CN102286099B - Human cytomegalovirus immunoglobulin for intravenous injection and preparation method thereof - Google Patents
Human cytomegalovirus immunoglobulin for intravenous injection and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a human cytomegalovirus immunoglobulin for intravenous injection and a preparation method thereof, and aims to improve the purity, yield and safety of the product. In the invention, the specific activity of the human cytomegalovirus immunoglobulin for intravenous injection is not less than 2.5 PEI-U/mg, the anti-CMV titer is not less than 100 PEI-U/ml, the purity is greater than 98.2%, and the protein content is 51-55 mg/ml. Caprylic acid precipitation and anion exchange chromatography are used instead of the partial ethanol precipitation step in the traditional low-temperature ethanol method, thereby keeping IgG in the supernate all the time so as to keep the IgG activity; and processes of caprylic acid virus inactivation and nano film virus removal are used, thereby effectively ensuring the safety of the product. Researches show that the preparation method disclosed by the invention improves the purity, yield and safety of the product, saves the energy and reduces the production cost.
Description
Technical field
The present invention relates to a kind of human normal immunoglobulin and preparation method thereof, particularly a kind of quiet note cytomegalovirus human normal immunoglobulin and preparation method thereof.
Background technology
Cytomegalovirus CMV (Cytomegalovirus) is the DNA virus that herpetoviridae β belongs to, and the pregnant woman who is caused by cytomegalovirus, newborn infant, Organ Transplantation Patients, immunosuppressed patient infect mortality ratio and can reach 50%～80%.Cytomegalovirus human normal immunoglobulin CMV-IgG, because of in can be special and cytomegalovirus, mainly treats with it cytomegalovirus severe infection causing in pregnant woman, newborn infant, immunosuppressed patient and organ transfer operation clinically.Cytomegalovirus natural infection rate in general population can reach more than 80%, from healthy population, screen and gather the blood plasma containing high-titer cytomegalovirus IgG antibody, adopt specific separation purification method to prepare cytomegalovirus human normal immunoglobulin medicine, the severe infection causing because of cytomegalovirus for treatment has irreplaceable clinical value.
At present, the specific human immunoglobulins medicine having gone on the market adopts cold ethanol method (Edwin J.Cohn mostly, L.E.Strong, W.L.Hughes JR., D.J.Mulford, J.N.Ashworthm, M.Melin and H.L.Taylor, J.Am.Chem.Soc., 68 (1946) 459-475.) carry out separating-purifying, professor Edwin.J.Cohn of Gai Fayou nineteen forty-six Harvard University invention, by the pH in adjusting process, protein concentration, temperature, five parameters of alcohol concn and ionic strength realize the separated of different albumen.Long-term practice shows, there is the defect of following several respects in cold ethanol method: first, ethanol is a kind of protein denaturant, easily causes IgG structural modification and deactivation in sepn process, the rate of recovery that causes tiring is lower, also may cause the formation of new antigenic determinant; Secondly, compare column chromatography technology, ethanol deposition and purification efficiency is lower, conventionally need to reduce the purity requirement that protein recovery reaches product, so technological process yield is on the low side, and product purity is not high; Again, because of sepn process, conventionally need under cold condition, carry out, also have the defects such as hardware and running cost are high, and labour intensity is large.
Summary of the invention
The object of this invention is to provide a kind of quiet note cytomegalovirus human normal immunoglobulin and preparation method thereof, the technical problem that solve is purity, yield and the security that improves product.
The present invention is by the following technical solutions: a kind of quiet note cytomegalovirus human normal immunoglobulin, described quiet note cytomegalovirus human normal immunoglobulin specific activity is not less than 2.5PEI-U/mg, anti-CMV tires and is not less than 100PEI-U/ml, and purity is greater than 98.2%, and protein content is 51～55mg/ml.
