CN105367651B - A kind of method of cold ethanol method removal human immunoglobulin(HIg) pyrogen - Google Patents
A kind of method of cold ethanol method removal human immunoglobulin(HIg) pyrogen Download PDFInfo
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- CN105367651B CN105367651B CN201510969585.2A CN201510969585A CN105367651B CN 105367651 B CN105367651 B CN 105367651B CN 201510969585 A CN201510969585 A CN 201510969585A CN 105367651 B CN105367651 B CN 105367651B
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 239000002510 pyrogen Substances 0.000 title claims abstract description 53
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 41
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 64
- 235000019441 ethanol Nutrition 0.000 claims abstract description 51
- 238000003825 pressing Methods 0.000 claims abstract description 43
- 239000000243 solution Substances 0.000 claims abstract description 33
- 238000001914 filtration Methods 0.000 claims abstract description 29
- 239000000047 product Substances 0.000 claims abstract description 28
- 230000001376 precipitating effect Effects 0.000 claims abstract description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000008215 water for injection Substances 0.000 claims abstract description 16
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 10
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims abstract description 9
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 9
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 238000010790 dilution Methods 0.000 claims abstract description 7
- 239000012895 dilution Substances 0.000 claims abstract description 7
- 239000008351 acetate buffer Substances 0.000 claims abstract description 4
- 238000001816 cooling Methods 0.000 claims description 21
- 239000000706 filtrate Substances 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 17
- 239000002158 endotoxin Substances 0.000 claims description 13
- 101000623895 Bos taurus Mucin-15 Proteins 0.000 claims description 12
- 230000006835 compression Effects 0.000 claims description 12
- 238000007906 compression Methods 0.000 claims description 12
- 239000012530 fluid Substances 0.000 claims description 11
- 238000007792 addition Methods 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- 229960000583 acetic acid Drugs 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 241000700605 Viruses Species 0.000 claims description 6
- 239000002826 coolant Substances 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 6
- 238000011010 flushing procedure Methods 0.000 claims description 6
- 238000004064 recycling Methods 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 4
- 239000000470 constituent Substances 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 230000000149 penetrating effect Effects 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 1
- -1 disodium hydrogen Chemical class 0.000 claims 1
- 238000001035 drying Methods 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 229910052698 phosphorus Inorganic materials 0.000 claims 1
- 239000011574 phosphorus Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000000502 dialysis Methods 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000036760 body temperature Effects 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 238000011046 pyrogen test Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000010513 Stupor Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 159000000021 acetate salts Chemical class 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000002391 anti-complement effect Effects 0.000 description 1
- 108010008730 anticomplement Proteins 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011082 depyrogenation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 231100000284 endotoxic Toxicity 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001698 pyrogenic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011265 semifinished product Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the methods of cold ethanol method removal human immunoglobulin(HIg) pyrogen, can effectively solve removal human immunoglobulin(HIg) pyrogen, need problem to human immunoglobulin(HIg) to meet.Method is, by the underproof human immunoglobulin(HIg) product normal saline dilution of pyrogen, components I+III is added or component III precipitates, adjusts solution ph with acetate buffer, is cooled to 0 DEG C ± 0.05 DEG C, add ethyl alcohol, adjust pH value；Diatomite is added, filters pressing obtains component III pressing filtering liquid, and sodium chloride is added, pH value is adjusted, ethanol synthesis is added, precipitating is collected in filters pressing, precipitating is dissolved with water for injection, adjusts pH value, stands filtering, ultrafiltration, maltose is added in dialysis, adjusts pH value, the method of the present invention section emulates the advanced, easy to operate, and effect is good, pyrogen in human immunoglobulin(HIg) product can be effectively removed, turns waste into wealth, reduces environmental pollution.
Description
Technical field
The present invention relates to field of biological pharmacy, especially a kind of side of cold ethanol method removal human immunoglobulin(HIg) pyrogen
Method.
Background technique
Pyrogen（pyrogen）The raised pyrogenic substance of homeothermal animal abnormal body temperature can be caused by meaning, the pyrogen of broad sense includes
Bacillary pyrogen, endogenous macromolecule pyrogen, endogenous low molecule pyrogen and chemical pyrogen etc., " pyrogen " in pharmacy is usual
Refer to bacillary pyrogen, is the metabolite that microorganism generates.Most of bacteriums, many moulds even virus can generate heat
Original, strongest pyrogenicity ability is pyrogen caused by gram negative bacilli.Endotoxin is the main pyrogenic substance for generating pyrogen.
