CN112480246A - Separation and purification method of dog immunoglobulin and application thereof - Google Patents
Separation and purification method of dog immunoglobulin and application thereof Download PDFInfo
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- CN112480246A CN112480246A CN202011529682.7A CN202011529682A CN112480246A CN 112480246 A CN112480246 A CN 112480246A CN 202011529682 A CN202011529682 A CN 202011529682A CN 112480246 A CN112480246 A CN 112480246A
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- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 96
- 238000000034 method Methods 0.000 title claims abstract description 83
- 238000000746 purification Methods 0.000 title claims abstract description 17
- 238000000926 separation method Methods 0.000 title abstract description 26
- 241000282465 Canis Species 0.000 claims abstract description 74
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 53
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 33
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 19
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- 239000006228 supernatant Substances 0.000 claims abstract description 19
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- 238000000108 ultra-filtration Methods 0.000 claims description 22
- 239000000872 buffer Substances 0.000 claims description 20
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- 238000002360 preparation method Methods 0.000 claims description 19
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- 238000001914 filtration Methods 0.000 claims description 14
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- 238000005086 pumping Methods 0.000 claims description 13
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- 238000005119 centrifugation Methods 0.000 claims description 5
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- 239000012501 chromatography medium Substances 0.000 description 7
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- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
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- GUPXYSSGJWIURR-UHFFFAOYSA-N 3-octoxypropane-1,2-diol Chemical compound CCCCCCCCOCC(O)CO GUPXYSSGJWIURR-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
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- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- IYLLULUTZPKQBW-UHFFFAOYSA-N Acrinol Chemical compound CC(O)C(O)=O.C1=C(N)C=CC2=C(N)C3=CC(OCC)=CC=C3N=C21 IYLLULUTZPKQBW-UHFFFAOYSA-N 0.000 description 1
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- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
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- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
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Images
Classifications
-
- 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
Abstract
The invention relates to a separation and purification method of canine immunoglobulin and application thereof, wherein the separation and purification method comprises the following steps: and (3) sequentially carrying out cation exchange chromatography and hydrophobic chromatography on the centrifugal supernatant of the canine plasma to obtain the canine immunoglobulin. The separation and purification method of the canine immunoglobulin creatively improves the purity of the canine immunoglobulin of the pretreated healthy canine plasma only by a specific two-step chromatography combination mode, namely cation exchange chromatography and hydrophobic chromatography, has higher product purity and lower content of impurity protein, and greatly improves the application safety. Compared with the traditional low-temperature ethanol precipitation process, the yield loss of the canine immunoglobulin caused by the processes of precipitation, depth filtration and dissolution is reduced, and the chromatography scale of 100L of medium can process the plasma raw material which is equivalent to 100-1000L, and can be completely enlarged to the actual production.
Description
Technical Field
The invention belongs to the technical field of blood products, particularly relates to a separation and purification method of canine immunoglobulin and application thereof, and particularly relates to a separation and purification method of canine immunoglobulin with high purity, high yield and simple operation and application thereof.
Background
Plasma immunoglobulins are the most abundant plasma proteins in plasma except albumin, and immunoglobulin G is the most important class of immunoglobulins, which contain various biological activities and mainly function to participate in immune defense reactions. Its indications include prevention of bacterial and viral infections, substitution of xenogenic serum products, suppression of primary immune reactions, etc.
With the increase in pet dogs, the demand for plasma products from dogs has increased. The current methods for purifying plasma immunoglobulins are mainly based on the Cohn-Oncley method or the Kistler-Nitschmamn cryoethanolic method, and furthermore, polyethylene glycol precipitation, ion exchange chromatography and pH4 treatment methods are also commonly used for purifying plasma immunoglobulins. For example, CN100569287 discloses an immunoglobulin for intravenous injection of dogs, a preparation method and a preparation thereof, which is the immunoglobulin which is prepared from blood plasma or blood serum from healthy dogs by a low-temperature ethanol method or a rivanol combined low-temperature ethanol method, has a pH value of 3.0-5.4 and a purity of not less than 95% and can be used for intravenous injection of dogs, and is prepared into an immunoglobulin injection containing 5-15% maltose, 5-10% glucose or one or two protective agents in 3-10% glucose saline, and simultaneously has a protein content of 1-15%.