A method for quiet note cytomegalovirus human normal immunoglobulin, comprises the following steps:
(1) FI+II+III, FII+III precipitation
The human plasma of the anti-CMV high-titer that the euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured, melts slurry at 2～30 ℃, merges and mixes;
1. FI+II+III precipitation
With physiological saline, regulate serum protein content to 45～55mg/ml, with glacial acetic acid, regulate pH to 6.0～6.5, add 95% ethanol or dehydrated alcohol to regulate alcohol concn to 20～25%, temperature of reaction is-5.5～-4.5 ℃, stirring reaction is 4～6 hours, reacts the complete centrifugal or separated acquisition of press filtration FI+II+III precipitation;
2. FII+III precipitation
With physiological saline, regulate serum protein content to 45～55mg/ml, with glacial acetic acid, regulate pH to 6.8～7.2, to add volume ratio mark be 95% ethanol or dehydrated alcohol and regulate alcohol concn to 7.5～8.5%, temperature of reaction is-2.5～-2.0 ℃, stirring reaction 4 hours, react complete through the centrifugal or separated removal of press filtration FI precipitation, obtain supernatant liquor, with glacial acetic acid, regulate supernatant liquor pH to 6.0～6.5, add 95% ethanol or dehydrated alcohol to regulate alcohol concn to 20～25%, temperature of reaction is-5.5～-4.5 ℃, stirring reaction is 4～6 hours, react complete through the centrifugal or separated acquisition of press filtration FII+III precipitation,
(2) FI+II+III or FII+III resolution of precipitate
FI+II+III or FII+III precipitation with the pH of 0.9～1.1 times of plasma volume be 4.8～5.2, concentration is that 20～80mM sodium acetate buffer stirs 8～16 hours at 2～8 ℃, makes to precipitate abundant dissolving, through centrifugal or press filtration separation of supernatant;
(3) sad precipitation
With 4mol/L acetic acid or 0.5～1mol/L sodium hydroxide, regulate supernatant liquor pH to 4.5～5.5, it is sad by 10～100mmol/L concentration, to add, and at 18～25 ℃, stirring reaction is 1～3 hour, through centrifugal or filtering separation supernatant liquor;
(4) sad inactivation of virus
Supernatant liquor is 1.0 μ m membrane filtrations with aperture, controlled filter pressure is not more than 0.25MPa, with 4mol/L acetic acid or 0.5～1mol/L sodium hydroxide, regulate filtrate pH to 4.5～5.5, add water for injection or sad concentration to the 20～80mmol/L of sad adjusting suspension, at 20～30 ℃, stir centrifugal or filtering separation supernatant liquor 1～2 hour;
(5) ethanol precipitation
With 0.5～1mol/L hydrochloric acid or sodium hydroxide, regulate supernatant liquor pH to 4.5～5.5, by 12～16% concentration, add 95% ethanol or dehydrated alcohol to carry out precipitin reaction, at-4.0～-2.5 ℃, stirring reaction is 2～8 hours, centrifugal or press filtration separation of supernatant;
(6) ultrafiltration
Supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, filtrate is concentrated with 15～20 times of 30KD ultra-filtration membranes, and through the phosphate buffered saline buffer ultrafiltration dialysis of 20～60mmol/L of 8～10 times of volume pH 6.0～7.1, after ultrafiltration, receiving sample, to control protein content be 25～40mg/ml;
(7) anion-exchange chromatography
With the phosphate buffered saline buffer that pH is 6.0～7.1, concentration is 20～60mmol/L as level pad balance chromatography column 8～10 column volumes, by every milliliter of filler, be not more than 70～80% of the maximum carrying capacity of filler and calculate loading protein content, after loading, collect and penetrate liquid, hanging column foreign protein is through the phosphate buffered saline buffer wash-out of the 20～60mmol/L of the pH 6.0～7.1 containing 2mol/LNaCl;
(8) nanometer film is except virus filtration
With the hydrochloric acid adjusting of 0.5～1mol/L, penetrate liquid pH to 4.2～5.0, after 0.1 μ m filter membrane pre-filtering, with Novasip DV20 nano-film filtration, except virus, controlled filter pressure is not more than 0.25Mpa;
(9) ultrafiltration
Except filtrate after virus is through 30KD ultra-filtration membrane protein concentrate content to 80～100mg/ml, with 8～10 times of water for injection ultrafiltration, after ultrafiltration, receiving sample control protein content is 80～150mg/ml;
(10) preparation
Measure stoste protein content after ultrafiltration, with water for injection dilution, adjust goods protein content to 51～55mg/ml, by 9～11% amount, add maltose, with 0.5～1mol/L salt acid for adjusting pH to 3.8～4.2 simultaneously.
Method of the present invention is degerming packing after described preparation steps, and through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa, by protein content 51～55mg/ml, tires and is not less than the packing of 100PEI-U/ml specification.
Goods protein content after method sampling mensuration of the present invention packing, anti-CMV tires, purity, molecular size distribution, sad residual quantity, the project quality index of osmotic pressure molar density.
Of the present inventionly in anion-exchange chromatography step, add filler, filler is selected from DEAE Sepharose Fast Flow, TOYOPEARL DEAE 650M or Macro-Prep DEAE Media.
The present invention compares with the whole process cold ethanol fractional separation processing method of prior art, and the technique effect having is as follows:
(1) adopt sad precipitation and the anion-exchange chromatography technique of mild condition, reduced the step of cold ethanol precipitation, when improving product yield, can effectively keep the activity of IgG, improved purity and yield.The technological process rate of recovery of tiring is 40～65%, and purification is that 5.30～8.14, the IgG rate of recovery is greater than 4.9g/L, and purity is greater than 98.2%, IgG polymer and is less than 0.1%.
(2) in technological process, adopted sad inactivation of virus and nano-film filtration except viral technique, effectively deactivation and removal are viral, and in conjunction with anion-exchange chromatography, technological process virus reducing amount is greater than 12log
10.
(3) sad precipitation can effectively precipitate NIg class impurity, and IgG, IgA, copper-protein be retained in supernatant liquor, and the precipitation process antibody titer rate of recovery can reach more than 90%.
(4) anion-exchange chromatography can effectively be removed polymer and acid foreign protein, and purified rear the finished product are not containing impurity such as polymer, albumin.
(5) production cycle of technique of the present invention is 5～7 days, and the Low-temperature Ethanol Processes production cycle of prior art is 28～30 days, has effectively improved production efficiency, has reduced the usage quantity of ethanol simultaneously, reduces energy consumption and labour intensity, can effectively save production cost.
In a word, the preparation technology of the cytomegalovirus human normal immunoglobulin that the present invention is used has not only improved product purity, yield and security, can also save energy consumption and reduce production costs.
