Summary of the invention
The present invention provides a kind of cleaning method of anion exchange chromatography, the cleaning refers to using or will
The unclean filler used is cleaned, and column chromatography next time is carried out after cleaning is qualified.
Anion exchange chromatography of the present invention, filler are any filling out with anion-exchange chromatography function
Material, preferably: Streamline DEAE, Streamline Q. are most preferably Streamline DEAE.
Above-mentioned filler can be by prior art preparation, can also be by being commercially available.
The present invention provides a kind of cleaning method of anion exchange chromatography, includes the following steps:
It by unclean anion exchange chromatography filler, washes with water, then with water-soluble containing sodium hydroxide and sodium chloride
Sodium hydroxide and sodium chloride is washed off with water, then is cleaned with isopropanol, then cleaned with acetic acid；
It washes with water, then with the aqueous cleaning containing sodium hydroxide and sodium chloride；
It is cleaned with equilibration buffer (pH5.0-8).
As needed, the invention also includes step, 5 and the step of step 6, as follows:
The cleaning solution that step 4 obtains is taken, phosphoric acid is dripped, whether there is or not precipitatings to generate for observation, such as without precipitating, uses total organic carbon analyzer
The cleaning solution is measured, measurement result is lower than 500ppm, then it represents that cleaning is qualified.
If there is precipitating to generate, after must occurring cleaning solution dilution without precipitating after phosphoric acid is added dropwise with water, after dilution
Cleaning solution measures the cleaning solution with total organic carbon analyzer, and measurement result is lower than 500ppm multiplied by the data that extension rate obtains,
Then indicate that cleaning is qualified.
Anion exchange chromatography of the present invention, preferred filler are Streamline DEAE；Column type number: BPG100/
500 or XK26/20 or XK50/20, preferably BPG100；The high ratio of pillar diameter: (0.5-1): 1, preferably (0.5-0.6): 1.Most preferably
Within the high 18cm of pillar, diameter: 10cm.
Cleaning method of the invention cleans in a reservoir after can removing filler from chromatographic column, can also be mounted in chromatography
It is cleaned in column, is preferably mounted in chromatographic column and cleans, wherein cleaning solution flow velocity is 120-150ml/min, preferably 120ml/min.This
Inventive step (1), (2), in (3), water consumption is that 3900~4200ml(is preferably 3900ml), the water is preferably injection
NaOH described in step (1) of the present invention, (3) refers to containing NaCl solution with 0.5~1M NaOH NaCl containing 1~2M
3900~4200ml is cleaned, preferably 0.5M NaOH NaCl containing 1M cleans 3900ml.It is specifically formulated as follows: weighing 160.0g respectively
Sodium hydroxide and 468g sodium chloride, are moved it into serum bottle or liquid storing bag with water for injection, are put into stirrer, are moved to magnetic force and are stirred
Mixing is uniformly dissolved solid all on device, by standardization of solution in serum bottle or liquid storing bag to 8L, continues stirring 2 minutes.After preparation
Be placed between the preparation of 2~12 DEG C of environment specify it is spare in region.
Sodium hydroxide has always been considered as capable of effectively removing albumen and nucleic acid, while it can also inactivate most of virus, thin
Bacterium, yeast, fungi and endotoxin.Sodium hydroxide energy saponified fat and it can dissolve albumen, sodium hydroxide is by albumen and nucleic acid
From the sample that the ability removed in chromatography media depends primarily on medium nature, sample and interference cleaning effect
Isopropanol described in step (2) of the present invention, acetic acid cleaning, preferably with clean 3900 with 30~40% isopropanols~
4200ml, then 3900~4200ml is cleaned with 25~30% acetic acid, most preferably 30% isopropanol cleans 3900ml, the cleaning of 25% acetic acid
3900ml.Specifically it is formulated as follows:
25% acetic acid (glacial acetic acid): taking acetic acid 2000ml, is moved it into serum bottle or liquid storing bag, is put into water for injection
Stirrer, moving on magnetic stirring apparatus is uniformly dissolved solid all, and standardization of solution in serum bottle or liquid storing bag to 8L continues
Stirring 2 minutes.Be placed in after preparation between the preparation of 2~12 DEG C of environment specify it is spare in region.
30% isopropanol: taking isopropanol 2400ml, is moved it into serum bottle or liquid storing bag with water for injection, is put into stirring
Son, moving on magnetic stirring apparatus is uniformly dissolved solid all, by standardization of solution in serum bottle or liquid storing bag to 8L, continues to stir
2 minutes.Be placed in after preparation between the preparation of 2~12 DEG C of environment specify it is spare in region.
