Oral chymotrypsin lyophilized powder and preparation method therefor

Abstract

An oral chymotrypsin lyophilized powder and a preparation method therefor. The oral chymotrypsin lyophilized powder comprises chymotrypsin, mannitol, sucrose, and dextran 20 in a ratio of 4000 U / mg:(167-222) mg:(167-222) mg:(83-111) mg. The prepared oral chymotrypsin lyophilized powder has the advantage that samples are stable under high-temperature conditions. The chymotrypsin samples are placed for 3 months at a high temperature of 40 °C and have no significant change in purity, with an activity recovery of greater than 90%.

Description

Chymotrypsin oral freeze-dried powder and preparation method thereof Technical Field

[0001] The invention belongs to the field of biological preparations, and particularly relates to a chymotrypsin oral lyophilized powder and a preparation method thereof. Background Art

[0002] Chymotrypsin, also known as chymotrypsin, is a proteolytic enzyme extracted from bovine or porcine pancreas. It acts as an endopeptidase, specifically hydrolyzing carboxyl-terminal aromatic amino acids (tyrosine, tryptophan, leucine) or bulky, hydrophobic residues such as methionine by cleaving the carboxyl-terminal peptides of tyrosine, tryptophan, and phenylalanine in protein peptides. Chymotrypsin also acts as a lipase, hydrolyzing certain lipids. As a proteolytic enzyme, chymotrypsin can be used to promote the digestion and clearance of blood clots, purulent secretions, and necrotic tissue. It is used in ophthalmic surgery to relax ligaments and alleviate traumatic iridocyclitis. It can also be applied to wounds or localized inflammation to reduce secretions and edema.

[0003] Gastric cancer is the most common digestive tract malignancy in my country, posing a serious threat to human health. According to statistics from the International Agency for Research on Cancer, approximately 1.089 million new cases of gastric cancer were reported worldwide in 2020, ranking fifth among malignant tumors. Approximately 769,000 deaths from gastric cancer were reported worldwide in 2020, ranking fourth among malignant tumors. Of these, 43.9% of cases and 48.6% of deaths occurred in China. Early-stage gastric cancer is curable, with a five-year survival rate exceeding 90% after resection. However, the five-year survival rate for advanced gastric cancer is only 10% to 20%. Therefore, early detection and treatment are crucial for a cure. Endoscopy is the most commonly used screening method for early-stage gastric cancer. Gastroscopy is a common diagnostic and treatment method for upper gastrointestinal diseases. It not only identifies lesions and defines their location, morphology, and nature, but also allows for endoscopic treatment. However, under normal circumstances, the upper gastrointestinal tract contains a large amount of mucus and foam, which significantly affects the endoscopic field of view and the detection of early lesions. Repeated flushing increases endoscopic examination time and increases patient pain. Propionase is used as a mucus remover to eliminate mucus caused by proteins on the surface of the gastrointestinal mucosa. However, due to its high price, propionase is not widely used. Chemotrypsin and N-acetylcysteine ​​are often used as substitutes.

[0004] Chymotrypsin preparations currently on the market are all injection chymotrypsin, and packaged form is a 2mL cillin bottle, needs to cooperate syringe to use, and is inconvenient to use when gastroscopy detects, and is inconvenient to take orally, and inclusion is lighter, is not suitable for bagged sub-packaging.The present invention has proposed a kind of new chymotrypsin oral preparation for the first time, can be weighed, realizes bagged sub-packaging, has stability high, conveniently stores, and uses easier, and the action time is long, can effectively remove gastric mucus, improves the advantage of visual field clarity, is the transformation and innovation that traditional technology is carried out.Said preparation has been declared as biopharmaceutical at present, and chymotrypsin oral preparation of the present invention has great innovative significance to the improvement of biopharmaceutical formulation.

[0005] Summary of the Invention

[0006] One object of the present invention is to provide a new chymotrypsin preparation, which is in the form of an oral lyophilized powder.

[0007] The chymotrypsin oral lyophilized powder comprises chymotrypsin, mannitol, sucrose and dextran 20, wherein the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 U / mg: (167-222) mg: (167-222) mg: (83-111) mg.

[0008] Preferably, the chymotrypsin is human chymotrypsin.

[0009] More preferably, the chymotrypsin is recombinant human chymotrypsin.

[0010] Preferably, the ratio of chymotrypsin, mannitol, sucrose and dextran 20 in the chymotrypsin oral lyophilized powder is 4000 U / mg: 200 mg: 200 mg: 100 mg.