However, the existing extraction and purification processes of the canine plasma immunoglobulin are complicated, the plasma components are separated, precipitated, prepared, dissolved, filtered and the like, the process steps are multiple, the period is long, the environmental requirement is high, the resource demand is high, the product purity is low, the lower purity can influence the stability of the product and the safety of clinical application, and more adverse drug reactions are caused.
At present, the production process of immunoglobulin products is optimized at home and abroad. The most common optimization mode of the low-temperature ethanol process is as follows: different components are obtained by a low-temperature ethanol process, and then are combined with different types of chromatographic methods to produce albumin, immunoglobulin and other various proteins. For example, CN111166877A discloses a method for preparing rabies immunoglobulin, which comprises separating and extracting immunoglobulin by low temperature ethanol method, purifying by Capto DEAE ion exchange chromatography, ultrafiltering, pasteurizing and inactivating virus, and preparing and packaging to obtain rabies immunoglobulin.
In addition, the combined chromatography is also used for purifying the plasma immunoglobulin, but the existing combined chromatography needs 3 or more steps, the steps are complicated, the medium requirement is high, the medium cost required in the actual production is huge, the production efficiency is low, and the method is difficult to be used for large-scale preparation.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for separating and purifying canine immunoglobulin and application thereof, in particular to a method for separating and purifying canine immunoglobulin with high purity, high yield and simple operation and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for separating and purifying canine immunoglobulin, comprising: and (3) sequentially carrying out cation exchange chromatography and hydrophobic chromatography on the centrifugal supernatant of the canine plasma to obtain the canine immunoglobulin.
The separation and purification method of the canine immunoglobulin creatively improves the purity of the canine immunoglobulin of the pretreated healthy canine plasma only by a specific two-step chromatography combination mode, namely cation exchange chromatography and hydrophobic chromatography, and the purity is more than or equal to 95 percent, the polymer content is less than or equal to 5 percent, the yield reaches 70 percent, the purity of the product is higher, the content of the foreign protein is lower, and the application safety is greatly improved. Compared with the traditional low-temperature ethanol precipitation process, the subsequent steps of low-temperature ethanol precipitation and deep filtration separation of components are reduced, the process time is shortened by 24-48h, and the yield loss of the canine immunoglobulin in the processes of precipitation, deep filtration and dissolution is reduced.
The cation exchange chromatography mainly plays a role in removing albumin in the supernatant of blood plasma, the hydrophobic chromatography directly elutes immunoglobulin by utilizing the hydrophobic property difference between proteins, so that the effective separation of the immunoglobulin and other proteins is realized, and the separation period is effectively shortened by two-step chromatographic purification. And the chromatography scale of 100L of medium can process the plasma raw material which is equivalent to 100-1000L, and can be completely enlarged to the actual production.
In the present invention, the method of cation exchange chromatography specifically comprises: pumping the centrifugal supernatant of the canine plasma into a balanced cation exchange chromatographic column, then eluting by adopting an elution buffer solution, and collecting the eluent.
Preferably, the packing medium of the cation exchange chromatography column may be exemplarily selected from CM QZT6FF, SP QZT6FF, CM QZT XL, SP QZT XL, CM Sepharose FF, SP Sepharose XL, Capto S, and the like.
Preferably, the ratio of the bed volume to the loading volume of the cation exchange chromatography column is 1 (2-40), such as 1:2, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, or 1:40, and other specific values in the value range can be selected, and are not described in detail herein.
Preferably, the cation exchange chromatography column is equilibrated in the following manner: the method comprises the steps of equilibrating 2-5 column volumes (such as 2 column volumes, 3 column volumes, 4 column volumes, 5 column volumes and the like) by using a buffer containing not more than 100mmol/L sodium citrate (such as 90mmol/L, 80mmol/L, 70mmol/L, 60mmol/L, 50mmol/L, 40mmol/L, 30mmol/L and the like), pH 4.0-6.9 (such as pH 4.0, pH 5.0, pH 6.0, pH 6.9 and the like), wherein other specific point values in each numerical value range can be selected, and are not repeated.
In the above-mentioned equilibrium treatment, the pH of the buffer is specifically selected in the range of 4.0 to 6.9 because if the pH is further raised, the adsorption capacity of the cation exchange medium is lowered, so that a large amount of immunoglobulin flows through, and the yield of cation exchange chromatography is lowered; if the pH value is further reduced, part of the hetero-protein is positively charged and adsorbed on the cation exchange medium, which greatly reduces the separation accuracy of the ion exchange chromatography.
