CN117069836A - Preparation method of anti-human T cell rabbit immunoglobulin - Google Patents
Preparation method of anti-human T cell rabbit immunoglobulin Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the technical field of biological products, in particular to a preparation method of anti-human T cell rabbit immunoglobulin. The method is that the rabbit plasma or serum immunized by human peripheral blood T lymphocytes and/or thymus cells is firstly absorbed by hydroformylation human erythrocytes and hydroformylation human placenta and then is sequentially subjected to first ion exchange chromatography, affinity chromatography and second ion exchange chromatography to prepare the anti-human T cell rabbit immunoglobulin which meets the standards of anti-human T cell rabbit immunoglobulin in the current edition of Chinese pharmacopoeia, and the molecular size purity detection result (monomer and dimer) of the final product can reach 100 percent, basically has no obvious impurity and is far higher than the standards of more than 90 percent in the current edition of Chinese pharmacopoeia. The purification method can not introduce aluminum and other impurities which are easy to cause clinical side reactions, has good process continuity, can well realize digital and automatic control, and is used for amplified production.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a preparation method of anti-human T cell rabbit immunoglobulin.
Background
Anti-human T-cell immunoglobulin (ATG) is a strong immunosuppressant, can inhibit lymphocyte activation process after antigen recognition, specifically destroy lymphocytes, and is mainly used for preventing and treating rejection reaction of organ inhibition, treating hormone tolerance and graft versus host disease and treating aplastic anemia in medical treatment.
The existing anti-human T cell immunoglobulin is mainly two kinds, namely anti-human T cell pig immunoglobulin and anti-human T cell rabbit immunoglobulin. At present, domestic research on anti-human T cell immunoglobulin is mainly focused on anti-human T cell pig immunoglobulin, while research on anti-human T cell rabbit immunoglobulin is very little, and the anti-human T cell rabbit immunoglobulin on the market mainly depends on import and has extremely high price. In recent years, research shows that the anti-human T cell rabbit immunoglobulin has better application effect in preventing graft versus host reaction of bone marrow transplantation, so that independent development of a preparation process of the anti-human T cell rabbit immunoglobulin has important significance.
The invention patent with publication number CN112111004A discloses a production process of immunoglobulin, which comprises inactivating complement with SPF rabbit serum, precipitating with octanoic acid to obtain clarified liquid, and sequentially passing the clarified liquid through the following chromatography: affinity chromatography, anion exchange chromatography and anti-A/anti-B chromatography, and finally obtaining the immunoglobulin product with high yield and purity. This method has at least the following problems: (1) The octanoic acid precipitation is easy to cause harm to human bodies, and residues such as aluminum ions and the like can exist when a filter aid (such as diatomite) is used, so that adverse reactions are easy to cause in clinic; (2) The ligand in the anti-A/anti-B chromatographic packing has the problem of easy falling off, and the potential risk of causing clinical adverse reaction can also exist; (3) it is inconvenient for automation control and industrialization.
Disclosure of Invention
Based on the above, the invention provides a new preparation method of anti-human T cell rabbit immunoglobulin, which can overcome the problems existing in the prior art and successfully prepare the anti-human T cell rabbit immunoglobulin meeting the standard requirements of anti-human T cell rabbit immunoglobulin of the current edition of Chinese pharmacopoeia, each theory, and is convenient for industrialized implementation.
The invention realizes the technical purposes through the following technical proposal: a method for preparing anti-human T cell rabbit immunoglobulin, comprising the following steps:
s1, obtaining rabbit plasma/serum immunized by human peripheral blood T lymphocytes and/or thymus cells;
s2, placing the mixture at the temperature of not higher than 37 ℃ to melt slurry;
s3, adding healthy human blood plasma into the rabbit blood plasma or serum after the plasma melting to obtain a feed liquid A; adding hydroformylation human red blood cells into the feed liquid A, adjusting the pH value to 7.0+/-1.0, keeping the reaction temperature at 0-8 ℃, separating the red blood cells after full reaction, and collecting supernatant B; adding an aldehyde human placenta into the supernatant B, adjusting the pH to 7.0+/-1.0, keeping the reaction temperature at 0-8 ℃, separating placenta slag after full reaction, and collecting the supernatant C;
s4, adjusting the pH value of the supernatant C to 6.0+/-1.0, performing deep filtration, performing first ion exchange chromatography by adopting a phosphate buffer system with the pH value of 6.0+/-1.0, and collecting the flow-through liquid D;
s5, filtering the flow-through liquid D, adjusting the concentration to 10-30 mg/mL, loading the flow-through liquid D on an affinity chromatography column, washing the flow-through liquid D by an acetate buffer system with the pH value of 4.0+/-1.0, and collecting an eluent E;
s6, filtering and sterilizing the eluent E, and incubating at 24+/-2 ℃ to obtain a solution F;
s7, regulating the pH value of the solution F to 6.0+/-1.0, performing ion exchange chromatography for the second time, and collecting the flow-through liquid G to obtain a product containing anti-human T cell rabbit immunoglobulin.
