CN1990039A - Modification method of polymeric hemoglobin - Google Patents
Modification method of polymeric hemoglobin Download PDFInfo
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- CN1990039A CN1990039A CN 200510136023 CN200510136023A CN1990039A CN 1990039 A CN1990039 A CN 1990039A CN 200510136023 CN200510136023 CN 200510136023 CN 200510136023 A CN200510136023 A CN 200510136023A CN 1990039 A CN1990039 A CN 1990039A
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Abstract
The invention relates to a modification method of polymmeric hemoglobin, it is characterized by: preparing purified hemoglobin from natural red blood cells in the condition of 0-4DEG C and nitrogen, first polymerizing them through same type bifunctional reagent, then modifying them with 5'-pyridoxal phosphate. During the modifying process, glutathione should be added after adding PLP 0.5-1.5 hours. The arts can control generation of ferrihemoglobin, but also ensuret the P50 value in appropriate range and reduce production costs greatly.
Description
(1) technical field:
The present invention relates to biomedicine field, particularly a kind of modification method of polymeric hemoglobin.
(2) background technology:
With the input whole blood is that the tradition blood transfusion mode of main means is being brought into play very important effect aspect the medical aid, but himself also there are the various shortcomings that are difficult to overcome simultaneously, mainly show: need carry out the short and special facility of needs of crossmatch, the danger that has multiple viral cross infection, blood source anxiety, storage period before the input.In order to solve above-mentioned contradiction, people have just begun the research of relevant red blood cell substitute as far back as 19 end of the centurys.Red blood cell substitute is meant to have and expands the blood volume, keeps the inside and outside osmotic pressure balance of blood vessel and the oxygen carrier function is arranged, in order to the general name of the various carriers of oxygen that replace human red blood cell.The blood vessel expander from initial no oxygen carrier function has been experienced in the development of blood substitute, as: hypertonic salt solution, hydroxyethyl starch solution, gelatin etc. have the various carrier solutions of function of carrying oxygen up till now.Various product and the whole blood developed at present relatively have plurality of advantages, as: but need not to carry out crossmatch the patient's that is specially adapted to bleed profusely in war, natural disaster and other accidents first aid before wide material sources, safety clean long preservation, the input human body.Hemoglobin is the intravital material of people, and its solution has more superiority as red blood cell substitute than chemosynthesis material.But also there are some problems in not modified hemoglobin solutions, is that not modified hemoglobin is cracked into dimer and monomer by the tetramer in vivo and filters from kidney as one, forms hemoglobinuria, causes renal damage; The 2nd, in the leaching process of hemoglobin, because 2, (2, losing 3-DPG) improves the oxygen combining power of hemoglobin to the 3-diphosphoglyceric acid, is unfavorable for to organizing effective oxygen supply; The 3rd, the hemoglobin of unmodified colloid osmotic pressure in blood plasma is higher, and circulating half-life is short.
Mainly concentrate on based on the research of the blood substitute of hemoglobin and to prolong circulating half-life, reduce aspects such as oxygen combining power and stable tetramer structure.Usually adopt method such as crosslinked, polymerization, liposome embedded and chemical modification or several method combined and reach this purpose.U.S. Northfield company ' after pyridoxal 5-phosphate (PLP) modification, has entered III phase clinical experiment with the polymeric product of glutaraldehyde with 5.People such as Shirley L., at HemoglobinPolymerization, Methods in enzymology, V (231), 287-308, in point out that cross-linked polymeric can make hemoglobin that oxygen combining power is improved greatly, modify and then can reduce oxygen combining power, so finished product will be these the two kinds results after the effect balance to the adhesion of oxygen.