Preferably, the centrifuged supernatant of canine plasma is filtered through a 0.45 μm microfiltration membrane before pumping.
Preferably, the elution is performed with a buffer of not more than 100mmol/L sodium citrate (e.g., 90mmol/L, 80mmol/L, 70mmol/L, 60mmol/L, 50mmol/L, 40mmol/L, 30mmol/L, etc.), not more than 1.5mol/L sodium chloride (e.g., 1.5mol/L, 1.0mol/L, 0.5mol/L, etc.), pH 4.0 to 6.9 (e.g., pH 4.0, pH 5.0, pH 6.0, pH 6.9, etc.); other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
In the elution process, the pH value of the buffer solution is specifically selected to be in the range of 4.0-6.9 because if the pH value is further increased, part of the hybrid protein is eluted at the same time, so that the purity of the immunoglobulin is reduced; if the pH is further lowered, the immunoglobulin is denatured and inactivated, and the yield of the immunoglobulin activity is lowered.
Preferably, the flow rate of the elution is 60-240cm/h, such as 60cm/h, 100cm/h, 150cm/h, 200cm/h or 240cm/h, and other specific values in the numerical range can be selected, and are not repeated herein.
In the present invention, the method of hydrophobic chromatography specifically comprises: pumping the eluent after the cation exchange chromatography into a hydrophobic chromatographic column after the balance, then adopting an elution buffer solution for elution, and collecting the eluent.
Preferably, the packing medium of the hydrophobic chromatography column may be selected from Butyl QZTFF, Butyl-S QZTFF, Octyl QZTFF, Phenyl QZTFF (HS), Phenyl QZTFF (LS), Butyl Sepharose 4FF, Butyl-S Sepharose6FF, Octyl Sepharose 4FF, Phenyl Sepharose6FF (HS), Phenyl Sepharose6FF (LS), Capto Butyl, Capto Octyl, Capto Phenyl ImpRes, Capto Phenyl (HS), and the like, as an example.
Preferably, the ratio of the bed volume to the loading volume of the hydrophobic chromatography column is 1 (2-20), 1:2, 1:5, 1:8, 1:10, 1:12, 1:15, 1:16, 1:18, or 1:20, and the like, and other specific points in the numerical range can be selected, which is not described in detail herein.
Preferably, the hydrophobic chromatography column has an equilibrium pattern of: the buffer solution containing not more than 100mmol/L (e.g., 90mmol/L, 80mmol/L, 70mmol/L, 60mmol/L, 50mmol/L, 40mmol/L, 30mmol/L, etc.) sodium citrate, not more than 2mol/L (e.g., 2mol/L, 1.5mol/L, 1mol/L, 0.5mol/L, etc.) ammonium sulfate, pH 4.0-6.0 (e.g., pH 4.0, pH 5.0, pH 6.0, etc.) is used to balance 2-5 column volumes (e.g., 2 column volumes, 3 column volumes, 4 column volumes, 5 column volumes, etc.), and other specific point values within the above value ranges can be selected, and will not be described in detail herein.
The effect of adding ammonium sulfate in the above operation is to increase the adsorption between the immunoglobulin and the hydrophobic chromatography medium and increase the adsorption capacity of the immunoglobulin on the hydrophobic chromatography medium, and the concentration range is not more than 2mol/L because if the concentration is further increased, the hetero-protein with weaker hydrophobicity is also adsorbed on the hydrophobic chromatography medium, thereby reducing the separation precision of the hydrophobic chromatography.
Preferably, the eluate after the cation exchange chromatography is adjusted to a conductivity of 50 to 150mS/cm (e.g., 50mS/cm, 80mS/cm, 100mS/cm, 120mS/cm, 150mS/cm, etc.) with ammonium sulfate before pumping, adjusted to a pH of 4.0 to 6.0 (e.g., pH 4.0, pH 5.0, pH 6.0, etc.) with hydrochloric acid or sodium hydroxide, and then filtered through a 0.45 μm microfiltration membrane. Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the elution is performed with not more than 100mmol/L (e.g., 90mmol/L, 80mmol/L, 70mmol/L, 60mmol/L, 50mmol/L, 40mmol/L, 30mmol/L, etc.) sodium citrate, not more than 1mol/L (e.g., 1mol/L, 0.8mol/L, 0.5mol/L, 0.3mol/L, etc.) ammonium sulfate, pH 4.0 to 6.0 (e.g., pH 4.0, pH 5.0, pH 6.0, etc.). Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
The effect of adding ammonium sulfate in the above operation is to adjust the adsorption strength of immunoglobulin and hydrophobic chromatography medium, and realize effective elution, and the concentration range is not more than 1mol/L because if the concentration is further increased, the interaction of immunoglobulin and hydrophobic medium is increased, so that the elution peak is trailing, and the separation selectivity is reduced.
