Guanzhong donkey plasma canine parvovirus immune globulin CPV-IgG, freeze-dried product, preparation process and application

Abstract

The invention discloses Guanzhong donkey plasma canine parvovirus immune globulin CPV-IgG, a freeze-dried product, a preparation process and application, and relates to the technical field of bioactive components and separation and purification. The method comprises the following steps: selecting a canine parvovirus CPV-XN1 low virulent strain as an immunogen to immunize an animal; the method comprises the following steps: collecting plasma of an immune animal, and purifying to obtain canine parvovirus immune globulin CPV-IgG. The SN titer of the obtained immune globulin CPV-IgG is not lower than 1: 256. The invention further provides a freeze-drying agent matched with the immune globulin CPV-IgG. The freeze-drying agent is prepared from, by mass, 6 parts of gelatin with the mass concentration being 2.6%, 2 parts of lactose with the mass concentration being 2.8%, 1 part of glycine with the mass concentration being 2.6% and 1 part of sodium chloride with the mass concentration being 2.34%. The lyophilized product obtained after mixing with immune globulin according to the volume ratio of 1: 1 has the potential of preparing the clinical treatment medicine for canine parvovirus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioactive ingredients and separation and purification freeze-drying technology, namely canine parvovirus immunoglobulin CPV-IgG prepared from Guanzhong donkey plasma, its freeze-dried product, preparation process and application. BACKGROUND

[0002] Human medical refined anti-tetanus (bacterial) immunoglobulin and anti-rabies virus immunoglobulin are prepared using grassland horses as plasma donors. Horses, mules and donkeys are all single-hoofed animals with different skin, meat and physical characteristics. There are great differences between horses and donkeys.

[0003] There is no specific and targeted treatment for viral diseases in pet dogs and cats, especially canine parvovirus CPV and feline parvovirus FPV.

[0004] So far, humans have used animals other than the current animals to prepare pharmaceutical plasma donors. Horses, cows, sheep, pigs, rabbits, camels and chickens have been used as antiviral plasma donors instead of the current animals. These animals occasionally have allergic reactions between different species, but no donkeys have been used as biological agents to treat viral diseases in humans and animals to save patients in clinical practice.

[0005] Guanzhong donkeys are tall and have high potential for blood donation. Their significant advantage is that donkey skin can be used to make Ejiao products with blood supplementing effects. These products have been used for a long time and are well recognized in the community. There is a saying in the community that "dragon meat in the sky and donkey meat on the ground", which shows the unique value of donkey meat. Therefore, donkey meat plays an important role in meat products, such as five-spice donkey meat. Guanzhong donkeys are relatively large in size and have little risk of transmitting foreign viruses, so they are easy to expand the scale of the herd in a confined environment. As a plasma donor, it has a considerable amount of blood donation, and the collection and supply of the required forage are relatively convenient. In addition, the probability of allergic reactions and side effects caused by donkey plasma used in humans and animals is extremely low or even non-existent. Therefore, it has potential application value and feasibility to develop it as a plasma preparation for treating viral diseases in pets.

[0006] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present disclosure, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0007] The present application aims to provide a kind of canine parvovirus (CPV) weakened strain cell culture antigen by program pre-inoculation, then isolated from horse plasma, followed by freeze-drying and extracted for capturing and neutralizing natural wild strain clinical application of canine parvovirus immunoglobulin CPV-IgG and its preparation method, provide an effective way for preparing freeze-dried antiviral immunoglobulin from the plasma of Guanzhong horse group.It is ensured that the immunoglobulin molecular structure is stable, the HI value of virus capture is not reduced, the SN value titer activity is not lost, the product activity can be extended, i.e.from 12 months to 24 months, and the cold chain transportation of intermediate traders to users is convenient, which ensures the stability of product activity.

[0008] To achieve the above purpose, the present application provides a preparation process of canine parvovirus immunoglobulin CPV-IgG, comprising the following steps: (12) preparing immunogen to immunize animals; (13) collecting the plasma of immunized animals, and obtaining canine parvovirus immunoglobulin CPV-IgG by purification; The immunogen is selected from canine parvovirus CPV-XN1 attenuated strain.

[0009] Further, in the preparation method provided by the present application, the immunogen contains antigen and adjuvant, and the titer of the virus strain in the immunogen is TCID 50 ≥10 6 / 0.1mL. Wherein, the adjuvant is preferably BCG, and the concentration of BCG in the immunogen is 5 μg / mL.

