CN111205363A - Chromatographic process for improving capacity of removing IgA in human immunoglobulin - Google Patents
Chromatographic process for improving capacity of removing IgA in human immunoglobulin Download PDFInfo
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- CN111205363A CN111205363A CN202010067301.1A CN202010067301A CN111205363A CN 111205363 A CN111205363 A CN 111205363A CN 202010067301 A CN202010067301 A CN 202010067301A CN 111205363 A CN111205363 A CN 111205363A
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- 238000000034 method Methods 0.000 title claims abstract description 39
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 36
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 36
- 230000008569 process Effects 0.000 title claims abstract description 22
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 67
- 239000000243 solution Substances 0.000 claims abstract description 62
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000000706 filtrate Substances 0.000 claims abstract description 21
- 238000001914 filtration Methods 0.000 claims abstract description 16
- 239000002244 precipitate Substances 0.000 claims abstract description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 45
- 239000007788 liquid Substances 0.000 claims description 34
- 238000011068 loading method Methods 0.000 claims description 34
- 102000004169 proteins and genes Human genes 0.000 claims description 33
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 239000000047 product Substances 0.000 claims description 15
- 238000001556 precipitation Methods 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- 238000005070 sampling Methods 0.000 claims description 9
- 239000001632 sodium acetate Substances 0.000 claims description 9
- 235000017281 sodium acetate Nutrition 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 238000011067 equilibration Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000005227 gel permeation chromatography Methods 0.000 claims description 3
- 239000008215 water for injection Substances 0.000 claims description 3
- 229940072221 immunoglobulins Drugs 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
- 239000011550 stock solution Substances 0.000 claims 1
- 238000010253 intravenous injection Methods 0.000 abstract description 5
- 230000000694 effects Effects 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 7
- 210000002381 plasma Anatomy 0.000 description 5
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 230000002391 anti-complement effect Effects 0.000 description 2
- 108010008730 anticomplement Proteins 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000012501 chromatography medium Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000027219 Deficiency disease Diseases 0.000 description 1
- 208000007924 IgA Deficiency Diseases 0.000 description 1
- 208000028622 Immune thrombocytopenia Diseases 0.000 description 1
- 208000031951 Primary immunodeficiency Diseases 0.000 description 1
- 206010039915 Selective IgA immunodeficiency Diseases 0.000 description 1
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 1
- 230000003171 anti-complementary effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 201000007156 immunoglobulin alpha deficiency Diseases 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 208000029138 selective IgA deficiency disease Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Abstract
The invention discloses a chromatographic process for improving IgA capacity in human immunoglobulin, which is used for separating and purifying secondary precipitated components obtained by a low-temperature ethanol method and comprises the following steps: s1: dissolving and filtering the secondary precipitate to obtain filtrate; s2: adjusting the pre-chromatography parameters of the filtrate to obtain a pre-chromatography solution; s3: and (4) carrying out chromatography on the solution before chromatography. The process can control the IgA content in the human immunoglobulin solution for intravenous injection within the national requirement range, save the cost and improve the removal of the IgA capacity in the human immunoglobulin.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to a chromatography process for improving the capacity of removing IgA in human immunoglobulin.
Background
Human immunoglobulin is fresh plasma or frozen plasma with a storage life of no more than 2 years from healthy blood donors, and each batch should be mixed with the plasma from at least 1000 healthy blood donors. The immunoglobulin component is separated by fractional precipitation by low-temperature ethanol protein separation method, and is prepared by the procedures of ultrafiltration or freeze drying dealcoholization, concentration, virus inactivation and the like, and the purity of the immunoglobulin is not lower than 90%. Then preparing into a solution with the protein concentration of 10 percent, adding a proper amount of stabilizer, sterilizing, filtering, and aseptically filling to prepare the protein feed.
