CN116574175A - Single plasma IgG product and preparation method thereof - Google Patents
Single plasma IgG product and preparation method thereof 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
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- 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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- 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)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of blood product production, in particular to a single-person plasma IgG product and a preparation method thereof. The method comprises the following steps of: centrifuging or filtering the plasma to obtain a liquid phase fraction; and sequentially performing a first anion exchange chromatography, a second anion exchange chromatography, a first cation exchange chromatography, a third anion exchange chromatography and a second cation exchange chromatography on the liquid phase part to obtain a plasma IgG product. The full-chromatographic process separation method can be used for primary separation, medium separation and fine separation of plasma globulin, has high chromatographic separation precision and convenient operation, and solves the technical problems of non-ideal purity, yield, activity, safety and preparation speed of the immunoglobulin obtained by the existing preparation process. The technology is used for purifying plasma IgG, is simple and convenient to operate, can meet the requirements of different production yields, and has ideal application prospect and popularization value.
Description
Technical Field
The invention relates to the technical field of blood product production, in particular to a single-person plasma IgG product and a preparation method thereof.
Background
The human immunoglobulin is a protein molecule which is produced by human plasma cells under the physiological and pathological conditions and is stimulated by antigen and has antibody activity or chemical structure, and the immunoglobulin is a tetrapeptide chain structure formed by connecting two identical light chains and two identical heavy chains through inter-chain disulfide bonds. It can be divided into five classes IgG, igA, igM, igD, igE, wherein the molecular weight of immunoglobulin G (immunoglobuling IgG) is about 150kDa, igG is the highest class in plasma immunoglobulin accounting for 75% -80% of total immunoglobulin, and the content of IgG in human plasma is about 7-16 g/L. IgG is one of the most durable and important antibodies in immune response, which has a broad spectrum of antibodies against viruses, bacteria, or other pathogens, and is the principal force of the body against infection.
The immunoglobulin is an immunomodulator, has the functions of immune replacement and immunity enhancement at low dosage, has anti-inflammatory activity and immunity regulation at high dosage, and is mainly used for primary and secondary immunoglobulin deficiency diseases and the like in clinic, such as hypoimmunoglobulin blood diseases, common variant immunodeficiency diseases, igG subtype deficiency diseases, severe infection, neonatal septicemia, primary thrombocytopenic purpura, kawasaki disease and the like. How to extract and isolate high quality immunoglobulins from plasma rapidly for the treatment of diseases is then a major challenge. At present, the existing domestic immunoglobulin production technology mainly adopts the means of a low-temperature ethanol method, a combination of the low-temperature ethanol method and a chromatography technology and the like, but the ethanol precipitation separation process is long in time consumption, the total recovery rate of the IgG protein is not high, and the efficiency of separating the IgG is low; the low-temperature ethanol precipitation at the temperature of minus 20 ℃ has relatively high requirements on equipment and environment; the low-temperature ethanol precipitation method has poor effect of removing the impurity proteins such as IgM and the like, and the obtained final product has higher IgM and IgA content.
In view of the foregoing, there is a need to develop a novel process for preparing immunoglobulins without relying on low-temperature ethanol precipitation, which improves the yield and purity of immunoglobulins and the preparation speed, so as to meet the market demand for enrichment and separation of high-quality immunoglobulins from convalescence plasma.
Disclosure of Invention
The invention aims to provide a preparation method of a single plasma IgG product, which aims to solve the technical problems of non-ideal purity, yield, activity, safety and preparation speed of immunoglobulin obtained by the existing preparation process.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of single-person plasma IgG product comprises centrifuging or filtering plasma to obtain liquid phase; and sequentially performing a first anion exchange chromatography, a second anion exchange chromatography, a first cation exchange chromatography, a third anion exchange chromatography and a second cation exchange chromatography on the liquid phase part to obtain a plasma IgG product.
The technical scheme also provides a plasma IgG product prepared by the single plasma IgG product preparation method.
The principle and the advantages of the scheme are as follows:
the full-layer separation process can be used for primary separation, medium separation and fine separation of plasma globulin, has high chromatographic separation precision, simple equipment and convenient operation, is widely applied to separation and purification of proteins according to different principles, is an effective separation means, is an important protein purification tool, and is one of purification technologies with highest precision in the downstream processing process of organisms. With the development of polymer chemistry, ion exchange chromatography technology is widely applied to separation and purification of proteins, polypeptides and other charged biomolecules, and has the advantages of high resolution, high protein binding capacity and the like. The gel chromatography process has the advantages of simple operation, simple required equipment, reusable separation medium, good separation effect, high repeatability and the like, and is most remarkable in that the recovery rate of the sample is high. Therefore, the technology is simple to operate when applied to purifying the IgG antibody by the blood plasma, can meet the requirements of different production yields, and can be used for small-scale extraction (for example, single-person blood plasma IgG protein extraction) and mass production and preparation. According to the technical scheme, igG purification and enrichment treatment can be performed on refrigerated raw material plasma and fresh plasma.
The prior art with application number CN202010893241.9 provides a method for extracting and separating IgM and IgG from plasma. The IgG obtained by 2-step ion exchange chromatography is low in product purity and contains more non-target proteins, so that the quality standard of the commercial product is not met, the problems of protein precipitation and the like in the chromatography process are not solved, and the large-scale production is not facilitated. The technical proposal can extract the required IgG protein from the blood plasma through multi-step chromatography separation, and the separated IgG product has high yield and purity, the quality reaches the standard of Chinese pharmacopoeia of 2020 edition, and the method can be used for clinic application. The separation of various proteins in the blood plasma can be increased through multi-step chromatography, so that the comprehensive utilization rate of the blood plasma is greatly improved, and the income is improved. Compared with the traditional low-temperature ethanol precipitation production process, the process removes the low-temperature environment produced by using the refrigerant during component precipitation production, thereby reducing energy consumption, reducing a large amount of ethanol consumption and further reducing production cost. Meanwhile, the process has good selectivity on sample throughput and high operability, can be used for single plasma customization treatment and also can be used for mass production application, and is also unrealizable by the traditional low-temperature ethanol process.
Further, in the first anion exchange chromatography, the pH value of the supernatant is regulated to be 6.0-7.6, the conductivity is 9.0-15.0 mS/cm, and the first to-be-chromatographed liquid is obtained after filtration; and loading the first chromatographic solution to be subjected to anion exchange chromatography column, and collecting the flow-through solution to obtain the first chromatographic flow-through solution.
Further, in the second anion exchange chromatography, the pH value of the first chromatography flow-through liquid is adjusted to be 5.5-6.5, the conductivity is 4.0-6.0 mS/cm, and a second to-be-chromatographed liquid is obtained after filtration; and loading the second chromatographic solution to be subjected to chromatography in an anion exchange chromatographic column, and collecting the flow-through solution to obtain a second chromatographic flow-through solution.