Preferably, the flow rate of the elution is 60-240cm/h, such as 60cm/h, 100cm/h, 150cm/h, 200cm/h or 240cm/h, and other specific values in the numerical range can be selected, and are not repeated herein.
In the invention, the manner of obtaining the centrifugal supernatant of the canine plasma is as follows: after centrifugation, the canine plasma is filtered through gauze, the pH is adjusted to 4.0-6.9 (for example, pH 4.0, pH 5.0, pH 6.0, pH 6.9, etc., and other specific values within the value range can be selected, which is not described herein again), centrifuged again, and the supernatant is collected and diluted.
Preferably, the centrifugation is performed at 2-8 deg.C (e.g., 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C, etc.) at 8000- > 12000r/min (e.g., 8000r/min, 9000r/min, 10000r/min, 11000r/min, 12000r/min, etc.) for 20-40min (e.g., 20min, 30min, 40min, etc.); other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the gauze is 8 layers and above.
Preferably, the pH adjusting agent is 0.5 to 3mol/L acid, such as 0.5mol/L, 1mol/L, 1.5mol/L, 2mol/L, 2.5mol/L, or 3 mol/L; the acid can be selected from acetic acid or hydrochloric acid.
Preferably, the dilution is performed with a buffer containing not more than 100mmol/L (e.g., 90mmol/L, 80mmol/L, 70mmol/L, 60mmol/L, 50mmol/L, 40mmol/L, 30mmol/L, etc.) sodium citrate, pH 4.0-6.9 (e.g., pH 4.0, pH 5.0, pH 6.0, pH 6.9, etc.); other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the dilution is performed until the protein concentration is 5-20g/L, such as 5g/L, 10g/L, 15g/L, 20g/L, and the like, and other specific values within the numerical range can be selected, which is not described in detail herein.
In a second aspect, the present invention provides a use of the method for separating and purifying canine immunoglobulin according to the first aspect in the preparation of a canine immunoglobulin preparation.
In a third aspect, the present invention provides a method for preparing a canine immunoglobulin preparation, the method comprising: performing ultrafiltration concentration and buffer replacement treatment on the canine immunoglobulin obtained by the separation and purification method of the first aspect: adjusting pH of the eluate to 3.7-4.7, ultrafiltering with 10-50kDa ultrafiltration membrane, adjusting protein concentration to 5-15%, pH to 3.7-4.7, sodium ion content below 100mmo/L, and conductivity to 2-10 mS/cm.
Preferably, the ultrafiltration concentration and buffer replacement treatment further comprises: glycine is added into the solution after ultrafiltration to 0.2-0.3mol/L, such as 0.2mol/L, 0.22mol/L, 0.25mol/L, 0.28mol/L or 0.3mol/L, and other specific values in the numerical range can be selected, and are not repeated herein.
The effect of the glycine addition is to increase the stability of the immunoglobulin and to reduce the loss of activity of the preparation during the subsequent low pH incubation virus inactivation procedure.
Preferably, the ultrafiltration concentration and buffer replacement treatment further comprise a virus inactivation treatment: adjusting pH of the treated dog immunoglobulin preparation to 3.8-4.2, and storing at 24-26 deg.C for more than 21 days.
Compared with the prior art, the invention has the following beneficial effects:
the separation and purification method of the canine immunoglobulin creatively improves the purity of the canine immunoglobulin of the pretreated healthy canine plasma only by a specific two-step chromatography combination mode, namely cation exchange chromatography and hydrophobic chromatography, and the purity is more than or equal to 95 percent, the polymer content is less than or equal to 5 percent, the yield reaches 70 percent, the purity of the product is higher, the content of the foreign protein is lower, and the application safety is greatly improved.
