CN102286099A - 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 notes 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, newborn infant, organ transplantation patient, the immunosuppressed patient infection mortality ratio that are caused by cytomegalovirus can reach 50%～80%.Cytomegalovirus human normal immunoglobulin CMV-IgG mainly treats the cytomegalovirus severe infection that causes in pregnant woman, newborn infant, immunosuppressed patient and the organ transfer operation with it clinically because of in can be special and cytomegalovirus.Cytomegalovirus natural infection rate among the general population can reach more than 80%, screening and gather contains the tire blood plasma of cytomegalovirus IgG antibody of height from healthy population, adopt specific separation purification method to prepare cytomegalovirus human normal immunoglobulin medicine, the severe infection that causes because of cytomegalovirus for treatment has irreplaceable clinical value.
At present, the specific human immunoglobulins medicine that has 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.) separate purification, this method is invented by nineteen forty-six professor Edwin.J.Cohn of Harvard University, by regulating the pH in the technology, protein concentration, temperature, five parameters of alcohol concn and ionic strength realize different proteic separation.Long-term practice shows, there is the defective of following several respects in cold ethanol method: at first, ethanol is a kind of protein denaturant, easily causes the IgG structural modification in the sepn process and the sex change inactivation, the rate of recovery that causes tiring is lower, also may cause the formation of new antigenic determinant; Secondly, compare column chromatography technology, the ethanol sedimentation purification efficiency is lower, need usually to reduce the purity requirement that protein recovery reaches product, so the technological process yield is on the low side, and product purity is not high; Once more,, also there are hardware and running cost height under cold condition, defective such as labour intensity is big because of sepn process usually needs carry out.
Summary of the invention
The purpose of this invention is to provide a kind of quiet notes 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 notes cytomegalovirus human normal immunoglobulin, described quiet notes 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 kind of method of quiet notes cytomegalovirus human normal immunoglobulin may further comprise the steps:
(1) FI+II+III, FII+III precipitate preparation
That the euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured is anti--human plasma that the CMV height is tired, melt slurry under 2～30 ℃, and merge and mix;
1. FI+II+III precipitation preparation
Regulate plasma proteins content to 45～55mg/ml with physiological saline, regulate pH to 6.0～6.5 with glacial acetic acid, add 95% ethanol or dehydrated alcohol and regulate alcohol concn to 20～25%, temperature of reaction is-5.5～-4.5 ℃, stirring reaction is 4～6 hours, and reaction finishes centrifugal or press filtration separates acquisition FI+II+III precipitation;
2. FII+III precipitation preparation
Regulate plasma proteins content to 45～55mg/ml with physiological saline, regulate pH to 6.8～7.2 with glacial acetic acid, adding the volume ratio mark is 95% ethanol or dehydrated alcohol and regulates alcohol concn to 7.5～8.5%, temperature of reaction is-2.5～-2.0 ℃, stirring reaction 4 hours, reaction finishes and separates removal FI precipitation through centrifugal or press filtration, obtain supernatant liquor, regulate supernatant liquor pH to 6.0～6.5 with glacial acetic acid, add 95% ethanol or dehydrated alcohol and regulate alcohol concn to 20～25%, temperature of reaction is-5.5～-4.5 ℃, and stirring reaction is 4～6 hours, and reaction finishes and separates acquisition FII+III precipitation through centrifugal or press filtration;
(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 stirred 8～16 hours down at 2～8 ℃, precipitation fully dissolved, through centrifugal or press filtration separation of supernatant;
(3) sad precipitation
Regulate supernatant liquor pH to 4.5～5.5 with 4mol/L acetate or 0.5～1mol/L sodium hydroxide, it is sad to add by 10～100mmol/L concentration, 18～25 ℃ of following stirring reactions 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 the aperture, controlled filter pressure is not more than 0.25MPa, regulate filtrate pH to 4.5～5.5 with 4mol/L acetate or 0.5～1mol/L sodium hydroxide, add water for injection or the sad concentration to 20 of sad adjusting suspension～80mmol/L, stirred 1～2 hour centrifugal or filtering separation supernatant liquor down at 20～30 ℃;
(5) ethanol sedimentation