[0010] Further, in the preparation method provided by the present application, the immunized animals are selected from Guanzhong horses, and the immunized animals are subjected to 2 times of basic immunization and 8 times of booster immunization, the first immunization is 2 mL, and each immunization is increased by 2 mL; when the HI titer of the immunized animals is not less than 1:128, blood is collected, and the plasma is collected.

[0011] Further, in the preparation method provided by the present application, the purification comprises pepsin digestion, ammonium sulfate salting-out and dialysis purification, and the SN titer of the filtrate after purification is not less than 1:256.

[0012] The present application also provides a canine parvovirus immunoglobulin CPV-IgG prepared by the above preparation method.

[0013] The present application also provides a freeze-drying agent matched with the above immunoglobulin CPV-IgG, which comprises: 6 parts of gelatin with a mass concentration of 2.6%, 2 parts of lactose with a mass concentration of 2.8%, 1 part of glycine with a mass concentration of 2.6%, and 1 part of sodium chloride with a mass concentration of 2.34%.

[0014] The application also provides a freeze-dried product of Guanzhong donkey plasma canine parvovirus immunoglobulin CPV-IgG, which comprises the immunoglobulin CPV-IgG and a freeze-drying agent.

[0015] The immunoglobulin CPV-IgG or the freeze-dried product thereof can be used for preparing a clinical treatment drug for canine parvovirus infection and has application potential in canine epidemic prevention.

[0016] The application can realize prevention and treatment of canine parvovirus infection through the following process, which comprises the following steps. 1. Pre-immunize Guanzhong donkeys with a purpose antigen component (prescription) according to a procedure, and strengthen the immunization; 2. Detect the CPV-IgG titer of the Guanzhong donkey group providing plasma, including HI determination, SN determination and OD value determination of the immunoglobulin, when the HI value reaches 1:128 and the SN value reaches 1:256, blood separation is performed; 3. Cut the FC fragment and f(ab)2 connection bond in the immunoglobulin molecular structure of CPV-IgG, so as to eliminate the FC fragment triggering interspecies allergic reaction;

[0017] 4. Technical points of two ammonium sulfate salting-out procedures (15% for the first time and 21% for the second time): collect the filtrate after the first salting-out, and collect the solid on the filter screen surface after the second salting-out; 5. Technical link: use special cotton paper for dialysis in the dialysis process, and dialyze for 72 hours at a flow rate of 50 ml per minute through pure water, so that the residual ammonium sulfate in the product is countercurrently folded out of the CPV-IgG, and a 60-month-old puppy is injected with 10 times the amount (10 ml), and should have no abnormal reaction; 6. Technical link: adopt a double system for filter sterilization, the filter plate is 0.45 mm, the filter membrane is 0.22 mm, the filter positive pressure pressure is not more than 0.25 atm, and four kinds of bacterial detection culture media (ordinary meat broth, ordinary slant, sabouraud slant and semi-solid) are cultured for 14 days, and there should be no bacterial growth; 7. Technical link of freeze-drying: adopt an optimal freeze-drying protective agent and mix the Guanzhong donkey plasma product freeze-dried product with an equal amount of components, and after freeze-drying for 30 hours, the product is diluted with sterile water for injection and quickly dissolved, easy to peel off, the freeze-drying surface is not concave, and after quality inspection and detection before and after freeze-drying, the HI value is 1:128, the SN value is 1:256, compared with the freeze-drying agent, the titer has no obvious deviation, the SN value has no reduction phenomenon compared with before freeze-drying, and the value is within the allowable range; 8. After the pregnant sick dog experiment, the preparation has no case of abortion of the sick dog in the clinical treatment of pregnant pets; 9. When administering intravenous infusions to sick dogs and cats, the addition of canine CPV-IgG may cause vomiting in a few dogs, but these reactions can be corrected. Following the principle of early detection and treatment, sick dogs are generally given intramuscular injections twice daily, morning and evening. If dehydration is a concern due to vomiting and diarrhea, a daily intravenous infusion (a three-in-one prescription consisting of one-third amino acids, one-third Ringer's solution, and one-third glucose saline) is recommended. One dose of canine CPV immunoglobulin is added to every 2.5 kg of animal, and the infusion is administered once daily for four consecutive days until recovery.

