CN1333296A - Process for preparing high-purity pyretogenless substrateless hematoglobin for red blood cell substitute - Google Patents
Process for preparing high-purity pyretogenless substrateless hematoglobin for red blood cell substitute Download PDFInfo
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- CN1333296A CN1333296A CN01108643A CN01108643A CN1333296A CN 1333296 A CN1333296 A CN 1333296A CN 01108643 A CN01108643 A CN 01108643A CN 01108643 A CN01108643 A CN 01108643A CN 1333296 A CN1333296 A CN 1333296A
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a preparation method of high-purity matrix-free pyretogen-less hematoglobin for substitute of red blood cell, and is characterized by that said preparation method includes the following steps: under the condition of 0-4 deg.C making natural blood cell undergo the process of hemolysis treatment to obtain crude hemoclastic liquor; adding anti-oxidant and regulating pH value of solution to 5.40-6.00, them removing oxygen from hematoglobin and adding polyhydric compound according to that the weight/volume ratio of said polyhydric compound and deoxyhemoglobin is 1-2 % and using said polyhydric compound as protection agent, heating for 1-3 hr. at 50-60 deg.c, then making conventional filtration and superfiltration treatment, and regulating pH value to 7.0-7.4 so as to obtain theinvented product.
Description
Technical field
That the present invention relates to is a kind of preparation method of the high purity pyrogen-free stroma-free hemoglobin that uses as red blood cell substitute.
Background technology
Because human blood group is very complicated, the blood transfusion immune response that may cause except that various antigens is difficult to overcome, hepatitis, acquired immune deficiency syndrome (AIDS) etc. are multiple can be also increasing through the transmissible disease threat that clinical direct use constitutes to blood that the blood approach is propagated, thereby in various clinical treatments and first aid, more and more higher to the requirement of quality and quantity of blood transfusion.In addition, the external storage and transport problem of human blood also is that influence will be gathered the important restraining factors that blood directly uses.Therefore, research and development and use blood substitute generally come into one's own.Clear and definite now, human blood group reaction derives from the difference of the antigen type on the erythrocyte membrane, can there be the sorrow of match type fully in stroma-free hemoglobin (Hb) class red blood cell substitute after having removed erythrocyte membrane and having gone up entrained various blood types antigen, and in preparation process by eliminating various possibilities with blood born diseases to the deactivation of virus.Test shows, the period of storage of this kind stroma-free hemoglobin class red blood cell substitute under general room temperature condition was more than 1 year, particularly its on performance, not only have similar to natural blood good take, the oxygen release function, and its viscosity is low, granularity is little, oxygen delivery capacity to the microcirculation surrounding tissue has also surpassed the neutral red cell, thereby is demonstrating bright development prospect aspect anoxic treatment of diseases such as cardiovascular and cerebrovascular and the emergency aid and treatment.
Though, have report to study success with the Recombinant Human Hemoglobin of escherichia coli expression for the oxyphorase that solves with human body is the limited problem in source of existing human hemoglobin in the feedstock production red blood cell substitute; Also have report tentatively to finish in the laboratory, but these technology all still have complex process and cost and cross problem aspect high dropping into suitability for industrialized production from the HRBC that people hemocytoblast vitro culture induces.Therefore, be that feedstock production hemoglobin red blood cell substitute is still approach and the mode of mainly taking directly at present with animal blood or blood of human body.In these areas, a kind of method of the existing report of data is mainly to handle by ultrafiltration with propylene sephadex column S-100, then through high-pressure liquid chromatography at present, by the method for affinity chromatography purification oxyphorase, can obtain highly purified hemoglobin solutions at last.But its method operation sequence is more loaded down with trivial details, causes cost too high, and the rate of recovery is also lower.Also have data report employing standard anion exchange method and base exchange method, can make the purity of prepared bovine hemoglobin reach 99%.Generally also there is the problem that the rate of recovery of oxyphorase is low and still be not suitable for large-scale industrial production in these methods.