Regulate supernatant liquor pH to 4.5～5.5 with 0.5～1mol/L hydrochloric acid or sodium hydroxide, add 95% ethanol or dehydrated alcohol carries out precipitin reaction by 12～16% concentration ,-4.0～-2.5 ℃ of following stirring reactions 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 concentrates for 15～20 times with the 30KD ultra-filtration membrane, and through the phosphate buffered saline buffer ultrafiltration dialysis of 20～60mmol/L of 8～10 times of volume pH 6.0～7.1, receiving sample control protein content after the ultrafiltration is 25～40mg/ml;
(7) anion-exchange chromatography
With pH is 6.0～7.1, concentration is 20～60mmol/L phosphate buffered saline buffer as level pad balance chromatography column 8～10 column volumes, be not more than 70～80% of the maximum carrying capacity of filler by every milliliter of filler and calculate upward sample protein content, collect behind the last sample and penetrate liquid, the phosphate buffered saline buffer wash-out of 20～60mmol/L of the pH 6.0～7.1 of hanging column foreign protein through containing 2mol/LNaCl;
(8) nanometer film is removed virus filtration
Hydrochloric acid adjusting with 0.5～1mol/L penetrates liquid pH to 4.2～5.0, after 0.1 μ m filter membrane pre-filtering, removes virus with Novasip DV20 nano-film filtration, and controlled filter pressure is not more than 0.25Mpa;
(9) ultrafiltration
Remove viral rear filtrate through 30KD ultra-filtration membrane protein concentrate content to 80～100mg/ml, with 8～10 times of water for injection ultrafiltration, receiving sample control protein content after the ultrafiltration is 80～150mg/ml;
(10) preparation
Stoste protein content after the mensuration ultrafiltration is adjusted goods protein content to 51～55mg/ml with the water for injection dilution, presses 9～11% amount adding maltose simultaneously, with 0.5～1mol/L hydrochloric acid adjusting pH to 3.8～4.2.
Method of the present invention is the degerming packing after described preparation steps, and through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa, presses protein content 51～55mg/ml, and tiring is not less than the packing of 100PEI-U/ml specification.
Goods protein content after the 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 add filler in the anion-exchange chromatography step, 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 that has is as follows:
(1) adopts the sad precipitation and the anion-exchange chromatography technology of mild condition, reduced the sedimentary step of cold ethanol, 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 the purifying multiple is 5.30～8.14, and the IgG rate of recovery is greater than 4.9g/L, and purity is greater than 98.2%, and the IgG polymer is less than 0.1%.
(2) adopted sad inactivation of virus and nano-film filtration to remove viral technology in the technological process, effectively deactivation and removal are viral, and in conjunction with anion-exchange chromatography, technological process virus reduction amount is greater than 12log
10
(3) sad precipitation can effectively precipitate NIg class impurity, and IgG, IgA, copper-protein be retained in the 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 back the finished product do not contain impurity such as polymer, albumin.
(5) production cycle of technology of the present invention is 5～7 days, and the cold ethanol explained hereafter cycle of prior art is 28～30 days, has effectively improved production efficiency, has reduced the alcoholic acid usage quantity simultaneously, cuts down the consumption of energy 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.
Description of drawings
Fig. 1 is the quiet notes of a present invention cytomegalovirus human normal immunoglobulin purifying process schema.
Fig. 2 is that the prior art cold ethanol method prepares the general population's immunoglobulin (Ig) process flow sheet.
Fig. 3 is embodiment 1 a quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretogram.
Fig. 4 is embodiment 2 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretograms.
Fig. 5 is embodiment 3 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretograms.
Fig. 6 is embodiment 4 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretograms.
Fig. 7 is embodiment 5 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis electrophoretograms.
Fig. 8 is Comparative Examples 1 a quiet notes human normal immunoglobulin 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.
Embodiment 1, as shown in Figure 1,
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured anti--human plasma 20 person-portions that the CMV height is tired, melts slurry under 25 ℃ of conditions, and mixing the back volume is 11540ml.
(2) add physiological saline 2310ml and regulate plasma proteins content to 49.53mg/ml, add glacial acetic acid and regulate pH to 6.18, add dehydrated alcohol 3926ml and regulate suspension alcohol concn to 22%, the conditioned reaction temperature is to-5.0 ℃, stirring reaction 4 hours reacts the centrifugation that finishes and obtains the FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 4.93, concentration is that 50mmol/L sodium acetate buffer 11500ml dissolves, 4 ℃ of stirrings 12 hours down, centrifugation supernatant liquor.
