CN111978394B - Preparation method of polyclonal preparation for COVID-19 patient specific immunotherapy - Google Patents
Preparation method of polyclonal preparation for COVID-19 patient specific immunotherapy Download PDFInfo
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Abstract
The invention discloses a preparation method of a polyclonal preparation for COVID-19 patient specific immunotherapy, which belongs to the technical field of biological medicine and comprises the following steps: s1, collecting blood of COVID-19 rehabilitation patient, adding heparin, and using StemSepTMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection; s2, culturing the separated CD19+ B lymphocytes by using an ACE solution, checking endotoxin in a culture solution every day, cleaning, obtaining a supernatant, and discarding a polluted sample; s3, freeze-drying and centrifuging the collected supernatant to obtain supernatant, and then concentrating the supernatant for endotoxin test; s4, combining the supernatants, centrifuging and collecting the supernatant; and S5, carrying out bacteria detection and toxin detection on the supernatant, and selecting qualified supernatant to obtain a finished product. Can prepare a polyclonal preparation for specific immunotherapy of patients with COVID-19, and the prepared polyclonal preparation is safe, nontoxic and low in cost.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly belongs to a preparation method of a polyclonal preparation for specific immunotherapy.
Background
The health of people all over the world, especially all medical researchers and practitioners, is seriously threatened by the virus SARS-CoV-2 causing the disease, namely, the novel coronavirus pneumonia, which is called new coronavirus pneumonia for short. The FDA has announced the safety and efficacy of treating patients with plasma from recovery patients and confirmed the efficacy through trials and studies on 10 COVID-19 patients. However, the number of convalescent patients is limited, the amount of plasma is extremely low, and at present, the treatment scheme can only be used in critical situations, which severely restricts the implementation and popularization of the immunotherapy treatment scheme using the plasma of the convalescent patients, and further research and application of preventive measures are difficult.
The culture of B-lymphocytes and T-lymphocytes is an ancient protocol, and since our first decade of the 21 st century began its use, many trials were conducted to use it as a specific immunotherapy for cancer patients. Polyclonal preparations of therapeutic immunoglobulins, i.e. intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used in the treatment of autoimmune and inflammatory diseases.
However, the reagent used for preparing the polyclonal preparation is very expensive and difficult to popularize, and can only be used for experiments in a laboratory theory, and no relevant practical product is really made at present, and more importantly, the preparation process is complex and severe, and various complex and even toxic substances are used, so that the prepared polyclonal preparation contains more impurities and toxic substances, and cannot be directly injected into a patient for treatment. For example, in routine experiments, phytohaemgluritin PHA is commonly used to stimulate emulsification of B lymphocytes, which can be used in the laboratory, but we cannot inject the finished product containing the substance into the human body, because PHA is toxic, which is one of the substances prohibited by the world health organization and other agencies.
Disclosure of Invention
Aiming at the defects and shortcomings in the background art, the invention provides a preparation method of a polyclonal preparation for specific immunotherapy of COVID-19 patients, the polyclonal preparation for specific immunotherapy of COVID-19 patients can be prepared, and the prepared polyclonal preparation is safe, nontoxic and low in cost.
In order to realize the purpose, the invention adopts the following technical scheme to realize the purpose:
a method of preparing a polyclonal preparation for use in COVID-19 patient specific immunotherapy, comprising the steps of:
s1, taking 45-55ml of blood of COVID-19 rehabilitation patients, adding 3800-TMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using an ACE solution for 28-35 days, checking endotoxin in the culture solution every day, cleaning the culture solution, obtaining a supernatant, and discarding any sample showing contamination;
s3, freeze-drying and centrifuging the supernatant collected in the step S2 to obtain the supernatant, concentrating the supernatant, and performing endotoxin test after the concentration is finished to control the safety and the quality;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging, and collecting the centrifuged supernatants;
s5, carrying out bacteria detection and toxin detection on the supernate obtained in the step S4, and selecting supernate qualified in detection to obtain a finished product;
the ACE solution is 100% bioactive peptide solution, and has pH of 7.2.