Endotoxin compound as composed by phosphatide, lipopolysaccharides and protein, wherein lipopolysaccharides is endotoxic main component, has spy
Not strong pyrogenicity activity, the molecular weight of general lipopolysaccharides is bigger, and its pyrogenic action is stronger.Injection containing pyrogen, it is especially defeated
After liquid injects human body, about after half an hour, human body generation will be made to feel cold, shiver with cold, body temperature raising, bodily pain, sweating, nausea and vomiting
Equal adverse reactions, body temperature can rise to 40 DEG C or so sometimes, and serious person will appear stupor, collapse or even threat to life, clinically claim
Above-mentioned phenomenon is " pyrogen reaction ", so pyrogen test is the project that injection must be examined in pharmacopoeia of each country.
Although increasingly strengthening in current GMP management, pyrogen contamination is so the big critical point of manufacturing enterprise faced.One
Loss caused by denier product pollutes is huge, therefore in blood product production process, strengthening apyrogeneity operation is always to produce
The emphasis of corporate training, generally speaking the channel of pyrogen contamination, following several nothing more than：Raw material blood plasma, is added reaction vessel
Reagent adding, manual operation and working environment.Open report removes the active carbon adsorption of the method for depyrogenation, and ion-exchange surpasses
Filter method, acid-alkali treatment method etc..But these methods more or less some deficiencies, such as active carbon adsorption, during addition
Cleaning shop can be potentially polluted, such as a large amount of additions will cause the loss of target protein.Ion-exchange is processed batch
Limitation.Ultrafiltration is only applicable in the product that molecular weight is small, the needs not being able to satisfy in actual production.
Summary of the invention
For above situation, for the defect for overcoming the prior art, the purpose of the present invention is just to provide a kind of cold ethanol method
The method for removing human immunoglobulin(HIg) pyrogen can effectively solve removal human immunoglobulin(HIg) pyrogen, immune to people to meet
Globulin needs problem.
The technical solution that the present invention solves is that the ethanol precipitation of volumetric concentration 15% is used based on cold ethanol method, removal heat
Protoplasm, steps are as follows：
（1）, by pyrogen underproof human immunoglobulin(HIg) product 0 DEG C of normal saline dilution to 20g/L, by every 100kg
Components I+III is added in the underproof human immunoglobulin(HIg) of pyrogen or component III precipitates 40-50Kg, stirring and dissolving, with the vinegar of pH 4.0
Phthalate buffer adjusts solution ph to 5.15-5.25, and solution is cooled to 0 DEG C ± 0.05 DEG C by unlatching coolant system, add-
The ethyl alcohol of 15 DEG C of volumetric concentrations 95% below to alcohol content volumetric concentration is 15%, and speed≤70L/h is added in ethyl alcohol, and ethyl alcohol finishes
Otherwise repetition measurement solution ph should be adjusted with the acetate buffer of pH 4.0 to pH5.15-5.25, Bian Jiayi in 5.15-5.25
Alcohol side is cooled to -4.0 DEG C ± 0.5 DEG C；
（2）, be added diatomite into the solution after cooling, diatomite 15Kg is added in the product after every 1000L cooling, stirs
It mixes 30 minutes, carries out first time filters pressing with filter press, operating pressure control is within 0.2MPa in pressure-filtering process, filtrate temperature control
System checks the temperature and clarity of first-time filtrate for every 30 minutes, is blown after filters pressing with cold compression air at -2.5~-4.5 DEG C
Filter press intracavity liquid to the greatest extent, then be pumped into -4.0 DEG C ± 0.5 DEG C of volumetric concentration and rinse filter press, every filters pressing for 15% equilibrium liquid
It is 150 ~ 200L that machine equilibrium liquid, which rinses dosage, then blows the intracavitary equilibrium liquid of filter press with cold compression air, is collected containing balance
The filtrate of liquid obtains component III pressing filtering liquid, discards precipitating；
