concentration of step (1) has a chymotrypsin content of more than 0.6mg/mL and an activity of more than 1000U/mL.
Preferably, the chymotrypsin activity of the chymotrypsin concentrate of step (1) is 1200U/mL-4000U/mL.
Preferably, the pH value of the mixed solution in the step (2) is 3.0-5.0.
Preferably, step (2) is sterile filtered using a 0.22 μm filter.
Preferably, the chymotrypsin content in the mixed solution in the step (2) is 0.4mg/mL-0.7mg/mL, and the activity is 1100-1300U/mL.
Preferably, the content of chymotrypsin in the filtrate of the step (3) is 0.4mg/mL-0.7mg/mL, and the activity is 1000-1300U/mL.
Preferably, step (4) comprises:
(a) Pre-freezing: the sample enters a freeze dryer at normal temperature, and after the temperature of the separator is reduced to-45 ℃, the sample is pre-frozen for more than 4 hours;
(b) Primary drying: the condensing temperature is reduced to below-45 ℃, and then the vacuum is pumped to below 10 pa; raising the temperature of the separator to-30 ℃ at a speed of 5 ℃/1.5h, and preserving the temperature for more than 48h until the crystal water disappears;
(c) And (3) secondary drying: raising the temperature of the separator to 20 ℃ at a speed of 5 ℃/1.5h, and preserving heat for more than 15 h; and (5) checking that the vacuum degree is unchanged, ending freeze-drying, and taking the sample out of the cabinet.
Preferably, the size of the sub-package in the step (5) is 0.5 g/bag.
Preferably, the preparation method comprises the following steps:
in the step (1), chymotrypsinogen liquid is taken as a raw material, the protein content is 1.0+/-0.2 mg/mL, a 5kDa membrane package is used for concentration and dialysis, the membrane package is firstly treated by 0.5M NaOH for at least 30min, then the membrane package is washed by water for injection, the chymotrypsinogen liquid is concentrated to 1/2-1/4 of the original volume after the washing is finished, the water for injection with the same volume is added, and the concentration and the dialysis are continuously repeated after the uniform mixing until the electric conductivity of the filtrate is lower than 0.5mS/cm; the concentration of protein is 1-4mg/mL in the concentration dialysis process, the concentration of protein is adjusted to be 0.6-2.0mg/mL after the dialysis is completed, and the pH of the solution is 3.0-5.0, preferably 4.0.
In the step (2), detecting the activity and the protein content of the chymotrypsin solution after dialysis concentration in the step (1), diluting the activity to 1000-1300U/mL with water for injection, adding 5% mannitol, 5% sucrose and 2.5% dextran 20 according to the final volume ratio, and stirring and mixing uniformly.
In the step (3), under the A-level laminar flow, the blending liquid in the step (2) is subjected to sterilization filtration by using a two-stage 0.22 mu m filter, and the obtained sample is a chymotrypsin semi-finished product, the activity is 1000-1300U/mL, and the protein content is 0.4-0.7mg/mL.
In the step (4), the chymotrypsin semi-finished product in the step (3) is subpackaged and freeze-dried according to 1L/tray under laminar flow, and the freeze-drying process comprises the following steps:
(a) Pre-freezing: the sample enters a freeze dryer at normal temperature, and after the temperature of the separator is reduced to-45 ℃, the sample is pre-frozen for more than 4 hours;
(b) Primary drying: the condensing temperature is reduced to below-45 ℃, and then the vacuum is pumped to below 10 pa; raising the temperature of the separator to-30 ℃ at a speed of 5 ℃/1.5h, and preserving the temperature for more than 48h until the crystal water disappears;
(c) And (3) secondary drying: raising the temperature of the separator to 20 ℃ at a speed of 5 ℃/1.5h, and preserving heat for more than 15 h; and (5) checking that the vacuum degree is unchanged, ending freeze-drying, and taking the sample out of the cabinet to obtain chymotrypsin oral freeze-dried powder.
In the step (5), the chymotrypsin oral freeze-dried powder in the step (4) is crushed into samples by a crusher, and is packaged according to 0.5 g/bag, and the packaging material is an opaque aluminum-containing composite film, preferably a polyester/aluminum/polyethylene medicinal composite film. And (3) confirming that the transverse seal and the longitudinal seal are horizontal and vertical, and the seal is perfect, thus completing the preparation of chymotrypsin oral freeze-dried powder.