Further measures taken are: in step S1, 50ml of blood of COVID-19 rehabilitation patients is taken, and 4000U of heparin is added.
Further measures taken are: the culture period in the step S2 is 30 days.
Further measures taken are: in the step S3, when freeze-drying centrifugation is performed at a centrifugal force of 1000g, the centrifugation time is 5 minutes.
Further measures taken are: in step S3, the mixture is concentrated by freeze-drying and then concentrated to 1/4 of the original volume by freeze-drying.
Further measures taken are: in step S4, centrifugation was performed at 1000g for 15 minutes.
Further measures taken are: the ACE culture solution is a culture medium which is extracted and purified from spleen, lymph node and umbilical cord of sheep embryo and is rich in lysine and arginine.
Further measures taken are: according to the clinical condition or experiment, the amount of immunoglobulin to be injected into a patient is determined, and the finished product is diluted, calibrated and adjusted to obtain the injection.
The invention adds proper amount of heparin into the blood of a COVID-19 rehabilitation patient and utilizes StemSepTMOr EasySepTMThe CD19 mixture is subjected to negative selection, so that CD19+ B lymphocytes can be efficiently and accurately separated from the PBMNCs; isolated CD19+ B lymphocytes were cultured using ACE medium, which is rich in natural excitatory growth factors and factors required to stimulate hyperactivity, via ACE solution as a rich medium and multiplicative stimulation using StemSepTMAnd EasySepTMThe isolated B lymphocytes were purified to 1 million fold in vitro proliferation within 40 days and maintained high viability.
In the whole process, toxic substances such as Phytohaemglurin (PHA) and the like are not needed to excite the emulsification of B lymphocytes, no toxic substance is added in the whole culture process, the endotoxin is checked every day, any sample showing pollution is discarded, finally, the endotoxin and bacteria are checked, the safety and the quality are strictly controlled, and the quality safety of a final finished product is guaranteed; the culture period of 28-35 days is the culture days with the best quality and effect finally determined after intensive study, and particularly when the culture days are 30 days, the obtained finished product has the most excellent quality and the obtained finished product has relatively large amount.
The freeze-drying centrifugation and concentration of the supernatant can keep the activity and the quality of the active ingredients, particularly the freeze-drying centrifugation is carried out for 5 minutes under the control of 1000g of centrifugal force, after the freeze-drying centrifugation is carried out to 1/4 with the original volume, the supernatant is combined, and then the centrifugation is carried out for 15 minutes under the 1000g of centrifugal force, so that the impurities can be effectively separated, the purity and the quality of the finished product are greatly improved, the loss is reduced, and the active state of immunoglobulin is effectively kept.
In addition, the amount of immunoglobulin to be injected into a patient is determined according to clinical conditions or experiments, and the prepared polyclonal preparation finished product is diluted, calibrated and adjusted, so that the obtained injection liquid is more suitable for the condition of each patient, and can exert more outstanding curative effect in specific immunotherapy.
The polyclonal preparation prepared by the invention breaks through the technical bottleneck that the blood plasma in the COVID-19 recovery period can not be popularized and used for immunotherapy, can greatly reduce the production cost, does not need to use very expensive reagents, is safe and nontoxic, and has guaranteed quality. The polyclonal preparation provided by the invention is a basic technical scheme for promoting the specific immunotherapy of patients with COVID-19 to realize landing application and popularization, and fills up the technical blank in the field; but also provides thinking and technical support for resisting SARS-CoV-2 virus, saving more COVID-19 patients and even providing future SARS-CoV-2 virus vaccine.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the invention can prepare a polyclonal preparation for specific immunotherapy of COVID-19 patients, and provides thinking and technical support for resisting SARS-CoV-2 virus and saving more COVID-19 patients.