In vivo, 2,3-DPG appreciable impact hemoglobin is to the adhesion of oxygen.Hemoglobin is in conjunction with 2, O behind the 3-DPG
2Adhesion reduce, the oxygen binding curve moves to right, P
50Increase to more than the 26mmHg.Endoerythrocytic 2,3-DPG has the important physical meaning, to the fortune O of hemoglobin
2Significant contribution is arranged, and hemoglobin is to O
2Adhesion and endoerythrocytic 2,3-DPG concentration is inversely proportional to, thereby in the erythrocyte 2,3-DPG concentration is higher, the O that blood flow discharges when organizing blood capillary
2Measure also the more, in conjunction with O
2The ability reduction helps carrying O on the contrary
2, human body can be by in the erythrocyte 2, and 3-DPG concentration changes the oxygen amount that obtains of regulating tissue, and this is to human body compensatory significant under some anaerobic condition.
External, in purge process 2,3-DPG loses in a large number, and polymerization process also can make hemoglobin that oxygen combining power is increased greatly, adopting PLP chemical modification hemoglobin is to solve 2, and 3-DPG loses, oxygen is in conjunction with one of method that increases, and has developed decades till now from the forties.Found at present and synthesized many modification cross-linking agent and method, pyridoxal 5-phosphate (PLP) is wherein a kind of, it is 2, the analog of 3-DPG, can generate schiff bases with valine (Val) reaction of hemoglobin β-subunit N-end, reduce the adhesion of hemoglobin, improved the P of hemoglobin
50
5 ' pyridoxal 5-phosphate (PLP) can generate imines with the valine generation aldol condensation of the subunit N-terminal of Hb, again through NaBH
4Reduction generates the amine key.Reaction is as Fig. 1.
It is reported, after PLP modifies no substrate purified hemoglobin, can be with its P
50Bring up to 30-32mmHg from 10-12mmHg, can reduce the oxygen combining power of hemoglobin after this explanation is modified with PLP, improve its P
50, recover it and give the ability of organizing oxygen supply.But consult in the document modified the PLP of hemoglobin and all is placed on (preceding modification) before the polymerization procedure, and unmanned the trial is placed on (modification afterwards) after the polymerization procedure with modification step.Because PLP brings a high price, preceding modification is modified about the big 5-10 of consumption times than the back, therefore guaranteeing to improve technology under the condition of hemoglobin to the adhesion of oxygen, will save cost, has very big economic benefit.
(3) summary of the invention:
The object of the present invention is to provide a kind of modification method of polymeric hemoglobin, it is at above-mentioned situation, invents a kind of easy and simple to handle, with low cost, modification process efficiently.
Technical scheme of the present invention: a kind of modification method of polymeric hemoglobin, it is characterized in that it may further comprise the steps: (1) is under 0～4 ℃ of condition, with the neutral red cell is that raw material obtains purified hemoglobin, at logical nitrogen and add under the Reducing agent protection, earlier carry out polymerization through the homotype bifunctional reagent, the reuse dressing agent is modified; (2) add Reducing agents such as adding glutathion behind the dressing agent and prevent that metahemoglobin from generating.
The used raw material neutral red cell source of preparation is people's placental blood or out of date stock's human blood in the above-mentioned steps (1).
Earlier purified hemoglobin is carried out polymerization in the above-mentioned steps (1), used bifunctional reagent is a glutaraldehyde.
Above-mentioned steps is modified hemoglobin after the polymerization in (1), and used dressing agent is 5 '-pyridoxal 5-phosphate (PLP).
The mol ratio of 5 '-pyridoxal 5-phosphate and hemoglobin is 4-8 in the above-mentioned steps (1): 1.
In the above-mentioned steps (2), after adding 5 '-pyridoxal 5-phosphate reacted 0.5-1.5 hour, add glutathion again.
In the above-mentioned steps (2), adding glutathione concentrations is 2%.
In the above-mentioned steps (2), add the glutathion final concentration by weight/volume ratio is 0.1～0.5%.
Operation principle of the present invention is: earlier modify post polymerization with PLP by the comparative study hemoglobin, and PLP modifies the P of two kinds of technology products obtained therefroms after the first polymerization
50Thereby, find out a kind of suitable modification process.And, in this process, add Reducing agent suppressing the generation of metahemoglobin, but the modification effect that the addition sequence of Reducing agent also can appreciable impact PLP.
