CN103087184B - Method for controlling prekallikrein activator in human serum albumin product - Google Patents
Method for controlling prekallikrein activator in human serum albumin product Download PDFInfo
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- CN103087184B CN103087184B CN201310012434.9A CN201310012434A CN103087184B CN 103087184 B CN103087184 B CN 103087184B CN 201310012434 A CN201310012434 A CN 201310012434A CN 103087184 B CN103087184 B CN 103087184B
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Abstract
The invention relates to a method for controlling prekallikrein activator in a human serum albumin product. The method comprises the following steps of in the process of separating and preparing human serum albumin through a low-temperature alcohol method: 1) adding activated kieselguhr subjected to sodium citrate pretreatment in a step of separating the component IV from the supernatant of components I+II+III, and adsorbing a blood coagulation factor XII so as to reduce the content of the blood coagulation factor XII; and 2) allowing the component V subjected to ultrafiltration and dialysis and purification to pass through a grease-removing filter element with positive charges, and adsorbing to remove the residual activation factor XIIa fragment in the human serum albumin solution. According to the two steps, the content of the prekallikrein activator in the human serum albumin product is reduced, the protein molecule activity, various physical and chemical indicators and protein yield are not influenced, and a reliable guarantee is provided for safe medication of a user.
Description
Technical field
The present invention relates to the preparation method of human blood albumin products, specifically relate to the control method of Prekallikrein (PKA) content in a kind of human serum albumin production process.
Background technology
Human serum albumin is that the human normal plasma of take through hepatitis b vaccination is raw material, through cold ethanol method separation, forms.Human normal plasma is a kind of very complicated egg white mixture, wherein, factor ⅫYou Chengjiechuyinzi in blood plasma is that the zymogen forms with non-activity exists, when vascular endothelial cell is impaired, the electronegative collegen filament that expose can activate factor ⅫYou Chengjiechuyinzi and become activation factor XII ɑ, and activation factor XII ɑ can activate prekallikrein (PK) and generate kallikrein (K
k), K
kmake prokinin discharge bradykinin, the factor fragment of these activation is called as Prekallikrein (PKA).The thawing repeatedly of human blood albumin products and freezing meeting cause protein component sex change in goods, can make PKA raise.
When containing the more human blood albumin products of PKA for the quick infusion of human vein, the prekallikrein of PKA in can human activin, generate kallikrein, cause kassinin kinin to produce, cause the untoward reactions such as capillary permeability increase, vasorelaxation, blood pressure drops, flushed face, headache, palpitaition, expiratory dyspnea.Therefore, detect the PKA content in blood products, and stipulate that it is limited the quantity of is necessary.The content of having stipulated Prekallikrein (PKA) in human blood albumin products in < < Chinese Pharmacopoeia > > should be higher than 35IU/ml.
1), raw blood plasma merge to melt now current blood plasma cold ethanol method separating technology is:; 2), adjust protein concentration 4.0 ± 0.5%, ethanol content 20 ± 1%, pH value 6.80 ± 0.10, temperature-4.5 ± 0.5 ℃, stirring, press filtration separation, collection components I+II+III precipitation; 3), the components I+II after precipitation separation+III supernatant liquor is adjusted protein concentration 1.25～1.50%, ethanol content 40 ± 1%, pH value 5.85 ± 0.05, temperature-4.5 ± 0.5 ℃; Stir, press filtration is separated, collects fraction IV precipitate; 4), the component IV supernatant liquor after precipitation separation is adjusted protein concentration 1.0～1.2%, ethanol content 40 ± 1%, pH value 4.80 ± 0.10, temperature-8.0 ± 0.5 ℃; Stir, press filtration is separated, collects component V precipitation; 5) 2～4 ℃ of 10% ethanolic soln stirring and dissolving, by component V precipitation with 6～8 times of precipitation volumes, filters, and filtrate should be clarified, without foreign matters such as fibers; 6), dialysis dealcoholysis, concentrated, dosing, 60 ± 0.5 ℃, 10 hours viral inactivation treatment.
Existing blood plasma cold ethanol separation method is preserved improper grade for other reasons because of raw blood plasma, easily occurs the too high phenomenon of PKA in human blood albumin products, and conventional method is that goods are heated repeatedly, and PKA is carried out to deactivation.Yet heat-processed has repeatedly caused potential threat to human blood albumin products.