2. The polyclonal preparation prepared by the invention is safe and nontoxic, has low cost and is suitable for popularization and application.
3. The preparation method of the invention uses simple reagents in the whole preparation process, has strict condition control, can avoid using complex, large-irritation and toxic and harmful substances, and jointly ensures the high activity and quality of the finished product.
Drawings
FIG. 1 is a statistical chart showing the results of lymphocyte proliferation in examples 1 to 5 of the present invention;
FIG. 2 is a statistical chart of the results of lymphocyte viability assays according to examples 1-5 of the present invention;
FIG. 3 is a graph showing the average secretion amounts of IgG, IgA and IgM, which are indispensable substances, after 20 hours, of CD19+ B lymphocytes obtained by culturing for 40 days according to the production methods of examples 1 to 5 of the present invention.
A, c, e, g, i in FIGS. 1 and 2 represent the CD19+ B lymphocytes of examples 1-5, respectively; b. d, f, h, j represent the T (CD38) lymphocytes obtained in blood of examples 1-5, respectively.
Detailed Description
In order to clearly understand the technical solutions adopted by the present invention, the following description is made on the preferred embodiments of the present invention, and it should be understood that the embodiments described herein are only used for illustrating and explaining the present invention, and are not used to limit the present invention.
Example 1: a method of preparing a polyclonal preparation for use in COVID-19 patient specific immunotherapy, comprising the steps of:
s1, collecting 45ml of blood of COVID-19 rehabilitation patient, adding 3800 heparin, subpackaging in 5 test tubes, and using StemSepTMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs (peripheral blood mononuclear cells) by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using ACE solution, wherein the culture period is 28 days, the endotoxin in the culture solution is checked every day, the culture solution is cleaned, supernatant liquid is obtained, and any sample showing pollution is discarded;
s3, when the supernatant collected in the step S2 is subjected to freeze drying centrifugation at the centrifugal force of 1000g, the centrifugation time is 5 minutes, then the supernatant is obtained, is subjected to concentration by cold freeze drying, is subjected to freeze drying concentration to 1/4 of the original volume, and is subjected to endotoxin test after the concentration is finished, so that the safety and the quality are controlled;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging for 15 minutes at the centrifugal force of 1000g, and collecting the centrifuged supernatants;
s5, performing bacteria detection and toxin detection on the supernatant obtained in the step S4, and selecting the supernatant qualified in detection to obtain a finished product 1.
Example 2: a method of preparing a polyclonal preparation for use in COVID-19 patient specific immunotherapy, comprising the steps of:
s1, collecting 55ml of blood of COVID-19 rehabilitation patient, adding 4200U heparin, and packagingIn 5 tubes, using StemSepTMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using ACE solution, wherein the culture period is 35 days, the endotoxin in the culture solution is checked every day, the culture solution is cleaned, supernatant liquid is obtained, and any sample showing pollution is discarded;
s3, when the supernatant collected in the step S2 is subjected to freeze drying centrifugation at the centrifugal force of 1000g, the centrifugation time is 5 minutes, then the supernatant is obtained, is subjected to concentration by cold freeze drying, is subjected to freeze drying concentration to 1/4 of the original volume, and is subjected to endotoxin test after the concentration is finished, so that the safety and the quality are controlled;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging for 15 minutes at the centrifugal force of 1000g, and collecting the centrifuged supernatants;
s5, performing bacteria detection and toxin detection on the supernatant obtained in the step S4, and selecting the supernatant qualified in detection to obtain a finished product 2.