[0011] Another object of the present invention is to provide a method for preparing a chymotrypsin oral lyophilized powder, the method comprising the steps of:

[0012] (1) Pretreatment: The chymotrypsin stock solution was concentrated and dialyzed until the conductivity of the filtrate was less than 0.5 mS / cm to obtain the chymotrypsin concentrate;

[0013] (2) preparing a mixed solution: adding 5% mannitol, 5% sucrose, and 2.5% dextran 20 to the chymotrypsin concentrate according to a volume ratio, and stirring to mix, thereby obtaining the mixed solution;

[0014] (3) Sterile filtration: sterilize and filter the mixed solution using a filter membrane;

[0015] (4) freeze drying: freeze drying the filtrate after filtering in step (3) to obtain freeze-dried powder;

[0016] (5) The freeze-dried powder is crushed and packaged to obtain the chymotrypsin oral freeze-dried powder.

[0017] Preferably, in step (1), the chymotrypsin stock solution is concentrated and dialyzed using a 5kD membrane package and water for injection at a temperature below 25°C.

[0018] Preferably, the content of chymotrypsin during the chymotrypsin concentration and dialysis process in step (1) is 1 mg / mL-4 mg / mL, and the activity is 2000 U / mL-9600 U / mL.

[0019] Preferably, the chymotrypsin content of the chymotrypsin concentrate in step (1) is greater than 0.6 mg / mL and the activity is greater than 1000 U / mL.

[0020] Preferably, the chymotrypsin activity of the chymotrypsin concentrate in step (1) is 1200 U / mL to 4000 U / mL.

[0021] Preferably, the pH value of the mixed solution in step (2) is 3.0-5.0.

[0022] Preferably, step (2) uses a 0.22 μm filter membrane for sterilization filtration.

[0023] Preferably, the content of chymotrypsin in the mixed solution of step (2) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1100-1300 U / mL.

[0024] Preferably, the content of chymotrypsin in the filtrate of step (3) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1000-1300 U / mL.

[0025] Preferably, step (4) comprises:

[0026] (a) Prefreezing: The sample enters the freeze dryer at room temperature, and the partition is cooled to -45°C and pre-frozen for more than 4 hours;

[0027] (b) Primary drying: After the condensation temperature drops below -45°C, evacuate to below 10 Pa; raise the temperature of the separator to -30°C at a rate of 5°C / 1.5h and keep the temperature for more than 48h until the crystal water disappears;

[0028] (c) Secondary drying: Raise the temperature of the shelf to 20°C at a rate of 5°C / 1.5h and keep warm for more than 15h; check that the vacuum degree does not change, terminate the freeze-drying process, and remove the sample from the cabinet.

[0029] Preferably, the packaging specification in step (5) is 0.5 g / bag.

[0030] Preferably, the preparation method comprises:

[0031] In step (1), chymotrypsin stock solution is used as raw material, and the protein content is 1.0±0.2 mg / mL. It is concentrated and dialyzed using a 5kDa membrane bag. The membrane bag is first treated with 0.5M NaOH for at least 30 minutes and then washed with water for injection. After washing, the chymotrypsin stock solution is concentrated to 1 / 2-1 / 4 of the original volume, and an equal volume of water for injection is added. After mixing, the concentration and dialysis are repeated until the conductivity of the filtrate is lower than 0.5mS / cm; the protein concentration during the concentration and dialysis process is 1-4 mg / mL, and after the dialysis is completed, the protein concentration is adjusted to 0.6-2.0 mg / mL, and the solution pH is 3.0-5.0, preferably 4.0.

[0032] In step (2), the activity and protein content of the chymotrypsin solution after dialysis and concentration in step (1) are detected, and its activity is diluted with water for injection to 1000-1300 U / mL and the protein content is 0.4-0.7 mg / mL. 5% mannitol, 5% sucrose, and 2.5% dextran 20 are added according to the final volume ratio and stirred to mix.

[0033] In step (3), the prepared solution of step (2) is sterilized and filtered using two-stage 0.22 μm filters under Class A laminar flow. The resulting sample is a chymotrypsin semi-finished product with an activity of 1000-1300 U / mL and a protein content of 0.4-0.7 mg / mL.

[0034] In step (4), the chymotrypsin semi-finished product in step (3) is packaged and freeze-dried at 1 L / tray under laminar flow. The freeze-drying process comprises the following steps:

[0035] (a) Prefreezing: The sample enters the freeze dryer at room temperature, and the partition is cooled to -45°C and pre-frozen for more than 4 hours;

[0036] (b) Primary drying: After the condensation temperature drops below -45°C, evacuate to below 10 Pa; raise the temperature of the separator to -30°C at a rate of 5°C / 1.5h and keep the temperature for more than 48h until the crystal water disappears;

[0037] (c) Secondary drying: Raise the temperature of the partition to 20°C at a rate of 5°C / 1.5h and keep it warm for at least 15h. Check that the vacuum degree remains unchanged, terminate the freeze-drying process, and remove the sample from the freezer to obtain chymotrypsin oral freeze-dried powder.