Further, in the first cation exchange chromatography, the pH value of the second chromatography flow-through liquid is adjusted to be 4.8-5.8, the conductivity is 0.5-2.0 mS/cm, and a third to-be-chromatographed liquid is obtained after filtration; and loading the third to-be-chromatographed liquid into a cation exchange chromatographic column, discarding the flow-through liquid, eluting the cation exchange chromatographic column by using the first eluent, and collecting the eluent to obtain the first chromatographic eluent.
Further, in the third anion exchange chromatography, the pH value of the first chromatography eluent is regulated to be 6.5-8.5, the conductivity is 0.5-2.0 mS/cm, and the fourth to-be-chromatographed liquid is obtained after filtration; and loading the fourth chromatographic solution to be subjected to chromatography in an anion exchange chromatographic column, and collecting the flow-through solution to obtain a third layer of flow-through solution.
Further, in the second cation exchange chromatography, the pH value of the third layer of chromatographic penetrating fluid is adjusted to be 4.0-6.0, the conductivity is 0.5-2.5 mS/cm, and a fifth to-be-chromatographed fluid is obtained after filtration; and loading the fifth to-be-chromatographed liquid into a cation exchange chromatographic column, discarding the flow-through liquid, eluting the cation exchange chromatographic column by using a second eluent, and collecting the eluent to obtain the second chromatographic eluent.
Experiments prove that the pH value and the conductivity value have larger influence on the enrichment and separation effect when the ion exchange chromatography experiments are carried out for a plurality of times, the purity of the extracted IgG can reach 100 percent within the pH and conductivity range selected in the experimental scheme, and the total yield of the IgG (calculated by five steps of chromatography) can reach more than 80 percent.
Further, the ligand of the filler used for the first cation exchange chromatography or the second cation exchange chromatography is carboxymethyl or sulfopropyl.
Further, the ligand of the filler used for the first anion exchange chromatography, the second anion exchange chromatography or the third anion exchange chromatography is diethylaminoethyl or quaternary aminoethyl.
Further, the pH value of the first eluent is 6.5-8.5 by taking sodium chloride as solute, and the conductivity is 2.5-5.0 mS/cm; the second eluent is a solution with sodium chloride and polysaccharide as solutes and pH of 4.0-7.0.
To sum up, the beneficial effects of this technical scheme lie in:
(1) Full-layer analysis process: avoiding the use of a low-temperature ethanol method and avoiding the preparation of component precipitation. The low-temperature ethanol method needs to use 95% ethanol, the high-concentration ethanol solution is inflammable and explosive, the safety risk is high, an explosion-proof factory building is needed to be specially designed, and the investment cost is high. In addition, the low-temperature ethanol precipitation method production process needs to form precipitation for many times in the intermediate production process, on one hand, the multi-step treatment of precipitation, redissolution, precipitation, redissolution and the like damages the protein structure and activity, and has low yield, on the other hand, the low-temperature ethanol precipitation method has low resolution ratio and low purity of the final finished product, and on the other hand, the high-concentration ethanol has a certain destructive effect on the protein, influences the natural activity of the protein and reduces the internal quality.
(2) The product quality is as follows: the IgG yield of the patent process can reach more than 80%, and the low-temperature ethanol method yield is only 50%. The purity can reach more than 95 percent, and the quality of the finished product meets the requirements of Chinese pharmacopoeia 2020 edition.
(3) The production time is saved: igG is extracted by a single plasma system (continuous flow production design) within 3-6 hours, and is particularly suitable for prevention and control of new epidemic infection epidemic situation. The production stage does not adopt continuous flow production design, and the production time can be reduced to 2-6 days to finish production. The low-temperature ethanol process has the advantages that the production time cannot be shortened because the flow rate can influence the temperature and the protein change of the product due to the addition of ethanol, and the production time is 12-15 days.
(4) The production scale is more flexible: the low-temperature ethanol method has the advantages that the volume can be continuously increased in the production process due to the need of adjusting five-variable parameters, the production is difficult to control as the production scale is increased, and the linear amplification is difficult. The chromatographic process meets the linear amplification requirement, the amplification and the reduction are more flexible, and the single blood plasma can also be used for production. Is especially suitable for sudden public health events, such as single blood plasma of COVID-19 rehabilitation, and IgG is prepared for treating severe patients.
Drawings
FIG. 1 is a flow chart of the process for preparing a plasma IgG preparation of example 1.
FIG. 2 is an overall schematic diagram of an apparatus for producing a plasma IgG preparation of example 1.
FIG. 3 is a schematic diagram showing the connection of a chromatographic column in the apparatus for producing a plasma IgG preparation of example 1.
FIG. 4 is a schematic diagram showing the connection of a chromatographic column in the apparatus for producing a plasma IgG preparation of example 2.
Fig. 5 is a front view of the plasma separation kit of example 2.
FIG. 6 is a front view of the cryoprecipitation separation unit of example 2
Fig. 7 is a front view of the modified plasma separation kit of example 2.
FIG. 8 is a typical elution profile of a first ion exchange chromatography.
FIG. 9 is a typical elution profile of a second ion exchange chromatography.
FIG. 10 is a typical elution profile of a third ion exchange chromatography.
FIG. 11 is a typical elution profile for a fourth ion exchange chromatography.
FIG. 12 shows the result of the molecular size distribution test of the sample after the fourth ion exchange chromatography.
FIG. 13 shows the protein electrophoresis patterns of samples before and after the fourth ion exchange chromatography.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
The reference numerals are specifically as follows: the first anion exchange chromatography column 1, the second anion exchange chromatography column 2, the first cation exchange chromatography column 3, the third anion exchange chromatography column 4, the second cation exchange chromatography column 5, the cryoprecipitation separation unit 6, the pump 7, the valve 8, the buffer bag 9, the prefilter 10, the nanofiltration 11, the computer 12, the chromatography unit casing 13, the fixing surface 14, the grille 15, the first pipeline 16, the second pipeline 17, the third pipeline 18, the fourth pipeline 19, the fifth pipeline 20, the sixth pipeline 21, the seventh pipeline 22, the eighth pipeline 23, the ninth pipeline 24, the tenth pipeline 25, the eleventh pipeline 26, the twelfth pipeline 27, the thirteenth pipeline 28, the fourteenth pipeline 29, the fifteenth pipeline 30, the sixteenth pipeline 31, the first container 32, the upper cover 33, the stirring assembly 34, the motor 35, the filter membrane 36, the pressure adjusting pipe 37, the second container 38, the delivery pipe 39, the cold box 40, the pressurizing filter membrane 41, and the third container 42.
Example 1: igG preparation flow for frozen raw material plasma
The preparation process of the plasma IgG comprises the following steps:
(1) Treatment of raw material plasma: after the raw material plasma is taken out of the warehouse, the surface of the plasma bag is sterilized by using 70% -75% ethanol solution, the plasma bag is broken, the temperature is controlled to be 0-4 ℃, the melting is carried out, the plasma is centrifuged at a speed of 10000rpm/min or more by using a centrifuge, and the supernatant is collected. The blood plasma (raw material blood plasma) used in the step is human blood plasma pointed out in Chinese pharmacopoeia: the human plasma for producing blood products is healthy human plasma for producing plasma protein products, which is collected by a single plasma collecting technology, is supernatant obtained by removing cells by blood centrifugation, contains proteins, inorganic salts, water and the like, and does not contain blood cells.