Example 3: a method of preparing a polyclonal preparation for use in COVID-19 patient specific immunotherapy, comprising the steps of:
s1, collecting 50ml of blood of COVID-19 rehabilitation patient, adding 4000U heparin, subpackaging in 5 test tubes, and using StemSepTMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using ACE solution for 30 days, checking endotoxin in the culture solution every day, cleaning the culture solution, obtaining supernatant, and discarding any sample showing pollution;
s3, when the supernatant collected in the step S2 is subjected to freeze drying centrifugation at the centrifugal force of 1000g, the centrifugation time is 5 minutes, then the supernatant is obtained, is subjected to concentration by cold freeze drying, is subjected to freeze drying concentration to 1/4 of the original volume, and is subjected to endotoxin test after the concentration is finished, so that the safety and the quality are controlled;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging for 15 minutes at the centrifugal force of 1000g, and collecting the centrifuged supernatants;
s5, performing bacteria detection and toxin detection on the supernatant obtained in the step S4, and selecting the supernatant qualified in detection to obtain a finished product 3.
Example 4: a method of preparing a polyclonal preparation for use in COVID-19 patient specific immunotherapy, comprising the steps of:
s1, collecting 52ml of blood of COVID-19 rehabilitation patient, adding 4100 heparin, subpackaging in 5 test tubes, and using StemSepTMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using ACE solution, wherein the culture period is 32 days, the endotoxin in the culture solution is checked every day, the culture solution is cleaned, supernatant liquid is obtained, and any sample showing pollution is discarded;
s3, when the supernatant collected in the step S2 is subjected to freeze drying centrifugation at the centrifugal force of 1000g, the centrifugation time is 5 minutes, then the supernatant is obtained, is subjected to concentration by cold freeze drying, is subjected to freeze drying concentration to 1/4 of the original volume, and is subjected to endotoxin test after the concentration is finished, so that the safety and the quality are controlled;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging for 15 minutes at the centrifugal force of 1000g, and collecting the centrifuged supernatants;
s5, performing bacteria detection and toxin detection on the supernatant obtained in the step S4, and selecting the supernatant qualified in detection to obtain a finished product 4.
Example 5: a method of preparing a polyclonal preparation for use in COVID-19 patient specific immunotherapy, comprising the steps of:
s1, collecting 42ml of blood of COVID-19 rehabilitation patient, adding 3900 heparin, subpackaging in 5 test tubes, and using StemSepTMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using ACE solution, wherein the culture period is 29 days, the endotoxin in the culture solution is checked every day, the culture solution is cleaned, supernatant liquid is obtained, and any sample showing pollution is discarded;
s3, when the supernatant collected in the step S2 is subjected to freeze drying centrifugation at the centrifugal force of 1000g, the centrifugation time is 5 minutes, then the supernatant is obtained, is subjected to concentration by cold freeze drying, is subjected to freeze drying concentration to 1/4 of the original volume, and is subjected to endotoxin test after the concentration is finished, so that the safety and the quality are controlled;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging for 15 minutes at the centrifugal force of 1000g, and collecting the centrifuged supernatants;
s5, performing bacteria detection and toxin detection on the supernatant obtained in the step S4, and selecting the supernatant qualified in detection to obtain a finished product 5.
Wherein the ACE culture solution in the above examples is 100% bioactive peptide solution, pH is 7.2, and purified culture medium rich in lysine and arginine is extracted from spleen, lymph node and umbilical cord of sheep embryo.
The ACE culture solution in the above examples was produced by ACE Cells Lab Limited, UK; StemSepTMAnd EasySepTMThe CD19 mixtures were purchased from Stem cell Technologies, Inc., Canada (e.g., STEMCELL Technologies Inc., Vancouver, BC, Canada).
The amount of immunoglobulin to be injected into a patient is determined according to clinical conditions or experiments, and the finished products of the above examples 1 to 5 are diluted, calibrated and adjusted to obtain an injection solution. After the dosage of the immunoglobulin is adjusted, the obtained routine dosage injection of the immunoglobulin can be injected into patients with COVID-19, the dosage of each injection is 400mg/Kg body weight/day, and the injection is given twice a day.