The diatomite first 180 DEG C it is dry 2.5 hours roasting, then be cooled to 0 DEG C it is used below；
The volumetric concentration is that the preparation method of 15% equilibrium liquid is ethyl alcohol containing volumetric concentration in every 100mL equilibrium liquid
15%, disodium hydrogen phosphate 0.005mol/L, with 2mol/L glacial acetic acid adjustment pH value to 5.20 ± 0.05, solution temperature temperature -4.0
℃±0.5℃；
The filter press is before use, first install filter plate, and every 100Kg human immunoglobulin(HIg) fills 25 filter plates, using note
Penetrating and being rinsed with water filter press to bacterial endotoxin is feminine gender, and diatomite 4Kg is added in 15% equilibrium liquid of every 500L, will after stirring
It is pumped into filter press and carries out circulating cooling, when filter press outlet temperature arrives -3 DEG C or less, stops recycling, -3 DEG C of compressions below skies
Gas dries up filter press spare；
（3）, component II precipitating preparation
1., metering component III pressing filtering liquid volume, by 5g/L amount addition sodium chloride, with 1mol/L sodium bicarbonate adjustment solution
For pH value to 7.10 ± 0.10, the ethyl alcohol that -15 DEG C of volumetric concentrations 95% below are added makes ethyl alcohol end body into component III pressing filtering liquid
Product concentration is 19%, is cooled to -6.0~-7.0 DEG C, is stirred to react 2 hours or more and carries out second of filters pressing；
2., second of filters pressing：During filters pressing, operating pressure control within 0.2MPa, filtrate temperature control-
6.0 DEG C ± 0.5 DEG C, the temperature and clarity of first-time filtrate are checked within every 30 minutes, with -3 DEG C with compressed air after filters pressing
Filter press intracavity liquid is blown, -6.0 DEG C ± 0.5 DEG C of 19% equilibrium liquid 200L of volumetric concentration is pumped into and rinses filter press, then with -3
DEG C filter press intracavity liquid is blown with compressed air, and continue to collect precipitating after blowing 30 minutes, is i.e. component II precipitating discards
Clear liquid；
19% equilibrium liquid is prepared：Ethyl alcohol containing volumetric concentration 19%, disodium hydrogen phosphate in every 100mL equilibrium liquid
0.005mol/L, sodium chloride 4g/L adjust pH 7.10 ± 0.10 with 2mol/L glacial acetic acid, and temperature is -6.0 DEG C ± 0.5 DEG C；
The filter press first installs filter plate before second of filters pressing, and every 100Kg human immunoglobulin(HIg) fills 25 filter plates,
Rinsing filter press to bacterial endotoxin with water for injection is feminine gender, and diatomite 5-6Kg is added in 19% equilibrium liquid of every 500L, stirs
Be pumped into after mixing filter press carry out circulating cooling, filter press outlet temperature arrive -4 DEG C or less when stopping recycle, -3 DEG C are below
Compressed air dries up filter press spare；
（4）, component II precipitating dissolution and filtering
It is precipitated with 0 DEG C of water for injection dissolved constituent II of 4~5 times of Sediment weights, lysate pH value is adjusted with 2mol/LHAc
To 3.80~4.10, filtering is stood within dissolution 4 hours or more, filtering terminates, and rinses sheet frame with 0 DEG C of water for injection, flushing liquor is incorporated to
Into filtered fluid；
（5）, ultrafiltration
Filtered fluid is squeezed into ultrafiltration tank, be concentrated into after being dialysed in equal volume 9 times with 0~4 DEG C of water-dialyzing protein concentration 60~
80g/L, then it is diluted to 51g/L with water for injection, the final content of addition maltose to maltose is 90-110g/L, uses 2mol/
L HAc adjusts solution ph 3.80~4.10, then is virus inactivated, and is the immune ball of people of removal pyrogen after degerming packing
Protein product.
The method of the present invention section emulates the advanced, easy to operate, and effect is good, can effectively remove pyrogen in human immunoglobulin(HIg) product
Matter turns waste into wealth, and reduces environmental pollution, and economic and social benefit is huge.
Detailed description of the invention
Fig. 1 is process flow chart of the invention.
Specific embodiment
It elaborates below in conjunction with drawings and examples to a specific embodiment of the invention.