The chymotrypsin oral freeze-dried powder can be used for gastroscopy to remove mucus in the stomach and improve visual field definition. The administration method comprises mixing 1 bag of chymotrypsin oral lyophilized powder with 1 bag of sodium bicarbonate 1g, dissolving in warm water, and administering within 1h, wherein the volume of warm water is preferably 50-100mL.
Drawings
FIG. 1 clarity observations of the solutions at different pH's during dialysis.
FIG. 2 is a diagram of spray-dried dry powder granulation of human chymotrypsin.
FIG. 3 SDS-PAGE detection of different formulations of human chymotrypsin oral particles; graph A shows the formulation of oral granule of human chymotrypsin, and graph B shows the optimization of the formulation of oral granule of human chymotrypsin, wherein M is a protein standard molecular weight Marker, groups 1-6 represent samples of different formulations, and 4 ℃/25 ℃/37 ℃ represent samples placed at different temperatures of 4 ℃/25 ℃/37 ℃.
FIG. 4 SDS-PAGE detection of samples of oral particles of human chymotrypsin prepared by spray process under different conditions; wherein M is a protein standard molecular weight Marker, groups 1-6 represent samples prepared by different spraying processes, 4 ℃/25 ℃/37 ℃ represent samples placed at different temperatures of 4 ℃/25 ℃/37 ℃, before spraying represent samples before spray drying, vacuum represents samples stored in vacuum package, and an aluminum bag represents samples stored in non-vacuum package aluminum bag package.
FIG. 5. Freeze-dried dry powder granulation of human chymotrypsin; wherein M is a protein standard molecular weight Marker, groups 1-6 represent samples prepared by different granulation processes, 100% represents samples prepared by 100% ethanol, 80% represents samples prepared by 80% ethanol, and pure water represents samples prepared by purified water.
FIG. 6, examination of 3 months stability of oral lyophilized powder of human chymotrypsin of different formulations, sample SDS-PAGE detection; wherein M is a protein standard molecular weight Marker, groups 1-2 represent freeze-dried samples prepared from different formulations, and 4 ℃/25 ℃/37 ℃ represent samples placed at different temperatures of 4 ℃/25 ℃/37 ℃.
FIG. 7 is a solution observation of the oral lyophilized powder of human chymotrypsin after mixing with sodium bicarbonate (30 min).
Fig. 8. Observation of mucus removal effects of different doses of human chymotrypsin oral lyophilized powder in beagle gastroscope.
Detailed Description
The technical scheme of the present invention will be described in detail below by examples and drawings, so that the features and advantages of the present invention will be better illustrated. The examples provided should be construed as illustrative of the method of the present invention and not limiting the disclosed technology in any way.
The reagents and apparatus used in the examples were all commercially available except for the specific descriptions.
Example 1 selection of dialysis conditions for human chymotrypsinogen
Concentrating human chymotrypsinogen liquid by using a 5kD membrane bag, adding purified water with the same volume, continuing dialysis concentration until the electric conductivity is 0.5mS/cm, sampling 8 mL/pipe, and respectively regulating the pH value to 4.0,4.5,5.0,5.5,6.0,7.0,8.1,9.5 and 10.0; when the pH is 6.0-7.0, the sample becomes turbid (slight turbidity starts to appear at the pH of 5.5), and when the pH is adjusted back to 3.5, the sample becomes clear; after adding 3%10xPBS to a turbid sample with pH 7.0, the sample became clear, and after standing for 2 hours (room temperature) and 20 hours (room temperature and 2-8 ℃), the sample was observed and activity was detected. The result shows that the electrical conductivity of chymotrypsin is reduced to 0.5mS/cm during dialysis, the sample is clear, and the sample becomes turbid after the pH is regulated to 6.0-7.0; the pH is adjusted back to 3.5, the sample is clarified again, and the activity is unchanged; after adding salt into a turbid sample with the pH of 7.0, the sample becomes clear, but the activity is reduced to about half, which indicates that the turbid sample cannot be dissolved again by adding salt during dialysis; chymotrypsin is more stable at a pH below 5.0, and is still more than 800U/mg in activity when left at room temperature for 20 hours, and the closer the pH is to 3.0, the more stable, so that the pH of the solution after dialysis is determined to be pH 3.0-5.0, preferably pH4.0.