(4) regulate supernatant pH to 4.57 with 4mol/L acetate, it is sad that 60mmol/L concentration adds, 21 ℃ of following stirring reactions 3 hours, and the centrifugation supernatant liquor.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, controlled filter pressure is not more than 0.25MPa, regulates pH to 4.74 with 0.5mol/L sodium hydroxide, adds water for injection and adjusts sad concentration to 22mmol/L, 22 ℃ are stirred 1 hour deactivation lipid-coated virus, centrifugation supernatant liquor down.
(6) regulate supernatant pH to 5.11 with 0.5mol/L sodium hydroxide, add dehydrated alcohol 2070ml by 14% concentration, reaction is 7 hours under-3.8 ℃, the centrifugation supernatant liquor.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate concentrates for 20 times with the 30KD ultra-filtration membrane, through 8 times of volume pH be 6.63, concentration is the ultrafiltration of 25mmol/L phosphate buffered saline buffer, receive sample 2000ml after the ultrafiltration, 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, the ultrafiltration rear filtrate is crossed post, collection penetrates liquid 7000ml, the hanging column foreign protein with the pH that contains 2mol/LNaCl be 6.63, concentration is the phosphate buffered saline buffer wash-out of 25mmol/L.
(9) penetrate liquid pH to 4.25 with the adjusting of 1mol/L hydrochloric acid, after 0.1 μ m filter membrane pre-filtering, remove virus with Novasip DV20 nano-film filtration again, controlled filter pressure is not more than 0.25MPa.
(10) removing viral rear filtrate concentrates for 10 times with the 30KD ultra-filtration membrane, through the ultrafiltration of 8 times of volume water for injection, results stoste 600ml, protein content is 107.21mg/ml, dilute and press the amount adding maltose of 10g/L with water for injection, regulate pH to 4.02 with 0.5mol/L hydrochloric acid, 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, sampling detects protein content with Kjeldahl determination after the packing, tire with euzymelinked immunosorbent assay (ELISA) method mensuration, non-reduced type E polyacrylamide gel electrophoresis SDS-PAGE method mensuration purity and albumin are residual, pH pH-value determination pH method is measured the pH value, high effective liquid chromatography for measuring IgG monomer, dimer, polymer, the cracking body, the sad residual quantity of gas chromatography determination, the osmotic pressure molar density assay method is measured osmotic pressure molar density, and detected result sees Table 6.
The technological process albumen and the rate of recovery of tiring see Table 1.
The table 1 technological process albumen and the rate of recovery of tiring
As shown in Figure 3, embodiment 1 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, the IgG molecular weight is 150KD～160KD, wherein, 1, sample-loading buffer, 2, anti--CMV height blood plasma of tiring, 3, FI+II+III supernatant, 4, FI+II+III resolution of precipitate, 5, sad deactivation supernatant, 6, the ethanol sedimentation supernatant, 7, before the DEAE chromatography, 8, penetrate 9, wash-out, 10, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG.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 impurity such as polymer, residual albumin, and penetrating IgG purity is 98.76%.
Embodiment 2
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured anti--human plasma 20 person-portions that the CMV height is tired, melts slurry under 30 ℃ of conditions, and mixing the back volume is 11410ml.
(2) add physiological saline 2280ml and regulate plasma proteins content to 50.30mg/ml, add glacial acetic acid and regulate pH to 6.48, add dehydrated alcohol 4603ml and regulate suspension alcohol concn to 25%, the conditioned reaction temperature is to-4.5 ℃, stirring reaction 6 hours reacts the centrifugation that finishes and obtains the 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 are through the centrifugation supernatant liquor.
(4) regulate supernatant pH to 5.07 with 4mol/L acetate, press 30mmol/L concentration add sad, at 25 ℃ of stirrings 2.5 hours down, centrifugation supernatant liquor.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, controlled filter pressure is not more than 0.25MPa, regulates pH to 5.17 with 1mol/L sodium hydroxide, adds the sad concentration of sad adjustment to 55mmol/L, 25 ℃ are stirred 1.5 hours deactivation lipid-coated virus, centrifugation supernatant liquor down.