Embodiment 1
The present invention in specific implementation, can be realized by following methods：
（1）, by pyrogen underproof human immunoglobulin(HIg) product 0 DEG C of normal saline dilution to 20g/L, by every 100kg
Components I+III is added in the underproof human immunoglobulin(HIg) of pyrogen or component III precipitates 40Kg, stirring and dissolving, with the acetate of pH4.0
Buffer adjusts solution ph to 5.2, opens coolant system for solution and is cooled to 0 DEG C ± 0.05 DEG C, add -15 DEG C it is below
95% ethyl alcohol of volumetric concentration to alcohol content volumetric concentration is 15%, and speed≤70L/h is added in ethyl alcohol, with the acetate salt buffer of pH4.0
Liquid adjusts solution ph to 5.2, -4.0 DEG C ± 0.5 DEG C is cooled to when adding ethyl alcohol, the product after must cooling down；
（2）, be added diatomite into the solution after cooling, diatomite 15Kg is added in the product after every 1000L cooling, stirs
It mixes 30 minutes, carries out first time filters pressing with filter press, operating pressure control is within 0.2MPa in pressure-filtering process, filtrate temperature control
System checks the temperature and clarity of first-time filtrate for every 30 minutes, is blown after filters pressing with cold compression air at -4.0 DEG C ± 0.5 DEG C
Filter press intracavity liquid to the greatest extent, then be pumped into -4.0 DEG C ± 0.5 DEG C of volumetric concentration and rinse filter press, every filters pressing for 15% equilibrium liquid
It is 150 ~ 200L that machine equilibrium liquid, which rinses dosage, then blows the intracavitary equilibrium liquid of filter press with cold compression air, is collected containing balance
The filtrate of liquid obtains component III pressing filtering liquid, discards precipitating；
The filter press is before use, first install filter plate, and every 100Kg human immunoglobulin(HIg) fills 25 filter plates, using note
Penetrating and being rinsed with water filter press to bacterial endotoxin is feminine gender, and diatomite 4Kg is added in 15% equilibrium liquid of every 500L, will after stirring
It is pumped into filter press and carries out circulating cooling, when filter press outlet temperature arrives -3 DEG C or less, stops recycling, -3 DEG C of compressions below skies
Gas dries up filter press spare；
（3）, component II precipitating preparation
1., metering component III pressing filtering liquid volume, by 5g/L amount addition sodium chloride, with 1mol/L sodium bicarbonate adjustment solution
PH value 7.10 ± 0.10, it is 19% that -15 DEG C of 95% ethyl alcohol below ethyl alcohol volumetric concentration into component III pressing filtering liquid, which is added, cooling
To -6.0~-7.0 DEG C, it is stirred to react 2 hours or more；
2., second of filters pressing：Filter plate, every 100Kg human immunoglobulin(HIg) dress are first installed on a filter press before second of filters pressing
25 filter plates, rinsing filter press to bacterial endotoxin with water for injection is feminine gender, and diatomite is added in 19% equilibrium liquid of every 500L
5-6Kg, be pumped into after stirring filter press carry out circulating cooling, filter press outlet temperature arrive -4 DEG C or less when stopping recycle, -3
DEG C compressed air below dries up filter press spare；
During filters pressing, operating pressure is controlled within 0.2MPa, and filtrate temperature is controlled at -6.0 DEG C ± 0.5 DEG C, often
The temperature and clarity for checking first-time filtrate for 30 minutes, blow filter press intraluminal fluid with -3 DEG C after filters pressing with compressed air
Body is pumped into -6.0 DEG C ± 0.5 DEG C of 19% equilibrium liquid 200L of volumetric concentration and rinses filter press, then blown with -3 DEG C with compressed air
Filter press intracavity liquid to the greatest extent, and continue to collect precipitating after blowing 30 minutes, i.e. component II precipitating discards supernatant liquid；
（4）, component II precipitating dissolution and filtering
It is precipitated with 0 DEG C of water for injection dissolved constituent II of 4~5 times of Sediment weights, 2mol/LHAc adjusting pH value is molten to 3.9
Stand filtering within solution 4 hours or more, filtering terminates, and rinses sheet frame with 0 DEG C of water for injection, flushing liquor is incorporated into filtered fluid；
（5）, ultrafiltration
Filtered fluid is squeezed into ultrafiltration tank, be concentrated into after being dialysed in equal volume 9 times with 0~4 DEG C of water-dialyzing protein concentration 60~
80g/L, then it is diluted to mass concentration 5.1% with water for injection, the final content of addition maltose to maltose is 90-110g/L,
2mol/L HAc adjusts pH3.90, then is virus inactivated, and as removes the human immunoglobulin(HIg) product of pyrogen.