Compared with the traditional low-temperature ethanol precipitation process, the subsequent steps of low-temperature ethanol precipitation and deep filtration separation of components are reduced, the process time is shortened by 24-48h, and the yield loss of the canine immunoglobulin caused by the processes of precipitation, deep filtration and dissolution is reduced.
The cation exchange chromatography mainly plays a role in removing albumin in the supernatant of blood plasma, the hydrophobic chromatography directly elutes immunoglobulin by utilizing the hydrophobic property difference between proteins, so that the effective separation of the immunoglobulin and other proteins is realized, and the separation period is effectively shortened by two-step chromatographic purification. And the chromatography scale of 100L of medium can process the plasma raw material which is equivalent to 100-1000L, and can be completely enlarged to the actual production.
Drawings
FIG. 1 is an HPLC chromatogram in example 1;
FIG. 2 is an HPLC chromatogram in example 3.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The method for determining the purity of the canine immunoglobulin and the method for determining the polymer content in the canine immunoglobulin referred to in the following examples are HPLC, and the column model thereof is TSK-GEL G3000 SWXL(300 mm. times.7.8 mm, I.D.), the mobile phase is 50mmol/L PB +0.1mol/L Na2SO4(pH 7.0); the purity is calculated by the second method of 0541 in the pharmacopoeia 2015 edition general rules of China; the determination method of the polymer content is the rule 3122 of the Chinese pharmacopoeia 2015 edition; the yield is calculated by the third method of the general rule 0731 in the Chinese pharmacopoeia 2015 edition.
Example 1
The present invention provides a method for separating and purifying canine immunoglobulin and a canine immunoglobulin product, which comprises the following steps:
(1) pretreatment of dog plasma: slowly thawing fresh frozen dog plasma at-20 deg.C in a refrigerator at 4 deg.C, centrifuging (4 deg.C, 10000r/min, 30min), and filtering the centrifuged supernatant with gauze (8 layers). The pH was adjusted to 5.0 with 0.5mol/L hydrochloric acid, centrifuged again (4 ℃, 10000r/min, 30min), the supernatant was taken and diluted with equilibration buffer (sodium citrate 50mmol/L, pH ═ 5.0) to a protein concentration of 5 g/L.
(2) Cation exchange chromatography: the CM QZT6FF medium was packed in a chromatography column with a ratio of bed volume to loading volume of 1:5, and the packed column was equilibrated for 5 column volumes with equilibration buffer (sodium citrate 50mmol/L, pH ═ 5.0); after centrifugal separation of dog plasma, filtering supernate by a microfiltration membrane of 0.45 mu m and pumping the supernate into a chromatographic column; eluting with buffer solution (sodium citrate 50mmol/L, sodium chloride 1.0mol/L, pH ═ 5.0), and collecting eluate during chromatography; the flow rate is controlled at 240cm/h during the chromatography.
(3) Hydrophobic chromatography: loading hydrophobic chromatography medium Phenyl QZTFF (HS) into a chromatography column, wherein the ratio of the volume of a column bed to the volume of a sample loading is 1:5, and balancing the loaded chromatography column by using an equilibrium buffer (50 mmol/L sodium citrate and 1mol/L, pH ammonium sulfate is 6.0) for 5 column volumes; adjusting the pH of the eluate of cation exchange chromatography to 6.0, adjusting the conductivity to 100mS/cm with ammonium sulfate, filtering with 0.45 μm microfiltration membrane, and pumping into chromatography column; eluting the chromatographic column by using a buffer solution (50 mmol/L sodium citrate and 0.5mol/L, pH-4.5 ammonium sulfate), and collecting eluent in the chromatographic process; the flow rate is controlled at 240cm/h during the chromatography.
(4) Ultrafiltration concentration and buffer replacement: the pH of the chromatography mixture was adjusted to 4.1 with 0.1mo1/L NaOH solution. And (3) performing ultrafiltration chromatography on the mixed solution by using a 10kDa ultrafiltration membrane package, wherein an ultrafiltration diluent is normal saline, and the concentration of the product protein is adjusted to be 10%, the pH value is 4.1, the content of sodium ions is 50mmol/L, and the conductivity is 4.0mS/cm to obtain the canine immunoglobulin product solution.
(5) Glycine was added to the above solution of the canine immunoglobulin preparation to a final concentration of 0.25mol/L, the pH was adjusted to 4.0, and the mixture was left at 25 ℃ for 21 days to inactivate the virus.