Accompanying drawing explanation
Fig. 1 is the quiet note of the present invention cytomegalovirus human normal immunoglobulin purifying process schema.
Fig. 2 is that prior art cold ethanol method is prepared the general population's immunoglobulin (Ig) process flow sheet.
Fig. 3 is the quiet note of embodiment 1 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Fig. 4 is the quiet note of embodiment 2 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Fig. 5 is the quiet note of embodiment 3 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Fig. 6 is the quiet note of embodiment 4 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Fig. 7 is the quiet note of embodiment 5 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Fig. 8 is the quiet note human normal immunoglobulin of comparative example 1 pH4 purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail.
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured human plasma 20 person-portions of anti-CMV high-titer, under 25 ℃ of conditions, melts slurry, and after mixing, volume is 11540ml.
(2) add physiological saline 2310ml to regulate serum protein content to 49.53mg/ml, add glacial acetic acid to regulate pH to 6.18, add dehydrated alcohol 3926ml to regulate suspension alcohol concn to 22%, regulate temperature of reaction to-5.0 ℃, stirring reaction 4 hours, reacts complete centrifugation and obtains FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 4.93, concentration is that 50mmol/L sodium acetate buffer 11500ml dissolves, and stirs 12 hours centrifugation supernatant liquor at 4 ℃.
(4) with 4mol/L acetic acid, regulate supernatant pH to 4.57, it is sad that 60mmol/L concentration adds, and at 21 ℃, stirring reaction is 3 hours, centrifugation supernatant liquor.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, controlled filter pressure is not more than 0.25MPa, with 0.5mol/L sodium hydroxide, regulates pH to 4.74, adds water for injection and adjusts sad concentration to 22mmol/L, at 22 ℃, stir 1 hour deactivation lipid-coated virus, centrifugation supernatant liquor.
(6) with 0.5mol/L sodium hydroxide, regulate supernatant pH to 5.11, by 14% concentration, add dehydrated alcohol 2070ml, at-3.8 ℃, react centrifugation supernatant liquor 7 hours.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate is concentrated with 20 times of 30KD ultra-filtration membranes, through 8 times of volume pH be 6.63, concentration is the ultrafiltration of 25mmol/L phosphate buffered saline buffer, after ultrafiltration, receive sample 2000ml, protein content is 36.81mg/ml.
(8) with pH be 6.63, concentration is 25mmol/L phosphate buffered saline buffer balance DEAE Sepharose Fast Flow (GE Healthcare Bio-Sciences AB, the U.S.) 10 column volumes of post, column volume is 200ml, filtrate after ultrafiltration is crossed to post, collection penetrates liquid 7000ml, the phosphate buffered saline buffer wash-out that containing the pH of 2mol/LNaCl is 6.63, concentration is 25mmol/L for hanging column foreign protein.
(9) with 1mol/L hydrochloric acid, regulate and penetrate liquid pH to 4.25, after 0.1 μ m filter membrane pre-filtering, then remove virus with Novasip DV20 nano-film filtration, controlled filter pressure is not more than 0.25MPa.
(10) except filtrate after virus is concentrated with 10 times of 30KD ultra-filtration membranes, through the ultrafiltration of 8 times of volume water for injection, results stoste 600ml, protein content is 107.21mg/ml, with water for injection, dilute and add maltose by the amount of 10g/L, with 0.5mol/L salt acid for adjusting pH to 4.02, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa, press protein content 51～55mg/ml, tire and be not less than the packing of 100PEI-U/ml specification, after packing, sampling detects protein content with Kjeldahl determination, by euzymelinked immunosorbent assay (ELISA) method, measure and tire, non-reduced type E polyacrylamide gel electrophoresis SDS-PAGE method mensuration purity and albumin are residual, pH pH-value determination pH method is measured pH value, high effective liquid chromatography for measuring IgG monomer, dimer, polymer, cracking body, the sad residual quantity of gas chromatography determination, osmotic pressure molar density assay method is measured osmotic pressure molar density, detected result is in Table 6.
Technological process albumen and the rate of recovery of tiring are in Table 1.
Table 1 technological process albumen and the rate of recovery of tiring
As shown in Figure 3, the quiet note of embodiment 1 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, IgG molecular weight is 150KD～160KD, wherein, 1, sample-loading buffer, 2, anti-CMV high-titer blood plasma, 3, FI+II+III supernatant, 4, FI+II+III resolution of precipitate, 5, sad deactivation supernatant, 6, ethanol precipitation supernatant, 7, before DEAE chromatography, 8, penetrate 9, wash-out, 10, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation.Collection of illustrative plates purity check result shows that the IgG purity of sad deactivation supernatant is 89.72%, shows that sad precipitation and inactivation technology can remove most of foreign proteins such as albumin, Fibrinogen.The electrophoretic band demonstration anion-exchange chromatography of wash-out can effectively be removed the impurity such as polymer, residual albumin, and penetrating IgG purity is 98.76%.
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured human plasma 20 person-portions of anti-CMV high-titer, under 30 ℃ of conditions, melts slurry, and after mixing, volume is 11410ml.