[0018] The present invention has the following advantages: This invention provides a method for producing a canine parvovirus immunoglobulin CPV-IgG lyophilized formulation by isolating and purifying donkey plasma, adding a (1:1) lyophilization protectant, and then selecting a lyophilization process through an optimization method. Verification has shown that this formulation can be used to treat acute enteritis and myocarditis in clinically ill dogs with canine parvovirus. This product can be used not only for intramuscular injection but also for intravenous administration in conjunction with immunoglobulin during dehydration infusions. Intramuscular injection twice a day for three days can keep the dog out of danger and promote recovery. For intravenous infusion, one dose of lyophilized CPV-IgG can be administered once a day (for a 2.5kg sick dog) for four consecutive days, resulting in recovery. The therapeutic and fluid effects are stable. The successful development of this lyophilized formulation extends the shelf life from one year to two years, improving its preservation performance during long-distance cold chain transportation. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] Example 1: Preparation of freeze-dried canine parvovirus immunoglobulin CPV-IgG 1. Selection of toxins: Source of attenuated antigen: F cells adapted to cat kidney passages 81 The cell's attenuated CPV-XN1 strain; The highly virulent strain used for testing was CPV-XN931.

[0022] The standards for the toxicant strains meet the requirements of Appendix III of the 2020 edition of the Chinese Veterinary Pharmacopoeia.

[0023] 2. Manufacture of immunoglobulin 1) Selection of immunized animals: Select 3-8 year old Guanzhong donkeys, well-nourished, healthy in shape, free of internal and external parasites, isolated for 15 days before vaccination with antigen, and should be prohibited from drinking a large amount of water within 6 hours when collecting blood from the donkey body.

[0024] 2) Preparation of antigen Select well-grown cat kidney passage cells (F 81 ), the cell growth solution is E-MEM containing 10% calf serum, 100 U / mL penicillin, and 100 ug / mL streptomycin, and the cells should be subcultured no more than 5 generations.

[0025] Inoculate the virus with 1% cell growth solution into the monolayer of well-grown F 81 cells, supplement the cell maintenance solution (same as the growth solution, but reduce the calf serum to 2%), and place it in a 37°C, 5% CO2 incubator with pH adjusted to 7.0 for 5 days. When the cell pathology (CPE) reaches 75%, harvest and store at -30°C.

[0026] Take the virus-containing cell culture solution, centrifuge at 3000 rpm / min for 20 min, discard the precipitate, and take the supernatant virus suspension for standby.

[0027] Take the previously inactivated BCG (100 μg / mL) at 60°C for 60 min (minutes) and mix it with the virus suspension to make the final concentration of BCG 5 μg / mL. Mix well after shaking and stand by; the antigen virus strain has a titer of TCID 50 ≥10 6 / 0.1 mL.

[0028] 3) Animal immunization The production Guanzhong donkey only performs one basic immunization procedure (2 injections) and multiple booster immunization procedures (8 injections) for its entire life, and also needs to perform pre-blood collection immunization and test before each blood collection.

[0029] Basic immunization: a total of 2 injections. The first time, inject 2 mL of immunization antigen into the neck and back muscles, 4 points, 0.5 mL per point, 9 days apart, and the second time, inject 4 mL. Booster immunization is performed 14 days after the second injection.

[0030] Booster immunization: inject 8 times into the neck muscles, with 3 days between each injection, starting with 6 mL of immunization antigen, increasing by 2 mL each time, and the last time being 20 mL. Pre-blood collection immunization is performed 9 days after the last injection.

[0031] Pre-immunization: Inject 2 times, 20 mL each time, in the neck muscle, with an interval of 9 days. Measure the HI titer on the 7th day after the second injection. When the HI is ≥1:128, perform blood collection. After blood collection, the donkey is rested for 9 days before the next pre-immunization, and the cycle of immunization and blood collection is repeated.

[0032] 4) Blood collection and separation of hyperimmune plasma Blood collection time: After completing the pre-immunization, select donkeys with an HI titer ≥1:128, and perform blood collection on the 9th day. Before each blood collection, weigh the immunized donkey, stop feeding concentrate 6 hours before blood collection, but do not limit water intake. The blood collection volume is 8-10 mL per kilogram of body weight.

[0033] Plasma separation: Collect the collected blood in a bottle containing 5% sodium citrate solution, with a blood to sodium citrate ratio of 9:1. Let it stand for 24-48 hours until the red blood cells settle. Then, extract the supernatant into a sterile bottle. Store at 2-8°C in a cool and dark place for future use.

[0034] 5) Immunoglobulin purification 14) Pepsin digestion: Add distilled water (1:2) at twice the amount of plasma, mix thoroughly, adjust the pH to 3.3-3.4 with 2 mol / L hydrochloric acid, then add 10 units of pepsin per milliliter, mix well, and water bath at 30°C±1 for 1 hour. This separates the FC fragment from the F(ab)2 in the IgG molecular structure.