The dog immunoglobulin prepared under the process condition is determined to have the purity of 96.1 percent, the polymer content of 2.0 percent and the yield of 70 percent. Its HPLC profile is shown in FIG. 1 (in the figure, 1 represents a canine immunoglobulin monomer, and 2 represents a canine immunoglobulin multimer).
Example 2
The present invention provides a method for separating and purifying canine immunoglobulin and a canine immunoglobulin product, which comprises the following steps:
(1) pretreatment of dog plasma: slowly thawing fresh frozen dog plasma at-20 deg.C in a refrigerator at 4 deg.C, centrifuging (4 deg.C, 10000r/min, 30min), and filtering the centrifuged supernatant with gauze (8 layers). The pH was adjusted to 6.0 with 0.5mol/L hydrochloric acid, centrifuged again (4 ℃, 10000r/min, 30min), the supernatant was taken and diluted with equilibration buffer (sodium citrate 50mmol/L, pH ═ 6.0) to a protein concentration of 10 g/L.
(2) Cation exchange chromatography: packing the SP QZT6FF medium in a chromatographic column, wherein the ratio of the volume of a column bed to the volume of a sample is 1:5, and balancing 5 column volumes of the packed chromatographic column by using an equilibrium buffer (sodium citrate 20mmol/L, pH ═ 6.0); after centrifugal separation of dog plasma, filtering supernate by a microfiltration membrane of 0.45 mu m and pumping the supernate into a chromatographic column; eluting with buffer solution (sodium citrate 20mmol/L, sodium chloride 1.0mol/L, pH ═ 6.0), and collecting eluate during chromatography; the flow rate is controlled to be 120cm/h in the chromatography process.
(3) Hydrophobic chromatography: loading the hydrophobic chromatography medium Octyl Sepharose 4FF into a chromatography column, wherein the ratio of the bed volume to the loading volume is 1:5, and the loaded chromatography column uses an equilibrium buffer (sodium citrate 100mmol/L, ammonium sulfate 1.5mol/L, pH is 5.5) to balance 5 column volumes; adjusting pH of the eluate of cation exchange chromatography to 5.5, adjusting conductivity to 120mS/cm with ammonium sulfate, filtering with 0.45 μm microfiltration membrane, and pumping into chromatography column; eluting the chromatographic column by using a buffer solution (100 mmol/L of sodium citrate and 1.0mol/L, pH-5.5 of ammonium sulfate), and collecting eluent in the chromatographic process; the flow rate is controlled to be 120cm/h in the chromatography process.
(4) Ultrafiltration concentration and buffer replacement: the pH of the chromatography mixture was adjusted to 4.1 with 0.1mo1/L NaOH solution. And (3) performing ultrafiltration chromatography on the mixed solution by using a 10kDa ultrafiltration membrane package, wherein an ultrafiltration diluent is normal saline, and the concentration of the product protein is adjusted to be 10%, the pH value is 4.1, the content of sodium ions is 50mmol/L, and the conductivity is 4.0mS/cm to obtain the canine immunoglobulin product solution.
(5) Glycine was added to the above solution of the canine immunoglobulin preparation to a final concentration of 0.25mol/L, the pH was adjusted to 4.0, and the mixture was left at 25 ℃ for 21 days to inactivate the virus.
The dog immunoglobulin prepared under the process condition is determined to have the purity of 95.5 percent, the polymer content of 3.5 percent and the yield of 69 percent.
Example 3
The present invention provides a method for separating and purifying canine immunoglobulin and a canine immunoglobulin product, which comprises the following steps:
(1) pretreatment of dog plasma: slowly thawing fresh frozen dog plasma at-20 deg.C in a refrigerator at 4 deg.C, centrifuging (4 deg.C, 10000r/min, 30min), and filtering the centrifuged supernatant with gauze (8 layers). The pH was adjusted to 6.5 with 0.5mol/L HCl, centrifuged again (4 ℃, 10000r/min, 30min), the supernatant was removed and diluted with equilibration buffer (sodium citrate 20mmol/L, pH ═ 6.5) to a protein concentration of 15 g/L.