Technique effect of the present invention is: done big quantity research on preferred suitable modification technology, the result shows that preceding modification is modified P with the back
50Be respectively 13.80mmHg and 15.07mmHg, no marked difference, but the PLP consumption latter lacks 5-10 doubly than the former.Reducing substances such as glutathion before PLP, add and after add P
50Be respectively 14.50mmHg and 22.11mmHg, two kinds add the mode ferrihemoglobin content and are respectively 6.8% and 6.6%, and ferrihemoglobin content is respectively 7.8% and 8.0% before modification, as can be seen, before adding PLP and add reducing substances afterwards high ferro is not made significant difference, but back adding group P
50Significantly improve.Therefore, summing up on the basis of a large amount of experimental results, we propose PLP modified and are put in after the polymerization, and will prevent the reducing substances adding that high ferro generates after adding PLP, can control the generation of metahemoglobin like this, can guarantee P again
50Value is in OK range, and can reduce production costs greatly.
(4) description of drawings:
Fig. 1 is 5, and ' pyridoxal 5-phosphate (PLP) can generate imines with the valine generation aldol condensation of the subunit N-terminal of Hb, again through NaBH
4Reduction generates the diagram of amine key.
(5) specific embodiment:
Below will again foregoing of the present invention be described in further detail by the form of the specific embodiment, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only is confined to the following specific embodiment, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Modify before the embodiment 1:PLP and the back modification
The hemoglobin that I will be purified into from placental blood (or becoming human peripheral), under 0～4 ℃ cryogenic conditions, feed noble gases such as oxygen-free high purity nitrogen, flow is per minute 1～5L, continue 15～30 minutes, and add conventional antibacterial medicines and abundant mix homogeneously such as penicillin and/or streptomycin by 100,000～1,000,000 units/1000 ml vols; With the Tris-Hcl dissolving of PLP with 1M, concentration is 2%, PH=7.3-7.4, press and hemoglobin mol ratio 4: 1-8: 1 adds, noble gas reactions such as the logical high purity nitrogen of continuation are after 0.5-1.5 hour, by weight/volume ratio be 0.1～0.5% glutathion that adds 2% concentration as antioxidant, noble gas reaction such as logical high purity nitrogen after 1-2 hour again, by and hemoglobin mol ratio 20: 1-80: 1 adds 1.56%NaBH
4(be dissolved in 10
-3The M sodium hydroxide) stops reaction 3-6 hour.The ultrafilter membrane that with hemoglobin solutions with the molecular cut off is 10KD again carries out hyperfiltration treatment in the usual way, makes concentration to 4-6g/dl, the 0.22um aseptic filtration.Press 5-9: 1 adds 1% glutaraldehyde, logical nitrogen reaction 0.5 hour, oozing with 10-15 times of volume etc. that PBS (PH=7.3-7.4) carries out with the molecular cut off is that the ultrafilter membrane of 100KD carries out hyperfiltration treatment in the usual way, be concentrated into 10-12g/dl, press and hemoglobin mol ratio 20: 1-80: 1 adds 1.56%NaBH4 (is dissolved in 10
-3The M sodium hydroxide) stops reaction 3-6 hour.The ultrafilter membrane that with hemoglobin solutions with the molecular cut off is 100KD again carries out hyperfiltration treatment in the usual way, makes concentration to 4-6g/dl, the 0.22um aseptic filtration, and P is carried out in sampling
50Detect.