TABLE 1 detection of the Activity of different pH solutions after dialysis of chymotrypsinogen solution (Unit: U/mL)
Example 2 spray drying Process for preparing oral granules of human chymotrypsin
1. Human chymotrypsin spray-dried dry powder granulation research
Spray drying a human chymotrypsin solution containing 10% mannitol and having an activity of 1000U/mL to obtain a human chymotrypsin dry powder, and granulating the dry powder with absolute ethanol, 90% ethanol, 80% ethanol, 70% ethanol and purified water respectively, wherein the result shows that: after granulation with ethanol and purified water of different concentrations, the appearance morphology showed that the higher the ethanol concentration, the better the particle dispersibility, the more uniform the formed particles (fig. 2), but the lower the recovery of particles (> 80 mesh), while the different wetting agents had no significant effect on the sample activity, thus confirming the subsequent particle stability sample granulation with 70% ethanol.
Table 2 recovery rate of pass particles of 10 mesh to 80 mesh:
| wetting agent | >80 mesh (g) | <80 mesh (g) | Recovery (%) |
| Absolute ethyl alcohol | 2.08 | 0.29 | 83.2 |
| 90% ethanol | 2.20 | 0.18 | 88.0 |
| 80% ethanol | 2.16 | 0.17 | 86.4 |
| 70% ethanol | 2.34 | 0.13 | 93.6 |
| Purified water | 0.95 | 0.02 | 76.0 |
TABLE 3 detection of sample Activity after granulation of different wetting agents
| Wetting agent | Sample concentration (mg/mL) | Sample Activity (U/mg) | Recovery (%) |
| Pre-granulation control | 2.0 | 2738 | 100 |
| Absolute ethyl alcohol | 2.0 | 2591 | 94.6 |
| 90% ethanol | 1.9 | 2498 | 91.2 |
| 80% ethanol | 1.9 | 2526 | 92.3 |
| 70% ethanol | 2.0 | 2680 | 97.9 |
| Purified water | 2.0 | 2827 | 103.3 |
2. Human chymotrypsin spray-dried formulations were sought
The dosage of 1g,0.5g and 0.2g of different specifications of medicine is designed, the concentration of the chymotrypsin contained in corresponding 10% mannitol is respectively 0.2mg/mL,0.4mg/mL and 1mg/mL, and the activity is 400U/mL,800U/mL and 2000U/mL.
1g of specification particles:adding 70% ethanol into 80g of sprayed 1g dry powder, mixing, adding 25.6mL volume, granulating with 20 mesh sieve, oven drying at 37deg.C overnight, vacuum/non-vacuum packaging according to 1g specification, and placing at 2-8deg.C, 25deg.C and 40deg.C for stability examination.
0.5g of specification particles:adding 70% ethanol into 40g sprayed 0.5g dry powder while mixing, and finally adding 12.8mL volume, and the rest is the same as above;
0.2g of specification particles:adding 70% ethanol into 16g sprayed 0.2g dry powder while mixing, and adding 5.12mL volume;
0.2g of standard OsrhCT dry powder and 1g of sodium bicarbonate powder are combined and granulated:adding 80g of sodium bicarbonate powder into 16g of sprayed 0.2g of dry powder, uniformly mixing, adding 70% ethanol while uniformly mixing, and finally adding 30.7mL of the mixture;
0.2g of standard OsrhCT particles+1 g of sodium bicarbonate powder:adding 70% ethanol into 16g sprayed 0.2g dry powder while mixing, adding 5.12mL volume, granulating by 20 mesh screen, oven drying overnight at 37deg.C, vacuum/non-vacuum packaging according to 0.2g specification, adding 1g sodium bicarbonate powder into each package, and placing at 2-8deg.C, 25deg.C and 40deg.C for stability examination.
The results show that: group 3 (0.2 g package format) proteins had the highest relative activities suggesting that 0.2g package format is better than 1g and 0.5g format; vacuum or non-vacuum packaging, the influence on the sample is not great at present; group 4 (combined granulation) had the lowest specific activity, followed by group 5 (packaged together) had lower specific activity, and SDS-PAGE detection found that these 2 groups of samples degraded largely (fig. 3A), suggesting that human chymotrypsin could not be packaged with sodium bicarbonate, with an impact on human chymotrypsin. Group 3 proteins had the highest relative specific activity, but the recovery of specific activity was less than 85% compared to the original liquid, and the specific activity of proteins tended to decrease with increasing temperature, suggesting that spray dried samples may not be as stable as freeze dried samples.
Table 4 2 results of the stability protein Activity test (Unit: U/mg)
3. Human chymotrypsin spray drying formulation optimization
The activity recovery of the early 0.2g specification sample is higher than that of the 1g and 0.5g specifications, so that the main sample specification uses 0.2g, and the protection effect of different stabilizer contents on the human chymotrypsin particles is examined.