(2) Cation exchange chromatography: the Capto S media was loaded into the column at a bed volume to loading volume ratio of 1:15, and the loaded column was equilibrated for 5 column volumes using equilibration buffer (sodium citrate 20mmol/L, pH ═ 6.5); after centrifugal separation of dog plasma, filtering supernate by a microfiltration membrane of 0.45 mu m and pumping the supernate into a chromatographic column; eluting with buffer solution (sodium citrate 20mmol/L, sodium chloride 1.0mol/L, pH ═ 6.5), and collecting eluate during chromatography; the flow rate is controlled to be 60cm/h in the chromatography process.
(3) Hydrophobic chromatography: filling a hydrophobic chromatography medium Capto Butyl into a chromatographic column, wherein the ratio of the volume of a column bed to the volume of a sample is 1:15, and balancing 5 column volumes of the filled chromatographic column by using an equilibrium buffer (20 mmol/L of sodium citrate and 2mol/L, pH of ammonium sulfate is 6.5); adjusting pH of the eluate of cation exchange chromatography to 6.5, adjusting conductivity to 150mS/cm with ammonium sulfate, filtering with 0.45 μm microfiltration membrane, and pumping into chromatography column; eluting the chromatographic column by using a buffer solution (sodium citrate is 20mmol/L, ammonium sulfate is 1.0mol/L, pH ═ 6.5), and collecting eluent in the chromatographic process; the flow rate is controlled to be 60cm/h in the chromatography process.
(4) Ultrafiltration concentration and buffer replacement: the pH of the chromatography mixture was adjusted to 4.1 with 0.1mo1/L NaOH solution. And (3) performing ultrafiltration chromatography on the mixed solution by using a 10kDa ultrafiltration membrane package, wherein an ultrafiltration diluent is normal saline, and the concentration of the product protein is adjusted to be 10%, the pH value is 4.1, the content of sodium ions is 50mmol/L, and the conductivity is 4.0mS/cm to obtain the canine immunoglobulin product solution.
(5) Glycine was added to the above solution of the canine immunoglobulin preparation to a final concentration of 0.25mol/L, the pH was adjusted to 4.0, and the mixture was left at 25 ℃ for 21 days to inactivate the virus.
The dog immunoglobulin prepared under the process condition is determined to have the purity of 96.1 percent, the polymer content of 3.8 percent and the yield of 70 percent. Its HPLC profile is shown in FIG. 2 (in the figure, 1 represents a canine immunoglobulin monomer, and 2 represents a canine immunoglobulin multimer).
Example 4
This example provides a method for separation and purification of canine immunoglobulin and a canine immunoglobulin preparation, which differs from example 3 only in that: in the cation exchange chromatography of step (2), the pH of the equilibration buffer was 7.5, the pH of the elution buffer was 7.5, and all other conditions were kept constant.
The dog immunoglobulin prepared under the process condition is determined to have the purity of 95.1 percent, the polymer content of 3.0 percent and the yield of 45 percent.
Example 5
This example provides a method for separation and purification of canine immunoglobulin and a canine immunoglobulin preparation, which differs from example 3 only in that: in the cation exchange chromatography of step (2), the pH of the equilibration buffer was 3.5, the pH of the elution buffer was 3.5, and all other conditions were kept constant.
The dog immunoglobulin prepared under the process condition is determined to have the purity of 75%, the polymer content of 2.9% and the yield of 65%.
Example 6
This example provides a method for separation and purification of canine immunoglobulin and a canine immunoglobulin preparation, which differs from example 3 only in that: in the hydrophobic chromatography in the step (3), the concentration of ammonium sulfate in the equilibrium buffer solution is 3mol/L, and other conditions are kept unchanged.
The dog immunoglobulin prepared under the process conditions was determined to have a purity of 87%, a polymer content of 5.2% and a yield of 62%.
Example 7
This example provides a method for separation and purification of canine immunoglobulin and a canine immunoglobulin preparation, which differs from example 3 only in that: in the hydrophobic chromatography in the step (3), the concentration of ammonium sulfate in the elution buffer solution is 2mol/L, and other conditions are kept unchanged.
The dog immunoglobulin prepared under the process condition is determined to have the purity of 88 percent, the polymer content of 4.8 percent and the yield of 40 percent.
The applicant states that the present invention is illustrated by the above examples to describe a method for separating and purifying canine immunoglobulin and the application thereof, but the present invention is not limited to the above examples, i.e., it is not meant that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for separating and purifying canine immunoglobulin, which comprises the following steps: and (3) sequentially carrying out cation exchange chromatography and hydrophobic chromatography on the centrifugal supernatant of the canine plasma to obtain the canine immunoglobulin.