(2) First anion exchange chromatography: the pH value of the supernatant fluid of the blood plasma after centrifugation is regulated to be 6.0-7.6, the conductivity is 9.0-15.0 mS/cm, and the supernatant fluid is filtered by a filter element with the terminal of 0.22 mu m (which can be omitted in a continuous production system) to obtain the first to-be-chromatographed liquid. The first chromatographic solution to be chromatographed can be prepared without using a 0.22 mu m filter element for filtration, and the inventor finds that the pH and the conductivity are adjusted to be in the above range in the step through a large amount of experimental researches, and the phenomenon of protein precipitation does not occur in the system. Particularly in a continuous production system, a 0.22 mu m filter element can be not used for filtering so as to accelerate the production speed, and the condition that the production continuity is affected due to protein precipitation can not occur. And (3) performing a first anion exchange chromatography experiment on the first to-be-chromatographed liquid, adsorbing IgM and coagulation factors, and collecting the flow-through liquid containing IgG (first chromatography flow-through liquid) for next chromatography.
(3) Second anion exchange chromatography: the first chromatographic flow-through liquid conductivity is regulated to 4.0-6.0 mS/cm, the pH value is regulated to 5.5-7.2 (more preferably 6.0-7.2), and the second chromatographic liquid to be obtained after the filtration is carried out by a filter element with the terminal of 0.22 mu m (the pH value and the conductivity are regulated to the above range, the protein precipitation phenomenon can not occur, and the filtration can be omitted in a continuous production system). And (3) carrying out anion exchange chromatography for the second time on the second to-be-chromatographed liquid to remove trace impurity proteins, and collecting the flow-through liquid containing IgG (second chromatography flow-through liquid) for next chromatography.
(4) First cation exchange chromatography: the pH value of the second chromatographic flow-through liquid is regulated to be 4.8-5.8, the conductivity is 0.5-2.0 mS/cm, and a filter element with the terminal of 0.22 mu m is used for filtering (the pH value and the conductivity are regulated to be in the above range, the phenomenon of protein precipitation can not occur, and the third chromatographic flow-through liquid can be omitted in a continuous production system) so as to obtain the third chromatographic flow-through liquid. Loading the third chromatographic solution to be subjected to cation exchange chromatographic column, adsorbing IgG, allowing other non-IgG proteins to flow through, eluting the IgG by using eluent (first eluent, 0.02-0.05M sodium chloride; pH value is adjusted to 6.5-8.5 by using hydrochloric acid or sodium hydroxide, and conductivity is 2.5-5.0 mS/cm), and collecting the eluent to obtain the first chromatographic eluent.
(5) Third anion exchange chromatography: the conductivity of the first chromatographic eluent in the last step is regulated to be 0.5-2.0 ms/cm, the pH value is 6.5-8.5, and a filter element with the terminal of 0.22 mu m is used for filtering (the pH value and the conductivity are regulated to be in the above range, the phenomenon of protein precipitation can not occur, and the fourth chromatographic liquid to be chromatographed can be obtained in a continuous production system). And loading the fourth chromatographic solution to be subjected to anion exchange chromatography column, removing trace impurity proteins, and collecting the flow-through solution to obtain a third chromatographic flow-through solution.
(6) Nano-membrane filtration; the previous step of flow through liquid (third chromatography flow through liquid) is filtered by a 0.1 mu m deep filter, and then is filtered by a 20nm nano membrane, so as to obtain the third layer of flow through liquid after filtration. If the final product is intended for intravenous injection, it is necessary to ensure that the product does not contain substances with particle sizes greater than 20 nm.
(7) Second cation exchange chromatography: the pH value of the third layer of the chromatographic penetrating liquid after filtration is adjusted to be 4.0-6.0, the conductivity is 0.5-2.5 mS/cm, and the fifth chromatographic liquid (the pH and the conductivity are adjusted to be in the above range, and the phenomenon of protein precipitation does not occur) is obtained. Loading the fifth chromatographic solution to be subjected to cation exchange chromatography, adsorbing IgG on the chromatography column, eluting the IgG by using an eluent (second eluent, 0.03-0.08M sodium chloride and 30-100 g/L polysaccharide, preferably glucose or maltose; more preferably 0.05mol/L sodium chloride and 50g/L maltose) and adjusting pH value by using an acid solution to 4.0-7.0, wherein an eluent outlet is connected with a degerming device, and subpackaging the second chromatographic eluent into molded bottles to obtain the IgG finished product. In other processes in the prior art, the preparation method generally comprises the steps of eluting, ultrafiltering, preparing, sterilizing and subpackaging; according to the technical scheme, the four steps are mixed together, so that the process steps are reduced, and continuous flow production is facilitated.
The detection of the IgG content adopts a Beckmann specific protein analysis system, the detection of the total protein content adopts a Yilanbei full-automatic biochemical analyzer, and the detection of the molecular size distribution is carried out by adopting liquid phase analysis. The IgG purity is calculated by adopting the ratio of the IgG content of the sample to the total protein content, and the IgG yield is calculated according to the ratio of the IgG content of different experimental steps to the IgG content of the IgG sample before chromatography to obtain the IgG yields of different steps.
The process can be used for chromatographic treatment of at least one part of blood plasma, and can be maximally amplified according to the scale of a chromatographic column, and is theoretically unlimited.
System for preparing plasma IgG
The system can be divided into two forms of step production system and continuous production system. The step production system comprises five chromatographic columns which are sequentially loaded according to the sequence of the process flow and the conventional operation means in the prior art. The chromatographic liquids (first to fourth) are obtained by filtration using a conventional 0.22 μm filter cartridge; the fifth chromatographic solution is filtered by a 0.1 mu m deep filter and a 20nm nano membrane before being loaded. The production time can be finished within 2-6 days by adopting a step-by-step production system.
If it is desired to speed up the production process, a continuous production system (continuous flow production design) can be used, see fig. 2 and 3. The continuous production system is designed and formed on the basis of the step production system. The phenomenon of protein precipitation of the first to fifth chromatographic liquids in the treatment process is avoided, and the chromatographic liquids are one of the bases which can be realized by a continuous production system.
As shown in fig. 2, the apparatus for preparing plasma IgG according to the present technical solution includes a computer 12 and a chromatography unit casing 13, where the chromatography unit casing 13 includes a casing, one surface of the casing is a fixing surface 14 for fixing a chromatography column and other accessories, and one surface of the casing is a grid 15 for assisting heat dissipation of an internal unit. The inside of the case shell is fixed with various units and storage tanks (used for storing various buffers and intermediate products). The above-mentioned arrangements are conventional arrangements of chromatographic apparatuses, which are all prior art and are not described in detail herein. The main improvement points of the technical scheme are as follows: in order to realize the rapid separation and purification of the plasma IgG, the connection mode and the sequence of the chromatographic columns are set, namely, the main improvement point is a chromatographic unit of the preparation equipment of the plasma IgG, which is fixed on the fixing surface 14 of the chassis shell of the chromatographic unit chassis 13.