[0038] In step (5), the chymotrypsin oral lyophilized powder in step (4) is crushed by a pulverizer and packaged at 0.5 g / bag. The material used for the packaging is an opaque aluminum-containing composite film, preferably a polyester / aluminum / polyethylene pharmaceutical composite film. Confirm that the horizontal seal and the vertical seal are horizontal and vertical, and the seal is intact, completing the preparation of the chymotrypsin oral lyophilized powder.

[0039] The chymotrypsin oral lyophilized powder of the present invention can be used for gastroscopy to remove gastric mucus and improve visual clarity. The method of taking is to mix one bag of chymotrypsin oral lyophilized powder with one bag of 1g sodium bicarbonate, dissolve it in warm water and take it within 1 hour, the volume of warm water preferably being 50-100mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1. Observation of solution clarity at different pH values ​​during dialysis.

[0041] Figure 2. Diagram of spray-dried dry powder granulation of human chymotrypsin.

[0042] Figure 3. SDS-PAGE analysis of human chymotrypsin oral granules with different formulations; Figure A shows the formulation exploration of human chymotrypsin oral granules, and Figure B shows the formulation optimization of human chymotrypsin oral granules, where M is the protein standard molecular weight marker, groups 1-6 represent samples with different formulations, and 4℃ / 25℃ / 37℃ represent samples stored at different temperatures of 4℃ / 25℃ / 37℃.

[0043] Figure 4. SDS-PAGE analysis of human chymotrypsin oral granule samples prepared by spray process under different conditions; where M is the protein standard molecular weight marker, groups 1-6 represent samples prepared by different spray processes, 4℃ / 25℃ / 37℃ represent samples placed at different temperatures of 4℃ / 25℃ / 37℃, pre-spray represents samples before spray drying, vacuum represents samples stored in vacuum packaging, and aluminum bag represents samples stored in non-vacuum packaging aluminum bags.

[0044] Figure 5. Granulation of freeze-dried human chymotrypsin powder; M is the protein standard molecular weight marker, groups 1-6 represent samples prepared by different granulation processes, 100% represents samples prepared with 100% ethanol, 80% represents samples prepared with 80% ethanol, and pure water represents samples prepared with purified water.

[0045] Figure 6. 3-month stability study of human chymotrypsin oral lyophilized powder with different formulations, sample SDS-PAGE analysis; where M is the protein molecular weight marker, groups 1-2 represent freeze-dried samples prepared with different formulations, and 4°C / 25°C / 37°C represent samples stored at different temperatures: 4°C / 25°C / 37°C.

[0046] Figure 7. Observation of the solution after mixing human chymotrypsin oral lyophilized powder with sodium bicarbonate (30 min).

[0047] Figure 8. Observation of mucus removal effects of different doses of human chymotrypsin oral lyophilized powder during gastroscopy in beagle dogs. DETAILED DESCRIPTION

[0048] The following examples and accompanying drawings are provided to illustrate the technical solutions of the present invention in detail, so as to better illustrate the characteristics and advantages of the present invention. The provided examples should be interpreted as illustrative of the method of the present invention, and should not limit the technical solutions disclosed by the present invention in any way.

[0049] Unless otherwise specified, the reagents and instruments used in the examples are commercially available.

[0050] [Example 1] Selection of dialysis conditions for human chymotrypsin stock solution

[0051] Take the human chymotrypsin stock solution, concentrate it using a 5kD membrane, add an equal volume of purified water, continue dialysis and concentration until the conductivity is 0.5mS / cm, sample 8mL / tube, and adjust the pH to 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.1, 9.5 and 10.0 respectively; when the pH is between 6.0 and 7.0, the sample becomes turbid (slight turbidity begins to appear at pH 5.5), and the sample becomes clear when the pH is adjusted to 3.5; after adding 3% 10xPBS to the turbid sample at pH 7.0, the sample becomes clear. After standing for 2h (room temperature) and 20h (room temperature and 2-8°C), the sample is observed and activity tested. The results showed that during dialysis, the conductivity of chymotrypsin dropped to 0.5 mS / cm, and the sample became clear. After adjusting the pH to 6.0-7.0, the sample became turbid; after adjusting the pH to 3.5, the sample regained clarity and the activity did not change; after adding salt to the turbid sample at pH 7.0, the sample also became clear, but the activity was reduced to about half, suggesting that salt should not be added for re-dissolution during dialysis; chymotrypsin is relatively stable when the pH is lower than 5.0. After being placed at room temperature for 20 hours, the activity is still above 800 U / mg, and the closer the pH is to 3.0, the more stable it is. Therefore, the pH of the solution after dialysis is determined to be pH 3.0-5.0, preferably pH 4.0.