The active component of the intravenous injection human immunoglobulin is human immunoglobulin, and more than 1000 human blood plasma is used as a raw material to separate and prepare IgG immunoglobulin. The intravenous injection human immunoglobulin contains broad-spectrum IgG antibody resisting virus, bacteria or other pathogens, and in addition, idiotype and idiotype antibodies of the immunoglobulin can form a complex immune network, so the intravenous injection human immunoglobulin has the double treatment effects of immune substitution and immune regulation, and is mainly used for primary immunoglobulin deficiency, secondary immunoglobulin deficiency and autoimmune diseases clinically.
The technology for extracting human immunoglobulin is mainly a low-temperature ethanol method, which starts in the 40 th century of 20 th. However, the method of isolating proteins using low temperature ethanol cannot be used for intravenous injection, which causes serious side effects, because, in addition to the lack of IgG in vivo, another important reason is the presence of anticomplementary activity associated with IgG polymers in the preparation. In order to treat diseases such as primary immunodeficiency, immune thrombocytopenic purpura, and cutaneous mucosal lymph node syndrome, which require the injection of large doses of immunoglobulin, research has focused on how to reduce the anti-complement activity. The main methods include enzymatic and chemical modification methods, which affect the full-activity expression of IgG while eliminating and reducing the anti-complement activity. Such as the function and effect of the Fc region on IgG. Through multiple improvements, the existing preparation technology mainly comprises a low-temperature ethanol method combined with a chromatography method. The low-temperature ethanol method uses human plasma as a raw material, and generally obtains a secondary precipitation component of immunoglobulin through secondary precipitation, wherein the secondary precipitation component comprises most IgG, a small amount of IgA and IgM, and also comprises polymers and PKA.
The immunoglobulin is mainly rich in monomeric and dimeric IgG, and simultaneously contains trace other globulins such as IgA and IgM, and products containing the trace globulins can generate adverse reactions when being used, for example, clinical IgA deficiency patients can generate adverse reactions when using products containing IgA, and serious patients even threaten life.
Ion exchange chromatography is a purification method widely applied in the biopharmaceutical industry, and is mainly used for separating and purifying proteins by adjusting pH value and conductivity according to the difference of surface net charges of various protein molecules. In a chromatography process, the protein content of the solution and the loading of the chromatography medium are important factors affecting the time and scale of the chromatography process. Many manufacturers currently adopt chromatography technology to remove IgA in immunoglobulin, but the loading capacity is too low and the cost is too high, and how to achieve the effect of removing IgA while improving the loading capacity is a technical difficulty.
Disclosure of Invention
The invention aims to provide a chromatography process for improving the capacity of removing IgA in human immunoglobulin, which can effectively remove IgA and improve the capacity of IgA.
In order to achieve the purpose, the invention adopts the following technical scheme:
a chromatographic process for improving the removal of IgA capacity in human immunoglobulin is used for separating and purifying a secondary precipitation component obtained by a low-temperature ethanol method and comprises the following steps:
a chromatographic process for improving the removal of IgA capacity in human immunoglobulin is used for separating and purifying a secondary precipitation component obtained by a low-temperature ethanol method and comprises the following steps:
s1: dissolving and filtering the secondary precipitate to obtain filtrate;
s2: adjusting the pre-chromatography parameters of the filtrate to obtain a pre-chromatography solution;
s3: and (4) carrying out chromatography on the solution before chromatography.
Wherein, step S1 is specifically as follows:
p1: dissolving the secondary precipitation component with injection water with the volume 5-10 times of that of the secondary precipitation component, and stirring for 2-4 hours at 2-8 ℃ to obtain a dissolved solution;
p2: when the dissolved solution was filtered, the solution was filtered through a filter cartridge having a terminal diameter of 0.2 μm to obtain a filtrate.
Wherein, in the step P2, the filtering pressure is less than or equal to 0.15MPa during the filtering.