As a preferred embodiment, the method further comprises the steps of:
s8, ultrafiltration displacement is carried out on the passing liquid G by adopting physiological sodium chloride solution, the pH value is regulated to 4.0+/-0.5, glycine is added to obtain a semi-finished product with the protein mass concentration of 1-3% and the glycine concentration of 10-30G/L, a 20nm virus-removing filter membrane is adopted to filter virus, and then virus-removing feed liquid is sterilized;
s9, sub-packaging the semi-finished product to obtain the finished product.
In a preferred embodiment, in step S2, the amount of healthy human plasma added is 1 to 5% of the amount of rabbit plasma added; in the step S5, the addition amount of the hydroformylation human red blood cells is 15-30% of the pulp addition amount, and the addition amount of the hydroformylation human placenta is 15-30% of the pulp addition amount.
As a preferred embodiment, the hydroformylation human placenta in the step S5 is prepared by hydroformylation reaction of a hydroformylation solution having a formaldehyde content of 0.8% -3% for 3-7 hours.
As a preferred embodiment, the method for preparing the hydroformylation human placenta comprises the following steps: obtaining human placenta dregs with negative endotoxin detection, and thawing; placing the unfrozen human placenta slag into placenta aldehyde liquid for carrying out an aldehyde reaction for 3-7 h, wherein the placenta aldehyde liquid is phosphate buffer solution containing 0.8% -3% formaldehyde; transferring the aldehyde placenta into a sodium chloride solution with physiological concentration for washing, transferring into a neutralization solution for neutralization reaction, wherein the neutralization solution is a glycine-containing sodium chloride solution, washing again, and drying by control to obtain the aldehyde human placenta.
As a preferred embodiment, the placental hydroformylation solution is a phosphate buffer solution containing 1% formaldehyde.
As a preferred embodiment, the pore size of the filter membrane used in the depth filtration in the steps S4 and S5 is 0.45. Mu.m.
In a preferred embodiment, the method for obtaining rabbit plasma in step S1 is as follows: the preparation method comprises the steps of mixing thymus cells and lymphocytes to immunize rabbits, and then collecting blood; wherein, the immunization program is as follows: performing primary immunization by subcutaneous multipoint injection at back, performing secondary boost immunization by intravenous injection or subcutaneous immunization at intervals of 14 days, and taking blood plasma 21 days after the secondary immunization.
The invention also aims to provide the anti-human T cell rabbit immunoglobulin prepared by the method, wherein the protein content is 10-30 g/L, the pH value is 4.0+/-0.2, the purity of P-ATG is not lower than 95% of the total protein, the sum of IgG monomer and dimer content is not lower than 95.0%, the polymer content is not higher than 5.0%, the E rose ring formation inhibition test titer is not lower than 1:4000, the lymphotoxin test titer is not lower than 1:2000, the anti-A and anti-B hemagglutinin is not higher than 1:64, the human platelet antibody is not higher than 1:4, the human plasma protein antibody and human plasma have no precipitation line, and the protein A residue is not higher than 0.001% of the total protein.