(2) add physiological saline 2280ml to regulate serum protein content to 50.30mg/ml, add glacial acetic acid to regulate pH to 6.48, add dehydrated alcohol 4603ml to regulate suspension alcohol concn to 25%, regulate temperature of reaction to-4.5 ℃, stirring reaction 6 hours, reacts complete centrifugation and obtains FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 5.16, concentration is that 50mmol/L sodium acetate buffer 10500ml dissolves, 2.2 ℃ of stirrings 12 hours, through centrifugation supernatant liquor.
(4) with 4mol/L acetic acid, regulate supernatant pH to 5.07, it is sad by 30mmol/L concentration, to add, and stirs centrifugation supernatant liquor at 25 ℃ 2.5 hours.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, controlled filter pressure is not more than 0.25MPa, with 1mol/L sodium hydroxide, regulates pH to 5.17, adds the sad concentration of sad adjustment to 55mmol/L, at 25 ℃, stir 1.5 hours deactivation lipid-coated virus, centrifugation supernatant liquor.
(6) with 1mol/L sodium hydroxide, regulate supernatant pH to 5.48, by 16% concentration, add dehydrated alcohol 2190ml, at-3.0 ℃, react centrifugation supernatant liquor 4 hours.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate is concentrated through 15 times of 30KD ultra-filtration membranes, through 8 times of volume pH be 6.93, concentration is the ultrafiltration of 50mmol/L phosphate buffered saline buffer, after ultrafiltration, receive sample 2000ml, protein content is 33.55mg/ml.
(8) with pH be 6.93, concentration is 10 column volumes of 50mmol/L phosphate buffered saline buffer balance DEAE Sepharose Fast Flow post, column volume is 200ml, filtrate after ultrafiltration is crossed to post, collection penetrates liquid 7000ml, the phosphate buffered saline buffer wash-out that containing the pH of 2mol/L NaCl is 6.93, concentration is 50mmol/L for hanging column foreign protein.
(9) with 1mol/L hydrochloric acid, regulate and to penetrate liquid pH to 5.07, after 0.1 μ m filter membrane pre-filtering, then Novasip DV20 nano-film filtration is except virus, and controlled filter pressure is not more than 0.25MPa.
(10) except filtrate after virus is concentrated with 10 times of 30KD ultra-filtration membranes, through the ultrafiltration of 8 times of volume water for injection, results stoste 750ml, protein content is 82.16mg/ml, with water for injection, dilute and add maltose by the amount of 10g/L, with 1mol/L salt acid for adjusting pH to 4.12, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa, after packing, sampling is measured protein content by the method described in embodiment 1, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result is in Table 6.
Technological process albumen and the rate of recovery of tiring are in Table 2.
Table 2 technological process albumen and the rate of recovery of tiring
As shown in Figure 4, the quiet note of embodiment 2 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, wherein, 1, anti-CMV high-titer blood plasma, 2, FI+II+III supernatant, 3, FI+II+III resolution of precipitate, 4, sad deactivation supernatant, 5, ethanol precipitation supernatant, 6, before DEAE chromatography, 7, penetrate, 8, wash-out, 9, cytomegalovirus human normal immunoglobulin CMV-IgG, 10. sample-loading buffer after preparation.Electrophoretogram shows that the present embodiment purifying process parameter effect is consistent with embodiment 1, IgG purity 99.10% after preparation.
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured human plasma 20 person-portions of anti-CMV high-titer, under 15 ℃ of conditions, melts slurry, and after mixing, volume is 11570ml.
(2) add physiological saline 2310ml to regulate serum protein content 49.69mg/ml, add glacial acetic acid to regulate pH to 6.32, add dehydrated alcohol 4603ml to regulate suspension alcohol concn to 20%, regulate temperature of reaction to-4.8 ℃, stirring reaction 6 hours, reacts complete centrifugation and obtains FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 5.02, concentration is that 80mmol/L sodium acetate buffer 12500ml dissolves, and stirs 14 hours centrifugation supernatant liquor at 6.0 ℃.
(4) with 1mol/L sodium hydroxide, regulate supernatant pH to 5.50, it is sad by 40mmol/L concentration, to add, and stirs centrifugation supernatant liquor at 23 ℃ 1 hour.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, and controlled filter pressure is not more than 0.25MPa, with 1mol/L sodium hydroxide, regulates pH to 5.50, it is sad to add, adjust sad concentration to 78mmol/L, stir 1 hour deactivation lipid-coated virus, centrifugation supernatant liquor at 30 ℃.
(6) with 1mol/L hydrochloric acid, regulate supernatant pH to 4.62, by 12% concentration, add 95% ethanol 1765ml, at-2.5 ℃, react centrifugation supernatant liquor 3 hours.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate is concentrated through 18 times of 30KD ultra-filtration membranes, through 10 times of volume pH be 6.87, concentration is the ultrafiltration of 40mmol/L phosphate buffered saline buffer, after ultrafiltration, receive sample 2000ml, protein content is 34.54mg/ml.
(8) with pH be 6.87, concentration is 40mmol/L phosphate buffered saline buffer balance DEAE Sepharose Fast Flow, column volume is 200ml, filtrate after ultrafiltration is crossed to post, collection penetrates liquid 6000ml, the phosphate buffered saline buffer wash-out that containing the pH of 2mol/LNaCl is 6.87, concentration is 40mmol/L for hanging column foreign protein.