[0035] 15) First salting-out: After pepsin digestion of the immunoglobulin, add 15% ammonium sulfate to the total amount of plasma, fully dissolve, adjust the pH to 5.3-5.4 with 2 mol / L NaOH solution, water bath warming, increase 1°C per minute, constantly stirring, until the temperature rises to 57-58°C, maintain stirring for 30 minutes, stand for 30 minutes, when the plasma cools to below 45°C, filter with pure cotton 32 cotton gauze, collect the filtrate, discard the waste.

[0036] 16) Second salting-out: Adjust the pH of the collected filtrate to 7.2-7.3 with 2 mol / L NaOH solution, add 21% ammonium sulfate at room temperature, fully stir to dissolve, stand for 30 minutes, filter with 32 pure cotton gauze, collect the precipitate, discard the filtrate.

[0037] 17) Pressing: Collect the solid material through the filter cloth, remove the water with a press dryer, and take out the dry material.

[0038] 18) Dialysis: Weigh the collected solid material, place it in a dialysis bag made of wood pulp paper, with a loading capacity of about 200-250g per bag, and dialyze in a stainless steel pure water tank with a flow rate of 50 mL per minute for 72 hours.

[0039] 19) Sampling quantitative dilution, titer pre-detection (HI) OD value determination and sterile filtration: according to the ratio of dry matter to dialysate volume, the dialysate is diluted with PBS (pH 7.1) to make the dry matter content in the diluent reach 180 mg / mL, and at the same time, HI detection is performed, if HI ≥ 1:128, the batch of products is diluted according to this ratio; if HI does not reach 128, add dry matter dialysate, calculate the dry matter content and perform HI detection, after HI reaches the standard, the batch is also diluted according to this ratio. During the dilution process, 2 mol / L NaOH solution is used to correct the pH to 7.0, and an appropriate amount of sodium chloride is added during the combination to make the final solution contain 0.85% sodium chloride, and it is precipitated at 2~8℃ for 48h. Finally, the obtained filtrate is the semi-finished product of canine parvovirus immunoglobulin CPV-IgG before freeze-drying.

[0040] Both the sterility test and the IgG titer determination are carried out according to the Appendix of the Chinese Veterinary Pharmacopoeia 2020 edition.

[0041] The obtained CPV-IgG is mixed with the pre-prepared optimized freeze-drying protectant at a volume ratio of 1:1 (freeze-drying agent and optimized ratio are shown in Table 1 and Table 2). After mixing, adjust the pH value to 7.2, and perform dispensing, 2 mL per vial (i.e. 1 head portion), after dispensing, quickly place the small vials in the freeze-drying machine box for freeze-drying, according to the pre-selected plasma freeze-drying process curve (see Table 3, Table 4, Table 5), the results show that after 30h, the semi-closed state of the semi-closed is pressed to full closed by the hydraulic system on the freeze-drying machine box, and the entire small vial is kept in a vacuum state. The freeze-dried product is prepared.

[0042] Table 1 Freeze-drying formula screening table of Guanzhong donkey plasma freeze-dried product

[0043] Table 2 Optimal freeze-drying protectant ratio of Guanzhong donkey

[0044] Table 3 Freeze-drying process curve of Guanzhong donkey plasma product (CPV-IgG) (38h)

[0045] Table 4 Freeze-drying process curve of Guanzhong donkey plasma product (CPV-IgG) (30h)

[0046] Table 5 Guanzhong donkey plasma product (CPV-IgG) freeze-drying process curve (26h)

[0047] In summary, the freeze-drying formulation screening evaluation results of the Guanzhong donkey plasma freeze-dried product in group C showed the best effect, i.e. the protective agent prescription was: mass concentration of 2.6% gelatin 6 parts, 2.8% lactose 2 parts, 2.6% glycine 1 part, and 2.34% sodium chloride 1 part; the freeze-drying process curve (30h) of Guanzhong donkey plasma product (CPV-IgG) showed the best effect.

[0048] Experimental Example 2 Application detection of finished product of immunoglobulin after freeze-drying 1. Three batches of canine CPV immunoglobulin G (IgG) freeze-dried products were obtained, respectively marked as 220303, 220316 and 220409. According to the "Chinese Veterinary Pharmacopoeia" 2020 edition third part appendix, the purity, safety and pyrogen test results meet the requirements.

[0049] 2. Determination of content: The OD values of the three batches of samples to be tested were measured at 280nm and 260nm, and the protein content was determined according to the general formula: and the protein content was checked on the OD value standard curve.