2. The method for separating and purifying canine immunoglobulin according to claim 1, wherein the cation exchange chromatography comprises: pumping the centrifugal supernatant of the canine plasma into a balanced cation exchange chromatographic column, then eluting by adopting an elution buffer solution, and collecting the eluent.
3. The method for separating and purifying canine immunoglobulin according to claim 2, wherein the ratio of the bed volume to the loading volume of the cation exchange chromatography column is 1 (2-40);
preferably, the cation exchange chromatography column is equilibrated in the following manner: equilibrating 2-5 column volumes with buffer containing no more than 100mmol/L sodium citrate at pH 4.0-6.9.
4. The method for separating and purifying canine immunoglobulin according to claim 2 or 3, wherein the centrifuged supernatant of canine plasma is filtered through a 0.45 μm microfiltration membrane before being pumped;
preferably, the elution is performed by using a buffer solution with the pH value of 4.0-6.9 and no more than 100mmol/L sodium citrate and no more than 1.5mol/L sodium chloride;
preferably, the flow rate of the elution is 60-240 cm/h.
5. The method for separating and purifying canine immunoglobulin according to any one of claims 1 to 4, wherein the hydrophobic chromatography specifically comprises: pumping the eluent after the cation exchange chromatography into a hydrophobic chromatographic column after the balance, then adopting an elution buffer solution for elution, and collecting the eluent.
6. The method for separating and purifying canine immunoglobulin of claim 5, wherein the ratio of the bed volume to the loading volume of the hydrophobic chromatography column is 1 (2-20);
preferably, the hydrophobic chromatography column has an equilibrium pattern of: equilibrating 2-5 column volumes with a buffer containing no more than 100mmol/L sodium citrate, no more than 2mol/L ammonium sulfate, pH 4.0-6.0;
preferably, the eluent after the cation exchange chromatography is adjusted to the conductivity of 50-150mS/cm by ammonium sulfate before pumping, the pH is adjusted to 4.0-6.0 by hydrochloric acid or sodium hydroxide, and then the eluent is filtered by a microfiltration membrane with the diameter of 0.45 mu m;
preferably, the elution is performed by using a buffer solution with the pH value of 4.0-6.0 and no more than 100mmol/L sodium citrate and no more than 1mol/L ammonium sulfate;
preferably, the flow rate of the elution is 60-240 cm/h.
7. The method for separating and purifying canine immunoglobulin according to any one of claims 1 to 6, wherein the canine plasma supernatant obtained by centrifugation is obtained by: centrifuging dog plasma, filtering with gauze, adjusting pH to 4.0-6.9, centrifuging again, collecting supernatant, and diluting;
preferably, the centrifugation mode is centrifugation for 20-40min at the speed of 8000-12000r/min at the temperature of 2-8 ℃;
preferably, the gauze is 8 layers or more;
preferably, the pH regulator is 0.5-3mol/L acid;
preferably, the dilution is performed with a buffer containing not more than 100mmol/L sodium citrate at pH 4.0-6.9;
preferably, the dilution is to a protein concentration of 5-20 g/L.
8. Use of the method according to any one of claims 1 to 7 for the isolation and purification of canine immunoglobulin for the preparation of canine immunoglobulin preparations.
9. A method for preparing a canine immunoglobulin preparation, the method comprising: subjecting the canine immunoglobulin obtained by the isolation and purification method according to any one of claims 1 to 7 to ultrafiltration concentration and buffer exchange treatment: adjusting pH of the eluate to 3.7-4.7, ultrafiltering with 10-50kDa ultrafiltration membrane, adjusting protein concentration to 5-15%, pH to 3.7-4.7, sodium ion content below 100mmo/L, and conductivity to 2-10 mS/cm.
10. The method of claim 9, wherein the ultrafiltration concentration and buffer exchange treatment further comprise: adding glycine to 0.2-0.3mol/L in the solution after ultrafiltration;
preferably, the ultrafiltration concentration and buffer replacement treatment further comprise a virus inactivation treatment: adjusting pH of the treated dog immunoglobulin preparation to 3.8-4.2, and storing at 24-26 deg.C for more than 21 days.
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