As shown in fig. 3, the chromatography unit includes a first anion exchange chromatography column 1, a second anion exchange chromatography column 2, a first cation exchange chromatography column 3, a third anion exchange chromatography column 4, a buffer bag 9, a prefilter 10, a nanofiltration 11, and a second cation exchange chromatography column 5, which are sequentially connected by a pipeline. The solid lines in fig. 3 represent the main material flow lines, the solid arrows represent the flow direction, and the dashed lines represent the waste flow direction. The pore size of the prefilter 10 is preferably 0.1 μm and the pore size of the nanofilter 11 is preferably 20nm. In the technical scheme, the chromatographic column, the pipeline, the liquid storage container and the like are all disposable equipment so as to ensure that no pollution source, viruses, bacteria and the like exist in the middle process. The materials continuously flow in the pipeline without stopping, thus realizing continuous production. The chromatographic column, the pipeline, the pump 7, the valve 8, the prefilter 10, the nanofiltration 11 and the like of the technical scheme are all conventional parts or devices for chromatographic analysis or production in the prior art.
The sample injection end of the first anion exchange chromatographic column 1 is communicated with a first pipeline 16 and a second pipeline 17, and a pump 7 and a valve 8 are arranged on the first pipeline 16 and the second pipeline 17 along the material flowing direction. The first line 16 is used for transporting the supernatant after centrifugation of the plasma, and the second line 17 is used for transporting a balancing liquid and a diluting liquid, which are used for adjusting the pH and the conductivity of the liquid, respectively. The balancing solution and the diluting solution can be selected from phosphate buffer, citric acid buffer, etc. By adding proper balance liquid and diluent to the materials, the pH and conductivity of the liquid to be chromatographed in each step can be ensured to be in a set range, and the dosage and solute concentration of the liquid to be chromatographed can be calculated through calculation and routine experiments. The balancing fluid and the dilution fluid are fed to the second line 17 via respective branch lines. The supernatant after centrifugation of the plasma is subjected to pH value and conductivity regulation (by mixing the balance liquid and the diluent liquid) to form a first to-be-chromatographed liquid, and the first to-be-chromatographed liquid enters the first anion exchange chromatographic column 1. The sample outlet end of the first anion exchange chromatographic column 1 is communicated with the second anion exchange chromatographic column 2 through a third pipeline 18, and the third pipeline 18 is communicated with a fourth pipeline 19. The first to-be-chromatographed liquid passes through the first anion exchange chromatographic column 1 to form a first chromatography flow-through liquid, and flows to the second anion exchange chromatographic column 2 through the third pipeline 18, and the fourth pipeline 19 is used for discharging waste liquid or collecting samples. The third pipeline 18 is provided with a valve 8, a pump 7 and a valve 8 in sequence, and the fourth pipeline 19 is provided with a valve 8.
The sample injection end of the second anion exchange chromatographic column 2 is communicated with a third pipeline 18 and also communicated with a fifth pipeline 20, and the third pipeline 18 and the fifth pipeline 20 are provided with a pump 7 and a valve 8 along the material flow direction. The third line 18 is used to deliver the first chromatography flow-through and the fifth line 20 is used to deliver the balancing and diluting liquids. The pH value and the conductivity of the first chromatographic flow-through liquid are regulated and controlled, a second liquid to be chromatographed is formed, and the second liquid enters a second anion exchange chromatographic column 2. The sample outlet end of the second anion exchange chromatographic column 2 is communicated with the first cation exchange chromatographic column 3 through a sixth pipeline 21, and the sixth pipeline 21 is also communicated with a seventh pipeline 22. The second to-be-chromatographed liquid passes through the second anion exchange chromatographic column 2 to form a second chromatography flow-through liquid, and flows to the first cation exchange chromatographic column 3 through a sixth pipeline 21, and a seventh pipeline 22 is used for discharging waste liquid or collecting samples. The sixth pipeline 21 is provided with a valve 8, a pump 7 and a valve 8 in sequence, and the seventh pipeline 22 is provided with a valve 8.
The sample injection end of the first cation exchange chromatographic column 3 is communicated with a sixth pipeline 21 and also communicated with an eighth pipeline 23, and the sixth pipeline 21 and the eighth pipeline 23 are provided with a pump 7 and a valve 8 along the material flow direction. The sixth line 21 is used for transporting the second chromatography flow-through and the eighth line 23 is used for transporting the balancing, diluting and eluting solutions. The pH value and the conductivity of the second chromatographic flow-through liquid are regulated and controlled, so that a third chromatographic liquid to be processed is formed, and the third chromatographic liquid enters the first cation exchange chromatographic column 3. The sample outlet end of the first cation exchange chromatographic column 3 is communicated with the third anion exchange chromatographic column 4 through a ninth pipeline 24, the ninth pipeline 24 is also communicated with a tenth pipeline 25, and the tenth pipeline 25 is used for discharging waste liquid or collecting samples. The second to-be-chromatographed liquid passes through the second anion exchange chromatography column 2, is discharged from the tenth pipeline 25, then conveys the first eluent from the eighth pipeline 23 into the first cation exchange chromatography column 3, forms the first chromatographic eluent through chromatography, and flows into the third anion exchange chromatography column 4 from the ninth pipeline. The ninth pipeline 24 is provided with a valve 8, a pump 7 and a valve 8 in sequence, and the tenth pipeline 25 is provided with a valve 8.
The sample injection end of the third anion exchange chromatographic column 4 is communicated with a ninth pipeline 24 and also communicated with an eleventh pipeline 26, and the ninth pipeline 24 and the eleventh pipeline 26 are provided with a pump 7 and a valve 8 along the material flow direction. A ninth line 24 is used to carry the first chromatographic eluent and an eleventh line 26 is used to carry the equilibration and dilution liquids. The pH value and the conductivity of the first chromatographic eluent are regulated, a fourth to-be-chromatographed liquid is formed, and the fourth to-be-chromatographed liquid enters a third anion exchange chromatographic column 4. The sample outlet end of the third anion exchange chromatographic column 4 is communicated with the second cation exchange chromatographic column 5 through a twelfth pipeline 27, and the twelfth pipeline 27 is also communicated with a thirteenth pipeline 28. The fourth to-be-chromatographed liquid passes through the third anion exchange chromatography column 4 to form a third layer of chromatography passing liquid, and flows to the second cation exchange chromatography column 5 through a twelfth pipeline 27, and a thirteenth pipeline 28 is used for discharging waste liquid or collecting samples. The twelfth line 27 is provided with a valve 8, a buffer bag 9, a pump 7, a prefilter 10, a nanofiltration 11, a pump 7 and a valve 8 in this order, and the thirteenth line 28 is provided with a valve 8.