Group 1 (2% mannitol+0.5% dextran 20) -0.25g specification:100mL of human chymotrypsinogen solution (4 mg/mL, 8000U/mL) is taken, added with 190 mL of purified water with pH of 3.4, 2L is added, 40g of mannitol and 10g of dextran 20 are added and mixed uniformly, and then a 0.22 mu m filter membrane is used for filtration, and spray drying is carried out;
group 2 (5% mannitol+0.5% dextran 20) -0.22g gauge:100 mL of human chymotrypsinogen solution (4 mg/mL, 8000U/mL) is taken, 700mL of purified water with pH of 3.4 is added, total 800mL is added, 40g of mannitol and 4g of dextran 20 are added and mixed uniformly, and the mixture is the same as above;
group 3 (5% mannitol+1.25% dextran 20) -0.25g gauge:100 mL of human chymotrypsinogen solution (4 mg/mL, 8000U/mL) is taken, 700mL of purified water with pH of 3.4 is added, total 800mL is added, 40g of mannitol and 10g of dextran 20 are added and mixed uniformly, and the mixture is the same as above;
group 4 (10% mannitol+0.1% dextran 20) -0.2g Specification:100 mL of human chymotrypsinogen solution (4 mg/mL, 8000U/mL) is taken, 300mL of purified water with pH of 3.4 is added, 400mL is added, 40g of mannitol and 0.4g of dextran 20 are added and mixed uniformly, and the mixture is the same as above;
group 5 (10% mannitol+0.5% dextran 20) -0.21g gauge:100 mL of human chymotrypsinogen solution (4 mg/mL, 8000U/mL) is taken, 300mL of purified water with pH of 3.4 is added, 400mL is added, 40g of mannitol and 2g of dextran 20 are added and mixed uniformly, and the mixture is the same as above;
group 6 (10% mannitol+2.5% dextran 20) -0.25g gauge:100 mL of human chymotrypsinogen solution (4 mg/mL, 8000U/mL) was added to 300mL of purified water at pH3.4, 400mL in total, and 40g of mannitol and 10g of dextran 20 were added to mix well, as above.
The stability test results for 2 months show that: the higher the temperature, the more the polymer content, the no obvious change in purity of the 4 ℃ sample (figure 3B), the recovery of the specific activity of the protein is about 80% (about 90% for 1 month), the recovery of the specific activity of the protein is about 70% (about 80% for 1 month) for the 25 ℃ sample; the specific activity of the 40 ℃ sample is recovered at 40% -50% (the specific activity is recovered at 50% -60% in 1 month), and compared with 1 month, the specific activity of the sample is obviously reduced, the total activity is reduced by about 10%, and the spray drying is possibly not suitable for sample stabilization.
Table 5 2 results of the measurement of the stability protein Activity (Unit: U/mg)
4. Human chymotrypsin spray drying process optimization
The spray drying process optimization was performed by selecting a 10% mannitol+0.1% dextran 20 formulation (higher recovery of activity than other formulations):
group 1: spray drying is carried out at the air inlet temperature of 90 ℃ and the air outlet temperature of 70 ℃ (uncontrollable, the actual temperature of 58 ℃ -60 ℃), the atomization frequency of 390-400 HZ and the sample injection flow rate of 10rpm (20 rpm cannot spray dry, the sample adheres to the wall, and water drops are obviously observed);
group 2: spray drying is carried out at the air inlet temperature of 100 ℃ and the air outlet temperature of 70 ℃ (uncontrollable, the actual temperature of 60 ℃ -62 ℃), the atomization frequency of 390-400 HZ and the sample injection flow rate of 12rpm (20 rpm cannot spray dry, the sample adheres to the wall, and water drops are obviously observed);
group 3: spray drying is carried out at the air inlet temperature of 120 ℃ and the air outlet temperature of 80 ℃ (uncontrollable, the actual temperature of 64 ℃ -66 ℃), the atomization frequency of 390-400 HZ and the sample injection flow rate of 20 rpm;
group 4: spray drying is carried out at the air inlet temperature of 120 ℃ and the air outlet temperature of 80 ℃ (uncontrollable, the actual temperature of 77 ℃ -80 ℃), the atomization frequency of 390-400 HZ and the sample injection flow rate of 10 rpm.