Embodiment 2
The present invention in specific implementation, can also be realized by following methods：
（1）, by pyrogen underproof human immunoglobulin(HIg) product 0 DEG C of normal saline dilution to 20g/L, by every 100kg
Components I+III is added in the underproof human immunoglobulin(HIg) of pyrogen or component III precipitates 50Kg, stirring and dissolving, with the acetic acid of pH value 4.0
Salt buffer adjusts solution ph to 5.25, opens coolant system for pH adjustment liquid and is cooled to 0 DEG C ± 0.05 DEG C, adds -15
DEG C 95% ethyl alcohol of volumetric concentration below to alcohol content volumetric concentration is 15%, and speed≤70L/h is added in ethyl alcohol, with pH value 4.0
Acetate buffer adjusts solution ph to 5.25, -4.0 DEG C ± 0.5 DEG C is cooled to when adding ethyl alcohol, the product after must cooling down；
（2）, be added diatomite into the product after cooling, diatomite 15Kg is added in the product after every 1000L cooling, stirs
It mixes 30 minutes, carries out first time filters pressing with filter press, operating pressure controls within 0.2MPa, and filtrate temperature is controlled -3~-4
DEG C, it checks within every 30 minutes the temperature and clarity of first-time filtrate, blows filter press intraluminal fluid with cold compression air after filters pressing
Body, then be pumped into -4.0 DEG C ± 0.5 DEG C of volumetric concentration and rinse filter press for 15% equilibrium liquid, every filter press equilibrium liquid, which rinses, to be used
Amount is 150 ~ 200L, blows the intracavitary equilibrium liquid of filter press with cold compression air, collects the filtrate containing equilibrium liquid, obtain component
III pressing filtering liquid discards precipitating；
The filter press is before use, first install filter plate, and every 100Kg human immunoglobulin(HIg) fills 25 filter plates, using note
Penetrating and being rinsed with water filter press to bacterial endotoxin is feminine gender, and diatomite 4Kg is added in 15% equilibrium liquid of every 500L, will after stirring
It is pumped into filter press and carries out circulating cooling, when filter press outlet temperature arrives -3 DEG C or less, stops recycling, -3 DEG C of compressions below skies
Gas dries up filter press spare；
（3）, component II precipitating preparation
1., metering component III pressing filtering liquid volume, by 5g/L amount addition sodium chloride, with 1mol/L sodium bicarbonate adjustment solution
For pH value to 7.10 ± 0.10, -15 DEG C of 95% ethyl alcohol below, which are added, makes ethyl alcohol volumetric concentration 19% into component III pressing filtering liquid,
- 6.0~-7.0 DEG C are cooled to, is stirred to react 2 hours or more；
2., second of filters pressing：Filter plate, every 100Kg human immunoglobulin(HIg) dress are first installed on a filter press before second of filters pressing
25 filter plates, rinsing filter press to bacterial endotoxin with water for injection is feminine gender, and diatomite is added in 19% equilibrium liquid of every 500L
5-6Kg, be pumped into after stirring filter press carry out circulating cooling, filter press outlet temperature arrive -4 DEG C or less when stopping recycle, -3
DEG C compressed air below dries up filter press spare；
During filters pressing, operating pressure is controlled within 0.2MPa, and filtrate temperature is controlled at -6.0 DEG C ± 0.5 DEG C, often
The temperature and clarity for checking first-time filtrate for 30 minutes, blow filter press intraluminal fluid with -3 DEG C after filters pressing with compressed air
Body is pumped into -6.0 DEG C ± 0.5 DEG C of 19% equilibrium liquid 200L of volumetric concentration and rinses filter press, then blown with -3 DEG C with compressed air
Filter press intracavity liquid to the greatest extent, and continue to collect precipitating after blowing 30 minutes, i.e. component II precipitating discards supernatant liquid；
（4）, component II precipitating dissolution and filtering
It is precipitated with 0 DEG C of water for injection dissolved constituent II of 4~5 times of Sediment weights, 2mol/LHAc adjusting pH value is molten to 3.9
Stand filtering within solution 4 hours or more, filtering terminates, and rinses sheet frame with 0 DEG C of water for injection, flushing liquor is incorporated into filtered fluid；
（5）, ultrafiltration
Filtered fluid is squeezed into ultrafiltration tank, be concentrated into after being dialysed in equal volume 9 times with 0~4 DEG C of water-dialyzing protein concentration 60~
80g/L, then it is diluted to mass concentration 5.1% with water for injection, the final content of addition maltose to maltose is 90-110g/L,
2mol/L HAc adjusts pH value 3.9, then is virus inactivated, and as removes the human immunoglobulin(HIg) product of pyrogen.