The sample inlet end of the second cation exchange chromatographic column 5 is communicated with a twelfth pipeline 27 and also communicated with a fourteenth pipeline 29, and the twelfth pipeline 27 and the fourteenth pipeline 29 are provided with a pump 7 and a valve 8 along the material flowing direction. Twelve lines are used to convey the third layer of the flow-through, fourteenth line 29 is used to convey the balancing, diluting and eluting solutions. And the third layer of chromatographic penetrating liquid completes the regulation and control of pH value and conductivity to form fifth to-be-chromatographed liquid, and the fifth to-be-chromatographed liquid enters the second cation exchange chromatographic column 5. The sample outlet end of the second cation exchange chromatographic column 5 is communicated with downstream equipment (can be a collecting device or can be directly connected with a sterilizing device) through a fifteenth pipeline 30, the fifteenth pipeline 30 is also communicated with a sixteenth pipeline 31, the sixteenth pipeline 31 is used for discharging waste liquid or collecting samples, and the sixteenth pipeline 31 is provided with a valve 8. The fifth to-be-chromatographed liquid passes through the second cation exchange chromatography column 5, is discharged from the sixteenth pipe 31, and then the second eluent is sent from the fourteenth pipe 29 to the second cation exchange chromatography column 5, the second eluent is formed by chromatography, and flows into the downstream equipment from the fifteenth pipe.
Wherein the flow rate is controlled by the pumping speed of the pump 7 and the valve opening percentage of the valve 8 of the first, second, third, fifth, sixth, eighth, ninth, eleventh, twelfth, and fourteenth lines 16, 17, 18, 20, 21, 23, 24, 26, 27, 29. For example, the ratio of flow rates may be controlled to be 5:1:1:2:1:1:1:2:1:1. under the technological conditions of the technical scheme, the parameters such as pH and conductivity of five to-be-chromatographed liquids can be ensured to be in the required range (the required range is referred to as (one) ") by adopting the flow rate proportion. Parameters can be preset according to the conventional means in the prior art, the flow rate is ensured, and the concentration and the dosage of the balance liquid, the diluent and the like are determined according to the conventional means in the art, so that real-time monitoring is not needed. The computer 12 can be used for automatically detecting and monitoring pH, conductivity and UV monitoring, and monitoring equipment such as pH, conductivity and the like can not be arranged in the technical scheme because parameters such as a constant flow rate and the like are preset.
The equipment of the technical proposal can process 1 to 10 parts of plasma, and the column specifications of five chromatographic columns are 10 to 100mm (diameter) multiplied by 20 to 30cm (length). The product (second chromatographic eluent) prepared by the scheme is in a solution state, can directly enter subsequent sterilization and split charging treatment, does not need to be prepared independently, and saves the running cost. In addition, the preparation time of the technical scheme is very short, and the purification of a single blood plasma can be completed within 3-6 hours.
Example 2: igG preparation procedure for fresh plasma
For sudden public health events, emergency plasma IgG preparation is required, and the preparation process is different from that of example 1. For example, severe patients may occur due to different physical conditions, as well as mild patients who recover faster. The new coronal antibody exists in the blood plasma of the recovered human population, and can neutralize the new coronavirus. So that the recovery personnel blood plasma (fresh blood plasma) is collected, the purified blood plasma IgG is enriched and injected into the body of the severe patient, the recovery of the severe patient can be quickly assisted, and the death rate is reduced. The above process has high speed requirement and reliable quality. Enrichment and purification of plasma IgG against fresh plasma requires a special procedure.
This example is basically the same as example 1, except that in "(1) raw material plasma treatment" of the preparation flow of plasma IgG preparation, a cryoprecipitate separation unit was used to separate the material to be column-fed.
As shown in fig. 4, in terms of equipment, the present embodiment adds a cryoprecipitation separation unit 6 at the beginning of the process of the continuous production system in example 1. The specific structure of the cryoprecipitation separation unit 6 is shown in fig. 5 and 6. The cryoprecipitation separation unit 6 includes a refrigeration unit, which in this embodiment is specifically a cold box 40, and a plasma separation kit. The plasma separation kit includes a first container 32 and a second container 38, the first container 32 being snapped over the second container 38 and sealed to each other. The lower side of the first container 32 is opened and a filter membrane 36 having a pore size of 0.22 μm is sealed at the opening by conventional means in the art. An upper cover 33 is arranged above the first container 32, a stirring assembly 34 is arranged in the upper cover 33 in a penetrating manner, the stirring assembly 34 comprises a rotating shaft and a stirring paddle, the rotating shaft penetrates through the upper cover 33 and is fixed with an output shaft of a motor 35 through a screw, and the motor 35 is fixed on the upper cover 33 through the screw. The second container 38 is communicated with a pressure regulating pipe 37, the bottom of the second container 38 is communicated with a delivery pipe 39, and the delivery pipe 39 is used for communicating with the first pipeline 16. The plasma separation kit may be placed entirely within the cold box 40, or as shown in fig. 6, only the first container 32 may be placed within the cold box 40, the cold box 40 being secured to the housing by means conventional in the art. When the device is used, fresh plasma is poured into the first container 32 under the sterile environment, the upper cover 33 is covered, and the motor 35 is started to stir the plasma after the temperature is reduced to 0-2 ℃. At the same time, the inside of the second container 38 is regulated to negative pressure by the pressure regulating tube 37, and after fresh plasma is filtered, the filtrate enters the second container 38. After filtration, the filtrate is sent to subsequent processes via outlet line 39, as described in detail in example 1.
In order to ensure that the filtered plasma can smoothly and sufficiently enter the subsequent process flow, a plasma separation kit may be provided as in fig. 7. The second container 38 is opened at the bottom, and a pressurized filter 41 is sealed. A third container 42 is clamped under the second container 38 and sealed from each other. The third container 42 has an inverted cone shape, and the lower portion of the third container 42 communicates with the delivery tube 39. With this device, after the filtration is completed, the second container 38 is pressurized by the pressure regulating pipe 37, and the whole filtrate is transferred to the third container 42, and the filtrate can be ensured to enter the delivery pipe 39 by the reverse taper shape of the third container 42. The subsequent process is described in detail in example 1.
Example 3: preparation of single-person plasma IgG preparation
The following describes the effects of the present apparatus and process using a single plasma IgG product as an example, and the raw plasma is specifically healthy human plasma. In practice, other types of raw plasma may be used, not limited to healthy human plasma, such as, for example, one-person plasma with a covd-19 rehabilitation, so as to obtain total IgG in the raw plasma for subsequent therapeutic purposes, where the conventional plasma collection standard is 600g/580ml per bag per person, i.e. one bag of raw plasma contains 600g/580ml of plasma per bag. The apparatus of the present scheme (step production system or continuous production system) can be used to process 1-10 human parts of raw plasma. Five chromatographic procedures, selected packing, eluent type, chromatographic column specification, and the like, see table 1. To investigate the process effect of each step, the following experiment was performed not using a continuous flow production system but using five-step chromatography steps (using a step production system).