(9) with 1mol/L hydrochloric acid, regulate and to penetrate liquid pH to 4.62, after 0.1 μ m filter membrane pre-filtering, then Novasip DV20 nano-film filtration is except virus, and controlled filter pressure is not more than 0.25MPa.
(10) except filtrate after virus is concentrated with 10 times of 30KD ultra-filtration membranes, through the ultrafiltration of 8 times of volume water for injection, results stoste 500ml, protein content is 126.57mg/ml, with water for injection, dilute and add maltose by the amount of 10g/L, with 1mol/L salt acid for adjusting pH to 3.85, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa.After packing sampling with the method described in embodiment 1 measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result is in Table 6.
Technological process albumen and the rate of recovery of tiring are in Table 3.
Table 3 technological process albumen and the rate of recovery of tiring
As shown in Figure 5, the quiet note of embodiment 3 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, wherein, 1, sample-loading buffer, 2., anti-CMV high-titer blood plasma, 3, FI+II+III supernatant, 4, FI+II+III resolution of precipitate, 5, sad deactivation supernatant, 6, ethanol precipitation supernatant, 7, before DEAE chromatography, 8, penetrate, 9, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 10, wash-out, 11, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 12, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 13, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 14, quiet note human normal immunoglobulin (abroad) has gone on the market, 15, sample-loading buffer.Electrophoretogram shows that the quiet note human normal immunoglobulin reference material IgG purity of having gone on the market is 86.6% abroad, polymer is containing 2.2%, and dimer is containing 7.52%, and albumin is containing 2.51%, the present embodiment purifying process parameter effect is consistent with embodiment 1, and after preparation, IgG purity is 98.89%.
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured human plasma 20 person-portions of anti-CMV high-titer, under 4 ℃ of conditions, melts slurry, and after mixing, volume is 11670ml.
(2) add physiological saline 2330ml to regulate serum protein content to 49.27mg/ml, add glacial acetic acid to regulate pH to 6.02, add 95% ethanol 4494ml to regulate suspension alcohol concn to 23%, regulate temperature of reaction to-5.5 ℃, stirring reaction 6 hours, reacts complete centrifugation and obtains FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 4.81, concentration is that 30mmol/L sodium acetate buffer 12000ml dissolves, and stirs 10 hours centrifugation supernatant liquor at 8.0 ℃.
(4) with 1mol/L sodium hydroxide, regulate supernatant pH to 4.96, it is sad by 10mmol/L concentration, to add, and stirs centrifugation supernatant liquor at 18 ℃ 3 hours.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, and controlled filter pressure is not more than 0.25MPa, with 1mol/L sodium hydroxide, regulates pH to 5.21, it is sad to add, adjust sad concentration to 46mmol/L, stir 2 hours deactivation lipid-coated virus, centrifugation supernatant liquor at 27 ℃.
(6) with 1mol/L salt acid for adjusting pH to 5.04, by 15% concentration, add 95% ethanol 2396ml, at-3.5 ℃, react centrifugation supernatant liquor 5 hours.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate is concentrated through 20 times of 30KD ultra-filtration membranes, through 10 times of volume pH be 7.02, concentration is the ultrafiltration of 60mmol/L phosphate buffered saline buffer, after ultrafiltration, receive sample 2000ml, protein content is 37.17mg/ml.
(8) with pH be 7.02, concentration is 60mmol/L phosphate buffered saline buffer balance Macro-Prep DEAE Media (Bio-Rad, the U.S.) 10 column volumes of post, column volume is 200ml, filtrate after ultrafiltration is crossed to post, collection penetrates liquid 8000ml, the phosphate buffered saline buffer wash-out that containing the pH of 2mol/LNaCl is 7.02, concentration is 60mmol/L for hanging column foreign protein.
(9) with 1mol/L hydrochloric acid, regulate and to penetrate liquid pH to 4.47, after 0.1 μ m filter membrane pre-filtering, then Novasip DV20 nano-film filtration is except virus, and controlled filter pressure is not more than 0.25MPa.
(10) except filtrate after virus is concentrated with 10 times of 30KD ultra-filtration membranes, through the ultrafiltration of 8 times of volume water for injection, results stoste 500ml, protein content is 134.27mg/ml, with water for injection, dilute and add maltose by the amount of 11g/L, with 1mol/L salt acid for adjusting pH to 4.08, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa.After packing sampling with the method described in embodiment 1 measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result is in Table 6.
Technological process albumen and the rate of recovery of tiring are in Table 4.
Table 4 technological process albumen and the rate of recovery of tiring
As shown in Figure 6, the quiet note of embodiment 4 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, wherein, 1, sample-loading buffer, 2, anti-CMV high-titer blood plasma, 3, FI+II+III supernatant, 4, FI+II+III resolution of precipitate, 5, sad deactivation supernatant, 6, ethanol precipitation supernatant, 7, before DEAE chromatography, 8, penetrate, 9, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 10, wash-out, 11, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 12, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 13, quiet note human normal immunoglobulin (abroad) has gone on the market, 14, quiet note human normal immunoglobulin (domestic) has gone on the market, 15, sample-loading buffer.Electrophoretogram has contrasted the purity of domestic, the external quiet note human normal immunoglobulin reference material of going on the market, and result shows that domestic quiet note human normal immunoglobulin IgG purity is 95.38%, and how concrete containing 0.8%, dimer is containing 2.37, and albumin is containing 0.52%.The present embodiment purifying process parameter effect is consistent with embodiment 1, and after preparation, IgG purity is 98.27%.