Wherein, step S2 is specifically as follows: adjusting the pH value of the filtrate to 5.70-5.90, the protein concentration to 10-30 g/L and the conductivity not higher than 0.30ms/cm to obtain a solution before chromatography.
Wherein, 1.0mol/L acetic acid solution is prepared, 300 to 400ml/min of the 1.0mol/L acetic acid solution is added into the filtrate, and the pH of the filtrate is adjusted to be 5.70 to 5.90; if the pH is lower than 5.70, adjusting by using 0.5mol/L sodium hydroxide solution; adjusting the concentration of the protein to be 10-30 g/L and the conductivity to be not higher than 0.30ms/cm by using low-temperature water for injection or balance liquid.
Wherein the equilibrium solution is 0.0025mol/L sodium acetate/acetic acid buffer solution, and the pH value is 5.70-5.90.
Wherein the chromatography comprises pre-gel equilibration, product chromatography and post-gel treatment; before chromatography, washing and balancing the gel until the difference between the pH value of the gel and the pH value of the solution before chromatography is-0.10; according to the protein concentration of the solution before chromatography, loading the product according to different gel chromatography loading capacity during chromatography, starting collecting flow-through liquid when the sample is loaded to an ultraviolet peak, and ejecting the product in a chromatographic column by using a balance liquid 2 after the sample loading is finished; uniformly stirring, measuring the volume of the flow-through liquid, and sampling to detect the concentration, pH and conductivity of the protein; during gel post-treatment, 3-5 times of bed volume of eluent, 3-5 times of bed volume of gel CIP liquid and 3-5 times of bed volume of gel sealing liquid are respectively flowed through linear flow velocity of 1.0-3cm/min, eluent is collected, volume is measured, and sampling is carried out to detect protein content and sample is reserved.
Wherein equilibrating the gel comprises: and respectively using 0.5mol/L sodium hydroxide solution to travel 3-5 times of the volume of the column bed, using balance liquid 1 to travel 3-5 times of the volume of the column bed and using balance liquid 2 to travel 3-5 times of the volume of the column bed according to the linear flow velocity of 1.5-3.0 cm/min, and balancing the pH until the pH value of the sample loading of the product is +/-0.10.
Wherein the equilibrium solution 1 is 0.025mol/L sodium acetate/acetic acid buffer solution with the pH value of 5.70-5.90; the equilibrium solution 2 is 0.0025mol/L sodium acetate/acetic acid buffer solution, and the pH value is 5.70-5.90.
The gel CIP liquid is a mixed liquid of 0.5mol/L sodium hydroxide, 1.0mol/L sodium chloride and 20% ethanol, and the gel sealing liquid is a mixed liquid of 0.15mol/L sodium chloride and 20% ethanol.
In conclusion, the invention has the following beneficial effects:
1. the protein concentration before chromatography is improved, the sample volume before chromatography is reduced, and the used production equipment is smaller under the production condition of the same scale, so that the waste of resources is reduced;
2. the loading capacity of unit gel is improved, the purchasing amount of the gel and the size of a chromatographic column are reduced under the same-scale production condition, and the cost is saved;
3. under the condition of high protein concentration and high loading capacity, the flow rate is reduced and the sample loading time of the equilibrium solution 1 is increased during chromatography, but the reduction amplitude of the sample chromatography time is greater than the increase time, so that the total chromatography time is reduced, and the pollution and denaturation possibly caused by long-time storage of a protein solution are avoided;
4. under the conditions that the protein content is 20g/L and the gel loading is 900g/L, the removal rate can reach more than 90 percent; under the conditions that the protein content is 30g/L and the gel loading is 1400g/L, the removal rate can reach more than 80 percent, and the residual IgA after removal is far above the requirements of people; that is, the loading can be increased to 1400g/L, i.e., 1400g protein per liter of chromatography medium can be chromatographed in solution.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
The first embodiment is as follows:
a chromatographic process for improving the removal of IgA capacity in human immunoglobulin is used for separating and purifying a secondary precipitation component obtained by a low-temperature ethanol method and comprises the following steps:
s1: dissolving and filtering the secondary precipitate to obtain filtrate;
specifically, dissolving the secondary precipitation component with injection water which is 5-10 times of the volume of the secondary precipitation component, and stirring for 2-4 hours at 2-8 ℃ to obtain a dissolved solution; sampling to detect endotoxin, and sampling to detect protein concentration, pH and conductivity after the endotoxin is qualified; filtering the dissolved solution with a filter element with a terminal diameter of 0.2 μm to obtain a filtrate, measuring the volume of the filtrate, sampling and detecting the protein concentration, pH and conductivity, and washing the filter with low-temperature water for injection. When filtering, the filtering pressure is less than or equal to 0.15 MPa. The specific process of the filtration can be firstly filtering by using a 0.45 mu m filter membrane, then filtering by using a 0.2 mu m filter membrane, so that the micro impurities in the dissolved solution can be removed step by step, and meanwhile, the pressure during the filtration is controlled, so that the filtration effect is ensured on one hand, and the activity of various effective proteins in the dissolved solution is ensured on the other hand.
S2: adjusting the pre-chromatography parameters of the filtrate to obtain a pre-chromatography solution;
specifically, adjusting the pH of the filtrate to 5.70-5.90, adjusting the protein concentration to 10-30 g/L, and adjusting the conductivity to be not higher than 0.30ms/cm to obtain a solution before chromatography; preparing 1.0mol/L acetic acid solution, adding the 1.0mol/L acetic acid solution into the filtrate at 300-400 ml/min, and adjusting the pH of the filtrate to 5.70-5.90; if the pH is lower than 5.70, adjusting by using 0.5mol/L sodium hydroxide solution; adjusting the protein concentration to 10-30 g/L and the conductivity to be not higher than 0.30ms/cm by using low-temperature injection water or equilibrium liquid (0.0025mol/L sodium acetate/acetic acid buffer solution, pH is 5.70-5.90).
S3: carrying out chromatography on the solution before chromatography;
specifically, the chromatography includes pre-gel equilibration, product chromatography and post-gel treatment; before chromatography, washing and balancing the gel until the difference between the pH value of the gel and the pH value of the solution before chromatography is-0.10; according to the protein concentration of the solution before chromatography, loading the product according to different gel chromatography loading capacity during chromatography, starting collecting flow-through liquid when the sample is loaded to an ultraviolet peak, and ejecting the product in a chromatographic column by using a balance liquid 2 after the sample loading is finished; uniformly stirring, measuring the volume of the flow-through liquid, and sampling to detect the concentration, pH and conductivity of the protein; during gel post-treatment, 3-5 times of bed volume of eluent, 3-5 times of bed volume of gel CIP liquid and 3-5 times of bed volume of gel sealing liquid are respectively flowed through linear flow velocity of 1.0-3cm/min, eluent is collected, volume is measured, and sampling is carried out to detect protein content and sample is reserved.
Wherein equilibrating the gel comprises: respectively using 0.5mol/L sodium hydroxide solution to walk 3-5 times of the volume of the column bed, balance liquid 1 to walk 3-5 times of the volume of the column bed and balance liquid 2 to walk 3-5 times of the volume of the column bed according to the linear flow velocity of 1.5-3.0 cm/min, and balancing the pH until the pH value of a sample of the product is +/-0.10; the balance liquid 1 is 0.025mol/L sodium acetate/acetic acid buffer solution with the pH value of 5.70-5.90, and the balance liquid 2 is 0.0025mol/L sodium acetate/acetic acid buffer solution with the pH value of 5.70-5.90; the gel CIP liquid is a mixed liquid of 0.5mol/L sodium hydroxide, 1.0mol/L sodium chloride and 20% ethanol, and the gel sealing liquid is a mixed liquid of 0.15mol/L sodium chloride and 20% ethanol.