The preparation method of the anti-human T cell rabbit immunoglobulin provided by the invention comprises the steps of firstly adding healthy human blood into rabbit immune plasma, then utilizing the hydroformylation human red blood cells and the hydroformylation human placenta to absorb the hybrid antibodies, and finally sequentially carrying out treatments such as ion exchange chromatography, affinity chromatography, low pH incubation, ion exchange chromatography, ultrafiltration dialysis, virus removal and the like, thereby obtaining the anti-human T cell rabbit immunoglobulin, wherein the preparation method at least has the following advantages:
(1) The three-step chromatography process is adopted, a low-temperature ethanol process is not adopted, a low-temperature environment is not needed, the energy consumption is low, the safety is realized, the environmental pollution risk is low, the process continuity is good, the digitization and the automatic control can be better realized, and the amplified production can be realized;
(2) The hybrid antibody absorption process is more comprehensive, so that the impurities with larger clinical influence such as anti-erythrocyte antibodies, anti-basement membrane antibodies, anti-plasma protein antibodies and the like can be comprehensively reduced, and the clinical safety is better;
(3) The low pH incubation process for inactivating viruses is more strict, so that various viruses can be effectively inactivated, and the biological load of the viruses is reduced;
(4) The method has the advantages that the method is free of octanoic acid precipitation and the like, is easy to generate operation of diving in the sea for human bodies, has better protection for personnel, is free of filter aid (diatomite) and the like, and avoids residues of impurities such as aluminum ions and the like, so that adverse reactions caused by the substances in clinical practice are avoided;
(5) The method can be used for separating and extracting the blood plasma and serum, and has wider separation coverage range;
(6) The prepared anti-human T cell rabbit immunoglobulin can meet the standard requirement of anti-human T cell rabbit immunoglobulin of each theory of the current edition of Chinese pharmacopoeia, the purity (monomer and dimer) of the final finished product can reach 100 percent, and the purity is basically no obvious impurity and is far higher than the standard of more than 90 percent of the current edition of Chinese pharmacopoeia, and 1 bottle of anti-human T cell rabbit immunoglobulin (25 mg/bottle) can be prepared by per 2-3ml of immune rabbit plasma.
Drawings
FIG. 1 is a graph of the average mortality analysis of complement-mediated lymphocytes in example 1;
FIG. 2 is a graph of percent pass analysis of complement-mediated lymphocytotoxicity in example 1;
FIG. 3 is a chart showing molecular size analysis of the affinity eluate of the first sample in example 2;
FIG. 4 is a chart showing molecular size analysis of the affinity eluate of the second sample in example 2;
FIG. 5 is a molecular size analysis chart of the affinity eluate of the third sample in example 2;
FIG. 6 is a chart showing the molecular size analysis of the second ion exchange chromatography flow-through of the first sample in example 2;
FIG. 7 is a chart showing the molecular size analysis of a second ion exchange chromatography flow-through for a second sample of example 2;
FIG. 8 is a chart showing molecular size analysis of a second ion exchange chromatography flow-through for a third sample of example 2;
FIG. 9 is a graph showing the detection criteria of protein A in example 2.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
The reagents used in the examples of the present invention are all commercially available, without specific explanation. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as molecular cloning, as described in Sambrook et al: conditions described in the laboratory Manual (New York: coldSpring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Example 1
The embodiment provides a method for obtaining anti-human T cell rabbit immunoglobulin plasma, which comprises the following steps:
(1) Preparation of antigen:
preparation of thymus cell immunogen: taking the waste thymus cells from the surgery, grinding and washing in a biosafety cabinet, and preparing into 7-10 multiplied by 10 by using physiological saline 7 After antigen epitope detection by a flow type detection analyzer, carrying out subsequent immunization according to the ratio of 7-10 multiplied by 10 7 The amount of immune cells per cell was kept.
Preparation of peripheral T lymphocyte immunogens: taking medical waste leukocyte filter disc, backflushing the leukocyte filter disc with 0.9% physiological saline in a biosafety cabinet, collecting backflushed saline containing leukocytes with a sterile bottle, separating mononuclear cells with lymphocyte separating liquid to obtain mononuclear cells with higher purity according to 1×10 7 Culture was performed at a density of/mL, and culture was performed using commercial lymphocyte serum-free medium. The lymphocytes obtained by proliferation culture were collected by centrifugation at 1500rpm for 5min, washed with 0.9% NaCl physiological saline 2 to 3 times, counted by a cell counter, and analyzed by flow assay for epitopes. The antigen cell amount was prepared according to the body weight of healthy rabbits and the antigen cell amount was 7 to 10X 10 7 The amount of immune cells per cell was kept.
