CN106800583A - It is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out - Google Patents
It is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out Download PDFInfo
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Abstract
The present invention relates to human fibrinogen preparation technology field, specifically a kind of instant cryodesiccant human fibrinogen preparation technology without precipitation is made up of, the first step, dissolving and the filtering of component I precipitations 5 operations；Second step, S/D inactivation of virus；3rd step, two step chilled alcohol precipitations are refined；4th step, freezes；5th step, xeothermic inactivation of virus.The present invention is by improved fine original production process, can prepare it is instant, without separate out, without opalescence, the fibrinogen without protein body, traditional human fibrinogen used for intravenous injection can be not only prepared by the technique, the high concentration human fibrinogen of compatibility in human fibrin adhesive can be also prepared.
Description
Technical field
The invention belongs to field of biological pharmacy, it is related to the preparation technology of blood product, is a kind of speed specifically
The molten cryodesiccant human fibrinogen preparation technology without precipitation.
Background technology
Human fibrinogen (human fibrinogen, referred to as fine former or Fg) is containing 2964 amino acid
Macromolecular glycoprotein, is a kind of protein with coagulation function synthesized by liver, and it is 13 kinds of human body
The maximum a kind of clotting factor of content in clotting factor, also known as factor I.Human fibrinogen is a kind of
It is also the indispensable first-aid medicine that stops blooding on battlefield, so clinically using very frequently blood product
And fibrinogen is clinically constantly in that supply falls short of demand state.Particularly in recent years, it is a kind of to have efficiently
The surgery blood product of hemostatic function --- human fibrin adhesive is increasingly subject to doctor and patient's
Favor, increases, so that the product as the demand of the high concentration human fibrinogen of one of the medicine compatibility
Product face shorter situation.Human fibrinogen used by human fibrin adhesive is defeated with conventional vein
The human fibrinogen of note is very different, and being mainly reflected in the former has protein concentration very high, at least
It is two times or i.e. 5-7% higher of the latter's (generally 2.5%).Human fibrinogen's product is maximum to ask
Topic is that freeze-dried powder need to be carried out when redissolving time long and dissolving in 30-37 DEG C of water-bath, and another problem is
Easily separate out albuminate and there is protein body to suspend, this brings inconvenience to Clinical practice, must for example carry
Preceding taking-up in refrigerator is placed in water-bath, and filter need to be equipped with during venoclysis.Low concentration human fibrin
Original is even in this way, the redissolution of high concentration human fibrinogen is just more difficult.Therefore with conventional people's fiber egg
White original production process is difficult to prepare qualified concentration cellulose albumen original product, for that purpose it is necessary to from technique
Source starts to include that the formula or even last lyophilized operation of stoste are both needed to take targetedly in links
Measure.
At present, the raw material for preparing human fibrinogen mainly has three kinds, and one is cryoprecipitate (also known as cold glue),
It is mainly used for preparing human blood coagulation FVIII, during because separating cold glue from blood plasma, due to being total to for albumen
Deposited phenomenon, part human fibrinogen and other protein in blood plasma are precipitated out (its with cold glue simultaneously
In fibrinogen content it is limited, only account for the very little ratio of whole plasma Fg)；Two is component I precipitations,
The precipitation is the precipitation separated and collected after certain density ethanol and cooling are added in removing cold glue blood plasma, is contained
The Fg of maximum ratio in blood plasma；Three is that cold glue is co-precipitated with component I, i.e., added in blood plasma melting certain dense
Make cold glue and component I coprecipitations after the ethanol of degree and cooling.