[0050] 3. SN titer determination: According to the fixed virus dilution serum method, take the IgG sample to be tested, dilute it by 2 times in series with M-MEM, take 1:64, 1:128 and 1:256 titers, and each dilution is inoculated with equal amount of 200TCID 50 CPV antigen solution, incubate at 37℃ for 1h, inoculate 5 rows of parallel monolayer F81 cell holes, and culture at 37℃ for 5d. The serum titer of 5 / 5 holes without CPE is taken as the neutralization titer, and the control group without serum virus liquid shows obvious CPE. The neutralization titer is calculated by Reed-Muench method, and the neutralizing antibody titer (SN) should be ≥1:128. According to the "Chinese Veterinary Pharmacopoeia" 2020 edition appendix 41.

[0051] 4. Potency test of three batches of freeze-dried products Fourteen puppies, aged 30-60 days and weighing 2-3 kg, obtained from the same source and having CPV neutralizing antibody titers of <1:4, were divided into six groups. Four dogs in each of three test groups were injected with 0.5 mL / kg of lyophilized immunoglobulin G and 1 mL of a suspension of 200 units of CPV-infected tissue. Two dogs in each of two control groups were injected with 1 mL of the suspension of tissue as a control. The control dogs should have a temperature rise within 24-72 hours after injection of the tissue suspension, followed by typical symptoms of CPV infection, and at least one of the control dogs should die of CPV infection within 7 days. The twelve test dogs were observed for 7-14 days and all were healthy and alive.

[0052] The results are as follows: The content of the three batches of immunoglobulin G was determined and is shown in Table 6. Table 6 OD values of three batches of lyophilized immunoglobulin G (160-220 mg / mL)

[0053] Note: The OD values were measured at 260-280 nm. The greater the dilution factor, the smaller the absorbance, and the greater the transmittance. The average of three dilutions (absorbance) was taken.

[0054] The protective effects of the three batches of product on dogs are shown in Table 7, and the criteria for determining the clinical symptoms of CPV in dogs are shown in Table 8. As can be seen, in the three batches of product, the SN titer was >1:256, the protein content was >0.25 mg, and none of the test dogs had clinical symptoms, with a protection rate of 100%, while the control dogs all had symptoms of CPV. This experiment proves that the lyophilized immunoglobulin G prepared in this project has a significant therapeutic effect and good specificity for canine parvovirus disease.

[0055] Table 7 Protective effects of three batches of product on beagle dogs

[0056] Table 8 Criteria for determining the clinical symptoms of CPV in dogs

[0057] Example 3 Safety verification of lyophilized immunoglobulin G (CPV-IgG) Twenty 15-day-old healthy experimental puppies were selected, six for each batch, and the SN titer for CPV was <1:4.

[0058] 1. Safety test on experimental animals (1) Safety test of single dose (0.5 mL / kg) Safety test of single dose (0.5 mL / kg)

[0059] Safety test of repeated use of single dose The experimental puppies were divided into 3 groups, 2 in each group (2.5 kg each), and each group was inoculated with two batches of lyophilized products with different batch numbers, 0.5 mL / kg each day, twice a day for 3 days. The control group of 2 experimental dogs was injected with normal saline, and the dose was the same as the test group. Two weeks after injection, the injection was repeated, and the dose and frequency were the same as the first time. Observe for 14 days and record changes every day.

[0060] Safety test of super-dose 1) Safety test of large dose (1.5 mL / kg) 36 experimental dogs aged 3-10 days were divided into 4 groups, 2 beagles in each group, and the first 3 groups were 3 batches of immunoglobulin lyophilized products. Group A is batch 220303, group B is batch 220316, group C is batch 220409, and group D is the control group. Each group is divided into two different dose subgroups, i.e. A1 (3 times the amount, i.e. 1.5 mL / kg, a total of three), A2 (5 times the amount, i.e. 2.5 mL / kg, a total of three), and the rest is similar. Inject twice a day for 3 days. Observe for 7 days after injection and record the situation every day; Group D is the control group, injected with normal saline. Observe for 14 days and record the situation every day.

[0061] 2) Safety test of super-dose (10 times the dose) The experimental puppies were divided into 2 groups, 2 in each group (1 kg each), and each group was inoculated with two batches of lyophilized products with different batch numbers, 5 mL / kg, three days in a row. The control group of 2 puppies was injected with normal saline, and the dose was the same as the test group. Twice a day, observe for 14 days and record changes every day.

[0062] 2、Results (1) Safety test of single dose 2 experimental puppies injected with CPV immunoglobulin injection, 2 times a day for 3 days, observed for 14 days, all healthy and no abnormal reactions.