The key distinction of the present embodiment and embodiment 1-4 is, blood plasma is first through the separated FI precipitation of 8% ethanol precipitin reaction, through 20% ethanol precipitin reaction, prepare FII+III precipitation for subsequent purification again, in addition, anion-exchange chromatography step replaces DEAESepharose Fast Flow filler with TOYOPEARL DEAE 650M (TOSOH, Japan) filler.
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured human plasma 2 person-portions of anti-CMV high-titer, under 20 ℃ of conditions, melts slurry, and after mixing, volume is 1150ml.
(2) add physiological saline 230ml to regulate serum protein content 50.72mg/ml, add glacial acetic acid to regulate pH to 7.00, add 95% ethanol 130ml to regulate suspension alcohol concn to 8%, regulate temperature of reaction to-2.5 ℃, stirring reaction 4 hours, reacts complete through centrifugation FI supernatant liquor, with glacial acetic acid, regulate supernatant pH to 6.25, add 95% ethanol 247ml adjusting suspension alcohol concn to 20%, at-5.0 ℃, react 6 hours, react complete and obtain FII+III precipitation through centrifugation.
(3) FII+III precipitation with pH be 4.98, concentration is that 40mmol/L sodium acetate buffer 1150ml dissolves, and stirs 12 hours centrifugation supernatant liquor at 3.0 ℃.
(4) with 4mol/L acetic acid, regulate supernatant pH to 4.78, it is sad by 100mmol/L concentration, to add, and stirs centrifugation supernatant liquor at 22 ℃ 2 hours.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, controlled filter pressure is not more than 0.25MPa, with 0.5mol/L sodium hydroxide, regulates pH to 5.08, injects water and adjusts sad concentration to 38mmol/L, at 23 ℃, stir 1 hour deactivation lipid-coated virus, centrifugation supernatant liquor.
(6) repetition measurement suspension pH to 5.03, adds 95% ethanol 208ml by 14% concentration, reacts centrifugation supernatant liquor at-4.0 ℃ 8 hours.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate is concentrated through 15 times of 30KD ultra-filtration membranes, through 10 times of volume pH be 6.46, concentration is the ultrafiltration of 25mmol/L phosphate buffered saline buffer, after ultrafiltration, receive sample 170ml, protein content is 38.09mg/ml.
(8) with pH be 6.46, concentration is 10 column volumes of 25mmol/L phosphate buffered saline buffer balance TOYOPEARL DEAE 650M post, column volume is 22ml, filtrate after ultrafiltration is crossed to post, collection penetrates liquid 328ml, the phosphate buffered saline buffer wash-out that containing the pH of 2mol/L NaCl is 6.46, concentration is 25mmol/L for hanging column foreign protein.
(9) with 1mol/L hydrochloric acid, regulate and to penetrate liquid pH to 4.75, after 0.1 μ m filter membrane pre-filtering, then Novasip DV20 nano-film filtration is except virus, and controlled filter pressure is not more than 0.25MPa.
(10) except filtrate after virus is concentrated with 10 times of 30KD ultra-filtration membranes, through the ultrafiltration of 8 times of volume water for injection, results stoste 50ml, protein content is 115.23mg/ml, with water for injection, dilute and add maltose by the amount of 9g/L, with 1mol/L salt acid for adjusting pH to 3.98, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa.After packing sampling with the method described in embodiment 1 measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result is in Table 6.
Technological process albumen and the rate of recovery of tiring are in Table 5.
Table 5 technological process albumen and the rate of recovery of tiring
As shown in Figure 7, the quiet note of embodiment 5 cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, wherein, 1, sample-loading buffer, 2, anti-CMV high-titer blood plasma, 3, FI supernatant, 4, FII+III supernatant, 5, FII+III resolution of precipitate, 6, ethanol precipitation supernatant, 7, before DEAE chromatography, 8, cytomegalovirus human normal immunoglobulin CMV-IgG after preparation, 9, wash-out, 10, quiet note human normal immunoglobulin (domestic) has gone on the market, 11, quiet note human normal immunoglobulin (abroad) has gone on the market, 12, quiet note human normal immunoglobulin (abroad) has gone on the market, 13, quiet note human normal immunoglobulin (abroad) has gone on the market, 14, quiet note human normal immunoglobulin (abroad) has gone on the market, 15, sample-loading buffer.Electrophoretogram shows that the present embodiment purifying process parameter effect is consistent with embodiment 1, and after preparation, IgG purity is 99.82%.
Comparative example 1,
The present embodiment for adopt existing cold ethanol method technique prepare the general population's immunoglobulin (Ig) (pharmacopeia name is called: quiet note human normal immunoglobulin pH4), as shown in Figure 2, concrete steps are as follows:
(1) get the general population's blood plasma 5 person-portions, under 20 ℃ of conditions, melt slurry, after mixing, volume is 2850ml.
(2) add physiological saline 640ml to regulate serum protein content to 47.36mg/ml, add glacial acetic acid to regulate pH to 6.28, add dehydrated alcohol 930ml to regulate suspension alcohol concn to 20%, regulate temperature of reaction to-4.5 ℃, stirring reaction 4 hours, has reacted centrifugation FI+II+III precipitation.