A series of fibre original preparation technology with cold glue as raw material, because the contained Fg in raw material the inside is very few, by behaviour
Make step, lose layer by layer, last yield is very limited, lack the value of commodity production；And with cold glue with
Component I co-precipitation is the fine original production process of raw material, there is also following problem, first, is contained in precipitation
8% ethanol, is a very unfavorable factor for the stability of the human blood coagulation factor VII I in precipitation,
The storage of precipitation must very with caution, and the holding time can not be long, and ethanol must be handled with great care when feeding intake to people
The degenerative lesion effect of blood coagulation factor VIII, so the presence of ethanol is undoubtedly to human blood coagulation factor VII I in raw material
Production technology propose harshness requirement；Secondly as precipitation in human blood coagulation factor VII I a large amount of presence,
Hidden danger is also brought to fine former production, causes production to fail because of fine former being easily activated, fine former activation
The reason for it is more complicated, but fibrin ferment, the reason for be wherein most direct, fibrin ferment is factor in Ca2+From
Son and other clotting factor include that blood coagulation factor VIII is swashed under participating in by the chain reaction of a series of complex
Living, so in fine former preparation process, the presence of human blood coagulation factor VII I is also a unfavorable factor,
Just should thoroughly be removed in the initial operation of technique as far as possible；In addition, from terms of fine former preparation technology, having
Technique process it is various, especially dissolving be repeated with precipitation, thus cause frequently heating, cooling,
The operations such as addition ethanol, soda acid, these operations all produce damage to albumen to some extent for albumen,
Once there is the situation of acute variation in operation, it is likely that may result in final products and produce opalescence or even precipitation
And the reason for redissolve overlong time etc..Further, it is too high to pursue fine former purity, it is also to cause some technique journeys
Sequence is various, the reason for the production cycle is long, it is many it was verified that too high product purity to stability on the contrary
It is unfavorable, it is easily caused fine former precipitation.For example albumin is famous protein protective agent to some albumen, is also one
The indispensable protective agent of a little biological products such as vaccines.So, for fine original product, should more pursue product
The stability of product, instant capacity target.
The content of the invention
It is an object of the invention to some shortcomings and defect that overcome the fine original preparation technology of tradition at present to exist, carry
For a kind of new instant, high concentration, without the cryodesiccant human fibrinogen's production technology for separating out, by the technique
Traditional human fibrinogen used for intravenous injection can be not only prepared, compatibility in human fibrin adhesive can be also prepared
High concentration human fibrinogen.
The present invention provides a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out, including following step
Suddenly：
A) component I is precipitated and is shredded, by 1：The thinner ratio of 10-20, dissolves, uniformly in input buffer solution 1
Stirring 1-2 hours；
B the filter element filtering suspension of one 1.0 μm of 30SP deep layers filter core (CUNO companies filter core) series connection) is used；
With filter wash core after above-mentioned buffer solution 1, filtrate is collected；
C S/D mother liquors) are added in filtrate；
D S/D solution) is cooled to -1 DEG C or so, cold ethanol is slowly added to (not higher than with spray pattern
- 20 DEG C) to concentration of alcohol be 8-10% (V/V)；Stirring 1-2 hours, (- 2-0 DEG C) centrifugation of low temperature, stream
Fast 1-2kg/min/ platforms, collect precipitation, abandon supernatant；
E 1) is pressed：The thinner ratio of 15-25 or so, the precipitation collected is dissolved with buffer solution 2, and 1-2 is small for stirring
When；
F) with a filter element filtering suspension for one 0.45 μm of 30SP deep layers filter core series connection, above-mentioned buffering is used
Filter wash core after liquid 2, collects filtrate；
G) filtrate is cooled to -1 DEG C, and it is 8-10% to be subsequently adding cold ethanol (not higher than -20 DEG C) to concentration of alcohol
(V/V), (- 2-0 DEG C) centrifugation of low temperature, flow velocity 1-2kg/min/ platforms, collect precipitation, abandon supernatant；
H 1) is pressed：The thinner ratio of 2-6, the precipitation collected is dissolved with buffer solution 3, is stirred at 20-30 DEG C
0.5-2 hours, with one 0.45 μm of filter element filtering suspension and with filter wash core after buffer solution 3, collect filter
Liquid；
I) regulation pH value is diluted protein concentration with above-mentioned buffer solution 3 according to required specification to 6.50-7.50
To 2.50-3.0% or 5.5-6.0%；It is aseptic subpackaged；
J) freeze, operated using precooling, quick-frozen and multiple " annealing ", finally obtain human fibrinogen's jelly
Dry powder；
K it is) xeothermic in boiling water bath to carry out inactivation of virus (100 DEG C, 30 minutes).
Described component I precipitations are classical Cohn plasma components I precipitations, and its preparation method is：
Cold 50% ethanol solution (temperature is not higher than -20 DEG C) to concentration of alcohol is added in cold glue blood plasma is removed
It is 8-10% (V/V) to reconcile blood plasma pH value to 6.85-7.15, keeps -2-2 DEG C of blood plasma temperature, uniformly stirs
Centrifugation obtains component I precipitations after mixing 1-3 hours.