(6) regulate supernatant pH to 5.48 with 1mol/L sodium hydroxide, add dehydrated alcohol 2190ml by 16% concentration, reaction is 4 hours under-3.0 ℃, the centrifugation supernatant liquor.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate concentrates 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, receive sample 2000ml after the ultrafiltration, 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, the ultrafiltration rear filtrate is crossed post, collection penetrates liquid 7000ml, the hanging column foreign protein with the pH that contains 2mol/L NaCl be 6.93, concentration is the phosphate buffered saline buffer wash-out of 50mmol/L.
(9) penetrate liquid pH to 5.07 with the adjusting of 1mol/L hydrochloric acid, after 0.1 μ m filter membrane pre-filtering, Novasip DV20 nano-film filtration removes virus again, and controlled filter pressure is not more than 0.25MPa.
(10) removing viral rear filtrate concentrates for 10 times with the 30KD ultra-filtration membrane, through the ultrafiltration of 8 times of volume water for injection, results stoste 750ml, protein content is 82.16mg/ml, dilute and press the amount adding maltose of 10g/L with water for injection, regulate pH to 4.12 with 1mol/L hydrochloric acid, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa, sampling is measured protein content with embodiment 1 described method after the packing, tire, purity, the pH value, the IgG monomer, dimer, polymer, the cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result sees Table 6.
The technological process albumen and the rate of recovery of tiring see Table 2.
The table 2 technological process albumen and the rate of recovery of tiring
As shown in Figure 4, embodiment 2 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretograms, wherein, 1, anti--CMV height blood plasma of tiring, 2, FI+II+III supernatant, 3, the FI+II+III resolution of precipitate, 4, sad deactivation supernatant, 5, ethanol sedimentation supernatant, 6, before the DEAE chromatography, 7, penetrate, 8, wash-out, 9, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 10. sample-loading buffer.Electrophoretogram shows that present embodiment purifying process parameter effect is consistent with embodiment 1, preparation back IgG purity 99.10%.
Embodiment 3
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured anti--human plasma 20 person-portions that the CMV height is tired, melts slurry under 15 ℃ of conditions, and mixing the back volume is 11570ml.
(2) add physiological saline 2310ml and regulate plasma proteins content 49.69mg/ml, add glacial acetic acid and regulate pH to 6.32, add dehydrated alcohol 4603ml and regulate suspension alcohol concn to 20%, the conditioned reaction temperature is to-4.8 ℃, stirring reaction 6 hours reacts the centrifugation that finishes and obtains the FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 5.02, concentration is that 80mmol/L sodium acetate buffer 12500ml dissolves, 6.0 ℃ of stirrings 14 hours down, centrifugation supernatant liquor.
(4) regulate supernatant pH to 5.50 with 1mol/L sodium hydroxide, press 40mmol/L concentration add sad, at 23 ℃ of stirrings 1 hour down, centrifugation supernatant liquor.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, and controlled filter pressure is not more than 0.25MPa, regulates pH to 5.50 with 1mol/L sodium hydroxide, and it is sad to add, and adjusts sad concentration to 78mmol/L, and 30 ℃ are stirred 1 hour deactivation lipid-coated virus, centrifugation supernatant liquor down.
(6) regulate supernatant pH to 4.62 with 1mol/L hydrochloric acid, add 95% ethanol 1765ml, reacted 3 hours the centrifugation supernatant liquor at-2.5 ℃ down by 12% concentration.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate concentrates 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, receive sample 2000ml after the ultrafiltration, 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, the ultrafiltration rear filtrate is crossed post, collection penetrates liquid 6000ml, the hanging column foreign protein with the pH that contains 2mol/LNaCl be 6.87, concentration is the phosphate buffered saline buffer wash-out of 40mmol/L.
(9) penetrate liquid pH to 4.62 with the adjusting of 1mol/L hydrochloric acid, after 0.1 μ m filter membrane pre-filtering, Novasip DV20 nano-film filtration removes virus again, and controlled filter pressure is not more than 0.25MPa.
(10) removing viral rear filtrate concentrates for 10 times with the 30KD ultra-filtration membrane, through the ultrafiltration of 8 times of volume water for injection, results stoste 500ml, protein content is 126.57mg/ml, dilute and press the amount adding maltose of 10g/L with water for injection, regulate pH to 3.85 with 1mol/L hydrochloric acid, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa.After the packing sampling with embodiment 1 described method measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result sees Table 6.
