CN102974217A - Method for sterilization and purification of protein solutions and serum solutions - Google Patents
Method for sterilization and purification of protein solutions and serum solutions Download PDFInfo
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- CN102974217A CN102974217A CN 201110263202 CN201110263202A CN102974217A CN 102974217 A CN102974217 A CN 102974217A CN 201110263202 CN201110263202 CN 201110263202 CN 201110263202 A CN201110263202 A CN 201110263202A CN 102974217 A CN102974217 A CN 102974217A
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Abstract
The present invention discloses a method for sterilization and purification of protein solutions and serum solutions, comprising the following steps: first making one pleated filter with closely arranged folds, wherein an upstream membrane pore size is of 0.45-1.2[mu]m, while a downstream membrane pore size is of 0.2[mu]m; then taking a serum protein solution, and under an input pressure of 0.1MPa, making the serum protein solution to pass through the pleated filter; determining the volume of bovine serum albumin passed though the pleated filter in 10min and calculating a filtration rate via a formula; analyzing the sterilization rate after the bovine serum albumin passes through the pleated filter; performing sterilization to the pleated filter for 30 minutes by using a steam sterilization method at 121 DEG C; and repeating the above steps to determine the number of effective using times of the pleated filter until a last time can still reach an effective sterilization rate (more than 99.99%). The sterilization and purification method disclosed by the present invention not only can ensure the sterilization rate to be 99.99%, but also is advantageous by being superior in filtration rate and reusable.
Description
Technical field
The present invention relates to a kind of bactericidal purifying method, particularly a kind of method of degerming and the purification for albumen, serum solution.
Background technology
Killing or remove all with methods such as physics or chemistry causes a disease and the brood body of non-pathogenic microorganism and brood cell's means are called sterilization.Sterilization can be divided into three kinds in the pharmacy: physical sterilization method, chemical sterilization and aseptic manipulation.The physical sterilization method mainly contains heat sterilization method, ray sterilizing method and sterilization by filtration; Chemical sterilization has gas sterilization and chemical agent sterilization, mainly is applicable to the sterilization of medical appliance, equipment and facility.The selection of sterilizing methods should take into account the chemical stability of medicine, the physical and chemical stability of preparation.Albumen, serum solution are solution owing to it, and have the heat of chance, the unsettled characteristic of ray, generally adopt the sterilization by filtration in the physical sterilization method.Sterilization by filtration is to make drug solution pass through aseptic specific filter, except deactivation or dead microorganism and do not contained the filtrate of microorganism.Research finds that reproduction type bacterium size is generally greater than 1 μ m, and gemma is greater than 0.5 μ m.
At present, the sterilising filter filter membrane aperture of haemocyanin on the market adopts 0.2 μ m, but has filtering velocity and the conflicting phenomenon of sterilization rate.Generally speaking, the filter membrane aperture is larger, and filtering velocity is faster, but simultaneously because the filter membrane aperture is large, the sterilization rate of bacterium can reduce.As under 0.1MPa air pressure, be the polyether sulfone millipore filter discovery of 0.2 μ m with haemocyanin by the aperture, the haemocyanin volume that 10min passes through is 47L, filtering velocity is 282l/h.Adopting the Chinese Pharmacopoeia Sterility Test to record sterilization rate is 99.995%.Under 0.1MPa air pressure, be the polyether sulfone millipore filter discovery of 1.2 μ m with haemocyanin by the aperture simultaneously, the haemocyanin volume that 10min passes through is 85L, and filtering velocity is 510l/h.Adopting the Chinese Pharmacopoeia Sterility Test to record sterilization rate is 96.875%, does not reach the sterilization requirement of high-end field.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the invention discloses and a kind ofly can process the albumen of different situations, the degerming of serum solution and the method for purification.