And respectively adding a mineral oil molecular adjuvant into the two cell antigens, taking out the mineral oil molecular adjuvant and the antigens under the aseptic condition, rapidly and uniformly mixing according to the mass ratio of 1:1, and vibrating for 10-20 minutes to form the oil-in-water antigen.
(2) Animal experiment:
the antigen prepared above is immunized with New Zealand white rabbits, and multi-point subcutaneous injections are carried out on both sides of the nape of the neck of the rabbits. Immunization program set 8 experimental groups and 1 blank group, 5 blank groups, 10 blank groups, and different immune antigens were set simultaneously for comparison study, and the specific table 1 is shown below. Groups 7 and 8 were immunized with thymocytes for the first time and boosted with peripheral T lymphocytes. The first immunization was followed by a 2 nd boost, which was administered by intravenous injection and subcutaneous immunization, respectively, 14 days and 21 days apart, and the last boost was followed by subcutaneous multipoint injection 21 days after the second immunization. The blood sampling detection titers are carried out before and after basic immunization and secondary immunization, the blood sampling is carried out once every 3 days after the last booster immunization, the fluctuation level of the antibody titer within 12 days is continuously monitored, and all blood sampling treatment is carried out for 14 days.
Table 1 immunization protocol formulation
(3) Potency detection
The collected rabbit serum was analyzed for antibody titer by lymphotoxin assay. The detailed description of lymphocyte toxicity test is described in the fourth part of the pharmacopoeia of 2020 edition (general rule 3516). Titers were detected and analyzed 14 days, 21 days after the second, and 3 days, 6 days, 9 days, 12 days after the third, and the results are shown in tables 2, 3 and fig. 1, 2:
TABLE 2 complement-mediated average mortality analysis of lymphotoxin
TABLE 3 complement mediated lymphocyte toxicity pass rate analysis
From the above results, it can be seen that: the lymphocyte immunized group thymus and cell mixed immunized group (i.e., experimental groups 7 and 8) were longer in potency duration and relatively stable, with the highest potency 21 days after the second immunization.
(4) Preparation of immune plasma
Immunization was performed in the manner of immunization of experimental groups 7 and 8, and immune plasma was obtained.
Example 2
The embodiment provides a preparation method of rabbit anti-human T cell immunoglobulin, which comprises the following steps:
s1, preparing immune rabbit plasma: human lymphocytes separated from human thymus cells or healthy human blood are used for immunizing healthy rabbits according to the immunization mode of the experimental groups 7 and 8 in the example 1, and immune rabbit plasma is collected and separated after the test is qualified.
S2, combining and melting plasma: mixing the thawed immune rabbit plasma according to the requirement of throwing the plasma, and placing the mixture into a plasma melting state under the condition of not higher than 37 ℃ to obtain the mixed rabbit plasma.
S3, absorbing hybrid antibody by healthy human blood plasma: adding healthy human plasma into the mixed rabbit plasma, wherein the adding amount of the healthy human plasma is 1-5% of the adding amount of the rabbit plasma, so as to obtain a feed liquid A; adding hydroformylation human red blood cells into the feed liquid A, adjusting the pH value to 7.0+/-1.0, keeping the reaction temperature at 0-8 ℃, stirring for at least 6 hours, press-filtering to separate the red blood cells, and collecting supernatant B; adding the hydroformylation human placenta into the supernatant B, adjusting the pH to 7.0+/-1.0, keeping the reaction temperature at 0-8 ℃, stirring for at least 6 hours, press-filtering to separate placenta slag, and collecting supernatant C;
wherein, the preparation method of the hydroformylation human placenta comprises the following steps: obtaining processed human placenta slag with negative endotoxin detection, and thawing; draining the thawed human placenta slag, placing the human placenta slag into placenta aldehyde liquid, wherein the placenta aldehyde liquid is phosphate buffer solution containing 0.8% -3% formaldehyde, and the ratio of the liquid to the solid is 1:2-1:5, and carrying out aldehyde reaction for 3-7 h; transferring the placenta after the hydroformylation reaction into a sodium chloride solution with physiological concentration, stirring for 10-30 min, and drying; transferring the washed aldehyde placenta into a neutralization solution, wherein the neutralization solution is sodium chloride solution containing 0.5% -2% glycine, and the material-liquid ratio is 1:2-1:5, and carrying out neutralization reaction for 1-3 h; transferring the neutralized placenta into a sodium chloride solution with physiological concentration, stirring for 10-30 min, and drying to obtain the aldehyde placenta.