(3) FI+II+III precipitation is that 20mmol/L phosphate buffered saline buffer 2300ml dissolves by pH 5.07, concentration, stirs 12 hours centrifugation supernatant liquor at 4 ℃.
(4) repetition measurement supernatant liquor pH to 5.25, adds 95% ethanol 456ml to regulate alcohol concn to 15%, reacts 3 hours centrifugation FI+III supernatant liquor at-3.5 ℃.
(5) with 1mol/L sodium hydroxide, regulate FI+III supernatant liquor pH to 7.05, add 95% ethanol 205ml and regulate alcohol concn to 20%, react 6 hours at-7.0 ℃, through centrifugation, obtain FII precipitation.
(6) FII precipitation is dissolved with 600ml water for injection, and 2～8 ℃ are stirred 12 hours, centrifugation supernatant liquor.(7) supernatant liquor is concentrated into 100ml with 30KD ultra-filtration membrane, through 8 times of volume water for injection ultrafiltration dealcoholysis, receives sample 150ml.
(8) by protein content be greater than 50mg/ml, maltose concentration 10g/L prepares, with 1mol/L salt acid for adjusting pH to 3.97, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa,
(9) after degerming, prepare sample and put between inactivation of virus, incubate for 23～25 ℃ and put 21 days, incubate to discharge to finish with Novasip DV50 nano-film filtration and remove virus, controlled filter pressure is less than 0.25MPa.After packing sampling with the method described in embodiment 1 measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, osmotic pressure molar density.Detected result is in Table 6.
Sample quality index after the preparation of table 6 cytomegalovirus human normal immunoglobulin
As shown in Figure 8, the quiet note human normal immunoglobulin of comparative example 1 pH4 purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, wherein, 1, sample-loading buffer, 2, the general population's blood plasma, 3, blood plasma after dilution, 4, FI+II+III supernatant, 5, FI+II+III resolution of precipitate, 6, FI+III supernatant, 7, FI+III resolution of precipitate, 8, FII resolution of precipitate, 9, after preparation.Electrophoretogram shows that the rear IgG purity of preparation is 96.7%.
Claims (4)
1. a preparation method for quiet note cytomegalovirus human normal immunoglobulin, comprises the following steps:
(1) FI+II+III, FII+III precipitation
It is the human plasma of 20.79～32.42PEI-U/ml that the anti-CMV that the euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured tires, and melts slurry at 2～30 ℃, merges and mixes;
1. FI+II+III precipitation
With physiological saline, regulate serum protein content to 45～55mg/ml, with glacial acetic acid, regulate pH to 6.0～6.5, add 95% ethanol or dehydrated alcohol to regulate alcohol concn to 20～25%, temperature of reaction is 4.5 ℃ of ﹣ 5.5～﹣, stirring reaction is 4～6 hours, reacts the complete centrifugal or separated acquisition of press filtration FI+II+III precipitation;
2. FII+III precipitation
With physiological saline, regulate serum protein content to 45～55mg/ml, with glacial acetic acid, regulate pH to 6.8～7.2, to add volume ratio mark be 95% ethanol or dehydrated alcohol and regulate alcohol concn to 7.5～8.5%, temperature of reaction is 2.0 ℃ of ﹣ 2.5～﹣, stirring reaction 4 hours, react complete through the centrifugal or separated removal of press filtration FI precipitation, obtain supernatant liquor, with glacial acetic acid, regulate supernatant liquor pH to 6.0～6.5, add 95% ethanol or dehydrated alcohol to regulate alcohol concn to 20～25%, temperature of reaction is 4.5 ℃ of ﹣ 5.5～﹣, stirring reaction is 4～6 hours, react complete through the centrifugal or separated acquisition of press filtration FII+III precipitation,
(2) FI+II+III or FII+III resolution of precipitate
FI+II+III or FII+III precipitation with the pH of 0.9～1.1 times of plasma volume be 4.8～5.2, concentration is that 20～80mM sodium acetate buffer stirs 8～16 hours at 2～8 ℃, makes to precipitate abundant dissolving, through centrifugal or press filtration separation of supernatant;
(3) sad precipitation
With 4mol/L acetic acid or 0.5～1mol/L sodium hydroxide, regulate supernatant liquor pH to 4.5～5.5, it is sad by 10～100mmol/L concentration, to add, and at 18～25 ℃, stirring reaction is 1～3 hour, through centrifugal or filtering separation supernatant liquor;
(4) sad inactivation of virus
Supernatant liquor is 1.0 μ m membrane filtrations with aperture, controlled filter pressure is not more than 0.25MPa, with 4mol/L acetic acid or 0.5～1mol/L sodium hydroxide, regulate filtrate pH to 4.5～5.5, add water for injection or sad concentration to the 20～80mmol/L of sad adjusting suspension, at 20～30 ℃, stir centrifugal or filtering separation supernatant liquor 1～2 hour;
(5) ethanol precipitation
With 0.5～1mol/L hydrochloric acid or sodium hydroxide, regulate supernatant liquor pH to 4.5～5.5, by 12～16% concentration, add 95% ethanol or dehydrated alcohol to carry out precipitin reaction, at 2.5 ℃ of ﹣ 4.0～﹣, stirring reaction is 2～8 hours, centrifugal or press filtration separation of supernatant;