Technical scheme: the invention discloses a kind of method of degerming and the purification for albumen, serum solution, may further comprise the steps:
1) formulate 1 compact arranged folder filter, its middle and upper reaches filter membrane aperture 0.45-1.2 μ m, filter membrane aperture, downstream is 0.2 μ m;
2) get haemocyanin, press as under the 0.1MPa condition in input, haemocyanin is passed through above-mentioned folder filter;
3) the bovine serum albumin volume that passes through in the folder filter in the time by formula mensuration 10min calculates filtering velocity;
4) analyze bovine serum albumin by the sterilization rate behind the folder filter;
5) sterilization that adopts 121 degree method of steam-sterilizing folder filter to be carried out 30 minutes;
6) repeating step 2 is to step 5 process, measures effective access times of filter, and effectively access times are with till still reaching for the last time effective sterilizing rate (more than 99.99%).
The described folder filter filter membrane of step 1 material adopts polyether sulfone, and on not impact of solution, existing problems are few in the use procedure, so miillpore filter is present most widely used aseptic filtration medium, and has become the basic tool of preparation sterile solution.The material of miillpore filter mainly contains cellulose acetate fat, celluloid fat, polyether sulfone and composite material.
The polyether sulfone chemical structural formula:
, neither contain the relatively poor aliphatic hydrocarbon chain link of heat endurance in the molecular structure, do not contain again the large biphenyl chain link of rigidity, and mainly formed by sulfuryl, ether and phenylene.The attached heat resistance of giving of sulfuryl; Ether makes the polymer chain link have good flowability when molten condition, is easy to processing and forming; Alternately link sulfuryl on to the penylene structure and ether can obtain noncrystalline polymer.Poly (ether sulfone) film has temperature classification height, good mechanical property, the characteristics of dimensionally stable, chemical proofing, heat-resisting water, hydrolytic resistance, anti-flammability.In view of the chemical characteristic of polyether sulfone, filter membrane material of the present invention adopts polyether sulfone.
Haemocyanin described in the step 2 is that molecular weight is 67000 bovine serum albumin.
The described formula of step 3 is: filtering velocity (l/h)=bovine serum albumin volume/10min.
The analysis of the described sterilization rate of step 4 adopts the Chinese Pharmacopoeia Sterility Test to measure.
Sterile detection method is as follows:
One, the preparation of culture medium and inoculation
(1) preparation of THIOGLYCOLLIC ACID salt fluid culture medium: junket peptone (pancreatin hydrolysis) 15.0g, yeast extract powder 5.0g, glucose 5.0g, sodium chloride 2.5g, CYSTINE 0.5g, 0.1% resazurin solution 1.0ml of new preparation, sodium thioglycollate 0.5g, agar 0.75g (or THIOGLYCOLLIC ACID, 0.3ml), water 1000ml.Except glucose and resazurin solution, get mentioned component and mix, the tepor dissolving, adjusting pH is alkalescent, boils, filtering adds glucose and resazurin solution, shakes up, and is 7.1 ± 0.2 after adjusting pH value makes sterilization.Divide to be filled in the suitable container, culture medium oxide layer (pink) was no more than 1/2 of the culture medium degree of depth after the ratio of its loading amount and container height should meet the cultivation end.Rearmounted 30 ℃～35 ℃ cultivations of sterilization.
(2) the applicability inspection of culture medium and sensitivity inspection: aseptic inspection: every batch of culture medium is got at random and is no less than 5 (bottles), cultivates 14 days, answers asepsis growth.Following test: bacterial classification: clostridium sporogenes (Clostridi μ m sporogenes) (CMCC (B) 64941), the fresh cultured thing of inoculation clostridium sporogenes were cultivated 18 hours～24 hours for 30 ℃～35 ℃ to THIOGLYCOLLIC ACID salt fluid culture medium.
Above-mentioned culture is made every 1ml with 0.9% aseptic sodium chloride solution and is contained the bacterium number less than the bacteria suspension of 100cfu (CFU).
Bacteria suspension is at room temperature placed and should be used in 2 hours, can use in 24 hours if be kept at 2～8 ℃.