The technological process albumen and the rate of recovery of tiring see Table 3.
The table 3 technological process albumen and the rate of recovery of tiring
As shown in Figure 5, embodiment 3 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretograms, wherein, 1, sample-loading buffer, 2., anti--CMV height blood plasma of tiring, 3, the FI+II+III supernatant, 4, the FI+II+III resolution of precipitate, 5, sad deactivation supernatant, 6, the ethanol sedimentation supernatant, 7, before the DEAE chromatography, 8, penetrate 9, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 10, wash-out, 11, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 12, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 13, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 14, quiet notes human normal immunoglobulin (abroad) has gone on the market, 15, sample-loading buffer.Electrophoretogram shows that the quiet notes human normal immunoglobulin reference material IgG purity of having gone on the market is 86.6% abroad, polymer contains 2.2%, and dimer contains 7.52%, and albumin contains 2.51%, present embodiment purifying process parameter effect is consistent with embodiment 1, and preparation back IgG purity is 98.89%.
Embodiment 4
(1) euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured anti--human plasma 20 person-portions that the CMV height is tired, melts slurry under 4 ℃ of conditions, and mixing the back volume is 11670ml.
(2) add physiological saline 2330ml and regulate plasma proteins content to 49.27mg/ml, add glacial acetic acid and regulate pH to 6.02, add 95% ethanol 4494ml and regulate suspension alcohol concn to 23%, the conditioned reaction temperature is to-5.5 ℃, stirring reaction 6 hours reacts the centrifugation that finishes and obtains the FI+II+III precipitation.
(3) FI+II+III precipitation with pH be 4.81, concentration is that 30mmol/L sodium acetate buffer 12000ml dissolves, 8.0 ℃ of stirrings 10 hours down, centrifugation supernatant liquor.
(4) regulate supernatant pH to 4.96 with 1mol/L sodium hydroxide, press 10mmol/L concentration add sad, at 18 ℃ of stirrings 3 hours down, centrifugation supernatant liquor.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, and controlled filter pressure is not more than 0.25MPa, regulates pH to 5.21 with 1mol/L sodium hydroxide, and it is sad to add, and adjusts sad concentration to 46mmol/L, and 27 ℃ are stirred 2 hours deactivation lipid-coated virus, centrifugation supernatant liquor down.
(6) regulate pH to 5.04 with 1mol/L hydrochloric acid, add 95% ethanol 2396ml, reacted 5 hours the centrifugation supernatant liquor at-3.5 ℃ down by 15% concentration.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate concentrates 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, receive sample 2000ml after the ultrafiltration, 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, the ultrafiltration rear filtrate is crossed post, collection penetrates liquid 8000ml, the hanging column foreign protein with the pH that contains 2mol/LNaCl be 7.02, concentration is the phosphate buffered saline buffer wash-out of 60mmol/L.
(9) penetrate liquid pH to 4.47 with the adjusting of 1mol/L hydrochloric acid, after 0.1 μ m filter membrane pre-filtering, Novasip DV20 nano-film filtration removes virus again, and controlled filter pressure is not more than 0.25MPa.
(10) removing viral rear filtrate concentrates for 10 times with the 30KD ultra-filtration membrane, through the ultrafiltration of 8 times of volume water for injection, results stoste 500ml, protein content is 134.27mg/ml, dilute and press the amount adding maltose of 11g/L with water for injection, regulate pH to 4.08 with 1mol/L hydrochloric acid, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa.After the packing sampling with embodiment 1 described method measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result sees Table 6.
The technological process albumen and the rate of recovery of tiring see Table 4.
The table 4 technological process albumen and the rate of recovery of tiring
As shown in Figure 6, embodiment 4 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretograms, wherein, 1, sample-loading buffer, 2, anti--CMV height blood plasma of tiring, 3, the FI+II+III supernatant, 4, the FI+II+III resolution of precipitate, 5, sad deactivation supernatant, 6, the ethanol sedimentation supernatant, 7, before the DEAE chromatography, 8, penetrate, 9, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 10, wash-out, 11, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 12, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 13, the quiet notes human normal immunoglobulin (abroad) that gone on the market, 14, the quiet notes human normal immunoglobulin (domestic) that gone on the market, 15, sample-loading buffer.Electrophoretogram has contrasted the purity of domestic, the external quiet notes human normal immunoglobulin reference material of going on the market, and the result shows that domestic quiet notes human normal immunoglobulin IgG purity is 95.38%, how specifically to contain 0.8%, and dimer contains 2.37, and albumin contains 0.52%.Present embodiment purifying process parameter effect is consistent with embodiment 1, and preparation back IgG purity is 98.27%.