[0063] (2) Safety test of repeated use of single dose The experimental puppies were divided into two groups, two in each group (2.0 kg each), each group was inoculated with 1 mL of freeze-dried product of different batch numbers, 0.5 mL / kg, twice a day, for 3 consecutive days. The control group of 2 puppies was injected with normal saline, the dose was the same as the test group. Two weeks after injection, repeat injection, the dose and frequency are the same as the first time, and observe for 14 days, record the changes every day.

[0064] The determination standard is that the diet is normal, the body temperature is normal, there is no allergic reaction or no side effect, and the sample used is 220303, 220316 and 220409, and the detailed results are shown in Table 9: Table 9 Safety of repeated multiple drug use

[0065] As can be seen from the results in Table 9, repeated injection of 9 experimental dogs with constant multiple times proves that the product has no toxic side effects when used repeatedly.

[0066] (3) Safety test of super-dose 1) Large dose safety test From the results of the three batches, it is proved that the three batches of IgG products, whether 3 times or 5 times the amount is used, the experimental dogs have no abnormal reaction in terms of injection site, diet and mental state, except that each of A2 and B2 groups has one animal with mild swelling block of apricot kernel size, which disappears after 6 hours, as shown in Table 10.

[0067] Table 10 Safety observation of CPV immunoglobulin G freeze-dried product in use

[0068] Note: Apricot kernel size, disappeared after 3 hours 2) Super large dose safety test The initial puppies (1 kg) used were healthy dogs, the usual amount was 0.5 mL / kg, and 10 times the amount was 5 mL / kg, as shown in Table 11, and the samples used were 220303, 220316 and 220409.

[0069] Table 11 Safety observation of CPV immunoglobulin G (IgG) freeze-dried product in use

[0070] Apricot kernel hardening reaction, disappeared at 6 hours.

[0071] As can be seen from the results in Table 11, 2 puppies were used with super 10 times the amount for 3 days, only 1 had mild swelling and hardening reaction, the animal showed depression, lying down, 6 hours within the diet, 8 hours later gradually recovered. This shows that the experimental puppies with 10 times the dose are also safe for the animals.

[0072] The above results can be seen that in the dog body, constant, repeated multiple, 3 times, 5 times and drug observation proved that 3 times, 5 times injection of experimental dogs of the IgG preparation of the experimental dogs is safe.

[0073] With 10 times the super constant injection of puppies, only one (4) dog showed a slight reaction, local apricot big lumps, about 3-6h after disappearance, but no death cases.

[0074] In summary, the experimental animals, puppies, muscle injection of CPV-IgG, whether it is constant 3 times, 5 times or 10 times large dose, in general, the freeze-dried product is safe for animals.

[0075] Experimental Example 4 CPV-IgG freeze-dried product shelf life verification To verify the shelf life of freeze-dried products, CPV-IgG freeze-dried product samples were selected, three batches of batch number 220303; 220316; 220409; SN titer ≥1:128, protein content ≥9.8mg / mL of sample were determined, including the following: Virus CPV--XN 931 Content determination (TCID 50 ) in 96 well micro plastic reaction vessel culture F81 cells, determination of CPV--XN 931 strain of infected cell TCID 50 value. That is, the virus stock solution is diluted with E-MEM with 10 as the base dilution to 10 -3 ~10 -7 Diluted 5 degrees, mixed with equal amount of F81 cell suspension, added to 10 -7 reverse dilution.

[0076] Cell test (SN) according to the same virus dilution serum method in 96 well plastic reaction plate, on F81 cell monolayer, culture medium is E-MEM, first on F81 cell monolayer to determine the TCID 50 titer of CPV antigen, then take the sample to be tested first with Hank's solution 5 times dilution, with M-MEM 2 times dilution, take 1:32; 1:64; 1:128; 1:256; 1:512 five dilutions for measurement each dilution, mixed with equal amount of 100 TCID 50 of CPV antigen, 2h at 37℃ environment, take 1:32; 1:64; 1:128; mixed sample culture 4-5d, 4 / 4 hole does not appear CPE sample dilution as the criterion, and virus liquid without sample as control, the results according to the conventional Reed-Muench method to calculate SN value.

[0077] Hemagglutination inhibition test (HI): Hemagglutinin agglutination titer determination, specifically for 96-well microplate method in Chinese Veterinary Pharmacopoeia 2020 Edition Appendix 39. Hemagglutinin is diluted with PBS (0.01 mol / L, pH=7.0~7.2) to different multiples, 1% dog red blood cell suspension is added, and the same amount of PBS as the serum in the inhibition test is added. Shake the 96-well microplate on the shaker, shake well, and place it at room temperature for 2h or at 2~8℃ overnight. When the red blood cells in the control well are significantly button-shaped, the result is determined. The highest dilution that causes complete agglutination of red blood cells is taken as the determination endpoint.