The composition of the buffer solution 1 in described step A is：Sodium citrate 2H2O 30-60mmol/L,
TRIS10-30mmol/L, lysine hydrochloric acid 10-30mmol/L, sucrose 1-3% (w/w), glycine
0.5-5% (w/w), sodium chloride 0.1-0.15mol/L, pH value are 6.50-7.50,20-30 DEG C of temperature.
The composition of the buffer solution 2 in described step E is：Sodium citrate 2H2O 30-60mmol/L, TRIS
10-30mmol/L, arginine monohydrochloride 1-3% (w/w), sucrose 1-3% (w/w), glycine 0.5-5%
(w/w), sodium chloride 0.1-0.15mol/L, pH value is 6.50-7.50,20-30 DEG C of temperature.
The Tween-80 of 0.015-0.025% (w/w) is additionally added in the buffer solution 2 of described step E, is promoted
The rapid dispersion of albumen and dissolving.
The composition of the buffer solution 3 in described step H is：Sodium citrate 2H2O 30-60mmol/L, essence
Propylhomoserin hydrochloride 1-5% (w/w), glycine 1-5% (w/w), sodium chloride 30-150mmol/L, pH value
It is 6.50-7.50.
The S/D mother liquors that prepare in advance are added in described step C in filtrate, makes Tween-80 in solution
Concentration to 1% (w/v), the concentration of TNBP is slowly stirred to 0.3% (w/v) at 24-26 DEG C, insulation 6
Hour.
Lyophilized operating procedure is in described step J：Operated using precooling, quick-frozen and multiple " annealing ",
3-5 DEG C of the rapid deep cooling of product will be pre-chilled to -50 DEG C or so, is kept for 1-2 hours, be then brought rapidly up to
- 30 DEG C or so, 0.5-1 hours (warming temperature referred to as " is annealed ") is kept, then be cooled to -50 DEG C, holding
1-2 hours, 1-2 annealing operation is repeated, finally start to vacuumize in -45 DEG C or so of products temperature, liter
China, main drying temperature is at least 5 DEG C lower than eutectic temperature or so of shelf temperature (or heat conduction oil temperature),
Ensure that products temperature is no more than eutectic temperature in the whole lyophilization stage, while taking measures to shorten product
Temperature duration at low temperature, unsuitable long, the maximum temperature control of product of last parsing-desiccation time
System finally obtains Fg freeze-dried powders at 26 DEG C or so.The quick-frozen crystal formation that can make product is fine and closely woven, and annealing then can
Make the crystal formation of whole product uniform, this can all significantly improve freeze-dried powder and redissolve property, shorten product at low temperature
The formation of protein body when duration then may consequently contribute to prevent from redissolving.
The invention has the advantages that：
(1) production procedure is succinct, with short production cycle, and the production time before freezing, less than 32 hours, freezes
Time 70-80 hours or so, 100 hours or so total production cycle；
(2) add glycine as protective agent in each step dissolving buffer solution, dissolving, filtering and
After wash, be centrifuged etc. in operation, Fg obtains the protection of glycine from start to finish, it is to avoid cause in operation
Egg white injury；
(3) plus ethanol link, using cold ethanol, temperature is less than -20 DEG C, while with spray pattern,
Reduce damage of the ethanol to albumen；
(4) lyophilized technique condition is optimized, quick-frozen and " annealing " measure is taken, while shortening jelly
Dry total time, shortened to 70-80 hours by traditional more than 100 hours, obtained freeze-dried powder is in uniform sponge
Shape, it is instant and without albumen separate out, without protein body.
Brief description of the drawings
Fig. 1 is to prepare cryodesiccant human fibrinogen's FB(flow block) from component I precipitations.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment to the present invention provide specific embodiment elaborate.
Embodiment 1
The first step, by thinner ratio 1：20, take fresh components I precipitation 2kg, chopping of making thinner immediately, then
Dissolved in input 40kg buffer solutions 1, uniform stirring 3 hours；The formula of buffer solution 1：Sodium citrate 2H2O
640g, TRIS 108g, lysine hydrochloric acid 160g, sucrose 600g, glycine 600g, sodium chloride 400g,
The pH value of buffer solution 1 is 6.95,20 DEG C of temperature；
Second step, with a filter element filtering first step suspension for one 1.0 μm of 30SP deep layers filter core series connection；
Filter pressure control, with filter wash core after the buffer solution 1 being formulated described in the first step, collects filter in 0.5bar or so
Liquid 50.1kg.