Table 1: process parameter setting
In order to study the effect of IgG preparation, the system of the present technical scheme is used to perform the following steps and parameter settings for enrichment and purification of IgG in healthy human plasma (if not specified, the following modes and parameter settings are adopted in the subsequent experiments):
(1) Taking a part of raw material plasma, sterilizing the surface by using 75% ethanol, breaking the plasma bag, controlling the temperature to be 3 ℃ for melting, centrifuging the plasma at a speed of more than 10000rpm/min by using a centrifuge after melting, and collecting supernatant.
(2) First anion exchange chromatography: the pH value of the supernatant of the centrifuged blood plasma is regulated to 7.6 (optional range is 6.0-7.6), the conductivity is 12.0mS/cm (optional range is 9.0-15.0 mS/cm), and the first to-be-chromatographed liquid is obtained after filtration by a filter element with the terminal of 0.22 mu m. Performing a first anion exchange chromatography on the first to-be-chromatographed liquid, adsorbing IgM and coagulation factors, and collecting the flow-through liquid containing IgG (first chromatography flow-through liquid) for the next chromatography. In a specific operation, the column used for the first anion exchange chromatography had a specification of 26mm×25cm and a packing of Fractgel EMD TMAE (M). After packing the column with the packing, the column was equilibrated with citric acid-sodium chloride buffer (pH 7.6). The loading amount of the first to-be-chromatographed liquid is 10mL of the first to-be-chromatographed liquid/mL of gel. The above operation is only one specific implementation form, in actual operation, other column specifications and strong anion fillers can be selected according to the process yield requirement, the column balance mode can be determined according to the specific filler selected, the loading quantity is also determined according to the filler property and the sample condition, and the details are not repeated later.
(3) Second anion exchange chromatography: the conductivity of the first chromatographic flow through liquid is regulated to 6.0mS/cm (the optional range is 4.0-6.0 mS/cm), the pH value is regulated to 6.5 (the optional range is 5.5-7.2), and the second chromatographic liquid to be obtained after filtration by a filter element with the terminal of 0.22 mu m. And (3) carrying out anion exchange chromatography for the second time on the second to-be-chromatographed liquid to remove trace impurity proteins, and collecting the flow-through liquid containing IgG (second chromatography flow-through liquid) for next chromatography. The selection of the second anion exchange chromatographic column and the packing, and the loading amount of the second chromatographic liquid to be processed are synchronous (2).
(4) First cation exchange chromatography: the pH value of the second chromatographic flow through liquid is adjusted to be 5.4 (optional range is 4.8-5.8), the conductivity is 2.0mS/cm (optional range is 0.5-2.0 mS/cm), and the third chromatographic liquid to be obtained is obtained by filtering with a filter element with the terminal of 0.22 mu m. Loading the third chromatographic solution to be subjected to cation exchange chromatographic column, adsorbing IgG, allowing other non-IgG proteins to flow through, eluting IgG by using eluent, and collecting eluent to obtain the first chromatographic eluent. In a specific operation, the first cation exchange chromatography used a column of 26mm by 25cm in size and with a packing of unigel 80sp. After packing the column with the packing, the column was equilibrated with citric acid-phosphoric acid buffer (ph 5.4). The loading amount of the third chromatographic liquid is 25mL of the third chromatographic liquid/mL of gel. The first chromatographic eluent comprises the following specific components: 0.05M sodium chloride (optional range 0.02-0.05M), pH 6.5 (optional range 6.5-8.5), conductivity 3.0mS/cm (optional range 2.5-5.0 mS/cm), and eluent amount of 2 column volumes.
(5) Third anion exchange chromatography: the conductivity of the first chromatographic eluent in the last step is adjusted to 1.5ms/cm (the optional range is 0.5-2.0 ms/cm), the pH value is 7.0 (the optional range is 6.5-8.5), and the fourth chromatographic liquid to be obtained is obtained by filtering with a filter element with the terminal of 0.22 mu m. And loading the fourth chromatographic solution to be subjected to anion exchange chromatography column, removing trace impurity proteins, and collecting the flow-through solution to obtain a third chromatographic flow-through solution. In a specific operation, the third anion exchange chromatography was performed using a column size of 16mm X25 cm and a packing of Unigel 80DEAE. After packing the column with the packing, the column was equilibrated with tromethamine-hydrochloric acid buffer (pH 7.0). The loading amount of the fourth to-be-chromatographed liquid is 20mL of the fourth to-be-chromatographed liquid/mL of gel.
(6) Nano-membrane filtration; the previous step of flow through liquid (third chromatography flow through liquid) is filtered by a 0.1 mu m deep filter, and then is filtered by a 20nm nano membrane, so as to obtain the third layer of flow through liquid after filtration.
(7) Second cation exchange chromatography: the pH value of the third layer of chromatographic penetrating liquid after filtration is adjusted to be 5.0 (the optional range is 4.0-6.0), the conductivity is 1.5mS/cm (the optional range is 0.5-2.5 mS/cm), a fifth to-be-chromatographed liquid is obtained, the fifth to-be-chromatographed liquid is loaded on a cation exchange chromatographic column, igG is adsorbed on the chromatographic column, and then the IgG is eluted by eluent (the second chromatographic eluent is obtained).
The subsequent operations were performed with reference to example 1, and subsequent experimental studies were performed using the above-described operation modes.
Experimental example 1
This experimental example tests the conditions of five chromatographic samples to be chromatographed, and the operation is described in example 2, and other parameter adjustments are shown in tables 2-6, unless otherwise specified. After adjustment of the pH and conductivity values, igG yields and purity were analyzed and the experimental results are shown in tables 2-6. A typical elution profile for the first ion exchange chromatography (exhibiting conductivity linear elution peaks) is shown in fig. 8, a typical elution profile for the second ion exchange chromatography is shown in fig. 9, a typical elution profile for the third ion exchange chromatography is shown in fig. 10, and a typical elution profile for the fourth ion exchange chromatography is shown in fig. 11. The detection result of the molecular size distribution of the sample after the fourth ion exchange chromatography is shown in fig. 12 (red is the result of the finished globulin product produced by the patent, and blue is the result of the finished globulin product produced by the low-temperature ethanol precipitation process), and the red is better than the blue). The fourth ion exchange chromatography is performed before and after the sample protein electrophoresis chart is shown in FIG. 13 (1 is Marker;2 is IgG product produced by adopting healthy human plasma and using the conventional low-temperature ethanol precipitation method in the prior art); 3 is 80DEAE chromatography; 4 is 80DEAE flow-through liquid 1;5 is 80DEAE flow-through liquid 2;6 is 80DEAE flow-through 3; wherein, the fluid passing through liquid 1-3 is the result obtained by each of the three repeated experiments).
Table 2: conditions and results of the first ion exchange chromatography (first anion exchange chromatography) (IgG yield = IgG content of first chromatography flow-through/IgG content of first solution to be chromatographed ×100%)
From the experimental results in table 2, it is clear that the conductivity of the first solution to be chromatographed has a relatively significant influence on the separation effect of IgG, igM, coagulation factors, etc. during the first ion exchange chromatography. In the range of 9.0 to 15.0mS/cm in conductivity, the IgG yield in the flow-through liquid is preferably 90% or more. However, too low a conductivity leads to a sharp drop in IgG yield in the flow-through to about 75%.