Human immunoglobulin(HIg) prepared by the present invention can dispense after semi-finished product preparation and inactivation of virus and ball is immunized for people
Albumen finished product, through detecting, quality meets claimed below：
1 purity：It presses《Chinese Pharmacopoeia》Cellulose acetate membrane electrophoresis method detects in three general rules of version in 2015,0,541 second method,
Its purity is not less than 95.0%；
2. protein content：It presses《Chinese Pharmacopoeia》Three general rules of version in 2015,0731 third method（Double contracting method urea method detections）,
Greater than 50g/L；
3.pH value：It presses《Chinese Pharmacopoeia》Three general rules 0631 of version in 2015, with physiological sodium chloride solution by test sample albumen
Matter content is diluted to 10g/L, and pH value is 3.80~4.40.
4. residual ethanol content：It presses《Chinese Pharmacopoeia》Three 3201 methods of general rule of version in 2015, the measurement of Kang Wei diffusion boat method, no
Higher than 0.025%.
5. pyrogen test：It presses《Chinese Pharmacopoeia》Three general rules 1142 of version in 2015, injection dosage are infused by rabbit weight 1Kg
0.5g protein is penetrated, regulation is met.
6. anti-complement activity：It presses《Chinese Pharmacopoeia》Three general rules 3410 of version in 2015 are not higher than 50%.
Loss late of the present invention is small, and through repeatedly testing repeatedly, protein losses are only 3% hereinafter, see the table below：
| Lot number | Remove prepyrogen | Pyrogen after removal | Product loss |
| 01 | The sum of heating 1.5 | 0.5 | 2.4% |
| 02 | The sum of heating 1.9 | 0.5 | 2.6% |
| 03 | The sum of heating 1.7 | 0.4 | 2.5% |
By above-mentioned it should be apparent that the present invention removes human immunoglobulin(HIg) pyrogen using cold ethanol method, for low
For the enterprise of warm ethyl alcohol production, without additionally adding equipment, and processing is of large quantities, and removing effect is obvious, is that one kind conscientiously may be used
Capable method has very strong practicability.After component I+III or FIII precipitating is added, five variable elements are adjusted, can be effectively reduced
Product pyrogen, protein losses only 3% hereinafter, the reason is that using I+III or FIII precipitating precipitate again, can
It precipitates pyrogen and FIII to combine and coprecipitated, achievees the purpose that removal heat source.Meanwhile component I+III or FIII precipitating are original
Human immunoglobulin(HIg) can achieve the purpose of recycling by dissolving again, this be it is double at one stroke, both eliminated original product
In pyrogen, while recycled component I+III or FIII precipitating in human immunoglobulin(HIg), realize waste utilization, change give up into
Treasured reduces environmental pollution, and economic and social benefit is huge.