[0078] MID minimum infectious dose determination and canine neutralization protection test, CPV-XN 931 strain.

[0079] Experimental puppies Each batch of experimental puppies is 3~10 days old, weighing 2-3 kg, and 4 healthy puppies with CPV-SN antibody ≤1:4.

[0080] CPV epidemic virulent strain is provided by Yangling Kaina Ying Polyclonal Animal Pharmaceutical Co., Ltd. The enteric suspension containing the virus is diluted 10 times successively with Hank's solution, i.e. 10 -3 ;10 -4 ;10 -5 ;10 -6 ;10 -7 Five dilutions, 2 for each dilution, each puppy orally administered 200 units of MID 1 mL of blood and intestinal poison 1 mL, through the digestive tract to inoculate puppies, of which 2 controls were only injected with 1 mL of Hank's solution. Isolate and observe the clinical changes for 14 days. The reciprocal of the dilution that causes 1 / 2 of the puppies in the group to become ill is taken as the determination standard, and the virus amount of 200 units of MID is calculated.

[0081] This animal (canine) protection test selects puppies of the same source with CPV-SN antibody <1:4 within 5~10 days old as test animals. In this test, IgG samples of different dilution multiples (1:32~512) are used as test samples, mixed with an equal amount of CPV virulent strain in each test tube for 2h, and orally inoculated into puppies. Observe for 14 days and calculate the protection value according to the method described in Chinese Veterinary Pharmacopoeia 2020 Edition.

[0082] Storage environment and temperature Select 20 sample portions of 3 batches of CPV-IgG produced, and store them at 2~8℃, 15℃, and 25℃ for 0, 4, 8, 12, 16, 20, 24, and 36 months, respectively. Store them at 15℃ for 0, 2, 4, 6, 8, 10, and 12 months, respectively. Store them at 25℃ for 0, 1, 2, 3, 4, 5, and 6 months, respectively. At the time, take them out for HI, SN, OD value determination and animal challenge protection test.

[0083] Results are as follows: (20) The CPV-IgG lyophilized samples stored for 0, 2, 4, 8, 12, 16, 20, 24, 36 months were taken for SN, HI determination and OD value determination. The results are shown in Table 12. It can be seen that the SN of the three batches of CPV-IgG samples stored for 24 months at 2-8°C was 1:256, the HI value was 1:128, and the OD value was 0.25. At 25°C for 24 months, the SN was 1:258, the HI value was 1:128, and the OD value was not lower than 0.25. Therefore, it can be seen from the results that the CPV-IgG stored at 2-8°C will be stored for 24 months, which is relatively conservative and reasonable.

[0084] Table 12: Effectiveness observation of CPV-IgG samples stored at 2-8°C for different periods

[0085] (2) The effectiveness determination results of CPV-IgG samples stored at 15°C for different periods are shown in Table 13. It can be seen that when the IgG samples are stored at 15°C, samples are taken for sampling inspection at 0, 2, 4, 6, 8, 10, 12, 24, 36 months, respectively. The SN value of the sample taken at 6 months is maintained at 1:256, the SN value of the sample taken at 10 months is 1:256, the protein content is maintained at 0.25 g / mL, and most of the animals are protected. Therefore, it can be proved that the quality effective period of the IgG lyophilized product of CPV-IgG at 15°C is 24 months, which provides strong evidence.

[0086] Table 13: Effectiveness observation of CPV-IgG samples stored at 15°C for different periods

[0087] (3) The titer determination results of CPV-IgG stored at 25°C for different periods are shown in Table 14. It can be seen that when the three batches of IgG lyophilized samples are stored at 25°C, samples are taken for sampling inspection at 0, 4, 8, 12, 16, 20, 24, 36 months, respectively. The SN value of the sample taken at 36 months is maintained at 1:128, one batch has an SN titer of 256, two batches have an SN titer of 128, and the OD value is maintained at more than 0.25. The SN value and the HI value of the sample taken at 24 months are not reduced, and the OD value is maintained at 0.25. All animals are protected. Therefore, it can be proved that the quality effective period of the IgG lyophilized product of CPV-IgG at 25°C is 24 months, and the titer is not significantly reduced, which provides strong evidence for the stability of the lyophilized product in cold storage for 24 months.