And (3) quality detection:
the quality performance index of the polyclonal preparation obtained by the preparation method of the polyclonal preparation used for the patient-specific immunotherapy of COVID-19 in examples 1 to 5 of the present invention was examined, and the test results are shown in Table 1 below.
Table 1: results of quality inspection of the polyclonal preparation products of examples 1 to 5
As can be seen from Table 1, the polyclonal preparation prepared by the present invention has the characteristics of high activity, high purity and good stability, and all indexes of the polyclonal preparation conform to the regulations.
And (3) safety inspection:
the finished preparations obtained in examples 1 to 5 were reduced and diluted to a specification of 1mg/ml with physiological saline for use.
One batch of 15 mice weighing 18-23 g are selected and divided into 5 groups on average, and 3 mice and 5 groups of mice in each group are subjected to safety tests corresponding to the finished products of examples 1-5 respectively. Each group of mice was orally administered with the preparation diluted to 1mg/ml in the above-mentioned corresponding examples 1 to 5, respectively, at a dose of 5ml per mouse, 2 times a day for 30 days, and observed and recorded.
And (II) selecting 15 grams of guinea pigs with the weight of 350-400 grams in the same batch, averagely dividing the guinea pigs into 5 groups, and carrying out safety tests on 3 and 5 groups of guinea pigs which respectively correspond to the finished products of the examples 1-5. Each group of guinea pigs was orally administered with the preparation diluted to 1mg/ml in the above-mentioned corresponding examples 1 to 5, respectively, at a dose of 10ml per one, 2 times daily for 30 days, and observed and recorded.
And (III) selecting 10 rabbits with the weight of 2.0-2.5 kg, averagely dividing the rabbits into 5 groups, and carrying out safety tests on 2 rabbits in each group and 5 rabbits in each group respectively corresponding to the finished products of the examples 1-5. Each group of rabbits was orally administered with the preparation diluted to 1mg/ml in the above-mentioned corresponding examples 1 to 5, respectively, at a dose of 15ml per one, 2 times a day for 30 days, and observed and recorded.
The results show that the mice, guinea pigs and rabbits in the above test have no abnormality after 30 days of administration, have normal activities, and normally grow and gain weight, so the polyclonal preparation of the present invention has safe quality, no toxicity and no harm.
The invention also detects the culture proliferation of CD19+ B (CD19) lymphocytes and T (CD38) lymphocytes in vitro. Culturing CD19+ B (CD19) lymphocytes and T (CD38) lymphocytes separated according to the steps of examples 1-5 with ACE solution for 60 days, detecting the proliferation and activity of the cells, and summarizing the data to obtain the result of the proliferation of the lymphocytes as shown in FIG. 1; the results of the lymphocyte viability assay are shown in FIG. 2.
As is evident from fig. 1, the direct relationship between CD19+ B (CD19) lymphocytes and T (CD38) lymphocytes with time increases the number of proliferating cells with time, almost linearly, indicating that the proliferation rate is almost proportional, and the proliferation amount of CD19+ B (CD19) lymphocytes and T (CD38) lymphocytes reaches one million at 40 days. From FIG. 2, it is evident that the relationship between the survival ability of lymphocytes and time is shown, and it is evident that the survival ability of lymphocytes of the treated CD19+ B lymphocytes is slightly reduced after 40 days, but within 40 days, the survival ability is still maintained at a high loading, and the survival ability is not substantially lost with time.
The CD19+ B lymphocytes can keep secreting 3 types of immunoglobulins, IgG, IgA and IgM, respectively, during the culture period, and 1X 10 cells can be obtained by culturing for 40 days according to the preparation methods of examples 1 to 5, respectively6CD19+ B cells cultured for 20 hours, the secretion amounts of IgG, IgA and IgM, which are the indispensable substances obtained after 20 hours in the preparation methods of the examples, were measured and averaged, and the final results are shown in FIG. 3, in which 1 million CD19+ B lymphocytes secreted 9000ng of IgG, 85ng of IgA and 85ng of Ig in 20 hoursM can reach 15 ng.