Claims (3)
1. a kind of method of cold ethanol method removal human immunoglobulin(HIg) pyrogen, which is characterized in that realized by following steps：
（1）, by pyrogen underproof human immunoglobulin(HIg) product 0 DEG C of normal saline dilution to 20g/L, by every 100kg pyrogen
Components I+III is added in underproof human immunoglobulin(HIg) or component III precipitates 40-50Kg, and stirring and dissolving is slow with pH4.0 acetate
Fliud flushing adjusts solution ph to 5.15-5.25, opens coolant system for solution and is cooled to 0 DEG C ± 0.05 DEG C, add -15 DEG C with
Under ethyl alcohol to the alcohol-containing final volume concentration of volumetric concentration 95% be 15%, speed≤70L/h is added in ethyl alcohol, and ethyl alcohol finishes repetition measurement
Otherwise solution ph should be adjusted with the acetate buffer of pH 4.0 to pH5.15-5.25, in 5.15-5.25 when adding ethyl alcohol
It is cooled to -4.0 DEG C ± 0.5 DEG C；
（2）, be added diatomite into the solution after cooling, diatomite 15Kg, stirring 30 is added in the product after every 1000L cooling
Minute, first time filters pressing is carried out with filter press, operating pressure control is within 0.2MPa in pressure-filtering process, the control of filtrate temperature
At -2.5~-4.5 DEG C, checks within every 30 minutes the temperature and clarity of first-time filtrate, blown after filters pressing with cold compression air
Filter press intracavity liquid, then be pumped into -4.0 DEG C ± 0.5 DEG C of volumetric concentration and rinse filter press, every filter press for 15% equilibrium liquid
It is 150 ~ 200L that equilibrium liquid, which rinses dosage, then blows the intracavitary equilibrium liquid of filter press with cold compression air, and collection contains equilibrium liquid
Filtrate, obtain component III pressing filtering liquid, discard precipitating；
The diatomite first 180 DEG C it is dry 2.5 hours roasting, then be cooled to 0 DEG C it is used below；
The volumetric concentration is that the preparation method of 15% equilibrium liquid is ethyl alcohol containing volumetric concentration 15%, phosphorus in every 100mL equilibrium liquid
Sour disodium hydrogen 0.005mol/L, with 2mol/L glacial acetic acid adjustment pH value to 5.20 ± 0.05, -4.0 DEG C of solution temperature temperature ± 0.5
℃；
The filter press is before use, first install filter plate, and every 100Kg human immunoglobulin(HIg) fills 25 filter plates, using injection
It is feminine gender that water, which rinses filter press to bacterial endotoxin, and diatomite 4Kg is added in 15% equilibrium liquid of every 500L, is pumped after stirring
Enter filter press and carry out circulating cooling, when filter press outlet temperature arrives -3 DEG C or less, stops recycling, -3 DEG C of compressed airs below generals
Filter press drying is spare；
（3）, component II precipitating preparation
1., metering component III pressing filtering liquid volume, by 5g/L amount addition sodium chloride, with 1mol/L sodium bicarbonate adjustment solution ph
To 7.10 ± 0.10, the ethyl alcohol that -15 DEG C of volumetric concentrations 95% below are added makes ethyl alcohol final volume into component III pressing filtering liquid
Concentration is 19%, is cooled to -6.0~-7.0 DEG C, is stirred to react 2 hours or more and carries out second of filters pressing；
2., second of filters pressing：During filters pressing, operating pressure is controlled within 0.2MPa, and filtrate temperature is controlled at -6.0 DEG C
It ± 0.5 DEG C, checks within every 30 minutes the temperature and clarity of first-time filtrate, pressure is blown with compressed air with -3 DEG C after filters pressing
Filter intracavity liquid is pumped into -6.0 DEG C ± 0.5 DEG C of 19% equilibrium liquid 200L of volumetric concentration and rinses filter press, then with -3 DEG C or less
Compressed air blows filter press intracavity liquid, and continues to collect precipitating after blowing 30 minutes, i.e. component II precipitating discards supernatant liquid；
19% equilibrium liquid is prepared：Ethyl alcohol containing volumetric concentration 19%, disodium hydrogen phosphate 0.005mol/ in every 100mL equilibrium liquid
L, sodium chloride 4g/L adjusts pH 7.10 ± 0.10 with 2mol/L glacial acetic acid, and temperature is -6.0 DEG C ± 0.5 DEG C；
The filter press first installs filter plate, every 100Kg human immunoglobulin(HIg) fills 25 filter plates, with note before second of filters pressing
Penetrating and being rinsed with water filter press to bacterial endotoxin is feminine gender, diatomite 5-6Kg is added in 19% equilibrium liquid of every 500L, after stirring
Be pumped into filter press carry out circulating cooling, filter press outlet temperature arrive -4 DEG C or less when stopping recycle, -3 DEG C of compressions below
Air dries up filter press spare；
（4）, component II precipitating dissolution and filtering
It is precipitated with 0 DEG C of water for injection dissolved constituent II of 4~5 times of Sediment weights, lysate pH value is adjusted with 2mol/L HAc and is arrived
3.80~4.10, stand filtering within dissolution 4 hours or more, filtering terminates, and rinses sheet frame with 0 DEG C of water for injection, flushing liquor is incorporated into
In filtered fluid；
（5）, ultrafiltration
Filtered fluid is squeezed into ultrafiltration tank, is concentrated into 60~80g/ of protein concentration after being dialysed in equal volume 9 times with 0~4 DEG C of water-dialyzing
L, then it is diluted to 51g/L with water for injection, addition maltose to the final content of maltose is 90-110g/L, with 2mol/L HAc
PH value of solution 3.80~4.10 is adjusted, then is virus inactivated, is the human immunoglobulin(HIg) system for removing pyrogen after degerming packing
Product.