Existing reported in literature makes oxyphorase be in the deoxidation state and can improve its tolerance to Heating temperature.For this reason, once after having report to adopt the supersaturation displacement analysis, oxyphorase is combined with carbon monoxide, to replace institute's bonded oxygen in the oxyphorase, through pasteurization inactivation of viruses and anion exchange chromatography and the processing of cation seperation column chromatography, can obtain the oxyphorase goods of high purifying then.Because oxyphorase is bigger approximately 150 times than oxygen with the bonding force of carbon monoxide, though therefore more or less freely with bonded oxygen in carbon monoxide expeling and the replacement oxyphorase, making after handling, the bonded carbon monoxide dissociates then obviously more difficult with oxyphorase again.Other has the data report, to handle the rough hemolysate that obtains through haemolysis, with feeding nitrogen, the mode of anaerobic rare gas elementes such as argon gas or vacuum, make after institute's bonded oxygen fully discharges in the oxyphorase, again at the gsh that adds reductibility, dithiothreitol (DTT) (DTT) or sulfoxylic acid compounds, the SODIUM HYDROSULPHITE compounds, the bisulfite compounds, sulphur hydrogen compounds etc. has the sulfocompound of reductibility, and keeping heating a few hours under the deoxidation state to a couple of days, with other protein component of removing non-hemoglobin class wherein and the method for preparing stroma-free hemoglobin of virus being carried out deactivation.Though the document is described Study on Conditions such as relevant deoxidation, heating and inactivation of virus, not clear and definite its has or not influence and/or changes to the purity of gained oxyphorase and to the relevant performance of goods.
Summary of the invention
At above-mentioned situation, the present invention will provide a kind of working method and required equipment greatly to simplify, and the preparation time cycle is shortened, the high purity pyrogen-free stroma-free hemoglobin preparation method who is used for red blood cell substitute that cost reduces.The oxyphorase goods that this method prepares gained not only can reach same high purity and pyrogen-free requirement, and the higher rate of recovery can be arranged.
The preparation method who is used for the high purity pyrogen-free stroma-free hemoglobin of red blood cell substitute of the present invention; be under 0 ℃ of-4 ℃ of condition; after in the rough hemolysate that the haemolysis processing obtains, adding oxidation inhibitor and regulator solution pH5.40-6.00 by natural hemocyte; fully remove be present in the oxyphorase in conjunction with behind oxygen and the dissolved oxygen; by with Reduced Hbs solution weight/volume ratio be the amount of 1-2% add polyol as protectant condition under; in 50 ℃-60 ℃ heating 1-3 hour, make conventional filtration and ultrafiltration membrance filter then and handle and adjust pH7.0-7.4.
The reported in literature and the inventor's test-results confirms, in the oxyphorase whether in conjunction with aerobic, to the Heating temperature height and/or heat-up time length tolerance on can show than evident difference.Therefore make oxyphorase be in the deoxidation state, to improving its tolerance to Heating temperature and/or heat-up time, loss in the minimizing process and raising goods yield obviously are favourable.In the above-mentioned preparation method of the present invention, make behind the centrifugal disgorging that institute's bonded oxygen fully removes in the hemolysate oxyphorase.For example, can adopt rare gas elementes such as feeding oxygen-free nitrogen, argon gas, or as carbonic acid gas etc. and indifferent other gas of oxyphorase, also can adopt the delivery mode under the vacuum condition, oxygen to be present in conjunction with various forms such as oxygen, dissolved oxygens in the oxyphorase can be discharged and be removed fully, be reduced to 0 mmhg until its oxygen partial pressure and end.
Be to improve the tolerance of oxyphorase, reduce the loss that causes because of heat denatured,, add in the oxyphorase treating processes that to have the oxidation inhibitor of reductive action and keep its deoxidation state be useful as existing reported in literature to heat treated.For this reason, except that the mode of existing report, the inventive method proposes to adopt with vitamins C as the oxidation inhibitor in the treating processes especially.For example, a kind of alternative mode be by weight/volume ratio is that the amount of 0.2-0.4% adds vitamins C, generally speaking, a step realizes simultaneously can also to make the operation of regulating pH5.40-6.00 this moment.In addition, compare with the various oxidation inhibitor that existing report in the document uses, use vitamins C except that the superiority that is had economically, the unrivaled remarkable superiority of another that it had also is the security that it uses in goods, and simplifies the follow-up work remove oxidation inhibitor from goods and require difficulty with quality control and inspection routine.