(6) ultrafiltration
Supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, filtrate is concentrated with 15～20 times of 30KD ultra-filtration membranes, and through the phosphate buffered saline buffer ultrafiltration dialysis of 20～60mmol/L of 8～10 times of volume pH6.0～7.1, after ultrafiltration, receiving sample, to control protein content be 25～40mg/ml;
(7) anion-exchange chromatography
With the phosphate buffered saline buffer that pH is 6.0～7.1, concentration is 20～60mmol/L as level pad balance chromatography column 8～10 column volumes, by every milliliter of filler, be not more than 70～80% of the maximum carrying capacity of filler and calculate loading protein content, after loading, collect and penetrate liquid, hanging column foreign protein is through the phosphate buffered saline buffer wash-out of 20～60mmol/L of pH6.0～7.1 containing 2mol/LNaCl;
(8) nanometer film is except virus filtration
With the hydrochloric acid adjusting of 0.5～1mol/L, penetrate liquid pH to 4.2～5.0, after 0.1 μ m filter membrane pre-filtering, with Novasip DV20 nano-film filtration, except virus, controlled filter pressure is not more than 0.25Mpa;
(9) ultrafiltration
Except filtrate after virus is through 30KD ultra-filtration membrane protein concentrate content to 80～100mg/ml, with 8～10 times of water for injection ultrafiltration, after ultrafiltration, receiving sample control protein content is 80～150mg/ml;
(10) preparation
Stoste protein content after mensuration ultrafiltration, with water for injection dilution, adjust goods protein content to 51～55mg/ml, by 9～11% amount, add maltose simultaneously, with 0.5～1mol/L salt acid for adjusting pH to 3.8～4.2, obtain quiet note cytomegalovirus human normal immunoglobulin;
Described quiet note cytomegalovirus human normal immunoglobulin specific activity is not less than 2.5PEI-U/mg, and anti-CMV tires and is not less than 100PEI-U/ml, and purity is greater than 98.2%, and protein content is 51～55mg/ml.
2. the preparation method of quiet note cytomegalovirus human normal immunoglobulin according to claim 1, it is characterized in that: degerming packing after described preparation steps, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa, press protein content 51～55mg/ml, tire and be not less than the packing of 100PEI-U/ml specification.
3. the preparation method of quiet note cytomegalovirus human normal immunoglobulin according to claim 2, is characterized in that: goods protein content after packing is measured in sampling, and anti-CMV tires, purity, molecular size distribution, sad residual quantity, the project quality index of osmotic pressure molar density.
4. the preparation method of quiet note cytomegalovirus human normal immunoglobulin according to claim 1, it is characterized in that: in anion-exchange chromatography step, add filler, filler is selected from DEAE Sepharose Fast Flow, TOYOPEARL DEAE650M or Macro-Prep DEAE Media.
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| CN111166879A (en) * | 2019-12-04 | 2020-05-19 | 昆明白马制药有限公司 | Pig immunoglobulin oral liquid |
| CN112375142B (en) * | 2020-11-18 | 2022-03-18 | 深圳市卫光生物制品股份有限公司 | Preparation method of novel coronavirus human immunoglobulin for intravenous injection |
| CN112500477B (en) * | 2020-12-05 | 2023-06-06 | 贵州泰邦生物制品有限公司 | Method for rapidly extracting human immunoglobulin from blood plasma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054415A (en) * | 2007-04-28 | 2007-10-17 | 中国人民解放军第三军医大学野战外科研究所 | Humanized single chain antibody for anti-human cytomegalovirus envelope glycoprotein |
| CN102143974A (en) * | 2008-07-16 | 2011-08-03 | 胡马斯有限公司 | Human cytomegalovirus neutralising antibodies and use thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7553938B2 (en) * | 2004-02-27 | 2009-06-30 | Octapharma Ag | Method of providing a purified, virus safe antibody preparation |
| CN101357228B (en) * | 2007-07-30 | 2011-03-16 | 深圳市卫武光明生物制品有限公司 | Preparation method of hepatitis b human immunoglobulin for intravenous injection |
-
2011
- 2011-08-05 CN CN201110223760.5A patent/CN102286099B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054415A (en) * | 2007-04-28 | 2007-10-17 | 中国人民解放军第三军医大学野战外科研究所 | Humanized single chain antibody for anti-human cytomegalovirus envelope glycoprotein |
| CN102143974A (en) * | 2008-07-16 | 2011-08-03 | 胡马斯有限公司 | Human cytomegalovirus neutralising antibodies and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 抗人巨细胞病毒中和性人源基因工程抗体的研究;段涛等;《中华实验和临床病毒学杂志》;20030930;第17卷(第3期);第247-250页 * |
| 段涛等.抗人巨细胞病毒中和性人源基因工程抗体的研究.《中华实验和临床病毒学杂志》.2003,第17卷(第3期),第247-250页. |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130172536A1 (en) * | 2011-08-16 | 2013-07-04 | Shenzhen Weiguang Biological Products Co.,Ltd. | Intravenous Cytomegalovirus Human Immune Globulin and Manufacturing Method Thereof |
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