2. the method for cold ethanol method removal human immunoglobulin(HIg) pyrogen according to claim 1, which is characterized in that institute
The step of stating（1）, by pyrogen underproof human immunoglobulin(HIg) product 0 DEG C of normal saline dilution to 20g/L, by every 100kg
Components I+III is added in the underproof human immunoglobulin(HIg) of pyrogen or component III precipitates 40Kg, stirring and dissolving, with the acetic acid of pH 4.0
Salt buffer adjusts solution ph to 5.2, opens coolant system for solution and is cooled to 0 DEG C ± 0.05 DEG C, adds -15 DEG C or less
95% ethyl alcohol of volumetric concentration to alcohol content volumetric concentration be 15%, speed≤70L/h is added in ethyl alcohol, slow with the acetate of pH 4.0
Fliud flushing adjusts solution ph to 5.2, -4.0 DEG C ± 0.5 DEG C is cooled to when adding ethyl alcohol, the product after must cooling down.
3. the method for cold ethanol method removal human immunoglobulin(HIg) pyrogen according to claim 1, which is characterized in that institute
The step of stating（1）, by pyrogen underproof human immunoglobulin(HIg) product 0 DEG C of normal saline dilution to 20g/L, by every 100kg
Components I+III is added in the underproof human immunoglobulin(HIg) of pyrogen or component III precipitates 50Kg, stirring and dissolving, with the acetic acid of pH 4.0
Salt buffer adjusts solution ph to 5.25, opens coolant system for solution and is cooled to 0 DEG C ± 0.05 DEG C, add -15 DEG C with
Under 95% ethyl alcohol of volumetric concentration to alcohol content volumetric concentration be 15%, ethyl alcohol be added speed≤70L/h, with the acetate of pH 4.0
Buffer adjusts solution ph to 5.25, -4.0 DEG C ± 0.5 DEG C is cooled to when adding ethyl alcohol, the product after must cooling down.
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| CN1699417A (en) * | 2005-06-21 | 2005-11-23 | 江永忠 | Human blood high density lipoprotein, its manufacturing method and application |
| CN101648998A (en) * | 2009-07-31 | 2010-02-17 | 郑州邦和生物药业有限公司 | Method for extracting human immune globulin from component I+III or component III |
| CN103333240A (en) * | 2013-07-22 | 2013-10-02 | 北海开元生物科技有限公司 | Method for reclaiming human albumin from component IV precipitate |
| CN104479011A (en) * | 2015-01-05 | 2015-04-01 | 深圳市卫光生物制品股份有限公司 | Method for preparing intravenous immunoglobulin |
| CN104558156A (en) * | 2015-01-23 | 2015-04-29 | 郑州莱士血液制品有限公司 | Method for extracting human serum albumin from plasma and increasing yield |
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| CN1699417A (en) * | 2005-06-21 | 2005-11-23 | 江永忠 | Human blood high density lipoprotein, its manufacturing method and application |
| CN101648998A (en) * | 2009-07-31 | 2010-02-17 | 郑州邦和生物药业有限公司 | Method for extracting human immune globulin from component I+III or component III |
| CN103333240A (en) * | 2013-07-22 | 2013-10-02 | 北海开元生物科技有限公司 | Method for reclaiming human albumin from component IV precipitate |
| CN104479011A (en) * | 2015-01-05 | 2015-04-01 | 深圳市卫光生物制品股份有限公司 | Method for preparing intravenous immunoglobulin |
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