IgA detection results are shown in Table 1, and IgA in 50g/L of finished product protein is controlled at 200 mu g/ml, namely the IgA content after chromatography is controlled at 4mg/g protein.
TABLE 1 IgA detection results
According to table 1, it can be known that, under the condition of high protein concentration and high loading capacity, although the flow rate is reduced and the sample loading time of the equilibrium solution 1 is increased during chromatography, the reduction range of the sample chromatography time is greater than the increase time, so that the total chromatography time is reduced, and the pollution and denaturation possibly caused by long-time storage of the protein solution are avoided; under the conditions of 20g/L protein content and 900g/L gel loading, the removal rate can reach over 90 percent, under the conditions of 30g/L protein content and 1400g/L gel loading, the removal rate can reach over 80 percent, and the residual IgA after removal is far above our requirements.
In the existing immunoglobulin production process, the low-load chromatography under low protein concentration is adopted in consideration of the influence of the chromatography back pressure and the chromatography effect, wherein the influence factors of the chromatography back pressure comprise: protein concentration, chromatography flow rate, chromatography column height; the influencing factors of the ion exchange chromatography effect include: pH, conductivity, protein concentration, chromatography flow rate, chromatography column height.
In order to achieve high-loading (400g/L to 1400g/L gel) chromatography under high protein concentration (from 10g/L to 30g/L), firstly, the height of a chromatographic column is arranged from 20cm to 25cm, secondly, the conductivity of a sample is reduced from 1.6ms/cm to below 0.30ms/cm, thirdly, the flow rate is reduced from 1.5cm/min to 1.0cm/min, fourthly, the pH is difficult to balance when a balancing solution 2 balances the chromatographic column due to the reduction of the conductivity and the flow rate (generally, the pH can be balanced to ten CV by 3-5 CV, but now, dozens of CV are needed), therefore, the balancing solution 1 is introduced, the pH of the balancing solution 1 is consistent with that of the balancing solution 2, but the conductivity of the balancing solution 1 is 10 times higher than that of the balancing solution 2, and the conductivity and the pH can be balanced within a required range within 10CV by balancing the balancing solution 1 and then the balancing solution 2.
In summary, the invention has the following advantages:
the method has the advantages that the protein concentration before chromatography is improved, the sample volume before chromatography is reduced, the used production equipment is smaller under the same scale production condition, and the waste of resources is reduced;
secondly, the loading capacity of unit gel is improved, the purchasing quantity of the gel and the size of a chromatographic column are reduced under the condition of the same-scale production, and the cost is saved;
thirdly, under the condition of high protein concentration and high loading capacity, the flow rate is reduced and the sample loading time of the equilibrium solution 1 is increased during chromatography, but the reduction amplitude of the sample chromatography time is greater than the increase time, so that the total chromatography time is reduced, and the pollution and denaturation possibly caused by long-time storage of the protein solution are avoided;
fourthly, under the conditions of 20g/L protein content and 900g/L gel loading, the removal rate can reach more than 90 percent, under the conditions of 30g/L protein content and 1400g/L gel loading, the removal rate can reach more than 80 percent, and the residual IgA after removal is far above the requirements of people.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A chromatographic process for improving the removal of IgA capacity in human immunoglobulin is used for separating and purifying a secondary precipitation component obtained by a low-temperature ethanol method, and is characterized by comprising the following steps of:
s1: dissolving and filtering the secondary precipitate to obtain filtrate;
s2: adjusting the pre-chromatography parameters of the filtrate to obtain a pre-chromatography solution;
s3: and (4) carrying out chromatography on the solution before chromatography.