Test shows; in the above-mentioned preparation method of the present invention; the sedimentary rough deoxidation state hemoglobin solutions of filtering is carried out heat treated under as protectant condition again adding polyol; for reducing the loss of oxyphorase in heat-treatment process; improving the rate of recovery, is significance and vital role.Can multiple choices can be arranged as the polyol that said protective material in the inventive method uses, wherein convenient with the polyol that adopts a 5-6 commonly used carbon atom.For example, adopting polyhydroxy-alcohol compounds such as N.F,USP MANNITOL, or adopt a kind of etc. in the multiple saccharide compound that contains 5-6 carbon atom such as sucrose, maltose, lactose or glucose, all is one of alternative concrete mode.Test-results shows, used above-mentioned protective material after, carry out heat treated in the same way after, the reduction of hemoglobin concentration is general all less than 10%, ferrihemoglobin content can be lower than 2%.And do not use protectant comparative test result to show, and the loss of its oxyphorase can surpass 30%, and the content of methemoglobin is higher than 10%.
By heat treated, making the non-hemoglobin proteinoid sex change precipitation in the rough hemolysate and be removed in preparation process, is required measure among this type of preparation method at present.Test shows, is adopting the adding polyol as protectant aforesaid method of the present invention, in 500-60 ℃ temperature range internal heating---generally can under 53 ℃-57 ℃ temperature, heat, satisfied effect can be arranged.
Based on the above method, for further improving efficient and the handled easily of handling, prepare in the treatment solution of conventional filtration after heat treated and cooling for said, can also after adding medical sorbent material and making its thorough mixing, carry out filtration treatment more in the usual way.Said medical sorbent material can be in general curative the most commonly used as selecting for use in the materials such as medical grade gac, chitosan, white bole.The add-on of said sorbent material generally need not strict restriction, can operate by general usual manner, for example, by weight/volume ratio is the amount use of 0.1-1% scope, is exactly general alternative a kind of mode.After taking this measure, help on the one hand further improving to being present in the pyrogen in the goods and the removal effect of non-hemoglobin class impurity, it can also have filtrating aid function on the other hand, can significantly improve filtration efficiency, also is useful to reducing the loss of goods in this step operation.
Prepare the raw material that the oxyphorase goods of above-mentioned red blood cell substitute are adopted, be reported to the animal blood of direct collection at present or be various forms of blood of human body.As described above, this type of raw material, when particularly being raw material with the blood of human body, the restriction of being originated is the matter of utmost importance that should face and actively solve.Owing to wherein remain the blood of contain a great deal of after human placenta is given birth to, and these valuable blood resources that are present among the human placenta fail still at present to be fully utilized.Proposed to can be used for gathering the device of placental blood in the once former relevant patent of the inventor, and the collection and utilization rate to placental blood is significantly improved, helped the abundant development and utilization of people's placental blood.Therefore, the used natural hemocyte raw material of the above-mentioned preparation method of the present invention at first and especially can be the placental blood of gathering.Compare with the collection of human peripheral blood, because placenta is vulnerable to the pollution of various factors in the process of giving birth to, therefore, when adopting placental blood to be raw material, generally can after adding conventional antibacterial medicines by the amount of 100,000-1,000,000 unit/1000 milliliter in the rough hemolysate that obtains through haemolysis, carry out handling procedure thereafter again.Said antibacterial medicines can be in the wide spectrum class be selected for use in the antibiotic and/or antibacterial medicines, and for example, Chang Yong penicillin, Streptomycin sulphate etc. are exactly one of alternative mode the most.In addition, also can adopt out of date stock's human blood etc., the waste that still can bring into play certain use value is fully utilized for the preparation raw material.Certainly, when adopting the natural hemocyte raw material in other source, as think and be necessary, also can handle by this same manner.