S4, performing first ion exchange chromatography: the pH of the supernatant C was adjusted to 6.0.+ -. 1.0, and after depth filtration (0.45 μm), the supernatant was subjected to a first ion exchange chromatography using a phosphate buffer system, washed with a phosphate buffer solution having a pH of 6.0.+ -. 1.0, and the flow-through D was collected.
S5, affinity chromatography: the flow-through solution D was filtered (0.45 μm) and the concentration was adjusted to 10 to 30mg/mL, applied to an affinity column, and the eluate E was collected using an acetate buffer system having a pH of 4.0.+ -. 1.0.
S6, virus inactivation and removal: filtering and sterilizing the eluent F, and incubating at 24+/-2 ℃ for 24 days to obtain a solution F.
S7, performing ion exchange chromatography for the second time: and (3) regulating the pH value of the solution F to 6.0+/-1.0, loading the solution F on an ion exchange chromatographic column, adding phosphate buffer solution with the pH value of 6.0+/-1.0 for washing, and collecting the flow-through liquid H.
S8, preparing a semi-finished product: the physiological sodium chloride solution is adopted to carry out ultrafiltration displacement on the flow-through liquid H, the pH value is regulated to 4.0+/-0.5, glycine is added to obtain a semi-finished product with the protein concentration of 1-3% and the glycine content of 10-30 g/L, a 20nm virus-removing filter membrane is adopted to filter and remove viruses, and then the virus-removing feed liquid is subjected to sterilization operation.
S9, verification and split charging: sampling, identifying the semi-finished product, and sub-packaging the semi-finished product into a medium boron silcillin bottle, wherein the sub-packaging amount is 1 mL/bottle, thus obtaining the R-ATG finished product.
2.1 according to the corresponding detection items and standards of the finished product of the anti-human T cell rabbit immunoglobulin of each theory of the current edition of Chinese pharmacopoeia, the finished product of the anti-human T cell rabbit immunoglobulin preparation prepared according to the method is detected, and the result is as follows:
the protein content is 10-30 g/L, the pH value is 4.0+/-0.2, the purity of the P-ATG is not lower than 95% of the total protein, the sum of the content of IgG monomers and dimer is not lower than 95.0%, the content of multimer is not higher than 5.0%, the E rose ring formation inhibition test titer is not lower than 1:4000, the lymphotoxin test titer is not lower than 1:2000, the anti-A and anti-B hemagglutinin are not higher than 1:64, the human platelet antibody is not higher than 1:4, the human plasma protein antibody and human plasma have no precipitation line, and the protein A residue is not higher than 0.001% of the total protein.
2.1 Process study
(1) Results of first ion exchange chromatography of samples of a batch in pilot experiments
TABLE 4 results of first ion exchange chromatography for a batch of samples
(2) The yields and purification of the affinity chromatography and the second ion exchange chromatography were studied in pilot experiments at different loading levels, and the results are shown in fig. 3 to 8 and table 5 below:
TABLE 5 yields and purification results of affinity chromatography and ion exchange chromatography under different loading conditions
From the above results, it can be seen that: the process has good stability, the purity (monomer and dimer) of the final product can reach 100 percent, and the product basically has no obvious impurity and is far higher than the standard of more than 90 percent of the current edition of Chinese pharmacopoeia.