Embodiment 5
The key distinction of present embodiment and embodiment 1-4 is, blood plasma precipitates through 8% ethanol sedimentation Reaction Separation FI earlier, be used for subsequent purification through 20% ethanol sedimentation prepared in reaction FII+III precipitation again, in addition, the 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 anti--human plasma 2 person-portions that the CMV height is tired, melts slurry under 20 ℃ of conditions, and mixing the back volume is 1150ml.
(2) add physiological saline 230ml and regulate plasma proteins content 50.72mg/ml, add glacial acetic acid and regulate pH to 7.00, add 95% ethanol 130ml and regulate suspension alcohol concn to 8%, extremely-2.5 ℃ of conditioned reaction temperature, stirring reaction 4 hours, reaction finishes through centrifugation FI supernatant liquor, regulate supernatant pH to 6.25 with glacial acetic acid, add 95% ethanol 247ml adjusting suspension alcohol concn to 20%, reacted 6 hours down at-5.0 ℃, reaction finishes and obtains the FII+III precipitation through centrifugation.
(3) FII+III precipitation with pH be 4.98, concentration is that 40mmol/L sodium acetate buffer 1150ml dissolves, 3.0 ℃ of stirrings 12 hours down, centrifugation supernatant liquor.
(4) regulate supernatant pH to 4.78 with 4mol/L acetate, press 100mmol/L concentration add sad, at 22 ℃ of stirrings 2 hours down, centrifugation supernatant liquor.
(5) supernatant liquor is through 1.0 μ m membrane filtrations, controlled filter pressure is not more than 0.25MPa, regulates pH to 5.08 with 0.5mol/L sodium hydroxide, adds the injection water and adjusts sad concentration to 38mmol/L, 23 ℃ are stirred 1 hour deactivation lipid-coated virus, centrifugation supernatant liquor down.
(6) repetition measurement suspension pH to 5.03 adds 95% ethanol 208ml by 14% concentration, reacts 8 hours the centrifugation supernatant liquor down at-4.0 ℃.
(7) supernatant liquor is through 0.45 μ m membrane filtration, controlled filter pressure is not more than 0.25MPa, and filtrate concentrates 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, receive sample 170ml after the ultrafiltration, 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, the ultrafiltration rear filtrate is crossed post, collection penetrates liquid 328ml, the hanging column foreign protein with the pH that contains 2mol/L NaCl be 6.46, concentration is the phosphate buffered saline buffer wash-out of 25mmol/L.
(9) penetrate liquid pH to 4.75 with the adjusting of 1mol/L hydrochloric acid, after 0.1 μ m filter membrane pre-filtering, Novasip DV20 nano-film filtration removes virus again, and controlled filter pressure is not more than 0.25MPa.
(10) removing viral rear filtrate concentrates for 10 times with the 30KD ultra-filtration membrane, through the ultrafiltration of 8 times of volume water for injection, results stoste 50ml, protein content is 115.23mg/ml, dilute and press the amount adding maltose of 9g/L with water for injection, regulate pH to 3.98 with 1mol/L hydrochloric acid, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa.After the packing sampling with embodiment 1 described method measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, sad residual quantity and osmotic pressure molar density, detected result sees Table 6.
The technological process albumen and the rate of recovery of tiring see Table 5.
The table 5 technological process albumen and the rate of recovery of tiring
As shown in Figure 7, embodiment 5 quiet notes cytomegalovirus human normal immunoglobulin purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretograms, wherein, 1, sample-loading buffer, 2, anti--CMV height blood plasma of tiring, 3, the FI supernatant, 4, the FII+III supernatant, 5, the FII+III resolution of precipitate, 6, the ethanol sedimentation supernatant, 7, before the DEAE chromatography, 8, preparation back cytomegalovirus human normal immunoglobulin CMV-IgG, 9, wash-out, 10, the quiet notes human normal immunoglobulin (domestic) that gone on the market, 11, quiet notes human normal immunoglobulin (abroad) has gone on the market, 12, quiet notes human normal immunoglobulin (abroad) has gone on the market, 13, the quiet notes human normal immunoglobulin (abroad) that gone on the market, 14, the quiet notes human normal immunoglobulin (abroad) that gone on the market, 15, sample-loading buffer.Electrophoretogram shows that present embodiment purifying process parameter effect is consistent with embodiment 1, and preparation back IgG purity is 99.82%.