Be understood that thus, the above-mentioned method for preparing the pyrogen-free stroma-free hemoglobin of the present invention can make the difficulty of operating process and technical qualification thereof and require greatly simplification, and required equipment is also simple relatively, compare with at present existing method, only the required expense in equipment aspect can reduce more than 50%; The required time cycle of whole process of preparation also can be shortened to about 1 day by 2-3 days of present routine, efficient is enhanced about more than once.Because the required time cycle of method for preparing process of the present invention shortens greatly, and taked to help further to reduce the loss and improved the corresponding measure of yield, make the total recovery of the pyrogen-free stroma-free hemoglobin that obtains can improve 10 percentage points approximately, the purity of goods can reach the requirement more than 99% equally.
Below will again foregoing of the present invention be described in further detail, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only is confined to following embodiment by the form of embodiment.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 is the HPLC collection of illustrative plates to oxyphorase goods purity detecting.
Embodiment
With the placental blood of gathering (or becoming human peripheral), under 0 ℃-4 ℃ cold condition, use according to a conventional method pyrogen-free physiological saline repetitive scrubbing, centrifugal after, obtain packed red cells, carry out haemolysis with usual manners such as hypotonic salt brine solution (is the phosphate buffered saline buffer of 15 m osmoles and pH7.4 as osmotic pressure) commonly used or ultrasonic wave again and handle, obtain rough hemolysate.In rough hemolysate by the amount of 100,000-1,000,000 unit/1000 milliliter add conventional antibacterial medicines such as penicillin and/or Streptomycin sulphate and thorough mixing evenly after, again by weight/volume ratio is that the amount of 0.2-0.4% adds vitamins C as oxidation inhibitor, and can makes pH value of solution be adjusted to 5.40-6.00 simultaneously.With feeding rare gas element such as oxygen-free nitrogen or the oxyphorase in the treatment solution being carried out deoxidation treatment with the mode of the indifferent carbon dioxide of oxyphorase, making to be present in removing and being removed in conjunction with oxygen and dissolved oxygen release fully in the oxyphorase, is that 0 mmhg ends to its oxygen partial pressure.In the oxyphorase treatment solution after deoxidation treatment by weight/amount of volume ratio 1-2% adds a kind of in the saccharide compound that contains 5-6 carbon atom commonly used such as sucrose, maltose, lactose, glucose; or add N.F,USP MANNITOL etc. contain 5-6 polyol such as carbon atom polyvalent alcohol as protective material and thorough mixing even after; temperature range in 50 ℃-60 ℃---for example can be incubated 1-3 hour---and for example generally can be 2 hours under 53 ℃ of-57 ℃ of temperature, make composition sex change such as the protein precipitation of non-hemoglobin class.Be cooled to 0 ℃-4 ℃ and remain under this temperature range, by weight/volume ratio is that the amount of 0.1-1% adds medical sorbent materials such as gac or white bole, kept 0.5-1 hour behind the thorough mixing, after doing centrifugation with 5000 rev/mins in the usual way then, filter in a usual manner again, eliminate pyrogen and remaining non-hemoglobin class impurity component in the treatment solution to greatest extent.After with weak base such as sodium bicarbonate or SODIUM PHOSPHATE, MONOBASIC supernatant liquor being adjusted to pH7.0-7.4, the ultra-filtration membrane that with the molecular weight cut-off is 10KD carries out 30 minutes uf processing in the usual way, through Sterile Filtration, promptly obtain the pyrogen-free stroma-free hemoglobin goods of high purity (〉=99%) again.The HPLC collection of illustrative plates that detects its purity by UV320 type Ultraviolet Detector as shown in Figure 1.This preparation cycle required time is about 1 day.As shown in table 1 to the leading indicator detected result that the red blood cell substitute by method for preparing carries out.