Group 5: spray drying is carried out at the air inlet temperature of 150 ℃ and the air outlet temperature of 80 ℃ (uncontrollable, the actual temperature is 75 ℃ -80 ℃), the atomization frequency is 390-400 HZ, the sample injection flow rate is 40rpm (50 rpm cannot spray dry, the sample adheres to the wall, and water drops are obviously observed);
group 6: spray drying is carried out at the air inlet temperature of 180 ℃ and the air outlet temperature of 80 ℃ (uncontrollable, the actual temperature of 80 ℃ -85 ℃), the atomization frequency of 390-400 HZ, the sample injection flow rate of 40rpm (50 rpm cannot spray dry, the sample adheres to the wall, and water drops are obviously observed);
TABLE 6 Activity measurements before and after spraying and after granulating (unit: U/mg)
Optimizing spray drying process, reducing air outlet temperature to (90 ℃ and 100 ℃), changing spray speed (120 ℃ and 10rpm/20 rpm), increasing air outlet temperature and spray speed (150 ℃ and 180 ℃ and 40 rpm), and the activity of samples after spraying and granulating is not obviously changed, and finally, the stability detection result for 1 month shows that: the vacuum package and the non-vacuum aluminum bag package have no obvious difference, the higher the temperature is, the more the polymer is (figure 4), the activity has obvious reduction trend, the specific activity of the 4 ℃ sample protein is recovered by 83 to 93 percent, the 25 ℃ sample activity is recovered by 70 to 80 percent, and the 40 to 64 percent of the 40 ℃ sample activity is recovered, which indicates that: the optimization of the spray drying process has no obvious improvement effect on the stability of the human chymotrypsin.
Table 7 1 results of the measurement of the stability protein Activity (Unit: U/mg)
Conclusion: from the data of the present case, the stability of the human chymotrypsin prepared by the spray drying process is poor, and the activity of the sample has a significant trend of decreasing along with the prolonged standing time.
Example 3 feasibility of preparing oral lyophilized powder of human chymotrypsin by freeze-drying
1. Human chymotrypsin freeze-dried dry powder granulation studies
Group 1 (2% mannitol+0.5% dextran 20) -0.25g gauge: wetting one part of freeze-dried powder with 80% ethanol, extruding and granulating; wetting one part of freeze-dried powder with 100% ethanol, extruding and granulating;
group 2 (5% mannitol+0.5% dextran 20) -0.22g gauge: extruding and granulating the freeze-dried powder with 80% ethanol;
group 3 (5% mannitol+1.25% dextran 20) -0.25g gauge: extruding and granulating the freeze-dried powder with 80% ethanol;
group 4 (10% mannitol+0.1% dextran 20) -0.2g Specification: freeze-drying fails;
group 5 (10% mannitol+0.5% dextran 20) -0.21g gauge: the freeze-dried powder is extruded and granulated after being wetted by pure water;
group 6 (10% mannitol+2.5% dextran 20) -0.25g gauge: extruding and granulating the freeze-dried powder after pure water wetting。
The results show that: the dry powder obtained by freeze drying is not suitable for granulating, the viscosity of the wet sample is high, the wet sample is not easy to mix uniformly, the particles are easy to adhere during granulating, and the dispersibility is poor (figure 5A); meanwhile, the electrophoresis detection particles have polymers and degradation zones (figure 5B), the activity recovery is lower than 80 percent, the particles are insoluble in the process of re-dissolution after 100 percent ethanol granulation, the particles cannot be selected from the dosage forms, and only powder split charging can be carried out.
Table 8 Activity detection after granulation of human chymotrypsin lyophilized powder (Unit U/mg)
2. Formulation investigation and stability investigation of human chymotrypsin freeze-dried powder
At present, all the chymotrypsin preparations for injection in the market are packaged in 2mL penicillin bottles, and are required to be matched with a syringe for use, so that the chymotrypsin preparations are not suitable for oral administration, the weight of the contents is light (within 50 mg), the contents are inconvenient to pack in bags, the freeze-drying formula ratio is tried to be optimized, and the freeze-drying formula is adjusted to be set 1:10% mannitol+2.5% dextran 20, group 2:5% mannitol, 5% sucrose and 2.5% dextran 20, wherein the concentration of human chymotrypsin protein is 0.5mg/mL, each 300mL is freeze-dried, and sub-packaged according to 0.5 g/bag after freeze-drying, namely 4000U/bag. The freeze-drying process is as follows:
after the human chymotrypsin freeze-dried powder is prepared, the human chymotrypsin freeze-dried powder is placed for 3 months at different temperatures, the aluminum bags at 25 ℃ and 40 ℃ except for the group 1 show dispersion bands and degradation bands in the non-vacuum packaging samples, and the purity of the rest samples is not obviously changed (figure 6). The activity detection results show that: the specific activity of the protein of the group 1 sample is obviously reduced along with the increase of the preservation temperature, and the recovery of the specific activity of the protein of the 40 ℃ sample is only 9% when the aluminum bag is not vacuum packed; the recovery of specific activity of all temperature (4 ℃,25 ℃ and 40 ℃ and vacuum and aluminum bag package) samples in group 2 is above 90%, which indicates that the formula in group 2 is more stable than the formula in group 1, and the formula is determined to be 5% mannitol, 5% sucrose and 2.5% dextran 20, the concentration of human chymotrypsin protein is 0.5mg/mL, and the activity is 1000U/mL.