Summary of the invention
The object of the invention is, for easily occurring the problem that Prekallikrein is too high in current human blood albumin products sepn process, provides the control method of Prekallikrein in a kind of human blood albumin products.
In human blood albumin products of the present invention, the control method of Prekallikrein is included in cold ethanol method separation and prepares in the technique of human serum albumin, described human serum albumin cold ethanol method separation preparation is common process, and the present invention increases following two step process in the process of human serum albumin is prepared in cold ethanol method separation:
1), utilize the adsorption of activation diatomite to factor ⅫYou Chengjiechuyinzi, in the step of separated portion IV from components I+II+III supernatant liquor, add through the pretreated activation diatomite of Sodium Citrate, factor ⅫYou Chengjiechuyinzi is adsorbed, to reduce factor ⅫYou Chengjiechuyinzi content;
2), by the component V after ultrafiltration dialysis purifying by positively charged grease removal filter core, electrostatic adhesion is removed the activation factor XII ɑ fragment remaining in human serum albumin solution;
By above two step process, reduce the Prekallikrein content in human blood albumin products.
Wherein, described through the preparation of the pretreated activation diatomite employing of Sodium Citrate following steps:
1), diatomite is added in the Sodium Citrate aqueous solution of 2g/L, stir 5～10 minutes, suspendible, standing 3～5 minutes, removes suspended substance and supernatant;
2), with the water that does not add Sodium Citrate, repeat aforesaid operations, repeatedly drain, be neutrality to washing lotion pH value;
3) add, again the glacial acetic acid aqueous solution of concentration expressed in percentage by volume 0.025%, stir 5～10 minutes, suspendible, standing 3～5 minutes, drains to obtain solid;
4), water repetitive scrubbing solid, to washing lotion pH value, be neutral, drain and obtain activating diatomite, stored refrigerated.
The water more than using is the water for injection of 20～30 ℃.
And then, the concrete separation condition of the present invention's separated portion IV from components I+II+III supernatant liquor is: the pH value 5.91～5.94 of adjusting supernatant liquor, protein mass concentration 1.0～1.5%, ionic strength 0.1, temperature-4～-5 ℃, ethanol volumetric concentration 40～42%, adds activation diatomite, making its concentration in supernatant liquor is 5～8g/L, with the speed of 60～120 revs/min, stirs 30～60 minutes.
The present invention obtains respectively fraction IV precipitate and component IV supernatant liquor by above-mentioned supernatant liquor with filter plate press filtration.Wherein, press filtration adopts the 50P filter plate of pall company, and required filter plate area is 5～8 square metres of/ton of blood plasma, and filter pressure is controlled at 0～3.0bar.
Particularly, the component V after purifying of the present invention is that pressure with 0～2.0bar is by described positively charged grease removal filter core.Described positively charged grease removal filter core is first with water for injection prewashing 15 minutes, then rinses with the ethanolic soln of volumetric concentration 12%, dries up rear use.
In control method of the present invention, the super-cell using has been removed molecule wherein by pre-treatment, has removed pyrogen, and diatomite is activated, and can not cause disadvantageous effect to human blood albumin products.
Control method of the present invention is on protein molecular activity, every physical and chemical index and protein yield all without impact, and in the human blood albumin products after controlling by the inventive method, PKA content is reduced greatly, for patient safety medication provides Reliable guarantee.
Accompanying drawing explanation
Fig. 1 adopts the cold ethanol method separation of control method of the present invention to prepare human serum albumin process flow sheet.
Embodiment
Embodiment 1
1, diatomite activating pretreatment
Get 100g Sodium Citrate and be dissolved in 20～30 ℃ of waters for injection of 50L, add 5kg diatomite, stir 5 minutes, suspendible, standing 5 minutes, removes suspended substance and supernatant;
Not add the water for injection of Sodium Citrate, repeat aforesaid operations, repeatedly drain, to washing lotion pH value, be neutral;
25mL Glacial acetic acid is dissolved in 20～30 ℃ of waters for injection of 10L, adds the above-mentioned solid of draining, stir 5 minutes, suspendible, standing 3 minutes, drains to obtain solid;
With water for injection repetitive scrubbing solid, to washing lotion pH value, be neutral, drain, obtain activating diatomite, be placed in cold operation room stand-by.