[0052] Table 1 Activity detection of chymotrypsin stock solution after dialysis in different pH solutions (unit: U / mL)

[0053] [Example 2] Preparation of human chymotrypsin oral granules by spray drying

[0054] 1. Study on the granulation of human chymotrypsin spray-dried powder

[0055] A human chymotrypsin solution containing 10% mannitol and having an activity of 1000 U / mL was spray-dried to obtain human chymotrypsin dry powder. The powder was granulated using anhydrous ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and purified water, respectively. The results showed that after granulation with different concentrations of ethanol and purified water, the appearance and morphology showed that the higher the ethanol concentration, the better the particle dispersibility and the more uniform the particles (Figure 2), but the lower the recovery rate of particles (>80 mesh). At the same time, different wetting agents had no significant effect on the activity of the sample. Therefore, 70% ethanol was determined to be used for subsequent particle stability sample granulation.

[0056] Table 2 Recovery rate of qualified particles from 10 to 80 meshes:

[0057] Table 3 Activity test of samples after granulation with different wetting agents

[0058] 2. Exploration of spray-drying formula for human chymotrypsin

[0059] Different dosages of 1g, 0.5g and 0.2g were designed, corresponding to human chymotrypsin concentrations of 0.2mg / mL, 0.4mg / mL and 1mg / mL in 10% mannitol, and activities of 400U / mL, 800U / mL and 2000U / mL, respectively.

[0060] 1g granules: Add 70% ethanol to 80g of the sprayed 1g dry powder while mixing, to a final volume of 25.6mL. Press through a 20-mesh sieve to granulate the granules. Dry in a 37°C oven overnight. Finally, vacuum / non-vacuum package according to the 1g specification and store at 2-8°C, 25°C, and 40°C to test stability.

[0061] 0.5g granules: Add 70% ethanol to 40g of the sprayed 0.5g dry powder while mixing, to a final volume of 12.8mL. All other preparations are the same as above.

[0062] 0.2g granules: Add 70% ethanol to 16g of the sprayed 0.2g dry powder while mixing, to a final volume of 5.12mL. All other procedures are the same as above.

[0063] Granulate 0.2g OsrhCT powder and 1g sodium bicarbonate powder: Add 80g sodium bicarbonate powder to 16g of the sprayed 0.2g powder and mix thoroughly. Add 70% ethanol while mixing to a final volume of 30.7mL. Other steps are the same as above.

[0064] 0.2g OsrhCT granules + 1g sodium bicarbonate powder: Add 70% ethanol to 16g of the sprayed 0.2g dry powder while mixing, to a final volume of 5.12mL. Press through a 20-mesh sieve to granulate the granules. Dry in a 37°C oven overnight and finally package in vacuum or non-vacuum configurations according to the 0.2g size. Add 1g of sodium bicarbonate powder to each package and test stability at 2-8°C, 25°C, and 40°C.

[0065] The results showed that Group 3 (0.2g packaging) had the highest protein specific activity, suggesting that the 0.2g packaging size was superior to the 1g and 0.5g sizes. Vacuum or non-vacuum packaging had little effect on the samples. Group 4 (combined pelleting) had the lowest specific activity, followed by Group 5 (packaged together). SDS-PAGE analysis revealed significant degradation in these two groups (Figure 3A), suggesting that human chymotrypsin should not be packaged together with sodium bicarbonate, as this affects it. Group 3 had the highest protein specific activity, but compared to the original solution, the specific activity recovery was less than 85%, and the protein specific activity tended to decrease with increasing temperature, suggesting that spray-dried samples may be less stable than freeze-dried samples.

[0066] Table 4 2-week stable protein activity test results (unit: U / mg)

[0067] 3. Optimization of human chymotrypsin spray drying formula

[0068] The activity recovery of the 0.2g sample was higher than that of the 1g and 0.5g samples in the early stage. Therefore, the main sample size was 0.2g to investigate the protective effect of different stabilizer contents on human chymotrypsin particles.

[0069] Group 1 (2% mannitol + 0.5% dextran 20) - 0.25 g: 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) was added to 1900 mL of pH 3.4 purified water (2 L), 40 g of mannitol and 10 g of dextran 20 were added, mixed, filtered through a 0.22 μm filter, and spray-dried.

[0070] Group 2 (5% mannitol + 0.5% dextran 20) - 0.22 g: 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) was added to 700 mL of pH 3.4 purified water, for a total of 800 mL. 40 g of mannitol and 4 g of dextran 20 were added and mixed, and the same mixture was used as above.

[0071] Group 3 (5% mannitol + 1.25% dextran 20) - 0.25 g: 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) was added to 700 mL of pH 3.4 purified water, for a total of 800 mL. 40 g of mannitol and 10 g of dextran 20 were added and mixed, and the mixture was repeated as above.