The hemoglobin that II will be purified into from placental blood (or becoming human peripheral), under 0～4 ℃ cryogenic conditions, feed noble gases such as oxygen-free high purity nitrogen, flow is per minute 1～5L, continue 15～30 minutes, and adding conventional antibacterial medicines such as penicillin and/or streptomycin and abundant mix homogeneously by the amount of 100,000～1,000,000 unit/1000 milliliter, logical high purity nitrogen etc. is noble gas 0.5-1 hour again.Press 5-9: 1 adds 1% glutaraldehyde, logical nitrogen reaction 0.5 hour, oozing with 10-15 times of volume etc. that PBS (PH=7.3-7.4) carries out with the molecular cut off is that the ultrafilter membrane of 100KD carries out hyperfiltration treatment in the usual way, be concentrated into 10-12g/dl, press and hemoglobin mol ratio 20: 1-80: 1 adds 1.56%NaBH
4(be dissolved in 10
-3The M sodium hydroxide) stops reaction 3-6 hour.The ultrafilter membrane that with hemoglobin solutions with the molecular cut off is 100KD again carries out hyperfiltration treatment in the usual way, is concentrated into 8-10g/dl, the 0.22um aseptic filtration.With the Tris-Hcl dissolving of PLP with 1M, concentration is 2%, PH=7.3-7.4, press and hemoglobin mol ratio 4: 1-8: 1 adds, noble gas reactions such as the logical high purity nitrogen of continuation are after 1-2 hour, by weight/volume ratio be 0.1～0.5% glutathion that adds 2% concentration as antioxidant, noble gas reaction such as logical high purity nitrogen after 1-2 hour again, by and hemoglobin mol ratio 20: 1-80: 1 adds 1.56%NaBH
4(be dissolved in 10
-3The M sodium hydroxide) stops reaction 3-6 hour.The ultrafilter membrane that with hemoglobin solutions with the molecular cut off is 100KD again carries out hyperfiltration treatment in the usual way, makes concentration to 4-6g/dl, the 0.22um aseptic filtration.P is carried out in sampling
50Detect.
Embodiment 2: the addition sequence of glutathion:
The hemoglobin that I will be purified into from placental blood (or becoming human peripheral), under 0～4 ℃ cryogenic conditions, feed noble gases such as oxygen-free high purity nitrogen, flow is per minute 1～5L, continue 15～30 minutes, add conventional antibacterial medicines such as penicillin and/or streptomycin and abundant mix homogeneously by the amount of 100,000～1,000,000 unit/1000 milliliter, more logical high purity nitrogen etc. noble gas 0.5-1 hour by weight/volume ratio is 0.1～0.5% to add the glutathion of 2% concentration as antioxidant.With the Tris-HCl dissolving of PLP with 1M, concentration is 2%, and PH=7.3-7.4 presses and hemoglobin mol ratio 4: 1-8: 1 adds, noble gas reactions such as the logical high purity nitrogen of continuation 2-3 hour.Press and hemoglobin mol ratio 20: 1-80: 1 adds 1.56%NaBH4 (is dissolved in 10
-3The M sodium hydroxide) cessation reaction 3-6 hour.The ultrafilter membrane that with hemoglobin solutions with the molecular cut off is 10KD again carries out hyperfiltration treatment in the usual way, is concentrated into 8-10g/dl, the 0.22um aseptic filtration, and P is carried out in sampling
50Detect.
The hemoglobin that II will be purified into from placental blood (or becoming human peripheral), under 0～4 ℃ cryogenic conditions, feed noble gases such as oxygen-free high purity nitrogen, flow is per minute 1～5L, continue 15～30 minutes, and adding conventional antibacterial medicines such as penicillin and/or streptomycin and abundant mix homogeneously by the amount of 100,000～1,000,000 unit/1000 milliliter, logical high purity nitrogen etc. is noble gas 0.5-1 hour again.With the Tris-Hcl dissolving of PLP with 1M, concentration is 2%, PH=7.3-7.4, press and hemoglobin mol ratio 4: 1-8: 1 adds, noble gas reactions such as the logical high purity nitrogen of continuation are after 1-2 hour, by weight/volume ratio is 0.1～0.5% to add 2% glutathion as antioxidant, noble gas reaction such as logical high purity nitrogen after 1-2 hour again, by and hemoglobin mol ratio 20: 1-80: 1 adds 1.56%NaBH4 (is dissolved in 10
-3The M sodium hydroxide) stops reaction 3-6 hour.The ultrafilter membrane that with hemoglobin solutions with the molecular cut off is 10KD again carries out hyperfiltration treatment in the usual way, is concentrated into 8-10g/dl, the 0.22um aseptic filtration, and P is carried out in sampling
50Detect.