TABLE 9 detection of Activity of human chymotrypsin lyophilized powder for 3 months stability sample (Unit: U/mg)
Conclusion: the data of the case show that the human chymotrypsin freeze-dried powder prepared by the freeze-drying method has the advantage of high temperature stability, the purity of the human chymotrypsin sample is not obviously changed when the human chymotrypsin freeze-dried powder is placed for 3 months at a high temperature of 40 ℃, and the activity recovery is more than 90%.
Example 4 stability investigation of human chymotrypsin oral lyophilized powder pilot-scale production samples
Taking 5L of human chymotrypsinogen liquid, concentrating and dialyzing by adopting a 5kD membrane package, adding 5% mannitol, 5% sucrose and 2.5% dextran 20, uniformly mixing, and performing sterilization and filtration by a 0.22 mu m filter to obtain a chymotrypsin semi-finished product; sub-packaging and freeze-drying are carried out according to 1L/tray under laminar flow, and the freeze-drying process comprises the following steps:
(a) Pre-freezing: the sample enters a freeze dryer at normal temperature, and after the temperature of the separator is reduced to-45 ℃, the sample is pre-frozen for more than 4 hours;
(b) Primary drying: the condensing temperature is reduced to below-45 ℃, and then the vacuum is pumped to below 10 pa; raising the temperature of the separator to-30 ℃ at a speed of 5 ℃/1.5h, and preserving the temperature for more than 48h until the crystal water disappears;
(c) And (3) secondary drying: raising the temperature of the separator to 20 ℃ at a speed of 5 ℃/1.5h, and preserving heat for more than 15 h; and (5) checking that the vacuum degree is unchanged, ending freeze-drying, and taking the sample out of the cabinet to obtain chymotrypsin oral freeze-dried powder. After pulverization, sub-packaging was performed at 0.5 g/bag, and accelerated stability at 25℃was examined.
Table 10 data were examined for accelerated stability (25±2 ℃/RH60% ±5%) of human chymotrypsin lyophilized powder
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Conclusion: the data of the case show that the freeze-dried powder of the human chymotrypsin prepared by the freeze-drying method has good consistency among batches, and each quality index of the sample is qualified after the sample is placed for 6 months, so that the freeze-dried powder of the human chymotrypsin can be used for large-scale sample production.
Example 5 detection of in vitro Activity of oral lyophilized powder of human chymotrypsin
1. Human chymotrypsin oral freeze-dried powder and sodium bicarbonate uniformly mixing mode
(1) 100mL of purified water is taken, 1g of sodium bicarbonate is added for uniform mixing, then 0.5g of human chymotrypsin oral freeze-dried powder is added for uniform mixing, the pH value is detected to be 8.32, and the solution and the activity change are observed.
(2) 1g of sodium bicarbonate and 0.5g of human chymotrypsin oral freeze-dried powder are added into a conical flask, 100mL of purified water is added for uniform mixing, the pH is detected to be 8.38, and the solution and the activity change are observed.
(3) 100mL of purified water is taken, 0.5g of human chymotrypsin oral freeze-dried powder is added and mixed uniformly, 1g of sodium bicarbonate is added and mixed uniformly, the pH value is detected to be 8.38, and the change of the solution is observed.
The results show that: the stability of the solutions in different redissolution modes is obviously different, after sodium bicarbonate and human chymotrypsin are added into the oral freeze-dried powder, 100mL of purified water is added for dissolution together, and the solution is always clear (also clear after overnight); whether the oral lyophilized powder of human chymotrypsin is added after the sodium bicarbonate is added for dissolving and mixing uniformly, or the oral lyophilized powder of human chymotrypsin is added after the sodium bicarbonate is added for dissolving and mixing uniformly, the solution becomes turbid about 30 minutes (figure 7), and the oral lyophilized powder of human chymotrypsin is suggested to be dissolved and mixed uniformly after the solid is poured and water is added together in clinical use. The activity detection result shows that the activity of the solution has no obvious change (recovery is more than 90%) in 4 hours.