[0072] Group 4 (10% mannitol + 0.1% dextran 20) - 0.2 g: 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) was added to 300 mL of pH 3.4 purified water, for a total of 400 mL. 40 g of mannitol and 0.4 g of dextran 20 were added and mixed, and the mixture was repeated as above.

[0073] Group 5 (10% mannitol + 0.5% dextran 20) - 0.21 g: 100 mL of human chymotrypsin stock solution (4 mg / mL, 8000 U / mL) was added to 300 mL of pH 3.4 purified water, for a total of 400 mL. 40 g of mannitol and 2 g of dextran 20 were added and mixed, and the mixture was repeated as above.

[0074] Group 6 (10% mannitol + 2.5% dextran 20) - 0.25g specification: Take 100mL of human chymotrypsin stock solution (4mg / mL, 8000U / mL) and add 300mL of pH 3.4 purified water, a total of 400mL, add 40g of mannitol and 10g of dextran 20, mix well, and proceed as above.

[0075] The results of the 2-month stability test showed that the higher the temperature, the more aggregates there were. The purity of the 4°C samples did not change significantly (Figure 3B), and the protein specific activity recovery was around 80% (about 90% after 1 month). For the 25°C samples, the protein specific activity recovery was around 70% (about 80% after 1 month). For the 40°C samples, the protein specific activity recovery was between 40% and 50% (50% to 60% after 1 month). Compared with 1 month, the activity showed a significant downward trend, with an overall decrease of about 10%, suggesting that spray drying may not be suitable for sample stability.

[0076] Table 5 2-month stable protein activity test results (unit: U / mg)

[0077] 4. Optimization of human chymotrypsin spray drying process

[0078] The 10% mannitol + 0.1% dextran 20 formulation (which has higher activity recovery than other formulations) was selected for spray drying process optimization:

[0079] Group 1: spray drying was performed at an air inlet temperature of 90°C, an air outlet temperature of 70°C (uncontrollable, actual temperature was 58°C to 60°C), an atomization frequency of 390 to 400 Hz, and a sample flow rate of 10 rpm (20 rpm was insufficient for spray drying, the sample adhered to the wall, and water droplets were clearly observed);

[0080] Group 2: Inlet temperature 100°C, outlet temperature 70°C (uncontrollable, actual temperature 60°C-62°C), atomization frequency 390-400 Hz, injection flow rate 12 rpm (20 rpm did not allow spray drying, the sample adhered to the wall, and water droplets were clearly observed), spray drying was performed;

[0081] Group 3: Inlet temperature 120°C, outlet temperature 80°C (uncontrollable, actual temperature 64°C-66°C), atomization frequency 390-400 Hz, injection flow rate 20 rpm, spray drying;

[0082] Group 4: Inlet temperature 120°C, outlet temperature 80°C (uncontrollable, actual temperature 77°C to 80°C), atomization frequency 390 to 400 Hz, injection flow rate 10 rpm, spray drying.

[0083] Group 5: Inlet temperature 150°C, outlet temperature 80°C (uncontrollable, actual temperature 75°C-80°C), atomization frequency 390-400 Hz, injection flow rate 40 rpm (50 rpm was not enough to spray dry, the sample adhered to the wall, and water droplets were clearly observed), spray drying was performed;

[0084] Group 6: Inlet temperature 180°C, outlet temperature 80°C (uncontrollable, actual temperature 80°C-85°C), atomization frequency 390-400 Hz, injection flow rate 40 rpm (50 rpm was not enough to spray dry, the sample adhered to the wall, and water droplets were clearly observed), spray drying was performed;

[0085] Table 6 Activity test results before and after spraying and granulation (unit: U / mg)

[0086] The spray drying process was optimized, the outlet temperature was lowered to (90℃, 100℃), the spray speed was changed (120℃, 10rpm / 20rpm), and the outlet temperature and spray speed were increased (150℃, 180℃, 40rpm). There was no significant change in the activity of the samples after spraying and granulation. The final 1-month stability test results showed that there was no significant difference between vacuum packaging and non-vacuum aluminum bag packaging. The higher the temperature, the more aggregates there were (Figure 4), and the activity showed a significant downward trend. The protein specific activity of the 4℃ sample was recovered by 83%-93%, the activity of the 25℃ sample was recovered by 70%-80%, and the activity of the 40℃ sample was recovered by 40%-64%. This suggests that optimizing the spray drying process has no significant effect on improving the stability of human chymotrypsin.

[0087] Table 7 1-month stable protein activity test results (unit: U / mg)

[0088] Conclusion: The data from this case show that human chymotrypsin prepared by spray drying process has poor stability, and the activity of the sample tends to decrease significantly with prolonged storage time.