[0088] Table 14: Titer results analysis of CPV-IgG stored at 25°C for different periods

[0089] (21) The potency of immunoglobulin lyophilized products stored at different temperatures for different times was determined, and the results are shown in Table 15. It can be seen that the SN activity of CPV-IgG remained at a decrease of 1 dilution from 1:256 to a stable value of 1:128 when stored at 2-8°C for 36 months, the protein content was 0.25 mg / mL, the HI and SN titers were not decreased at 24 months, the SN and HI of the 24th month could maintain in the effective range of 1:128 and 1:256, respectively, and the CPV-IgG sample maintained the original value of ≥0.2 mg. At 25°C (room temperature), the SN also remained at 1:256, and the HI value was in the effective activity range of 1:128. The results showed that the HI, SN values and protein activity content of the preparation were in the effective range whether stored at 2-8°C, 15°C or 25°C, so the storage period of this procedure was determined to be 24 months.

[0090] Table 15 Analysis of the storage results of immunoglobulin lyophilized products at different temperatures

[0091] In summary, through the storage period test observation of three batches of CPV-IgG plasma lyophilized products produced in the Guanzhong donkey group, it was proved that the HI, SN, and protein content activity values of the antiviral neutralization titer could be maintained for 24 months at a temperature environment of 2-8°C, the protein content value of the 220303 batch was 0.26, the SN value was 1:256, and the HI value was 128; the protein content value of the 220316 batch was 0.25, the SN value was 256, and the HI value was 256; the protein content value of the 220409 batch was 0.26, the SN value was 256, and the HI value was 256. At 15°C and 25°C, the three batches of samples were tested for 24 months, and it can be seen from the three quality test results that the HI and SN activity values were not found to be decreased by one base dilution. The CPV-IgG was stored at 15°C-25°C, and the storage period was determined to be 24 months. At 25°C, the HI, SN, and OD values were unevenly decreased by one dilution for 36 months. According to the test results, the storage period of CPV-IgG is temporarily determined to be 24 months at 2-8°C, which can maintain the protein form in the stable measurable range, proving that the IgG structure remains complete, and the SN value and HI value are also in the standard effective range, so the storage period of the preparation is determined to be 24 months.

[0092] Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present application. After reading the above content, various modifications and alternatives of the present application will be apparent to those skilled in the art. Therefore, the protection scope of the present application should be defined by the appended claims.

Claims

1. A preparation process for canine parvovirus immunoglobulin CPV-IgG, characterized in that, It includes the following steps: 1) Prepare immunogens to immunize animals; 2) Collect plasma from immunized animals and purify it to obtain canine parvovirus immunoglobulin CPV-IgG; The immunogen is a weakened strain of canine parvovirus CPV-XN1.

2. The preparation method according to claim 1, characterized in that, The immunogen comprises an antigen and an adjuvant, and the viral strain in the immunogen has a TCID value of [missing value]. 50 ≥10 6 / 0.1mL.

3. The preparation method according to claim 2, characterized in that, The adjuvant is BCG, and the concentration of BCG in the immunogen is 5 μg / mL.

4. The preparation method according to claim 1, characterized in that, The immunized animals were selected from Guanzhong donkeys. The immunized animals underwent two basic immunizations and eight booster immunizations. The first immunization was 2 mL, and the amount increased by 2 mL with each subsequent immunization. Blood was collected from the immunized animals when their HI titer was not lower than 1:128, and plasma was collected.

5. The preparation method according to claim 1, characterized in that, The purification process includes gastric enzyme digestion, ammonium sulfate precipitation, and dialysis purification, and the SN titer of the purified filtrate is not less than 1:

256.

6. Canine parvovirus immunoglobulin CPV-IgG prepared by the preparation method according to claim 1.

7. A lyophilizing agent compatible with the immunoglobulin CPV-IgG of claim 6, characterized in that, The freeze-drying agent comprises: 6 parts of 2.6% gelatin, 2 parts of 2.8% lactose, 1 part of 2.6% glycine, and 1 part of 2.34% sodium chloride, with a mass concentration of 2.6%.

8. A freeze-dried product of canine parvovirus immunoglobulin CPV-IgG from Guanzhong donkey plasma, characterized in that, The lyophilized product comprises the immunoglobulin CPV-IgG of claim 6 and the lyophilizing agent of claim 7.

9. The lyophilized immunoglobulin CPV-IgG product according to claim 8, characterized in that, The volume ratio of immunoglobulin CPV-IgG to the lyophilizing agent in the lyophilized product is 1:

1.

10. The use of the immunoglobulin CPV-IgG of claim 6 or the lyophilized immunoglobulin CPV-IgG of claim 8 in the preparation of a clinical treatment for canine parvovirus infection.

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