Isopropanol and acetic acid of the invention can effectively remove albumen and lipid chemical combination not soluble in water in chromatographic column
Buffer described in step (4) of the present invention is phosphate buffer, and preferably equilibration buffer is 0.01mol/L phosphoric acid
Buffer, pH6.0, NaCl containing 0.1-0.2M.Specially (pH6.0 ± 0.1 contains 0.1M to 0.01mol/L equilibration buffer
The preparation of the 0.01mol/L equilibration buffer (pH6.0 ± 0.1 NaCl containing 0.1mol/L): sodium chloride is weighed
58.9g, sodium dihydrogen phosphate 12g, disodium hydrogen phosphate 2.0g are moved it into serum bottle or liquid storing bag with water for injection, are put into and stir
Son is mixed, moving on magnetic stirring apparatus is uniformly dissolved solid all, and standardization of solution in serum bottle or liquid storing bag to 10L continues
Stirring 2 minutes, determines its pH value in 6.0 ± 0.1 ranges.It is placed in after preparation between the preparation of 2~12 DEG C of environment and specifies region
It is interior spare.
Drop phosphoric acid described in step (5) of the present invention, is that cleaning solution is added in container, and the phosphoric acid of concentration is added, and is stood,
Visually observe that whether there is or not Precipitations.The purpose is to tentatively confirm phosphorus content in cleaning solution, if there is precipitating illustrate phosphorus content compared with
Height may can block total organic carbon analyzer pipeline in checkout procedure.
It is described to be measured with total organic carbon analyzer, it is with organic carbon concentration this hair in total organic carbon analyzer cleaning solution
Cleaning solution must be diluted with water described in bright step (6), the method is as follows:, must be straight by cleaning solution dilution with water if there is precipitating to generate
To after being added dropwise after phosphoric acid without precipitating appearance, the cleaning solution after dilution is measured into the cleaning solution, measurement knot with total organic carbon analyzer
The data that fruit obtains multiplied by extension rate are lower than 500ppm, then it represents that cleaning is qualified.
The explanation of relational language of the present invention:
1, anion-exchange chromatography: it is a kind of technology of Protein Separation, relies primarily on the interaction between charge, utilize band
Charge fine difference carries out Separation of Proteins in electric molecule.
2, endotoxin: it is one of gram-negative bacterial cell wall ingredient, is called lipopolysaccharides.Lipopolysaccharides is to host
It is virose.Endotoxin is only worked as bacterial death dissolution or is just released after destroying bacterium cell by artificial means.
3, total organic carbon (TOC): refer to the total amount that dissolubility and suspension organic matter are carbon containing in water body.TOC is one fast
The overall target of speed calibrating, it indicates that the total amount in water containing organic matter, unit are ppm or ppb with the quantity of carbon.In the world
TOC is by an important reference indicator as organic pollution degree in evaluation water body.
Test example one, the selection of technique
The cleaning method and parameter of anion exchange chromatography of the invention are obtained by screening, and screening process is such as
1, the selection of cleaning step:
The present invention passes through the cleaning of 5 cleaning solutions, and endotoxin and total organic carbon comply with standard.
2, the selection of concentration of lotion
The following table 2 cleaning solution is according to the sequential irrigation of embodiment, and as a result as follows: 0.5M NaOH NaOH containing 2MNaCl and 1M contains
3MNaCl cleaning effect is similar, considers for energy consumption is reduced, preferably 0.5M NaOH contains 1-2MNaCl
3, the selection of cleaning solution dosage
|Cleaning solution title
||Clean volume 1
||Clean volume 2
||Clean volume 3
||Clean volume 4
|Sodium hydroxide sodium chloride-containing
|Acetic acid solution
|As a result
|Total organic carbon
From upper table as it can be seen that cleaning solution is identical with the effect of volume 4200 and 4500, for saving principle, preferably cleaning solution volume
Beneficial effects of the present invention are proved below by way of comparative experiments:
Art methods 3:
Used anion exchange chromatography is cleaned with 2600ml water for injection, contains 1M with 2600ml0.5M NaOH
NaCl cleaning, is cleaned with 2600ml water for injection, is cleaned with 2600ml20% isopropanol, cleaned with 2600ml20% acetic acid, is used
The cleaning of 2600ml water for injection, is cleaned with 2600ml0.5M NaOH NaCl containing 1M, is finally balanced with 6500ml0.01mol/L slow
Fliud flushing (pH6.0 ± 0.1 NaCl containing 0.1M) rushes pillar；It takes pillar efflux to carry out endotoxin and TOC detection, does not as a result meet
It is required that.