3rd step, adds the S/D mother liquor 2.5kg that prepare in advance in filtrate, makes Tween-80 in solution
Concentration is incubated 6 hours to the concentration of 1%, TNBP to being slowly stirred at 0.3%, 24-26 DEG C；
4th step, -1 DEG C or so is cooled to by the solution after S/D, and low temperature is slowly added to spray pattern
(- 22 DEG C) 50% ethanol solution 8.2kg, stirs 1.5 hours, (- 2-0 DEG C) centrifugation of low temperature, flow velocity
1.5-1.8kg/min/ platforms, collect precipitation 1.62kg, abandon supernatant；
5th step, precipitates chopping of making thinner, by thinner ratio 1 by more than：25, put into 40.5kg buffer solutions 2
Middle dissolving, uniform stirring 2 hours；Buffer solution 2 is formulated：Sodium citrate 2H2O 640g, TRIS 108g,
Arginine monohydrochloride 600g, sucrose 600g, glycine 607g, sodium chloride 400g, Tween-80,8g,
The pH value of buffer solution 2 is 7.05,20 DEG C of temperature；
6th step, under normal temperature, more than one 0.45 μm of filter element filtering of being connected with 30SP deep layers filter core suspension
Liquid, with filter wash core after the buffer solution 2 being formulated described in the 5th step, collects filtrate 55.2kg；
7th step, -1 DEG C is cooled to by above-mentioned filtrate, and low temperature (- 21 DEG C) 50% is slowly added to spray pattern
Ethanol solution 9.4kg, stirs 1.5 hours, (- 2~-0 DEG C) centrifugation of low temperature, flow velocity 1.5-1.8kg/min/ platforms,
Precipitation 1.32kg is collected, supernatant is abandoned；
8th step, precipitates chopping of making thinner, by thinner ratio 1 by more than：4, then put into 5.28kg buffer solutions
Dissolved in 3, then uniform stirring 1.5 hours is used in combination with one 0.45 μm of filter element filtering above suspension
Filter wash core after buffer solution 3；Buffer solution 3 is formulated：Sodium citrate 2H2O 80g, arginine monohydrochloride 186g,
Glycine 158g, sodium chloride 32g, the pH value of buffer solution 3 is 7.13,20 DEG C of temperature；
9th step, 2.8% is diluted to the buffer solution 3 being formulated described in the 8th step by protein concentration；Repetition measurement pH
7.08, send to aseptic subpackaged；
Tenth step, the tamponade of product half that will have been dispensed, is put into freeze dryer, and product is cooled to 3 DEG C in advance first,
Then shelf temperature is set as -55 DEG C, it is quick-frozen to -45 DEG C of products temperature, kept for 2 hours afterwards, Ran Houxun
Speed raises shelf temperature makes products temperature rise to -25 DEG C, is kept for 1 hour, then sets shelf temperature again
It is set to -55 DEG C, it is quick-frozen to -45 DEG C of products temperature, kept for 2 hours；Afterwards after -30 DEG C of shelf temperature of setting, open
Beginning vacuumizes, distillation.About 40 hours, trunk constipation beam；Final 28 DEG C of the shelf temperature of setting parsing-desiccation section,
Start to warm up, after about 16 hours, parsing-desiccation terminates, tamponade, shutdown, outlet.Complete whole lyophilized
Operation.
11st step, product is xeothermic in boiling water bath (100 DEG C, 30 minutes), inactivation of virus is carried out,
After be placed in the storage of 2-8 DEG C of freezer.
Embodiment 2
The first step, takes fresh components I precipitations 10kg and makes thinner immediately chopping, by thinner ratio 1：20, input
Dissolved in 200kg buffer solutions 1, uniform stirring 1.5 hours；Buffer solution 1 is formulated：Sodium citrate 2H2O 3.2kg,
TRIS 540g, FE-5 800g, sucrose 3kg, glycine 2.0kg, sodium chloride 2.0kg, delay
The pH value of fliud flushing 1 is 6.95,25 DEG C of temperature；
Second step, with 30SP deep layers filter core connect the one 1.0 μm filter element filterings of (10 inches) more than hang
Supernatant liquid；Filter pressure control in 0.5bar or so, with filter wash core after the about 50kg of buffer solution 1 of above-mentioned formula,
Collect filtrate 251kg.