Hemoglobin P by method for preparing
50Testing result is as shown in table 1.
Table 1 hemoglobin P
50Testing result
| P 50 | Preceding (mmHg) | Back (mmHg) |
| The preceding modification with the back modified | 13.80 | 15.07 |
| GSH before PLP and afterwards | 14.50 | 22.11 |
Claims (8)
1, a kind of method that is used for the polymeric hemoglobin modification, it is characterized in that it may further comprise the steps: (1) is under 0～4 ℃ of condition, with the neutral red cell is that raw material obtains purified hemoglobin, at logical nitrogen and add under the Reducing agent protection, earlier carry out polymerization through the homotype bifunctional reagent, the reuse dressing agent is modified; (2) behind the adding dressing agent, Reducing agents such as adding glutathion prevent that metahemoglobin from generating.
2, a kind of modification method of polymeric hemoglobin according to claim 1 is characterized in that preparing used raw material neutral red cell source and is people's placental blood or out of date stock's human blood.
3, a kind of modification method of polymeric hemoglobin according to claim 1 is characterized in that earlier purified hemoglobin being carried out polymerization, and used bifunctional reagent is a glutaraldehyde.
4, a kind of modification method of polymeric hemoglobin according to claim 1 is characterized in that hemoglobin after the polymerization is modified, and used dressing agent is 5 '-pyridoxal 5-phosphate.
5, a kind of modification method of polymeric hemoglobin according to claim 1 is characterized in that adding Reducing agents such as glutathion again after adding 5 '-pyridoxal 5-phosphate.
6, a kind of modification method of polymeric hemoglobin according to claim 4, the mol ratio that it is characterized in that PLP and hemoglobin is 4-8: 1.
7, a kind of modification method of polymeric hemoglobin according to claim 5 is characterized in that adding glutathion again after adding 5 '-pyridoxal 5-phosphate reacts 0.5-1.5 hour.
8, a kind of modification method of polymeric hemoglobin according to claim 5, it is characterized in that adding the glutathion final concentration by weight/volume ratio is 0.1～0.5%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107137699A (en) * | 2017-06-15 | 2017-09-08 | 中国医学科学院输血研究所 | The method of deoxidation and preparation technology of a kind of natural hemoglobin class blood substitute |
| CN110522904A (en) * | 2019-09-09 | 2019-12-03 | 润方（北京）生物医药研究院有限公司 | A kind of raised polymeric hemoglobin of inhibition blood pressure |
| CN114099648A (en) * | 2021-12-27 | 2022-03-01 | 西安血氧生物技术有限公司 | Cosolvent, hemoglobin oxygen carrier freeze-dried powder containing cosolvent, injection and preparation method |
-
2005
- 2005-12-28 CN CN 200510136023 patent/CN1990039A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107137699A (en) * | 2017-06-15 | 2017-09-08 | 中国医学科学院输血研究所 | The method of deoxidation and preparation technology of a kind of natural hemoglobin class blood substitute |
| CN107137699B (en) * | 2017-06-15 | 2021-02-09 | 中国医学科学院输血研究所 | Deoxygenation method and preparation process of natural hemoglobin blood substitute |
| CN110522904A (en) * | 2019-09-09 | 2019-12-03 | 润方（北京）生物医药研究院有限公司 | A kind of raised polymeric hemoglobin of inhibition blood pressure |
| CN114099648A (en) * | 2021-12-27 | 2022-03-01 | 西安血氧生物技术有限公司 | Cosolvent, hemoglobin oxygen carrier freeze-dried powder containing cosolvent, injection and preparation method |
| CN114099648B (en) * | 2021-12-27 | 2023-08-08 | 西安血氧生物技术有限公司 | Cosolvent, hemoglobin-oxygen carrier freeze-dried powder and injection containing cosolvent and preparation method of hemoglobin-oxygen carrier freeze-dried powder and injection |
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