2. The chromatographic process for improving the IgA loading removal from human immunoglobulin according to claim 1, wherein step S1 comprises the following steps:
p1: dissolving the secondary precipitation component with injection water with the volume 5-10 times of that of the secondary precipitation component, and stirring for 2-4 hours at 2-8 ℃ to obtain a dissolved solution;
p2: when the dissolved solution was filtered, the solution was filtered through a filter cartridge having a terminal diameter of 0.2 μm to obtain a filtrate.
3. The chromatographic process for improving the IgA loading in human immunoglobulin according to claim 2, wherein in step P2, the filtering pressure is lower than or equal to 0.15 MPa.
4. The chromatographic process for improving the IgA loading removal from human immunoglobulin according to claim 1, wherein step S2 comprises the following steps: adjusting the pH value of the filtrate to 5.70-5.90, the protein concentration to 10-30 g/L and the conductivity not higher than 0.30ms/cm to obtain a solution before chromatography.
5. The chromatography process for improving the IgA loading removal in human immunoglobulin according to claim 4, wherein 1.0mol/L acetic acid solution is prepared, 300-400 ml/min of the 1.0mol/L acetic acid solution is added into the filtrate, and the pH of the filtrate is adjusted to 5.70-5.90; if the pH is lower than 5.70, adjusting by using 0.5mol/L sodium hydroxide solution; adjusting the concentration of the protein to be 10-30 g/L and the conductivity to be not higher than 0.30ms/cm by using low-temperature water for injection or balance liquid.
6. The chromatographic process for improving the removal of IgA load from human immunoglobulin of claim 5, wherein the equilibration solution is 0.0025mol/L sodium acetate/acetic acid buffer solution with pH 5.70-5.90.
7. The chromatographic process for increasing the removal of IgA load in human immunoglobulins according to claim 1 wherein said chromatography comprises pre-gel equilibration, product chromatography and post-gel treatment; before chromatography, washing and balancing the gel until the difference between the pH value of the gel and the pH value of the solution before chromatography is-0.10; according to the protein concentration of the solution before chromatography, loading the product according to different gel chromatography loading capacity during chromatography, starting collecting flow-through liquid when the sample is loaded to an ultraviolet peak, and ejecting the product in a chromatographic column by using a balance liquid 2 after the sample loading is finished; uniformly stirring, measuring the volume of the flow-through liquid, and sampling to detect the concentration, pH and conductivity of the protein; during gel post-treatment, 3-5 times of bed volume of eluent, 3-5 times of bed volume of gel CIP liquid and 3-5 times of bed volume of gel sealing liquid are respectively flowed through linear flow velocity of 1.0-3cm/min, eluent is collected, volume is measured, and sampling is carried out to detect protein content and sample is reserved.
8. The chromatographic process for increasing the capacity of IgA removal from human immunoglobulins of claim 7 wherein equilibrating the gel comprises: and respectively using 0.5mol/L sodium hydroxide solution to travel 3-5 times of the volume of the column bed, using balance liquid 1 to travel 3-5 times of the volume of the column bed and using balance liquid 2 to travel 3-5 times of the volume of the column bed according to the linear flow velocity of 1.5-3.0 cm/min, and balancing the pH until the pH value of the sample loading of the product is +/-0.10.
9. The chromatographic process for improving the removal of the IgA load in human immunoglobulin according to claim 8, wherein the equilibrium solution 1 is 0.025mol/L sodium acetate/acetic acid buffer solution, and has a pH value of 5.70-5.90; the equilibrium solution 2 is 0.0025mol/L sodium acetate/acetic acid buffer solution, and the pH value is 5.70-5.90.
10. The chromatographic process for improving the removal of IgA load from human immunoglobulin of claim 7, wherein the gel CIP solution is a mixture of 0.5mol/L NaOH, 1.0mol/L NaCl and 20% ethanol and the gel stock solution is a mixture of 0.15mol/L NaCl and 20% ethanol.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN103087188A (en) * | 2005-05-10 | 2013-05-08 | 墨累古尔本合作有限公司 | Immunoglobulin fraction and process therefor |
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