Comparative Examples 1,
Present embodiment for adopt existing cold ethanol method prepared the general population immunoglobulin (Ig) (the pharmacopeia name is called: quiet notes 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, melt slurry under 20 ℃ of conditions, mixing the back volume is 2850ml.
(2) add physiological saline 640ml and regulate plasma proteins content, add glacial acetic acid and regulate pH to 6.28, add dehydrated alcohol 930ml and regulate suspension alcohol concn to 20% to 47.36mg/ml, the conditioned reaction temperature is to-4.5 ℃,, stirring reaction 4 hours has reacted centrifugation FI+II+III precipitation.
(3) the FI+II+III precipitation is that 20mmol/L phosphate buffered saline buffer 2300ml dissolves with pH 5.07, concentration, and 4 ℃ were stirred the centrifugation supernatant liquor 12 hours down.
(4) repetition measurement supernatant liquor pH to 5.25, the ethanol 456ml of adding 95% regulates alcohol concn to 15%, and-3.5 ℃ were reacted centrifugation FI+III supernatant liquor 3 hours down.
(5) regulate FI+III supernatant liquor pH to 7.05 with 1mol/L sodium hydroxide, add 95% ethanol 205ml adjusting alcohol concn to 20% ,-7.0 ℃ were reacted 6 hours down, obtained the FII precipitation through centrifugation.
(6) the FII precipitation is dissolved with 600ml water for injection, and 2～8 ℃ were stirred the centrifugation supernatant liquor 12 hours.(7) supernatant liquor is concentrated into 100ml with the 30KD ultra-filtration membrane, through 8 times of volume water for injection ultrafiltration dealcoholysis, receives sample 150ml.
(8) prepare greater than 50mg/ml, maltose concentration 10g/L by protein content, regulate pH to 3.97 with 1mol/L hydrochloric acid, through 0.2 μ m membrane filtration degerming, controlled filter pressure is not more than 0.25MPa,
(9) the preparation sample is put between inactivation of virus after the degerming, incubates for 23～25 ℃ and puts 21 days, incubates putting the usefulness Novasip DV50 nano-film filtration that finishes except that virus, and controlled filter pressure is less than 0.25MPa.After the packing sampling with embodiment 1 described method measure protein content, tire, purity, pH value, IgG monomer, dimer, polymer, cracking body, albumin, osmotic pressure molar density.Detected result sees Table 6.
Table 6 cytomegalovirus human normal immunoglobulin preparation back sample quality index
As shown in Figure 8, Comparative Examples 1 quiet notes human normal immunoglobulin pH4 purification of samples polyacrylamide gel electrophoresis SDS-PAGE electrophoretogram, wherein, 1, sample-loading buffer, 2, the general population's blood plasma, 3, dilution back blood plasma, 4, the FI+II+III supernatant, 5, the FI+II+III resolution of precipitate, 6, FI+III supernatant, 7, FI+III resolution of precipitate, 8, the FII resolution of precipitate, 9, after the preparation.Electrophoretogram shows that preparation back IgG purity is 96.7%.
Claims (5)
1. quiet notes cytomegalovirus human normal immunoglobulin, it is characterized in that: described quiet notes 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.