Test example 2
Multiple real example experiment is carried out to the method for the embodiment of the present invention 1, it was demonstrated that in the conditions of the invention, use embodiment
1 method, endotoxin content, total content of organic carbon, content of microorganisms, qualification rate can achieve 99%.
It tests as follows:
■ anion exchange chromatography use, which finishes, to start to clean
Flow rate of liquid when ■ is cleaned are as follows: 120ml/min
■ cleans 3900ml with water for injection
■ cleans 3900~4200ml with 0.5M NaOH NaCl containing 1M
■ cleans 3900ml with water for injection
■ cleans 3900ml with 30% isopropanol
■ cleans 3900ml with 25% acetic acid
■ cleans 3900~4200ml with water for injection
■ cleans 3900~4200ml with 0.5M NaOH NaCl containing 1M
Then ■ rushes pillar 13000ml with phosphate buffer (0.01MPBS(pH6.0 NaCl containing 0.1~0.2M))；
After ■ is cleaned, chromatographic column is stored in 2~12 DEG C of chromatography cabinet
Wherein, the detection method of endotoxin content is as follows:
1.1 examine preceding prepare
1.1.1 prepare reagents, positive control solution, baterial endotoxin test water, sample and experiment phase before experiment starts
Close articles (except heat source pipette tips, pipettor, alcohol, scissors, sealed membrane etc.)；
1.1.2 dry powder-shaped reagents ampoule bottle top is touched, dry powder in bottle is made to be retained in bottom of bottle portion, avoids losing when corkage
1.1.3 ampoule bottle outer wall is wiped one time with 75% alcohol, after alcohol volatilization, carefully breaks ampoule bottle into two with one's hands.
1.2 sample treatment
1.2.1 it is tested using the reagents of λ=0.25EU, it is positive that sample treatment carries out positive control, test sample simultaneously
And negative control experiment, as experiment whether true foundation.
1.2.2 positive control: taking reagents 1, and 0.1ml baterial endotoxin test is added thereto and is redissolved with water, then plus
Enter 0.1ml bacterial endotoxin positive control solution (0.5EU/ml)；
1.2.3 negative control: taking reagents 1, and 0.2ml baterial endotoxin test water is added thereto.
1.2.4 test sample is positive: taking reagents 1,0.1ml purification solution sample is added thereto and redissolves, adds
0.1ml bacterial endotoxin positive control solution (0.5EU/ml)；
1.2.5 sample treatment: taking reagents 1, and 0.1ml baterial endotoxin test is added thereto and is redissolved with water, then plus
Enter 0.1ml sample (concrete operations can see the table below)；
||Test sample is positive
|Baterial endotoxin test water
|Positive control solution
1.2.6 sample is kept the temperature: after the reagents ampoule bottle after sample-adding is mixed gently, being sealed with sealed membrane, is placed on test tube
On frame；This rack for test tube is put into 37 DEG C ± 1 DEG C of constant temperature, keeps the temperature 60 ± 2min, insulating process, which should be avoided, to be shaken.
1.2.7 result is observed
22.214.171.124 after keeping the temperature, the rack for test tube for being placed with reagents ampoule bottle is taken out from constant incubator；
126.96.36.199 reagents ampoule bottle is carefully picked up from rack for test tube, slowly reverses 180 °；
If 188.8.131.52 pipe in formed gel, and gel it is indeformable, not from tube wall slippage person be the positive be denoted as (+)；
184.108.40.206 not formed gel or the gel of formation be not solid, deforms and is that feminine gender is denoted as (-) from tube wall slippage person；
1.2.8 inspection result determines
220.127.116.11 test establishment condition: positive control pipe and the test sample positive are (+), and negative control pipe is (-), should
Experiment can be set up.
18.104.22.168 sample cell is (-), then shows that sample detection is qualified (< 0.25EU/ml)
If 22.214.171.124 sample is (+), ready sample is taken to carry out retrial 2, two results show (-), then show sample
Qualified (<0.25EU/ml) is detected, otherwise shows that sample detection is unqualified (>0.25EU/ml)
The detection method of total content of organic carbon is as follows:
Pillar cleaning solution drips phosphoric acid, and whether there is or not precipitatings to generate for observation, and such as without precipitating, it is clear to measure this with total organic carbon analyzer
Washing lotion, measurement result are lower than 500ppm, then it represents that cleaning is qualified；If there is precipitating to generate, MilliQ water must be used cleaning solution dilution
Until the cleaning solution after dilution is measured the cleaning solution with total organic carbon analyzer after occurring after phosphoric acid is added dropwise without precipitating, measure
As a result it is lower than 500ppm multiplied by the data that extension rate obtains, then it represents that cleaning is qualified.