[0089] [Example 3] Feasibility of preparing human chymotrypsin oral lyophilized powder by freeze drying

[0090] 1. Study on the granulation of freeze-dried dry powder of human chymotrypsin

[0091] Group 1 (2% mannitol + 0.5% dextran 20) - 0.25g: one portion of the lyophilized powder was moistened with 80% ethanol and then extruded into granules; one portion of the lyophilized powder was moistened with 100% ethanol and then extruded into granules;

[0092] Group 2 (5% mannitol + 0.5% dextran 20) - 0.22g Specification: lyophilized powder was extruded and granulated with 80% ethanol;

[0093] Group 3 (5% mannitol + 1.25% dextran 20) - 0.25g Specification: lyophilized powder was extruded and granulated with 80% ethanol;

[0094] Group 4 (10% mannitol + 0.1% dextran 20) - 0.2g strength: lyophilization failure;

[0095] Group 5 (10% mannitol + 0.5% dextran 20) - 0.21g Specification: freeze-dried powder moistened with pure water and then extruded into granules;

[0096] Group 6 (10% mannitol + 2.5% dextran 20) - 0.25g Specification: freeze-dried powder was moistened with pure water and then extruded into granules.

[0097] The results showed that the dry powder obtained by freeze-drying was not suitable for granulation. The sample had high viscosity after wetting and was difficult to mix. The particles were more likely to stick together during granulation and had poor dispersibility (Figure 5A). At the same time, electrophoresis detection showed aggregates and degradation bands in the particles (Figure 5B), the activity recovery was less than 80%, and the particles did not dissolve when re-dissolved after granulation in 100% ethanol. Granules were not an option for the dosage form and only powder packaging was possible.

[0098] Table 8 Activity detection of human chymotrypsin lyophilized powder after granulation (unit U / mg)

[0099] 2. Formulation exploration and stability study of human chymotrypsin lyophilized powder

[0100] Currently, human chymotrypsin preparations for injection on the market are all packaged in 2mL vials and require use with a syringe. They are not suitable for oral administration. The contents are light in weight (less than 50mg), making bag packaging inconvenient. We attempted to optimize the freeze-dried formula ratio and adjusted the freeze-dried formula to Group 1: 10% mannitol + 2.5% dextran 20, Group 2: 5% mannitol + 5% sucrose + 2.5% dextran 20, with a human chymotrypsin protein concentration of 0.5mg / mL. 300mL of each was freeze-dried and packaged at 0.5g / bag after freeze-drying, i.e. 4000U / bag. The freeze-drying process is as follows:

[0101] After preparation, human chymotrypsin freeze-dried powder samples were stored at different temperatures for three months. Except for the samples in Group 1 packaged in aluminum bags at 25°C and 40°C, which showed diffuse bands and degradation bands, the purity of the remaining samples remained unchanged (Figure 6). Activity assay results showed that the specific activity of the protein in Group 1 decreased significantly with increasing storage temperature, with only 9% recovery of the specific activity in the sample packaged in aluminum bags at 40°C. In Group 2, the specific activity recovery was above 90% at all temperatures (4°C, 25°C, and 40°C, vacuum-packed and aluminum-bag-packed), indicating that the Group 2 formulation was more stable than that in Group 1. The formulation was determined to be 5% mannitol + 5% sucrose + 2.5% dextran 20, with a human chymotrypsin protein concentration of 0.5 mg / mL and an activity of 1000 U / mL.

[0102] Table 9 Activity test results of human chymotrypsin lyophilized powder for 3 months (unit: U / mg)

[0103] Conclusion: The data from this case show that the freeze-dried powder of human chymotrypsin prepared by the freeze-drying method of the present invention has the advantage of high-temperature stability. After being placed at a high temperature of 40°C for 3 months, the purity of the human chymotrypsin sample did not change significantly, and the activity recovery was greater than 90%.

[0104] [Example 4] Stability study of pilot production samples of human chymotrypsin oral lyophilized powder

[0105] 5 L of human chymotrypsin stock solution was concentrated and dialyzed using a 5 kD membrane cassette. 5% mannitol, 5% sucrose, and 2.5% dextran 20 were added and mixed. The mixture was then sterilized and filtered through a 0.22 μm filter to obtain the chymotrypsin semi-finished product. The product was then packaged and freeze-dried at 1 L per tray under laminar flow. The freeze-drying process included the following steps:

[0106] (a) Prefreezing: The sample enters the freeze dryer at room temperature, and the partition is cooled to -45°C and pre-frozen for more than 4 hours;

[0107] (b) Primary drying: After the condensation temperature drops below -45°C, evacuate to below 10 Pa; raise the temperature of the separator to -30°C at a rate of 5°C / 1.5h and keep the temperature for more than 48h until the crystal water disappears;

[0108] (c) Secondary Drying: Heat the shelf to 20°C at a rate of 5°C / 1.5h and maintain for at least 15h. Verify that the vacuum level remains stable, terminate the freeze-drying process, and remove the sample from the freezer to obtain chymotrypsin oral freeze-dried powder. After pulverization, distribute the powder in 0.5g / bags and examine the accelerated stability at 25°C.