2. the method for a quiet notes cytomegalovirus human normal immunoglobulin may further comprise the steps:
(1) FI+II+III, FII+III precipitate preparation
That the euzymelinked immunosorbent assay (ELISA) of learning from else's experience is measured is anti--human plasma that the CMV height is tired, melt slurry under 2～30 ℃, and merge and mix;
1. FI+II+III precipitation preparation
Regulate plasma proteins content to 45～55mg/ml with physiological saline, regulate pH to 6.0～6.5 with glacial acetic acid, add 95% ethanol or dehydrated alcohol and regulate alcohol concn to 20～25%, temperature of reaction is-5.5～-4.5 ℃, stirring reaction is 4～6 hours, and reaction finishes centrifugal or press filtration separates acquisition FI+II+III precipitation;
2. FII+III precipitation preparation
Regulate plasma proteins content to 45～55mg/ml with physiological saline, regulate pH to 6.8～7.2 with glacial acetic acid, adding the volume ratio mark is 95% ethanol or dehydrated alcohol and regulates alcohol concn to 7.5～8.5%, temperature of reaction is-2.5～-2.0 ℃, stirring reaction 4 hours, reaction finishes and separates removal FI precipitation through centrifugal or press filtration, obtain supernatant liquor, regulate supernatant liquor pH to 6.0～6.5 with glacial acetic acid, add 95% ethanol or dehydrated alcohol and regulate alcohol concn to 20～25%, temperature of reaction is-5.5～-4.5 ℃, and stirring reaction is 4～6 hours, and reaction finishes and separates acquisition FII+III precipitation through centrifugal or press filtration;
(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 stirred 8～16 hours down at 2～8 ℃, precipitation fully dissolved, through centrifugal or press filtration separation of supernatant;
(3) sad precipitation
Regulate supernatant liquor pH to 4.5～5.5 with 4mol/L acetate or 0.5～1mol/L sodium hydroxide, it is sad to add by 10～100mmol/L concentration, 18～25 ℃ of following stirring reactions 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 the aperture, controlled filter pressure is not more than 0.25MPa, regulate filtrate pH to 4.5～5.5 with 4mol/L acetate or 0.5～1mol/L sodium hydroxide, add water for injection or the sad concentration to 20 of sad adjusting suspension～80mmol/L, stirred 1～2 hour centrifugal or filtering separation supernatant liquor down at 20～30 ℃;
(5) ethanol sedimentation
Regulate supernatant liquor pH to 4.5～5.5 with 0.5～1mol/L hydrochloric acid or sodium hydroxide, add 95% ethanol or dehydrated alcohol carries out precipitin reaction by 12～16% concentration ,-4.0～-2.5 ℃ of following stirring reactions 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 concentrates for 15～20 times with the 30KD ultra-filtration membrane, and through the phosphate buffered saline buffer ultrafiltration dialysis of 20～60mmol/L of 8～10 times of volume pH 6.0～7.1, receiving sample control protein content after the ultrafiltration is 25～40mg/ml;
(7) anion-exchange chromatography
With pH is 6.0～7.1, concentration is 20～60mmol/L phosphate buffered saline buffer as level pad balance chromatography column 8～10 column volumes, be not more than 70～80% of the maximum carrying capacity of filler by every milliliter of filler and calculate upward sample protein content, collect behind the last sample and penetrate liquid, the phosphate buffered saline buffer wash-out of 20～60mmol/L of the pH 6.0～7.1 of hanging column foreign protein through containing 2mol/LNaCl;
(8) nanometer film is removed virus filtration
Hydrochloric acid adjusting with 0.5～1mol/L penetrates liquid pH to 4.2～5.0, after 0.1 μ m filter membrane pre-filtering, removes virus with Novasip DV20 nano-film filtration, and controlled filter pressure is not more than 0.25Mpa;
(9) ultrafiltration
Remove viral rear filtrate through 30KD ultra-filtration membrane protein concentrate content to 80～100mg/ml, with 8～10 times of water for injection ultrafiltration, receiving sample control protein content after the ultrafiltration is 80～150mg/ml;
(10) preparation
Stoste protein content after the mensuration ultrafiltration is adjusted goods protein content to 51～55mg/ml with the water for injection dilution, presses 9～11% amount adding maltose simultaneously, with 0.5～1mol/L hydrochloric acid adjusting pH to 3.8～4.2.
3. the method for quiet notes cytomegalovirus human normal immunoglobulin according to claim 2, 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, tiring is not less than the packing of 100PEI-U/ml specification.
4. the method for quiet notes cytomegalovirus human normal immunoglobulin according to claim 3 is characterized in that: goods protein content after the packing of sampling mensuration, and anti--CMV tires, purity, molecular size distribution, sad residual quantity, the project quality index of osmotic pressure molar density.
5. the method for quiet notes cytomegalovirus human normal immunoglobulin according to claim 2, it is characterized in that: add filler in the anion-exchange chromatography step, filler is selected from DEAE Sepharose Fast Flow, TOYOPEARL DEAE 650M or Macro-Prep DEAE Media.
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