3rd step, adds the S/D mother liquor 12.5kg that prepare in advance in filtrate, makes Tween-80 in solution
Concentration is incubated 6 hours to the concentration of 1%, TNBP to being slowly stirred at 0.3%, 24-26 DEG C；；
4th step, -1 DEG C or so is cooled to by solution after S/D, and it is (not high to be slowly added to low temperature with spray pattern
In -20 DEG C) 50% ethanol solution 42kg, stirs 1.5 hours, (- 2-0 DEG C) centrifugation of low temperature, flow velocity
1.5-1.8kg/min/ platforms, collect precipitation 8.9kg, abandon supernatant；
5th step, precipitates chopping of making thinner, by thinner ratio 1 by more than：20, put into 178kg buffer solutions 2
Middle dissolving, uniform stirring 1.5 hours；Buffer solution 2 is formulated：Sodium citrate 2H2O 2.85kg, TRIS 480g,
Arginine monohydrochloride 2.67kg, sucrose 2.67kg, glycine 1.78kg, sodium chloride 1.78kg, Tween-80,
35.6g, the pH value of buffer solution 2 is 7.05,25 DEG C of temperature；
6th step, under normal temperature, more than one 0.45 μm of filter element filtering of being connected with 30SP deep layers filter core suspension
Liquid, with filter wash core after the buffer solution 2 of above-mentioned formula, collects filtrate 249kg；
7th step, -1 DEG C is cooled to by above-mentioned filtrate, and low temperature (- 22 DEG C) 50% is slowly added to spray pattern
Ethanol solution 42.8kg, stirs 1.5 hours, (- 2-0 DEG C) centrifugation of low temperature, flow velocity 1.5-1.8kg/min/ platforms,
Precipitation 7.1kg is collected, supernatant is abandoned；
8th step, precipitates chopping of making thinner, by thinner ratio 1 by more than：In 4, input 28.4kg buffer solution 3
Dissolving, uniform stirring 1.0 hours, then with one 0.45 μm of filter element filtering above suspension and after wash
Filter core；Buffer solution 3 is formulated：Sodium citrate 2H2O 430g, arginine monohydrochloride 1.00kg, glycine 568g,
Sodium chloride 170g, the pH value of buffer solution 3 is 7.20,25 DEG C of temperature；
9th step, with embodiment 1；
Tenth step, the tamponade of product half that will have been dispensed, is put into freeze dryer, and product is cooled to 5 DEG C in advance first,
Then baffle temperature is set as -55 DEG C, it is quick-frozen, to -45 DEG C of products temperature, keep 2 hours afterwards, then
It is brought rapidly up to -30 DEG C, is kept for 1 hour, then is cooled to -55 DEG C, holding 1 hour；It is repeated once and " moves back
Fire " operation, after kept for 2 hours at -45 DEG C of products temperature, after -28 DEG C of baffle temperature of setting, start to take out
Vacuum, distillation.After about 48 hours, terminate trunk dry；28 DEG C of final baffle temperature is set, is started to warm up,
Into the parsing-desiccation stage, after about 18 hours, parsing-desiccation terminates, tamponade, shutdown, outlet, completes
Entirely freeze operation.
11st step, with embodiment 1.
Embodiment 3
The first step, takes fresh components I precipitation 10kg, chopping of making thinner immediately, by thinner ratio 1：10, so
Dissolved in input 100kg buffer solutions 1 afterwards, uniform stirring 3 hours；Buffer solution 1 is formulated：Sodium citrate 2H2O
1.6kg, TRIS 270g, FE-5 400g, sucrose 1.5kg, glycine 3.0kg, sodium chloride 1.0kg,
The pH value of buffer solution 1 is 6.80,30 DEG C of temperature；
Second step, with a filter element filtering above suspension for one 1.0 μm of 30SP deep layers filter core series connection；
Filter pressure control in 0.5bar or so, with filter wash core after the about 50kg of buffer solution 1 being formulated described in the first step,
Collect filtrate 152kg.