The detection method of content of microorganisms is as follows:
1.0 methods of inspection:
1.0.1 experimental implementation carries out in biological clean bench.
1.0.2 before experiment starts, 1 block of soybean casein fine jade of each placement in left and right in the working region of biological clean bench
The prefabricated plate of rouge culture medium 90mm is used for monitoring operation environment.
1.0.3 the preparation of test liquid (1:10):
126.96.36.199 water-soluble liquid test sample: test sample 10ml is pipetted in the sterile triangular flask of 250ml with Sterile pipette
In, pH7.0 sterile NaCl-peptone buffer agent is added to 100ml(graduation mark), it mixes, the test liquid as 1:10.
188.8.131.52 water-soluble solid or semisolid test sample: weighing test sample 10g in the sterile triangular flask of 250ml, be added pH7.0 without
Bacterium sodium chloride-peptone buffer agent is to 100ml(graduation mark), light shaking makes to be completely dissolved, it is mixed with suitable method, as 1:
10 test liquid.
184.108.40.206 water-insoluble test sample and the test sample of test liquid need to be prepared with specific process: advising according to pharmacopeia correlation
Surely it is prepared.
1.0.4 the filtering of test liquid and pad pasting:
220.127.116.11 filling filter bowl and filter membrane:
◆ peristaltic pump: opening the packaging with membrane filtration cup, be buckled on the buckle of peristaltic pump, opens cup lid.
◆ microorganism detection filters system:
● alcolhol burner is lighted, cotton ball soaked in alcohol is clamped with haemostatic clamp and lights, three stainless steel stent filtering heads alcohol swab fire
Flame over-fires disinfection one by one, and flame is allowed to come into full contact with inside filtering head and edge.Sanitized puts into the cotton ball soaked in alcohol of burning
In the beaker for collecting waste.
● the sterilization packaging for opening filter core takes haemostatic clamp calcination 1min or so on alcolhol burner flame to sterilize, from packaging bag
In press from both sides out sterilizing filter element, be placed in filtering head one by one.
● it takes anodontia tweezers calcination 1min or so on alcolhol burner flame to sterilize, opens sterilised membrane filter packaging and (be careful not to
Filter membrane is encountered with hand), clamping filter membrane is placed on the filtering mouth of suction filtration system.
● one bag of sterile filter bowl is opened from the bottom of packaging, holds the filter mouth for being carefully buckled in stainless steel stent in the middle part of filter bowl
On, and confirm filter bowl clamping.
18.104.22.168 filter membrane is soaked
◆ filter membrane should be first soaked before water-soluble test liquid filtering, takes about 10~20ml flushing liquor (whole filter membrane can be soaked
Subject to the amount on surface) it is poured into filter bowl along filter bowl mouth；Peristaltic pump or vacuum diaphragm pump are opened, will be closed after solution filter to the greatest extent.
◆ oils test sample, filter membrane and filter should be dried sufficiently before use, must not moisten film.
22.214.171.124 test liquid filters:
◆ 10ml test liquid (being equivalent to test sample of the every filter membrane containing 1g or 1ml), which is drawn, with 10ml Sterile pipette is added
In suitable diluent (about 100ml), mix；
◆ after poured into filter bowl along filter bowl mouth；Peristaltic pump or empty diaphragm pump are opened, will be closed after solution filter to the greatest extent.
126.96.36.199 rinsing filter membrane:
◆ non-biocidal property product to be checked: flushing liquor is poured into the 100ml graduation mark in filter bowl to filter bowl along filter bowl mouth, is opened compacted
Dynamic pump or vacuum diaphragm pump will close after solution filter to the greatest extent.It rinses 2 times altogether, each 100ml.
◆ biocidal property product to be checked: its flushing dose should be confirmed by verifying, examining every time should be by authenticated flushing dose to filter
Film is rinsed, and each flushing dose of every filter membrane is 100ml, and total flushing dose must not exceed 1000ml, to avoid micro- on filter membrane
Biology is damaged.
◆ it should be noted that keeping test liquid and flushing liquor to cover entire filter membrane surface, to play the maximum of filter membrane in flushing process
188.8.131.52 pad pasting:
◆ remove filter bowl:
● peristaltic pump: flicking band membrane filtration cup cup body makes filter bowl and UF membrane, removes filter bowl.