[0109] Table 10 Accelerated stability (25±2°C / RH60%±5%) of human chymotrypsin lyophilized powder

[0110] Conclusion: The data from this case show that the freeze-dried powder of human chymotrypsin prepared by the freeze-drying method of the present invention has good batch consistency. The quality indicators of the samples are still qualified after accelerated storage for 6 months, and can be used for large-scale sample production.

[0111] [Example 5] In vitro activity detection of human chymotrypsin oral lyophilized powder

[0112] 1. Exploration of the mixing method of human chymotrypsin oral lyophilized powder and sodium bicarbonate

[0113] (1) Take 100 mL of purified water, add 1 g of sodium bicarbonate and mix well. Then add 0.5 g of human chymotrypsin oral lyophilized powder and mix well. Detect the pH at 8.32 and observe the changes in solution and activity.

[0114] (2) First, add 1g of sodium bicarbonate and 0.5g of human chymotrypsin oral lyophilized powder into a conical flask, then add 100mL of purified water and mix well. Detect the pH at 8.38 and observe the changes in solution and activity.

[0115] (3) Take 100 mL of purified water, add 0.5 g of human chymotrypsin oral lyophilized powder and mix well, then add 1 g of sodium bicarbonate and mix well. Detect the pH at 8.38 and observe the changes in the solution.

[0116] Results showed significant differences in solution stability between reconstitution methods. When sodium bicarbonate and human chymotrypsin oral lyophilized powder were added first, followed by 100 mL of purified water, the solution remained clear (even after overnight). However, regardless of whether sodium bicarbonate was added first and then human chymotrypsin oral lyophilized powder was added, or whether human chymotrypsin oral lyophilized powder was added first and then dissolved and mixed, followed by sodium bicarbonate, the solution became turbid after approximately 30 minutes (Figure 7). Therefore, for clinical use, it is recommended to pour the solid solution out first and then add water to dissolve and mix. Activity assays showed no significant change in solution activity within 4 hours (recovery greater than 90%).

[0117] Table 11 Changes in activity of human chymotrypsin oral lyophilized powder after dissolution with sodium bicarbonate

[0118] 2. Detection of in vitro activity changes of human chymotrypsin oral lyophilized powder in a simulated gastric fluid environment

[0119] This experiment refers to the national standard "Safety Testing of Genetically Modified Organisms and Their Products for Edible Use - Test Method for Digestion Stability of Exogenous Proteins in Simulated Gastrointestinal Fluid". The test was conducted based on a gastric fluid volume of 50-100mL, 0.5g human chymotrypsin oral lyophilized powder + 1g sodium bicarbonate solution volume of 100mL, simulated gastric digestive fluid: oral solution = 1:1 and 1:2, and reaction times: 5min, 10min, 20min, 30min, 60min, 90min, 2h, 3h, and 4h.

[0120] To two 50mL centrifuge tubes, add 5mL and 10mL of simulated gastric digestive fluid (SGF), respectively, and incubate in a 37°C water bath for 5 minutes. Add 10mL of human chymotrypsin oral lyophilized powder + sodium bicarbonate solution to each tube, rapidly vortex to mix, and quickly place in a 37°C water bath. Accurately record the time. At each reaction time point, quickly aspirate the reaction solution for activity testing. Results showed that after mixing different ratios of simulated gastric digestive fluid with human chymotrypsin oral lyophilized powder + sodium bicarbonate solution, the solution pH ranged from 5.7 to 6.8. Activity testing results showed no significant change in activity within 3 hours (recovery was above 90%).

[0121] Table 12 Simulated gastric digestive fluid: oral solution = 1:1 activity test results

[0122] Table 13 Simulated gastric digestive fluid: oral solution = 1:2 activity test results

[0123] [Example 6] Detection of mucus removal effect of human chymotrypsin oral lyophilized powder during gastroscopy in beagle dogs

[0124] Ten dogs (of either sex) were randomly assigned to four groups: a control group (1 dog) and three dogs in each low-, medium-, and high-dose groups. Each group received a blank control (sodium bicarbonate solution) and a test product (recombinant human chymotrypsin oral lyophilized powder) at 60, 150, and 750 U / kg, respectively, by single gavage. The animals were euthanized seven days later. General anesthesia was administered before endoscopy. The initial endoscopy was performed to examine the stomach for foam and preserve the images. Using an endoscope, 4000 U of recombinant human chymotrypsin oral lyophilized powder and 1 g of sodium bicarbonate were added to 100 mL of drinking water (20-40°C). After vortexing, the dogs were administered orally in a fixed amount based on their body weight. Endoscopy was performed 30 minutes later, and images were preserved. During the trial, clinical observations, adverse events, survival rate, body temperature / weight, blood biochemistry, visual field clarity, safety assessment, gross anatomical observations, and histopathological examinations were monitored.