3rd step, adds the S/D mother liquor 7.5kg that prepare in advance in filtrate, makes Tween-80 in solution
Concentration is incubated 6 hours to the concentration of 1%, TNBP to being slowly stirred at 0.3%, 24-26 DEG C；；
4th step, -1 DEG C or so is cooled to by solution after S/D, and it is (not high to be slowly added to low temperature with spray pattern
In -20 DEG C) 50% ethanol solution 25.0kg, stirs 1.5 hours, (- 2-0 DEG C) centrifugation of low temperature, flow velocity
1.5-1.8kg/min/ platforms, collect precipitation 9.1kg, abandon supernatant；
5th step, precipitates chopping of making thinner, by thinner ratio 1 by more than：During 15 put into 137kg buffer solutions 2
Dissolving, uniform stirring 1 hour；Buffer solution 2 is formulated：Sodium citrate 2H2O 2.2kg, TRIS 366g,
Arginine monohydrochloride 2.1kg, sucrose 2.1kg, glycine 4.2kg, sodium chloride 1.37kg, Tween-80,28g,
The pH value of buffer solution 2 is 7.35,30 DEG C of temperature；
6th step, with embodiment 2, obtains filtrate 152kg；
7th step, -1 DEG C is cooled to by above-mentioned filtrate, and low temperature (- 22 DEG C) 50% is slowly added to spray pattern
Ethanol solution 26.1kg, stirs 1.5 hours, (- 2-0 DEG C) centrifugation of low temperature, flow velocity 1.5-1.8kg/min/ platforms,
Precipitation 7.5kg is collected, supernatant is abandoned；
8th step, precipitates chopping of making thinner, by thinner ratio 1 by more than：2, then put into 15.0kg buffer solutions
Dissolved in 3, uniform stirring 2 hours, then with one 0.45 μm of filter element filtering above suspension and after
Filter wash core；Buffer solution 3 is formulated：Sodium citrate 2H2O 272g, arginine monohydrochloride 645g, glycine
600g, sodium chloride 132g, the pH value of buffer solution 3 is 6.85,30 DEG C of temperature；
9th step, 5.8% is diluted to the buffer solution 3 being formulated described in the 8th step by protein concentration；Repetition measurement pH
6.88, send to aseptic subpackaged；
Ten, the 11 steps, with embodiment 2.
Above is the specific descriptions that the preferred embodiment to the invention is carried out, but implementation of the invention
The embodiment is not limited to, those of ordinary skill in the art are in the premise without prejudice to the invention spirit
A variety of equivalent modification or replacements can be also made down, and these equivalent modifications or replacement are all contained in the application
In claim limited range.
The product of present invention process is compareed with domestic certain product that lists a company, see the table below 1.
The product of the present invention process of table 1 and domestic certain product contrast that lists a company
Claims (7)
1. it is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is characterised in that including following
Step：
A) component I is precipitated and is shredded, by 1：The thinner ratio of 10-20, dissolves, uniformly in input buffer solution 1
Stirring 1-2 hours；The mass percent of glycine is 0.5-5% in described buffer solution 1；
B the filter element filtering suspension of one 1.0 μm of 30SP deep layers filter core series connection) is used；Described in step A
Buffer solution 1 after filter wash core, collect filtrate；
C S/D mother liquors) are added in filtrate；
D S/D solution) is cooled to -1 DEG C or so, not higher than -20 DEG C of low temperature is slowly added to spray pattern
Ethanol to concentration of alcohol is 8-10% (V/V)；Stirring 1-2 hours, -2-0 DEG C of low-temperature centrifugation, flow velocity
1-2kg/min/ platforms, collect precipitation, abandon supernatant；
E 1) is pressed：The thinner ratio of 15-25 or so, the precipitation collected is dissolved with buffer solution 2, and 1-2 is small for stirring
When；
F) with a filter element filtering suspension for one 0.45 μm of 30SP deep layers filter core series connection, step E is used
Filter wash core after described buffer solution 2, collects filtrate；
G) filtrate is cooled to -1 DEG C, and it is 8-10% to be subsequently adding not higher than -20 DEG C of cold ethanol to concentration of alcohol
(V/V), -2-0 DEG C of low-temperature centrifugation, flow velocity 1-2kg/min/ platforms collect precipitation, abandon supernatant；
H 1) is pressed：The thinner ratio of 2-6, the precipitation collected is dissolved with buffer solution 3, and 0.5-2 is stirred at 20-30 DEG C
Hour, with one 0.45 μm of filter element filtering suspension and with filter wash core after buffer solution 3, collect filtrate；
I) regulation pH value is to 6.50-7.50, according to required specification, with the buffer solution 3 described in step H by egg
White concentration dilution is to 2.50-3.0% or 5.5-6.0%；It is aseptic subpackaged；
J) freeze, using precooling, quick-frozen, annealing and lyophilization and parsing-desiccation, finally obtain people's fiber
Proteinogen freeze-dried powder；
K it is) xeothermic in boiling water bath to carry out inactivation of virus, 100 DEG C of temperature, 30 minutes time.