Table 11 human chymotrypsin oral lyophilized powder and sodium bicarbonate after mixing and dissolving solution activity change
| Detection time | 10min | 30min | 60min | 2h | 3h | 4h |
| Activity (U/mL) | 37.9 | 37.0 | 36.1 | 36.1 | 36.8 | 36.3 |
| Recovery (%) | 100 | 97.7 | 95.3 | 95.1 | 97.1 | 95.9 |
2. Detection of human chymotrypsin oral freeze-dried powder in-vitro activity change by simulating gastric juice environment
The experiment refers to national standard 'method for testing the digestion stability of exogenous protein of gastric juice by detecting edible safety of transgenic organisms and products thereof-simulated gastric juice', wherein gastric juice is simulated according to the volume of gastric juice of 50-100mL,0.5g of oral lyophilized powder of human chymotrypsin and the volume of sodium bicarbonate solution of +1g of 100 mL: oral solution = 1:1 and 1:2, test, reaction time: 5min, 10min, 20min, 30min, 60min, 90min, 2h, 3h, 4h.
5mL and 10mL simulated gastric digest (SGF) were added to each of the 2 50mL centrifuge tubes, and incubated in a 37℃water bath for 5min. 10mL of human chymotrypsin oral freeze-dried powder and sodium bicarbonate solution are added into each, the mixture is rapidly and evenly mixed, the mixture is rapidly placed in a water bath at 37 ℃, the time is accurately recorded, and at each reaction time point, the reaction liquid is rapidly sucked for activity detection. The results show that: after the simulated gastric digestion liquid, the oral freeze-dried powder of the human chymotrypsin and the sodium bicarbonate oral solution are uniformly mixed in different ratios, the pH of the solution is 5.7-6.8, and the activity detection result shows that the activity does not change obviously within 3 hours (the recovery is more than 90%).
Table 12 simulates gastric digest: oral solution = 1:1 Activity assay results
Table 13 simulates gastric digest: oral solution = 1:2 Activity assay results
| Detection time | 10min | 30min | 60min | 2h | 3h | 4h |
| Activity (U/mL) | 35.8 | 36.0 | 34.5 | 33.1 | 32.5 | 31.1 |
| Recovery (%) | 100 | 100.6 | 96.5 | 92.6 | 90.7 | 87.0 |
Example 6 detection of mucus removal Effect of human chymotrypsin oral lyophilized powder in beagle gastroscope observations
The test uses 10 dogs (with unlimited sexuality) which are randomly distributed into 4 groups, 1 control group, 3 low, medium and high dose groups, and a blank control (sodium bicarbonate solution) and 60, 150 and 750U/kg test products (recombinant human chymotrypsin oral freeze-dried powder) are respectively administrated, and the test uses are euthanized after 7 days after single gastric lavage administration. The animals are subjected to general anesthesia before gastroscopy, whether foams exist in the stomach or not is checked by the gastroscopy for the first time, and images are stored. 4000U of recombinant human chymotrypsin oral freeze-dried powder and 1g of sodium bicarbonate are added into 100mL of drinking water (20-40 ℃) through a gastroscope, after shaking and dissolving, the oral freeze-dried powder and the sodium bicarbonate are respectively and quantitatively taken into dogs according to the weight of the dogs, and after 30 minutes, gastroscope observation is carried out, and images are stored. During the test, indexes such as clinical daily observation, adverse events, survival rate, body temperature/body weight, blood biochemistry, visual field definition evaluation, safety evaluation, general anatomic observation, histopathological examination and the like are detected.
The results show that: compared with the preoperative and control groups, the gastric mucus and foam can be obviously removed after the administration of the low, medium and high dose groups (figure 8), and the gastroscope has good visual field definition after 30 minutes and no adverse reaction.
TABLE 14 visual field clarity score before and after administration
Remarks: evaluation criteria: the image is excellent, and the gastric mucosa has no adhesion of mucus and foam, so that the score is 1; the image is good, a small amount of mucus and foam of the gastric mucosa are attached, the observation is not affected, and the score is 2; image difference, gastric mucosa has mucus and foam adhesion, and needs to be washed with water (flushing amount is less than 50 mL), so as to obtain 3 points; the images are extremely poor, a large amount of mucus and foam of the gastric mucosa are attached, and the gastric mucosa is required to be washed by water (the flushing amount is more than or equal to 50 mL), so that the gastric mucosa is 4 minutes.