Table 3: conditions and results of the second ion exchange chromatography (second anion exchange chromatography) (IgG yield = IgG content of second chromatography flow-through/IgG content of second solution to be chromatographed ×100%)
From the experimental results in table 3, it is clear that the conductivity of the first solution to be chromatographed has a relatively significant effect on the separation of IgG and other hetero proteins during the second ion exchange chromatography. In the range of 4.0 to 6.0mS/cm in conductivity, the IgG yield in the flow-through liquid is preferably 90% or more. However, too low a conductivity leads to a sharp drop in IgG yield in the flow-through to about 80%.
Table 4: third ion exchange chromatography (first cation exchange chromatography) experimental conditions and results (IgG purity: igG content in first chromatography eluent/total protein content in first chromatography eluent. Times.100%; igG yield = IgG content in first chromatography eluent/IgG content in fourth to-be-chromatographed liquid. Times.100%)
From the experimental results in table 4, it is clear that the conductivity of the third solution to be chromatographed has a certain influence on the separation effect of IgG and has a very significant influence on the purity of IgG in the eluent in the first cation exchange chromatography. The sample conductivity higher than 2.0 can influence the yield of IgG in the eluent to a small extent. The sample conductivity lower than 0.5 can have a great influence on the purity of IgG in the eluent, so that the purity is reduced by about 50%.
Table 5: fourth time ion exchange chromatography (third anion exchange chromatography) experimental conditions and results (IgG purity: igG content in third chromatography flow-through/total protein content in third chromatography flow-through×100%; igG yield = third chromatography flow-through IgG content/fourth to-be-chromatographed liquid IgG content×100%)
From the experimental results in Table 5, it is clear that the conductivity and pH of the fourth chromatographic solution have a certain influence on the separation effect of IgG and the purity of IgG in the flow-through solution during the third anion exchange chromatography. The sample conductivity is less than 0.5, which can greatly influence the yield of IgG in the flow-through liquid, and "/" indicates that the purity is far lower than the process requirement. Sample conductivities above 2 negatively affect IgG purity in the fluid and reduce yields. In addition, pH values below the range required for this protocol will similarly reduce yield and purity.
Table 6: fifth ion exchange chromatography (second cation exchange chromatography) experimental conditions and results (IgG purity: igG content in second chromatography eluent/total protein content in second chromatography eluent×100%; igG yield = IgG content in second chromatography eluent/IgG content in fifth to-be-chromatographed liquid×100%)
As shown in the experimental results of Table 6, after the second cation exchange chromatography, the purity of the eluent IgG can reach 100%, which meets the requirement of the subsequent application.
Comparative example 1
This comparative example was substantially the same as example 3, and the parameter settings of experiment 24 of Table 6 (hereinafter, the operation mode of experiment 24 of Table 6 will be referred to as the present method) were adopted, except that the second anion chromatography (i.e., omitting step "(3) second anion exchange chromatography") was omitted. When parameters (pH and conductivity) are adjusted before the first cationic chromatography, more products are precipitated, which can cause the blockage of the first cationic chromatography column, greatly affect the production continuity and are not suitable for the process flow for rapidly and continuously producing IgG, so the inventor does not use the scheme of the comparative example. The first two chromatographic steps of this comparative example are similar to the applicant's prior patent CN111961130B (a method of extracting and separating IgM and IgG from plasma). Although the precipitate can be removed by filtration after the precipitation phenomenon occurs, the subsequent preparation process is continued, but the production expansion is not facilitated, the production continuity is ensured, and the purity and the yield of IgG are also affected to a certain extent.
There are a large number of labile proteins in the plasma that need to be removed to ensure the purity of IgG in the final product and to avoid precipitation during production affecting process continuity. The variety of labile proteins is large, and the inventors have found that these labile proteins need to be removed by different conditions. The first step of anion chromatography removes macromolecules and part of coagulation factors (part of unstable protein components), and the rest of unstable protein components need to be further removed by a second step of anion chromatography to ensure that IgG is sufficiently enriched and purified and process continuity is ensured.
Comparative example 2
This comparative example was substantially the same as example 3, and the parameter settings of experiment 24 of Table 6 (hereinafter, the operation mode of experiment 24 of Table 6 will be referred to as the present method) were adopted, except that the third anion chromatography (i.e., omitting step "(5) the third anion exchange chromatography") was omitted, and the experimental results were shown in Table 7. Omitting the third anion chromatography step results in the influence of purity, molecular size distribution and other indexes, and the product has heat stability and tiny hair precipitation.
Comparative example 3
This comparative example was essentially the same as example 3 and employed the experimental 24 parameter set of table 6 (hereinafter the manner of operation of experiment 24 of table 6 is referred to as the present method) except that the order of the first anion-exchange chromatography and the second anion-exchange chromatography was reversed (i.e., steps "(2) first anion-exchange chromatography" and "(3) second anion-exchange chromatography" were reversed). The sequence of the first anion chromatography and the second anion chromatography and the corresponding operation process are exchanged, which can lead to a large amount of protein precipitation in the initial stage of preparation and can not be used for subsequent experiments.
Comparative example 4
This comparative example was substantially the same as example 3, and the parameter settings of experiment 24 of Table 6 (hereinafter, the operation mode of experiment 24 of Table 6 will be referred to as the present method) was adopted, except that the order of the first cation chromatography and the second anion chromatography was reversed (i.e., the "first cation exchange chromatography and the" second anion exchange chromatography "of step" (4)) and the equipment setting order was adjusted, and the experimental results were shown in Table 7. The second anion chromatography was not performed before the first cation chromatography, and similar to the case of example 1, the product had more precipitation and the first cation chromatography column was blocked when the parameters (pH and conductivity) were adjusted before the first cation chromatography. Although the precipitated protein affects the production continuity, in the case of small-volume production and after completion of the whole production flow, the detection revealed that the IgG purity of the second chromatographic eluate was lowered and the molecular size distribution was deteriorated.
Comparative example 5
This comparative example was substantially the same as example 3, and the parameter settings of experiment 24 of Table 6 (hereinafter, the operation mode of experiment 24 of Table 6 will be referred to as the present method) were adopted, except that the order of the second cation chromatography and the third anion chromatography was reversed (i.e., the "third anion exchange chromatography of step" (5) and the "second cation exchange chromatography of step" (7) "were reversed), and the equipment setting order was adjusted, and the experimental results were shown in Table 7. The experimental results demonstrate that the order of the second cationic chromatography and the third anionic chromatography affects the purity and molecular size distribution of the final product. The third anion chromatography realizes the precise IgG and the removal of the impurity protein, and the second cation chromatography is used for enriching the IgG. If the two are sequentially exchanged, the effect of rapid purification cannot be achieved, and the final product has large volume and complex buffering components and is not suitable for human injection.