(3) culture medium inoculated: 9 of the THIOGLYCOLLIC ACID salt fluid culture mediums that to get every pipe loading amount be 12ml, inoculate respectively each 2 of staphylococcus aureuses less than 100 CFUs, pseudomonas aeruginosa, bacillus subtilis, clostridium sporogenes, do not inoculate as blank for 1 in addition, cultivated 3 days;
(4) result judges: the blank pipe is answered asepsis growth, if add the equal well-grown of cultivation parent tube of bacterium, the following test of declaring this culture medium is up to specification.
(5) method validation test: adopt direct inoculation, get THIOGLYCOLLIC ACID salt fluid culture medium 8 pipes that meet the requirement of direct inoculation culture medium consumption, access respectively staphylococcus aureus, EHEC, bacillus subtilis, each 2 pipe of clostridium sporogenes less than 100 CFUs, wherein 1 pipe accesses the test sample amount of every culture medium ormal weight, 1 pipe was cultivated 3～5 days by the temperature of putting regulation in contrast in addition.
(6) result judges: compare with control tube, as contain the equal well-grown of test organisms in each container of test sample, this inspected number that test sample then is described can be ignored without bacteriostasis or its bacteriostasis under this test condition, and inspection method and inspection condition are carried out the sterility test of test sample like this.As contain test organisms growth in arbitrary container of test sample faint, slowly or not grow, this inspected number that test sample then is described has bacteriostasis under this test condition, can adopt the consumption, use nertralizer or the methods such as inactivator, replacing filter membrane kind that increase flushing dose, increase culture medium, eliminate the bacteriostasis of test sample, and re-start the method validation test.
Two, the sterility test of test sample (bovine serum albumin)
Adopt direct inoculation that the bovine serum albumen solution before and after the micro-filtration is carried out sterility test.
(1) gets the ormal weight test sample and be seeded to respectively sulfur-bearing glycollate broth.Get 9 pipes 12ml THIOGLYCOLLIC ACID salt fluid culture medium is housed, every pipe inoculation bovine serum albumin 1ml.
(2) cultivate and observe: the above-mentioned container temperature in accordance with regulations that contains culture medium was cultivated 14 days.Should observe day by day and record between culture period whether bacteria growing is arranged.Adopt the CFU of microscopy record bacterium.
As after adding test sample or in incubation, culture medium occurs muddy, cultivate after 14 days, can not have or not growth of microorganism from judging in appearance, the an amount of transferred species of desirable this nutrient solution is to fresh culture of the same race, Bacteria Culture 2 days, fungal culture 3 days, whether the fresh culture of the same race of observing inoculation occurs muddy again; Or get the nutrient solution smear, and dyeing, microscopy has judged whether bacterium.
The calculating of sterilization rate:
The bovine serum albumin bacterium forms units (cfu/ml) * 100% before sterilization rate=(the bovine serum albumin bacterium formed units (cfu/ml) after the bovine serum albumin bacterium formed units (cfu/ml)-micro-filtration before the sterilization)/sterilization
Principle of the present invention is for sterilizing to albumen, serum solution by MF method and purifying.
The present invention at first adopts the large aperture coarse filtration, adopts the smart method of filtering in small-bore to reach the haemocyanin sterilizing methods that filtering velocity is fast, sterilization is high again.The reproduction type bacterium is mainly removed in the large aperture coarse filtration, and smart filtration the in small-bore mainly removed gemma and reproduction type bacterium.Large aperture, upstream filter membrane can carry out pre-filtering to the reproduction type bacterium to adopt upstream coarse filtration, downstream fine to filter on the one hand, can protect the small-bore filter membrane in downstream on the one hand.
Beneficial effect: the method for a kind of degerming and purification for albumen, serum solution disclosed by the invention, adopt aperture, upstream 0.45-1.2 μ m, the Combination filter element of aperture, downstream 0.2 μ m not only can guarantee 99.99% sterilization rate, and has superior filtering velocity and reusable advantage.