● microorganism detection filters system: it holds filter bowl edge or less and lightly filter is allowed to tilt backwards, it will from bracket
Filter bowl is removed.Anodontia tweezers are taken, cooling a moment (in order to avoid burning out filter membrane), careful to clamp on filter after alcolhol burner flame sterilization
Filter membrane is affixed on agar medium.
◆ when pad pasting, after filter membrane one end is contacted with media surface (bacterium is face-up), filter membrane slowly close to media surface,
Ensure that filter membrane inner ring does not have bubble, until whole filter membrane is affixed on culture medium, then gently drives filter membrane outer ring out of with anodontia tweezers
Bubble, make whole there is no bubble formation between filter membrane and media surface.
◆ by the standard requirements of test sample, it is affixed on the prefabricated plate of soybean casein agar medium or the training of Rose Bengal Sodium agar
It supports on the prefabricated plate of base.
◆ after pad pasting, culture ware lid is covered, sample ID, lot number is recorded in culture dish bottom and is inverted in biology after the date
On clean bench.
1.0.5 the filtering of negative control and pad pasting: referring to 6.4.4 lower content operations, after profit film, diluent 10ml is taken to add
Enter in suitable diluent (about 100ml), filtered after mixing, then with flushing liquor rinse filter membrane after pad pasting.
1.0.6 Check-Out Time: test liquid must not exceed 1h from preparation to inspection culture medium, time is added.
1.0.7 in experimentation, at interval of 15 minutes or so, disappeared again with sterile 75% ethanol solution sprinkling hand
1.0.8 experiment all finishes
184.108.40.206 2 pieces of prefabricated plates of soybean casein agar medium 90mm are taken, each one piece of right-hand man, the five fingers are pressed
In 5 seconds on soybean casein agar medium face, culture ware lid is covered.
220.127.116.11 the prefabricated plate of 90mm on biological clean bench is packed up, covers culture ware lid.
1.1 cultures: it unless otherwise specified, is cultivated according to following temperature and times.
1.1.1 the prefabricated plate of soybean casein agar medium is inverted in incubator, 30~35 DEG C are cultivated 5 days, by
Day point inspection.
1.1.2 the prefabricated plate of Rose Bengal Sodium agar medium is inverted in incubator, 23~28 DEG C cultivate 5 days, day by day
1.2 count: in culture period, point examines clump count day by day
If 1.2.1 asepsis growth is calculated as 0.
If 1.2.2 having 2 or 2 or more bacterium colony overlappings on plate, when distinguishable still in terms of 2 or 2 or more bacterium colonies
Number；Bacterium colony sprawling, which grows sheet of plate, to be counted, and TNTC is filled in.If there is TNTC, that is, terminate culture.
1.2.3 soybean casein agar medium is used for count of bacteria, and Rose Bengal Sodium agar medium is used for mould and ferment
Female bacterium counts；If on soybean casein agar medium on yeast and mold, Rose Bengal Sodium agar medium with thin
Bacterium, should put respectively meter yeast and mold, bacterium clump count, then by soybean casein agar medium mould and ferment
Bacterial population on female bacterium or Rose Bengal Sodium agar medium, with yeast and mold number on Rose Bengal Sodium agar medium or big
Bacterial population on beans casein agar culture medium is compared, and the clump count in the culture medium high using bacterium number is as count results.
1.3 result judgements:
1.3.1 experiment effectiveness:
18.104.22.168 after cultivation (incubation time is consistent with sample), clump count should be 0(i.e. < 1 to negative control plate).
22.214.171.124 the clump count on every filter membrane answers≤100CFU.
1.3.2 result calculates:
126.96.36.199 result report value: point inspection most all day, point meter plate as a result, compare soybean casein agar culture medium and
Bacterium, saccharomycete clump count and the mold colony number of meter are put on Rose Bengal Sodium agar medium.Wherein the larger value conduct is taken respectively
Final bacterium colony numerical value.
188.8.131.52 every 1g(ml is calculated) micro organism quantity of test sample, formula is as follows:
Inspection product result (CFU/g(ml)) each bacterium of=test sample result report value (CFU) ÷ test sample filtration yield g(ml) ×
Test sample report amount g(ml).
Anion exchange chromatography is cleaned by above-mentioned cleaning method, so that anion exchange chromatography cleaning effect
Fruit is ensured, the endotoxin in anion exchange chromatography is eliminated, and microorganism and last time use rear remaining egg
It is white.