The red blood cell substitute leading indicator detected result of table 1 pair preparation
| Test item | The inventive method resulting product | Existing reported in literature level |
| Oxyphorase (Hb) content | ????7-14％ | ????7-14％ |
| MetHb content | ????＜5％ | ????＜3-10％ |
| Oxyphorase (Hb) purity | ?????99％ | ????99％ |
| Molecular weight | ????120-600KD | ????120-700KD |
| Polymerization Hb not | ????＜1％ | ????＜1％ |
| PH value | ????7.0-7.4 | ????7.0-7.4 |
| ????P50 | ????20-28mmHg | ????20-30mmHg |
| Osmotic pressure (COP) | ????20-25mmHg | ????23-28mmHg |
| Intracellular toxin | Qualified | Qualified |
| Bacterium | Qualified | Qualified |
| Thermal source | Negative | Negative |
| ????Ma + | ????130-140Eg/L | ????130-135Eg/L |
| ????K + | ????3.5-4.0Eg/L | ????3.5-2.8Eg/L |
| ????Cl - | ????95-106Eg/L | ????100-105Eg/L |
Claims (10)
1. the preparation method who is used for the high purity pyrogen-free stroma-free hemoglobin of red blood cell substitute; it is characterized in that under 0 ℃ of-4 ℃ of condition; after in the rough hemolysate that the haemolysis processing obtains, adding oxidation inhibitor and regulator solution pH5.40-6.00 by natural hemocyte; after fully removing the oxygen that is present in the oxyphorase; by with Reduced Hbs solution weight/volume ratio be the amount of 1-2% add polyol as protectant condition under; in 50 ℃-60 ℃ heating 1-3 hour, make conventional filtration and ultrafiltration membrance filter then and handle and adjust pH7.0-7.4.
2. preparation method as claimed in claim 1 is characterized in that said protective material is the polyol of 5-6 carbon atom.
3. preparation method as claimed in claim 2 is characterized in that said polyol protective material is a N.F,USP MANNITOL.
4. preparation method as claimed in claim 1 is characterized in that said polyol protective material is the saccharide compound that contains 5-6 carbon atom.
5. preparation method as claimed in claim 4 is characterized in that said polyol protective material is a kind of in sucrose, maltose, lactose or the glucose.
6. preparation method as claimed in claim 1, it is characterized in that said in rough hemolysate, add oxidation inhibitor and regulate pH5.40-6.00 adopt by weight/volume ratio is that the amount of 0.2-0.4% adds ascorbic one step of mode and finishes.
7. preparation method as claimed in claim 1 is characterized in that the heat treated temperature after said adding polyol is as protective material is 53 ℃-57 ℃.
8. preparation method as claimed in claim 1, it is characterized in that in the treatment solution after said heating by weight/volume ratio is after the amount of 0.1-1% adds medical sorbent material and thorough mixing, to remake conventional filtration and ultrafiltration membrance filter and handle.
9. preparation method as claimed in claim 1 is characterized in that preparing placental blood or the out of date stock human blood of raw materials used natural hemocyte for gathering.
10. as the described preparation method of claim 1 to 9, after adding conventional antibacterial medicines, the amount by 100,000-1,000,000 unit/1000 milliliter of it is characterized in that in said rough hemolysate remakes processing thereafter.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011086432A CN1238380C (en) | 2001-07-17 | 2001-07-17 | Process for preparing high-purity pyretogenless substrateless hematoglobin for red blood cell substitute |
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| CNB011086432A CN1238380C (en) | 2001-07-17 | 2001-07-17 | Process for preparing high-purity pyretogenless substrateless hematoglobin for red blood cell substitute |
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| CN1238380C CN1238380C (en) | 2006-01-25 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786018B (en) * | 2005-12-12 | 2011-06-22 | 天津协和生物科技发展有限公司 | Separation and purification of high purity hemoglobin and virus inactivation technology |
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2001
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786018B (en) * | 2005-12-12 | 2011-06-22 | 天津协和生物科技发展有限公司 | Separation and purification of high purity hemoglobin and virus inactivation technology |
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