[0125] The results showed that compared with the preoperative and control groups, the low-, medium-, and high-dose groups were able to significantly remove gastric mucus and foam after administration (Figure 8). After 30 minutes, the endoscopic field of view was clear and no adverse reactions occurred.

[0126] Table 14 Visual field clarity scores before and after drug administration Note: Evaluation criteria: 1 point for excellent images with no mucus or foam attached to the gastric mucosa; 2 points for good images with a small amount of mucus and foam attached to the gastric mucosa that does not affect observation; 3 points for poor images with mucus and foam attached to the gastric mucosa requiring flushing (flushing volume less than 50 mL); 4 points for extremely poor images with a large amount of mucus and foam attached to the gastric mucosa requiring flushing (flushing volume greater than or equal to 50 mL).

Claims

1. A chymotrypsin oral lyophilized powder, characterized in that: The chymotrypsin oral lyophilized powder comprises chymotrypsin, mannitol, sucrose and dextran 20, wherein the ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 U / mg: (167-222) mg: (167-222) mg: (83-111) mg.

2. The chymotrypsin oral lyophilized powder according to claim 1, characterized in that The chymotrypsin is human chymotrypsin.

3. The chymotrypsin oral lyophilized powder according to claim 2, characterized in that The chymotrypsin is recombinant human chymotrypsin.

4. The chymotrypsin oral lyophilized powder according to claim 1, characterized in that The ratio of chymotrypsin, mannitol, sucrose and dextran 20 is 4000 U / mg: 200 mg: 200 mg: 100 mg.

5. A method for preparing the chymotrypsin oral lyophilized powder according to claims 1 to 4, comprising the following steps: (1) Pretreatment: The chymotrypsin stock solution is concentrated and dialyzed until the conductivity of the filtrate is lower than 0.5 mS / cm to obtain a chymotrypsin concentrate; (2) preparing a mixed solution: adding 5% mannitol, 5% sucrose and 2.5% dextran 20 to the chymotrypsin concentrate according to a volume ratio, and stirring to mix, to obtain the mixed solution; (3) sterilizing and filtering: sterilizing and filtering the mixed solution using a filter membrane to obtain a filtrate; (4) freeze drying: freeze drying the filtrate after filtering in step (3) to obtain freeze-dried powder; (5) The freeze-dried powder is crushed and packaged to obtain the chymotrypsin oral freeze-dried powder.

6. The preparation method according to claim 5, characterized in that: Step (1) uses a 5kD membrane package and / or injection water with a temperature below 25°C to concentrate and dialyze the chymotrypsin stock solution.

7. The preparation method according to claim 5, characterized in that: The content of chymotrypsin in the chymotrypsin concentration and dialysis process of step (1) is 1 mg / mL-4 mg / mL, and the activity is 2000 U / mL-9600 U / mL.

8. The preparation method according to claim 5, characterized in that: The chymotrypsin content of the chymotrypsin concentrate in step (1) is greater than 0.6 mg / mL and the activity is greater than 1000 U / mL.

9. The preparation process of chymotrypsin oral lyophilized powder according to claim 8, characterized in that: The chymotrypsin activity of the chymotrypsin concentrate is 1200U / ml-4000U / ml.

10. The preparation method according to claim 5, characterized in that: The pH value of the mixed solution in step (2) is 3.0-5.

0.

11. The preparation method according to claim 5, characterized in that: The content of chymotrypsin in the mixed solution of step (2) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1100 U / mL-1300 U / mL.

12. The method for preparing minced meat according to claim 5, characterized in that: The content of chymotrypsin in the filtrate of step (3) is 0.4 mg / mL-0.7 mg / mL, and the activity is 1000 U / mL-1300 U / mL.

13. The preparation method according to claim 5, characterized in that: Step (4) comprises: (a) Pre-freezing: The sample enters the freeze dryer at room temperature, and the partition is cooled to -45°C and pre-frozen for more than 4 hours; (b) Primary drying: After the condensation temperature drops below -45°C, evacuate to below 10 Pa; raise the temperature of the partition to -30°C at a rate of 5°C / 1.5h and keep the temperature for more than 48h until the crystal water disappears; (c) Secondary drying: Raise the temperature of the partition to 20°C at a rate of 5°C / 1.5h and keep warm for more than 15h; check that there is no change in the vacuum degree, end freeze-drying, and take the sample out of the cabinet.

14. The preparation method according to claim 5, characterized in that: The packaging specification of step (5) is 0.5g / bag.

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