(3) Protein a residue detection: protein A residue detection was performed on the material collected by the affinity and second ion exchange chromatography in the pilot experiment, and the results are shown in FIG. 9 and the following tables 6 and 7:
TABLE 6 detection and analysis of protein A residues in samples from various purification stages
TABLE 7 protein A residual detection fit standard curve equation
Curve name | Curve formula | A | B | C | D | R 2 |
Standard curve | Y=(A-D)/(1+(X/C)^B)+D | 0.0318 | 1.13 | 10.1 | 26.2 | 0.986 |
Therefore, after the second ion exchange chromatography treatment, the method provided by the invention has the advantages that the fallen protein A is not detected in the final finished product (ion flow through liquid), the requirement that the residual amount of the protein A is not higher than 0.001% (10 ppm) of the total amount of protein in Chinese pharmacopoeia is completely met, and the method provided by the invention is reliable and good in repeatability.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for preparing anti-human T cell rabbit immunoglobulin, which is characterized by comprising the following steps:
s1, obtaining rabbit plasma or serum immunized by human peripheral blood T lymphocytes and/or thymus cells;
s2, placing the mixture at the temperature of not higher than 37 ℃ to melt slurry;
s3, adding healthy human blood plasma into the rabbit blood plasma or serum after the plasma melting to obtain a feed liquid A; adding hydroformylation human red blood cells into the feed liquid A, adjusting the pH value to 7.0+/-1.0, keeping the reaction temperature at 0-8 ℃, separating the red blood cells after full reaction, and collecting supernatant B; adding aldehyde human placenta dregs into the supernatant B, adjusting the pH to 7.0+/-1.0, keeping the reaction temperature at 0-8 ℃, separating the placenta dregs after full reaction, and collecting the supernatant C;
s4, adjusting the pH value of the supernatant C to 6.0+/-1.0, performing deep filtration, performing first ion exchange chromatography by adopting phosphate buffer solution with the pH value of 6.0+/-1.0, and collecting the flow-through liquid D;
s5, filtering the flow-through liquid D, adjusting the protein concentration to 10-30 mg/mL, loading the solution on an affinity chromatography column, washing the solution by an acetate buffer system with the pH value of 4.0+/-1.0, and collecting an eluent E;
s6, filtering and sterilizing the eluent E, and incubating at 24+/-2 ℃ to obtain a solution F;
s7, regulating the pH value of the solution F to 6.0+/-1.0, performing ion exchange chromatography for the second time, and collecting the flow-through liquid G to obtain a product containing anti-human T cell rabbit immunoglobulin.
2. The method for preparing anti-human T cell rabbit immunoglobulin according to claim 1, further comprising the steps of:
s8, ultrafiltration displacement is carried out on the passing liquid G by adopting physiological sodium chloride solution, the pH value is regulated to 4.0+/-0.5, glycine is added to obtain a semi-finished product with the protein mass concentration of 1-3% and the glycine concentration of 10-30G/L, a 20nm virus-removing filter membrane is adopted to filter virus, and then virus-removing feed liquid is sterilized;
s9, sub-packaging the semi-finished product to obtain the finished product.
3. The method for producing anti-human T cell rabbit immunoglobulin according to claim 1 or 2, wherein in step S2, the amount of healthy human plasma added is 1 to 5% of the amount of rabbit plasma added; in the step S5, the addition amount of the hydroformylation human red blood cells is 15-30% of the pulp addition amount, and the addition amount of the hydroformylation human placenta is 15-30% of the pulp addition amount.
4. The method for preparing anti-human T cell rabbit immunoglobulin according to claim 1, wherein the hydroformylation human placenta in step S5 is prepared by hydroformylation reaction of an hydroformylation solution with formaldehyde content of 0.8% -3% for 3-7 hours.
5. The method of claim 4, wherein the method of preparing the hydroformylation human placenta comprises the steps of: obtaining human placenta dregs with negative endotoxin detection, and thawing; placing the unfrozen human placenta slag into placenta aldehyde liquid for carrying out an aldehyde reaction for 3-7 h, wherein the placenta aldehyde liquid is phosphate buffer solution containing 0.8% -3% formaldehyde; transferring the aldehyde placenta into a sodium chloride solution with physiological concentration for washing, transferring into a neutralization solution for neutralization reaction, wherein the neutralization solution is a glycine-containing sodium chloride solution, washing again, and drying by control to obtain the aldehyde human placenta.
6. The method for preparing anti-human T cell rabbit immunoglobulin according to claim 5, wherein the placental hydroformylation solution is phosphate buffer solution containing 1% formaldehyde.