2, factor ⅫYou Chengjiechuyinzi absorption
Get cold ethanol method separation and prepare the components I+II+III supernatant liquor 100L obtaining in human serum albumin process, this supernatant liquor has been adjusted to pH value 5.91, protein concentration 1.2%, and ionic strength is 0.1, temperature-4.4 ℃, alcohol concn 40.2%.In supernatant liquor, add above-mentioned pretreated activation diatomite 0.5kg, with the speed of 60 revs/min, stir after 30 minutes, upper filter plate press filtration, obtains respectively fraction IV precipitate and component IV supernatant liquor.Pressure-filtering process adopts the 50P filter plate of pall company, and required filter plate area is 5～8 square metres of/ton of blood plasma, and filter pressure is controlled at 0～3.0bar.
3, PKA electrostatic adhesion
The ZETAPLUS grease removal filter core of mounting strap positive charge in strainer, with cold water for injection prewashing strainer 15 minutes, then uses 12% the about 50L flush filter of ethanolic soln (pH value 4.56), dries up.
Get cold ethanol method separation and prepare the component V after purifying in human serum albumin process, with the pressure of 0～2.0bar, by above-mentioned strainer, filter.
Embodiment 2
1, diatomite activating pretreatment
Get 100g Sodium Citrate and be dissolved in 20～30 ℃ of waters for injection of 50L, add 5kg diatomite, stir 8 minutes, suspendible, standing 4 minutes, removes suspended substance and supernatant;
Not add the water for injection of Sodium Citrate, repeat aforesaid operations, repeatedly drain, to washing lotion pH value, be neutral;
25mL Glacial acetic acid is dissolved in 20～30 ℃ of waters for injection of 10L, adds the above-mentioned solid of draining, stir 8 minutes, suspendible, standing 4 minutes, drains to obtain solid;
With water for injection repetitive scrubbing solid, to washing lotion pH value, be neutral, drain, obtain activating diatomite, be placed in cold operation room stand-by.
2, factor ⅫYou Chengjiechuyinzi absorption
Get cold ethanol method separation and prepare the components I+II+III supernatant liquor 100L obtaining in human serum albumin process, this supernatant liquor has been adjusted to pH value 5.93, protein concentration 1.4%, and ionic strength is 0.1, temperature-4.3 ℃, alcohol concn 40.8%.In supernatant liquor, add above-mentioned pretreated activation diatomite 0.7kg, with the speed of 90 revs/min, stir after 45 minutes, upper filter plate press filtration, obtains respectively fraction IV precipitate and component IV supernatant liquor.Pressure-filtering process adopts the 50P filter plate of pall company, and required filter plate area is 5～8 square metres of/ton of blood plasma, and filter pressure is controlled at 0～3.0bar.
3, PKA electrostatic adhesion
The ZETAPLUS grease removal filter core of mounting strap positive charge in strainer, with cold water for injection prewashing strainer 15 minutes, then uses 12% the about 50L flush filter of ethanolic soln (pH value 4.62), dries up.
Get cold ethanol method separation and prepare the component V after purifying in human serum albumin process, with the pressure of 0～2.0bar, by above-mentioned strainer, filter.
Embodiment 3
1, diatomite activating pretreatment
Get 100g Sodium Citrate and be dissolved in 20～30 ℃ of waters for injection of 50L, add 5kg diatomite, stir 10 minutes, suspendible, standing 5 minutes, removes suspended substance and supernatant;
Not add the water for injection of Sodium Citrate, repeat aforesaid operations, repeatedly drain, to washing lotion pH value, be neutral;
25mL Glacial acetic acid is dissolved in 20～30 ℃ of waters for injection of 10L, adds the above-mentioned solid of draining, stir 10 minutes, suspendible, standing 5 minutes, drains to obtain solid;
With water for injection repetitive scrubbing solid, to washing lotion pH value, be neutral, drain, obtain activating diatomite, be placed in cold operation room stand-by.