Based on the fact that 1ml of blood contains 4000 White Blood Cells (WBC), 80% of which are lymphocytes, 3200 lymphocytes are expected to be obtained, 10ml of a sample is available for 32000 lymphocytes, and 1X 32000X 10 in 3 weeks of culture6＝32×1010One cell, or 2X 32000X 106＝64×1010One cell, i.e., 480 hundred million cells on average. 1X 106Each cell can produce 9000ng of IgG, 85ng of IgA and 15ng of IgM; then 480 million cells can produce 432000g IgG, 4080g IgA and 720g IgM. It is thus understood that the present invention enables the preparation of polyclonal preparations of relatively large amounts of immunoglobulin, which are required for immunotherapy, using a small amount of blood of a rehabilitee.
In conclusion, the embodiment of the invention can effectively improve the preparation of the polyclonal preparation for specific immunotherapy of patients with COVID-19, and the prepared polyclonal preparation is safe, nontoxic, high in activity and high in yield, overcomes the technical bottleneck that the plasma in the recovery period of COVID-19 cannot be popularized for treatment, also provides thought and technical support for resisting SARS-CoV-2 virus, saving more COVID-19 patients and even providing thought and technical support for the subsequent SARS-CoV-2 virus vaccine.
The above description is only for the purpose of illustrating the embodiments of the present invention and not for the purpose of limiting the same, and equivalent modifications and variations of the embodiments of the present invention will be apparent to those skilled in the art without departing from the overall spirit of the invention.
Claims (6)
1. A method of preparing a polyclonal preparation for use in COVID-19 patient-specific immunotherapy, comprising: the method comprises the following steps:
s1, taking 45-55ml of blood of COVID-19 rehabilitation patients, adding 3800-TMOr EasySepTMCD19 mixture CD19+ B lymphocytes were isolated from PBMNCs by negative selection;
s2, culturing the CD19+ B lymphocytes separated in the step S1 by using an ACE solution for 28-35 days, checking endotoxin in the culture solution every day, cleaning the culture solution, obtaining a supernatant, and discarding any sample showing contamination;
s3, freeze-drying and centrifuging the supernatant collected in the step S2 to obtain the supernatant, concentrating the supernatant, and performing endotoxin test after the concentration is finished to control the safety and the quality;
s4, combining all the supernatants collected in the step S3 during the culture period, centrifuging, and collecting the centrifuged supernatants;
s5, carrying out bacteria detection and toxin detection on the supernate obtained in the step S4, and selecting supernate qualified in detection to obtain a finished product;
the ACE solution is a 100% bioactive peptide solution, and the pH value is 7.2; the ACE culture solution is a culture medium which is extracted and purified from spleen, lymph node and umbilical cord of sheep embryo and is rich in lysine and arginine.
2. The method of claim 1, wherein the polyclonal preparation is administered by a patient specific immunotherapy with COVID-19, comprising: in step S1, 50ml of blood of COVID-19 rehabilitation patients is taken, and 4000U of heparin is added.
3. The method of claim 1, wherein the polyclonal preparation is administered by a patient specific immunotherapy with COVID-19, comprising: the culture period in the step S2 is 30 days.
4. The method of claim 1, wherein the polyclonal preparation is administered by a patient specific immunotherapy with COVID-19, comprising: in the step S3, when freeze-drying centrifugation is performed at a centrifugal force of 1000g, the centrifugation time is 5 minutes.
5. The method of claim 4 for the preparation of a polyclonal preparation for use in COVID-19 patient specific immunotherapy, wherein: in step S3, the mixture is concentrated by freeze-drying and then concentrated to 1/4 of the original volume by freeze-drying.
6. The method of claim 1, wherein the polyclonal preparation is administered by a patient specific immunotherapy with COVID-19, comprising: in step S4, centrifugation was performed at 1000g for 15 minutes.
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