The quality evaluation is carried out by adopting the second chromatographic eluent (obtained after five times of chromatography), and the evaluation method adopts the detection standard method of three intravenous injection human immunoglobulin finished products in Chinese pharmacopoeia of 2020 edition.
Table 7: purification and enrichment of IgG (detection of second chromatography eluent, igG purity: igG content in second eluent; igG yield = second chromatography eluent IgG content/plasma supernatant IgG content)
Comparative example 6:
see inventor's prior patent CN111961130B (a method of extracting and separating IgM and IgG from plasma). The prior patent mainly considers that IgM and IgG are separated and relatively good product purity and yield are obtained, so the technical scheme of the prior patent is that two chromatography steps are carried out step by step, and the problem of process continuity is not considered. Since the prior patent does not have continuous production requirements, the inventor does not pay attention to whether protein precipitation occurs in the material or not, and does not form any prior art records. In order to develop a continuous production system, the inventor conducts more intensive research, and discovers that the pH value and the conductivity of a material are adjusted to the pH value of 5.4-5.6 and the conductivity of 2.0-4.0 mS/cm level of the patent before cationic chromatography, so that the phenomenon of protein precipitation of the system can be caused, and the realization of continuous production is seriously influenced.
To solve the above problems, the inventors have further added one step of anion chromatography between anion chromatography and cation chromatography. Before the second anion chromatography is not added, the pH value of the material before cation chromatography is 5.5, and the conductivity is 3.0-4.0 mS/cm, so that the optimal effect (IgG yield and yield) can be obtained. However, after the addition of the second anion chromatography, the process flow is changed considerably, and the empirical values of the prior patents are no longer suitable for the technical solution of the present invention, requiring the recourse to optimal conditions. In this patent scheme, since there are two subsequent chromatographic steps, the parameter setting principle of the third step of chromatography (first cationic chromatography) is: igG is recovered as much as possible, so that the purity is sacrificed to achieve the effect of improving the protein yield and reducing the loss. On the premise of meeting the requirement of yield, the purity of the IgG is ensured as much as possible. The conductivity and pH range of the technology of this patent can be selected to meet the above criteria, see experimental data in table 4. In the scheme of the patent, the conductivity of the sample is higher, and the yield of IgG in the eluent can be influenced to a certain extent; the sample conductivity is too low, and the purity of IgG in the eluent is seriously affected under the condition that the yield is not obviously improved.
The technical scheme of the patent is developed on the basis of the prior patent technology of comparative example 6, and solves the problem that protein is easy to separate out. Due to the addition of the new chromatography step, technical parameters need to be adjusted, thereby reducing IgG loss and ensuring the efficiency of the whole production flow.
In summary, the number of times of chromatography, the chromatography mode and the arrangement sequence of the chromatography columns are all obtained after a great number of experimental attempts by the inventor, each step of chromatography has specific parameters and requirements, and along with the progress of different chromatography steps, the pH and the conductivity of the chromatography all show regular changes, the product volume can be better controlled, and the effective injection for people can be produced in the shortest time. If the positions are randomly changed or a certain procedure is reduced, the whole process can be damaged, igG can not be effectively extracted, and the process continuity is affected.
The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
1. A method for preparing a single plasma IgG product, which is characterized in that: centrifuging or filtering the plasma to obtain a liquid phase fraction; and sequentially performing a first anion exchange chromatography, a second anion exchange chromatography, a first cation exchange chromatography, a third anion exchange chromatography and a second cation exchange chromatography on the liquid phase part to obtain a plasma IgG product.
2. The method of claim 1, wherein the step of preparing a single-serving plasma IgG preparation comprises: in the first anion exchange chromatography, the pH value of a liquid phase part is regulated to be 6.0-7.6, and the conductivity is regulated to be 9.0-15.0 mS/cm, so as to obtain a first to-be-chromatographed liquid; and loading the first chromatographic solution to be subjected to anion exchange chromatography column, and collecting the flow-through solution to obtain the first chromatographic flow-through solution.
3. The method of claim 2, wherein the step of preparing a single-serving plasma IgG preparation comprises: in the second anion exchange chromatography, the pH value of the first chromatography flow-through liquid is adjusted to be 5.5-7.2, and the conductivity is adjusted to be 4.0-6.0 mS/cm, so as to obtain a second to-be-chromatographed liquid; and loading the second chromatographic solution to be subjected to chromatography in an anion exchange chromatographic column, and collecting the flow-through solution to obtain a second chromatographic flow-through solution.
4. A method of preparing a single-serving plasma IgG preparation as claimed in claim 3, wherein: in the first cation exchange chromatography, the pH value of the second chromatography flow-through liquid is adjusted to be 4.8-5.8, and the conductivity is adjusted to be 0.5-2.0 mS/cm, so as to obtain a third to-be-chromatographed liquid; and loading the third to-be-chromatographed liquid into a cation exchange chromatographic column, discarding the flow-through liquid, eluting the cation exchange chromatographic column by using the first eluent, and collecting the eluent to obtain the first chromatographic eluent.
5. The method of claim 4, wherein the step of preparing a single-serving plasma IgG preparation comprises: in the third anion exchange chromatography, the pH value of the first chromatography eluent is regulated to be 6.5-8.5, and the conductivity is regulated to be 0.5-2.0 mS/cm, so as to obtain a fourth to-be-chromatographed liquid; and loading the fourth chromatographic solution to be subjected to chromatography in an anion exchange chromatographic column, and collecting the flow-through solution to obtain a third layer of flow-through solution.
6. The method of claim 5, wherein the step of preparing a single-serving plasma IgG preparation comprises: in the second cation exchange chromatography, regulating the pH value of the third layer of chromatographic penetrating fluid to be 4.0-6.0 and the conductivity to be 0.5-2.5 mS/cm to obtain fifth to-be-chromatographed fluid; and loading the fifth to-be-chromatographed liquid into a cation exchange chromatographic column, discarding the flow-through liquid, eluting the cation exchange chromatographic column by using a second eluent, and collecting the eluent to obtain the second chromatographic eluent.
7. The method of claim 6, wherein the step of preparing a single-serving plasma IgG preparation comprises: the ligand of the filler used for the first cation exchange chromatography or the second cation exchange chromatography is carboxymethyl or sulfopropyl.
8. The method of claim 7, wherein the step of preparing a single-serving plasma IgG preparation comprises: the ligands of the packing used for the first anion exchange chromatography, the second anion exchange chromatography or the third anion exchange chromatography are diethylaminoethyl or quaternary aminoethyl.
9. The method of claim 8, wherein the step of preparing a single-serving plasma IgG preparation comprises: the first eluent is a solution with sodium chloride as solute, pH value of 6.5-8.5 and conductivity of 2.5-5.0 mS/cm; the second eluent is a solution with sodium chloride and polysaccharide as solutes and pH of 4.0-7.0.
10. A plasma IgG preparation according to any one of claims 1 to 9, prepared by a method for preparing a single-serving plasma IgG preparation.
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