7. The method for preparing anti-human T cell rabbit immunoglobulin according to claim 1, wherein the pore size of the filter membrane used in the depth filtration in the steps S4 and S5 is 0.45. Mu.m.
8. The method for preparing anti-human T cell rabbit immunoglobulin according to claim 1, wherein the method for obtaining rabbit plasma in step S1 comprises the steps of: the preparation method comprises the steps of mixing thymus cells and lymphocytes to immunize rabbits, and then collecting blood; wherein, the immunization program is as follows: performing primary immunization by subcutaneous multipoint injection at back, performing secondary boost immunization by intravenous injection or subcutaneous immunization at intervals of 14 days, and taking blood plasma 21 days after the secondary immunization.
9. The anti-human T cell rabbit immunoglobulin prepared by the method of any one of claims 1-8, wherein the protein content is 10-30 g/L, the pH value is 4.0+ -0.2, the purity of P-ATG is not lower than 95% of the total protein, the sum of IgG monomer and dimer content is not lower than 95.0%, the multimer content is not higher than 5.0%, the E rose ring formation inhibition test titer is not lower than 1:4000, the lymphotoxin test titer is not lower than 1:2000, the anti-A and anti-B hemagglutinin is not higher than 1:64, the human platelet antibody is not higher than 1:4, the human plasma protein antibody and human plasma have no precipitation line, and the protein A residue is not higher than 0.001% of the total protein.
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---|---|---|---|---|
CN118011006A (en) * | 2024-01-29 | 2024-05-10 | 武汉中生毓晋生物医药有限责任公司 | Kit for detecting concentration of anti-human thymus/lymphocyte rabbit immunoglobulin total IgG antibody in plasma of patient to be dosed and application |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101084923A (en) * | 2007-06-22 | 2007-12-12 | 岳阳博康生物技术有限公司 | Method for preparing rabbit against human T lymphocytes immune serum |
CN101570576A (en) * | 2009-06-08 | 2009-11-04 | 王太林 | Method for preparing antihuman lymphocyte antibodies |
US20100330071A1 (en) * | 2009-05-27 | 2010-12-30 | Baxter International Inc. | Method to produce a highly concentrated immunoglobulin preparation for subcutaneous use |
US20170073396A1 (en) * | 2014-03-11 | 2017-03-16 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Method for preparing human plasma proteins |
CN112111004A (en) * | 2020-08-27 | 2020-12-22 | 成都生物制品研究所有限责任公司 | Production process of rabbit immunoglobulin |
CN115232783A (en) * | 2022-08-03 | 2022-10-25 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell pig immunoglobulin and hydroformylation placenta |
-
2023
- 2023-09-22 CN CN202311241053.8A patent/CN117069836A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101084923A (en) * | 2007-06-22 | 2007-12-12 | 岳阳博康生物技术有限公司 | Method for preparing rabbit against human T lymphocytes immune serum |
US20100330071A1 (en) * | 2009-05-27 | 2010-12-30 | Baxter International Inc. | Method to produce a highly concentrated immunoglobulin preparation for subcutaneous use |
CN101570576A (en) * | 2009-06-08 | 2009-11-04 | 王太林 | Method for preparing antihuman lymphocyte antibodies |
US20170073396A1 (en) * | 2014-03-11 | 2017-03-16 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Method for preparing human plasma proteins |
CN112111004A (en) * | 2020-08-27 | 2020-12-22 | 成都生物制品研究所有限责任公司 | Production process of rabbit immunoglobulin |
CN115232783A (en) * | 2022-08-03 | 2022-10-25 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell pig immunoglobulin and hydroformylation placenta |
Non-Patent Citations (2)
Title |
---|
余健等: "传代人淋巴细胞免疫猪的免疫效果初探", 微生物学免疫学进展, vol. 47, no. 6, 14 November 2019 (2019-11-14), pages 47 - 51 * |
曹澍泽等: "兽医微生物学及免疫学技术", 31 May 1992, 北京农业大学出版社, pages: 288 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118011006A (en) * | 2024-01-29 | 2024-05-10 | 武汉中生毓晋生物医药有限责任公司 | Kit for detecting concentration of anti-human thymus/lymphocyte rabbit immunoglobulin total IgG antibody in plasma of patient to be dosed and application |
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