2. according to claim 1 instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is special
Levy and be, the composition of the buffer solution 1 of described step A is：Sodium citrate 2H2O 30-60mmol/L,
TRIS 10-30mmol/L, lysine hydrochloric acid 10-30mmol/L, sucrose 1-3% (w/w), glycine
0.5-5% (w/w), sodium chloride 0.1-0.15mol/L, pH value are 6.50-7.50,20-30 DEG C of temperature.
3. according to claim 1 instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is special
Levy and be, the composition of the buffer solution 2 of described step E is：Sodium citrate 2H2O 30-60mmol/L,
TRIS 10-30mmol/L, arginine monohydrochloride 1-3% (w/w), sucrose 1-3% (w/w), glycine
0.5-5% (w/w), sodium chloride 0.1-0.15mol/L, pH value are 6.50-7.50,20-30 DEG C of temperature.
4. according to claim 3 instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is special
Levy and be, the Tween-80 of 0.015-0.025% (w/w) is additionally added in the buffer solution 2 of described step E.
5. according to claim 1 instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is special
Levy and be, the composition of the buffer solution 3 of described step H is：Sodium citrate 2H2O 30-60mmol/L,
Arginine monohydrochloride 1-5% (w/w), glycine 1-5% (w/w), sodium chloride 30-150mmol/L, pH
It is 6.50-7.50 to be worth.
6. according to claim 1 instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is special
Levy and be, add the advance S/D mother liquors for preparing in described step C in filtrate, make tween in solution
- 80 concentration to 1% (w/v), the concentration of TNBP is slowly stirred to 0.3% (w/v) at 24-26 DEG C, protects
Temperature 6 hours.
7. according to claim 1 instant without the cryodesiccant human fibrinogen preparation technology for separating out, it is special
Levy and be, the operating procedure freezed in described step J is：3-5 DEG C of the rapid deep cooling of product will be pre-chilled to
To -50 DEG C or so, kept for 1-2 hours, be then brought rapidly up to -30 DEG C or so, kept for 0.5-1 hours, should
Warming temperature referred to as " is annealed ", then is cooled to -50 DEG C, is kept for 1-2 hour, repeatedly 1-2 annealing operation,
It is last to start to vacuumize in -45 DEG C or so of products temperature, distil, main drying temperature is shelf temperature or leads
At least 5 DEG C lower than eutectic temperature or so of hot oil temperature, the maximum temperature of product is controlled at 26 DEG C or so, most
After obtain human fibrinogen's freeze-dried powder.
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Cited By (2)
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| CN111518197A (en) * | 2020-03-30 | 2020-08-11 | 哈尔滨派斯菲科生物制药股份有限公司 | Production method of fibrinogen |
| CN112354002A (en) * | 2020-11-12 | 2021-02-12 | 广东深蓝生物科技有限公司 | Hemostatic sealant and preparation method thereof |
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| CN111518197A (en) * | 2020-03-30 | 2020-08-11 | 哈尔滨派斯菲科生物制药股份有限公司 | Production method of fibrinogen |
| CN111518197B (en) * | 2020-03-30 | 2024-01-05 | 哈尔滨派斯菲科生物制药有限公司 | Production method of fibrinogen |
| CN112354002A (en) * | 2020-11-12 | 2021-02-12 | 广东深蓝生物科技有限公司 | Hemostatic sealant and preparation method thereof |
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