Claims (14)
1. An oral chymotrypsin lyophilized powder, which is characterized by comprising chymotrypsin, mannitol, sucrose and dextran 20, wherein the ratio of the chymotrypsin, the mannitol, the sucrose and the dextran 20 is 4000 units: 167-222mg:167-222mg:83-111mg.
2. The chymotrypsin oral lyophilized powder of claim 1 wherein the chymotrypsin is human chymotrypsin.
3. The chymotrypsin oral lyophilized powder of claim 2 wherein the chymotrypsin is a recombinant human chymotrypsin.
4. The chymotrypsin oral lyophilized powder according to claim 1, wherein the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 units: 200mg:200mg:100mg.
5. A method for preparing the chymotrypsin oral freeze-dried powder according to claims 1 to 4, comprising the following steps:
(1) Pretreatment: concentrating and dialyzing chymotrypsinogen liquid until the electric conductivity of the filtered liquid is lower than 0.5mS/cm to obtain chymotrypsin concentrated liquid;
(2) Preparing a mixed solution: adding 5% mannitol, 5% sucrose and 2.5% dextran 20 to the chymotrypsin concentrate according to the volume ratio, and stirring and uniformly mixing to obtain a mixed solution;
(3) And (3) sterilizing and filtering: sterilizing and filtering the mixed solution by adopting a filter membrane to obtain filtrate;
(4) And (3) freeze drying: freeze-drying the filtered filtrate in the step (3) to obtain freeze-dried powder;
(5) And (3) crushing the freeze-dried powder, and sub-packaging to obtain the chymotrypsin oral freeze-dried powder.
6. The method of claim 5, wherein step (1) comprises concentrating and dialyzing the chymotrypsinogen solution with a 5kD membrane pack and/or water for injection having a temperature below 25 ℃.
7. The method according to claim 5, wherein the chymotrypsin concentration dialysis in step (1) has a chymotrypsin content of 1mg/mL to 4mg/mL and an activity of 2000U/mL to 9600U/mL.
8. The method of claim 5, wherein the chymotrypsin concentration in step (1) has a chymotrypsin content of greater than 0.6mg/mL and an activity of greater than 1000U/mL.
9. The process for preparing the chymotrypsin oral freeze-dried powder according to claim 8, wherein the chymotrypsin activity of the chymotrypsin concentrate is 1200U/ml to 4000U/ml.
10. The chymotrypsin oral lyophilized powder of claim 5, wherein the pH of the mixture of step (2) is 3.0-5.0.
11. The oral lyophilized powder of chymotrypsin according to claim 5, wherein the content of chymotrypsin in the mixed solution in the step (2) is 0.4mg/mL-0.7mg/mL, and the activity is 1100-1300U/mL.
12. The oral lyophilized powder of chymotrypsin according to claim 5, wherein the content of chymotrypsin in the filtrate in the step (3) is 0.4mg/mL-0.7mg/mL, and the activity is 1000-1300U/mL.
13. The chymotrypsin oral lyophilized powder of claim 5, wherein step (4) comprises:
(a) Pre-freezing: the sample enters a freeze dryer at normal temperature, and after the temperature of the separator is reduced to-45 ℃, the sample is pre-frozen for more than 4 hours;
(b) Primary drying: the condensing temperature is reduced to below-45 ℃, and then the vacuum is pumped to below 10 pa; raising the temperature of the separator to-30 ℃ at a speed of 5 ℃/1.5h, and preserving the temperature for more than 48h until the crystal water disappears;
(c) And (3) secondary drying: raising the temperature of the separator to 20 ℃ at a speed of 5 ℃/1.5h, and preserving heat for more than 15 h; and (5) checking that the vacuum degree is unchanged, ending freeze-drying, and taking the sample out of the cabinet.
14. The method of claim 5, wherein the size of the sub-package in step (5) is 0.5 g/bag.
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| CN202311437415.0A CN117338730A (en) | 2023-10-30 | 2023-10-30 | Chymotrypsin oral freeze-dried powder and preparation method thereof |
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| CN202311437415.0A CN117338730A (en) | 2023-10-30 | 2023-10-30 | Chymotrypsin oral freeze-dried powder and preparation method thereof |
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