The specific embodiment
Below in conjunction with embodiment the present invention is further explained.
Embodiment one:
Formulate 1 microporous filtration, micro-filtration membrane adopts compact arranged collapsible, and material is polyether sulfone.Filter membrane aperture, filter upstream is 0.45 μ m, and filter membrane aperture, downstream is 0.2 μ m, and the upstream filter membrane is 1:1 with the effective filtration area ratio of downstream filter membrane.Get molecular weight and be 67000 haemocyanin, press as under the 0.1MPa condition in input, with haemocyanin by above-mentioned filter.Determining the haemocyanin volume that the 10min time filter passes through is 60L, and calculating filtering velocity according to formula is 360l/h.According to the Chinese Pharmacopoeia Sterility Test haemocyanin by the filter front and back is carried out sterility test.Calculate the sterilization rate of haemocyanin.At first prepare THIOGLYCOLLIC ACID salt fluid culture medium, and this culture medium is carried out applicability inspection and sensitivity inspection, and then culture medium is carried out inoculated and cultured, and carry out method validation.THIOGLYCOLLIC ACID salt fluid culture medium is through behind the method validation, is inoculated into haemocyanin on this culture medium and cultivates observation, record filter before the haemocyanin bacterial population be 5 * 10
5Cfu/ml, the bacterial population of haemocyanin is 22cfu/ml after filtering, calculating sterilization rate is 99.996%.Adopt 121 degree method of steam-sterilizing to the filter sterilization that carries out disinfection, each 30 minutes.Repeat above-mentioned test, with till still reaching for the last time effective sterilizing rate (more than 99.99%), the number of times of reusing that records this filter is 34 times.Main result sees the following form:
| Numbering | Aperture combination (μ m) | Membrane material | Effective filtration area (m 2) | Filtering velocity (l/h) | Sterilization rate | Reuse number of times |
| 1 | (0.45+0.2)μm | Polyether sulfone | 0.7 | 360 | 99.996% | 34 |
Embodiment two:
Formulate 1 microporous filtration, micro-filtration membrane adopts compact arranged collapsible, and material is polyether sulfone.Filter membrane aperture, filter upstream is 0.8 μ m, and filter membrane aperture, downstream is 0.2 μ m, and the upstream filter membrane is 1:1 with the effective filtration area ratio of downstream filter membrane.Get molecular weight and be 67000 haemocyanin, press as under the 0.1MPa condition in input, with haemocyanin by above-mentioned filter.Determining the haemocyanin volume that the 10min time filter passes through is 80L, and calculating filtering velocity according to formula is 480l/h.According to the Chinese Pharmacopoeia Sterility Test haemocyanin by the filter front and back is carried out sterility test.Calculate the sterilization rate of haemocyanin.At first prepare THIOGLYCOLLIC ACID salt fluid culture medium, and this culture medium is carried out applicability inspection and sensitivity inspection, and then culture medium is carried out inoculated and cultured, and carry out method validation.THIOGLYCOLLIC ACID salt fluid culture medium is through behind the method validation, is inoculated into haemocyanin on this culture medium and cultivates observation, record filter before the haemocyanin bacterial population be 5.4 * 10
5Cfu/ml, the bacterial population of haemocyanin is 20cfu/ml after filtering, calculating sterilization rate is 99.996%.Adopt 121 degree method of steam-sterilizing to the filter sterilization that carries out disinfection, each 30 minutes.Repeat above-mentioned test, with till still reaching for the last time effective sterilizing rate (more than 99.99%).The number of times of reusing that records this filter is 34 times.Main result sees the following form:
| Numbering | Aperture combination (μ m) | Membrane material | Effective filtration area (m 2) | Filtering velocity (l/h) | Sterilization rate | Reuse number of times |
| 2 | (0.8+0.2)μm | Polyether sulfone | 0.7 | 480 | 99.996% | 35 |
Embodiment three:
Formulate 1 microporous filtration, micro-filtration membrane adopts compact arranged collapsible, and material is polyether sulfone.Filter membrane aperture, filter upstream is 1.2 μ m, and filter membrane aperture, downstream is 0.2 μ m, and the upstream filter membrane is 1:1 with the effective filtration area ratio of downstream filter membrane.Get molecular weight and be 67000 haemocyanin, press as under the 0.1MPa condition in input, with haemocyanin by above-mentioned filter.Determining the haemocyanin volume that the 10min time filter passes through is 81L, and calculating filtering velocity according to formula is 486l/h.According to the Chinese Pharmacopoeia Sterility Test haemocyanin by the filter front and back is carried out sterility test.Calculate the sterilization rate of haemocyanin.At first prepare THIOGLYCOLLIC ACID salt fluid culture medium, and this culture medium is carried out applicability inspection and sensitivity inspection, and then culture medium is carried out inoculated and cultured, and carry out method validation.THIOGLYCOLLIC ACID salt fluid culture medium is through behind the method validation, is inoculated into haemocyanin on this culture medium and cultivates observation, record filter before the haemocyanin bacterial population be 5.2 * 10
5Cfu/ml, the bacterial population of haemocyanin is 58cfu/ml after filtering, calculating sterilization rate is 99.989%.Main result sees the following form:
| Numbering | Aperture combination (μ m) | Membrane material | Effective filtration area (m 2) | Filtering velocity (l/h) | Sterilization rate |
| 3 | (1.2+0.2)μm | Polyether sulfone | 0.7 | 486 | 99.989% |
The invention provides thinking and the method for a kind of degerming for albumen, serum solution and purification; method and the approach of this technical scheme of specific implementation are a lot; the above only is preferred embodiment of the present invention; should be understood that; for those skilled in the art; under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications; these improvements and modifications also should be considered as protection scope of the present invention, in the present embodiment not clear and definite each part all available prior art realized.
Claims (5)
1. one kind is used for albumen, the degerming of serum solution and the method for purification, it is characterized in that, may further comprise the steps:
1) formulate 1 compact arranged folder filter, its middle and upper reaches filter membrane aperture 0.45-1.2 μ m, filter membrane aperture, downstream is 0.2 μ m;
2) get haemocyanin, press as under the 0.1MPa condition in input, haemocyanin is passed through above-mentioned folder filter;
3) the bovine serum albumin volume that passes through in the folder filter in the time by formula mensuration 10min calculates filtering velocity;
4) analyze bovine serum albumin by the sterilization rate behind the folder filter;
5) sterilization that adopts 121 degree method of steam-sterilizing folder filter to be carried out 30 minutes;
6) repeating step 2 is to step 5 process, measures effective access times of filter, and effectively access times are with till still reaching for the last time effective sterilizing rate (more than 99.99%).
2. the method for described a kind of degerming and purification for albumen, serum solution according to claim 1 is characterized in that the described folder filter filter membrane of step 1 material adopts polyether sulfone.
3. the method for described a kind of degerming and purification for albumen, serum solution according to claim 1 is characterized in that haemocyanin described in the step 2 is that molecular weight is 67000 bovine serum albumin.
4. the method for described a kind of degerming and purification for albumen, serum solution according to claim 1 is characterized in that the described formula of step 3 is: filtering velocity (l/h)=bovine serum albumin volume/10min.
5. the method for described a kind of degerming and purification for albumen, serum solution according to claim 1 is characterized in that the analysis of the described sterilization rate of step 4 adopts the Chinese Pharmacopoeia Sterility Test to measure.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104922938A (en) * | 2014-03-20 | 2015-09-23 | 上海天士力药业有限公司 | Washing method of affinity chromatography column |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104922938A (en) * | 2014-03-20 | 2015-09-23 | 上海天士力药业有限公司 | Washing method of affinity chromatography column |
| CN104922938B (en) * | 2014-03-20 | 2018-12-04 | 上海天士力药业有限公司 | A kind of cleaning method of affinity column |
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Application publication date: 20130320 |