2, factor ⅫYou Chengjiechuyinzi absorption
Get cold ethanol method separation and prepare the components I+II+III supernatant liquor 100L obtaining in human serum albumin process, this supernatant liquor has been adjusted to pH value 5.94, protein concentration 1.4%, and ionic strength is 0.1, temperature-4.9 ℃, alcohol concn 42.0%.In supernatant liquor, add above-mentioned pretreated activation diatomite 0.8kg, with the speed of 90 revs/min, stir after 60 minutes, upper filter plate press filtration, obtains respectively fraction IV precipitate and component IV supernatant liquor.Pressure-filtering process adopts the 50P filter plate of pall company, and required filter plate area is 5～8 square metres of/ton of blood plasma, and filter pressure is controlled at 0～3.0bar.
3, PKA electrostatic adhesion
The ZETAPLUS grease removal filter core of mounting strap positive charge in strainer, with cold water for injection prewashing strainer 15 minutes, then uses 12% the about 50L flush filter of ethanolic soln (pH value 4.69), dries up.
Get cold ethanol method separation and prepare the component V after purifying in human serum albumin process, with the pressure of 0～2.0bar, by above-mentioned strainer, filter.
In the separated human serum albumin process of cold ethanol method, adopt technique of the present invention.Above-described embodiment respectively carries out 10 batches of tests, and before and after implementing, product P KA detected result is relatively in Table 1.
As can be seen from Table 1, before implementing, mean P KA content is 24.2 IU/mL, is reduced to respectively 4.8IU/mL, 4.9IU/mL and 5.0 IU/mL after enforcement.By above data, can prove, adopt technique of the present invention can effectively reduce PKA content in human blood albumin products, its content well below in < < Chinese Pharmacopoeia > > not higher than the requirement of 35IU/mL, guaranteed the security of goods.
Claims (4)
1. the control method of Prekallikrein in human blood albumin products, is included in cold ethanol method separation and prepares in the technique of human serum albumin, it is characterized in that increasing following two step process in the process of human serum albumin is prepared in cold ethanol method separation:
1), in the step of separated portion IV from components I+II+III supernatant liquor, add through the pretreated activation diatomite of Sodium Citrate, factor ⅫYou Chengjiechuyinzi is adsorbed, to reduce factor ⅫYou Chengjiechuyinzi content;
Wherein, describedly through the pretreated activation diatomite of Sodium Citrate, adopt following steps preparation:
A), diatomite is added in the Sodium Citrate aqueous solution of 2g/L, stir 5～10 minutes, suspendible, standing 3～5 minutes, removes suspended substance and supernatant;
B), with the water that does not add Sodium Citrate, repeat aforesaid operations, repeatedly drain, be neutrality to washing lotion pH value;
C), again add the glacial acetic acid aqueous solution of concentration expressed in percentage by volume 0.025%, stir 5～10 minutes, suspendible, standing 3～5 minutes, drains to obtain solid;
D), water repetitive scrubbing solid, to washing lotion pH value, be neutral, drain and obtain activating diatomite, stored refrigerated;
The water more than using is the water for injection of 20～30 ℃;
2), by the component V after ultrafiltration dialysis purifying by positively charged grease removal filter core, the activation factor XII ɑ fragment remaining in human serum albumin solution is removed in absorption;
By above two step process, reduce the Prekallikrein content in human blood albumin products.
2. the control method of Prekallikrein in human blood albumin products according to claim 1, described in it is characterized in that, from components I+II+III supernatant liquor, the separation condition of separated portion IV is: pH value 5.91～5.94, protein mass concentration 1.0～1.5%, ionic strength 0.1, temperature-4～-5 ℃, ethanol volumetric concentration 40～42%, adding activation diatomite to make its concentration in supernatant liquor is 5～8g/L, with the speed of 60～120 revs/min, stirs 30～60 minutes.
3. the control method of Prekallikrein in human blood albumin products according to claim 1, it is characterized in that described positively charged grease removal filter core first with water for injection prewashing 15 minutes, with the ethanolic soln of volumetric concentration 12%, rinse again, dry up rear use.
4. the control method of Prekallikrein in human blood albumin products according to claim 1, is characterized in that the component V after purifying with the pressure of 0～2.0bar by described positively charged grease removal filter core.
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| CN105582883B (en) * | 2015-12-25 | 2017-12-15 | 北海开元生物科技有限公司 | A kind of activated diatomaceous earth and the application in blood product production |
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| EP2640413B1 (en) * | 2010-11-16 | 2019-06-12 | Octapharma AG | A process for reduction and/or removal of fxi and fxia from solutions containing said coagulation factors |
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