WO2011122647A1 - Hemoglobin material intermediate product used in artificial oxygen carrier manufacturing - Google Patents
Hemoglobin material intermediate product used in artificial oxygen carrier manufacturing Download PDFInfo
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- WO2011122647A1 WO2011122647A1 PCT/JP2011/057906 JP2011057906W WO2011122647A1 WO 2011122647 A1 WO2011122647 A1 WO 2011122647A1 JP 2011057906 W JP2011057906 W JP 2011057906W WO 2011122647 A1 WO2011122647 A1 WO 2011122647A1
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
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- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
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- the present invention relates to a hemoglobin raw material intermediate product used in the production of a hemoglobin-based artificial oxygen carrier.
- hemoglobin-based one there are a non-capsule type (chemically modified hemoglobin and the like) and a capsule type (hemoglobin-containing liposome and the like).
- a method is known in which blood cells are separated by red blood cells by a process such as centrifugation, and then the red blood cell membrane is removed and hemoglobin is extracted and purified (see, for example, Patent Document 1 and Patent Document 2).
- those containing a reducing agent in order to suppress the methemoglobin formation of hemoglobin are known. (For example, see Patent Document 3)
- hemoglobin raw materials used in hemoglobin-based artificial oxygen carriers such as hemoglobin-containing liposome-type artificial oxygen carriers have been studied by extracting and purifying from erythrocyte concentrates, which are blood products for blood transfusions derived from humans. .
- erythrocyte concentrates which are blood products for blood transfusions derived from humans.
- human blood as a hemoglobin raw material
- using an expired erythrocyte concentrate that has passed the expiration date of blood collected from a human (21 days after blood collection in Japan) Be acceptable. It is a reasonable method to clean and remove plasma, white blood cells, and platelets from the concentrated red blood cell as much as possible, and then cryopreserve and prepare for the next red blood cell membrane removal and purification step.
- the concentrated erythrocyte solution that has been washed and cryopreserved is thawed, passed through a erythrocyte membrane removal and purification step to become erythrocyte membrane-removed hemoglobin, and further encapsulated with liposomes by a known method, such as a hemoglobin-containing liposome suspension. It becomes a hemoglobin-based artificial oxygen carrier.
- the conventional method using physiological saline as a washing solution is: After thawing the above-contained concentrated erythrocyte liquid, there was a problem that the hemoglobin metholysis rate in the liquid increased.
- the method of using a reducing agent for the purpose of preventing this has a problem that it becomes difficult to remove the reducing agent or a decomposition product of the reducing agent, a metabolite, etc. in the subsequent manufacturing process, and is adopted. Was difficult.
- the present invention provides an intermediate product of hemoglobin raw material stored for use in the production of a hemoglobin-based artificial oxygen carrier, and does not use a reducing agent as an additive. It is an object of the present invention to provide a method for preventing an increase in the intermediate product hemoglobin metration rate.
- the present invention provides a hemoglobin raw material for a hemoglobin-based artificial oxygen carrier, in a series of production steps for extracting and purifying the red blood cell concentrate represented by blood products for blood transfusion, from the red blood cell concentrate, After washing and removing plasma, leukocytes, and platelets, it is a hemoglobin raw material intermediate used in the method for preparing for the subsequent red blood cell membrane removal and purification step, and is the hemoglobin raw material at the time of thawing after the above cryopreservation This prevents an increase in the rate of hemoglobin methalation in the production intermediate product.
- the removal of plasma, leukocytes, and platelets from the erythrocyte concentrate means that the sample does not substantially contain plasma, leukocytes, and platelets. Specifically, the amount of erythrocyte concentrate is doubled. After adding the washing solution, the mixture is stirred, centrifuged at 2630 G for 12 minutes, and the supernatant is discarded. Preferably, 4 times the amount of washing solution is added to the erythrocyte concentrate, and after stirring, centrifuged at 2630 G for 12 minutes, and the supernatant is discarded. Say.
- An intermediate product of a hemoglobin raw material used in the production of a hemoglobin-based artificial oxygen carrier, washing and removing plasma, platelets and leukocytes from an erythrocyte concentrate using an isotonic washing solution containing a pH adjusting agent is obtained.
- the intermediate product of the hemoglobin raw material used for the production of a hemoglobin-based artificial oxygen carrier, wherein the pH of the solution obtained by freezing and thawing the erythrocyte solution obtained in the above is 7.1 to 7.5.
- Hemoglobin-containing characterized in that hemoglobin is extracted and purified from the hemoglobin raw material intermediate product used for the production of the hemoglobin-based artificial oxygen carrier according to (1) to (5), and the hemoglobin is produced into liposomes Liposome suspension.
- the erythrocyte concentrate used in the present invention is typified by erythrocyte concentrate, which is a blood product for blood transfusion.
- erythrocyte storage is carried out in an erythrocyte layer from which most of leukocytes, platelets and plasma have been removed from human blood mixed with blood storage solution.
- the additive solution is mixed. From the viewpoint of effective use of blood products, those that have passed the expiration date of the erythrocyte concentrate (21 days or more after blood collection) are used.
- the hemoglobin raw material of the hemoglobin-based artificial oxygen carrier is extracted and purified from the erythrocyte concentrate
- Physiological saline solution that has been used in the past has no pH buffering ability, has an unstable pH, and the pH varies between 4.5 and 8.0 depending on the external environment.
- the pH varies between 4.5 and 8.0 depending on the external environment.
- the pH is low, the erythrocyte concentrate is washed and then frozen and stored for a predetermined period of time, and then the pH of the thawed erythrocyte solution is also lowered.
- a washing solution having a higher pH such as an aqueous sodium hydrogen carbonate solution is used so that the pH of the production intermediate of the hemoglobin raw material after the melting operation does not become too low. That is, an aqueous solution of sodium bicarbonate was added to the erythrocyte concentrate after the expiration date and centrifuged to remove leukocytes, platelets and plasma as much as possible, and the erythrocyte layer was collected with the pH kept weakly alkaline. Store frozen. Centrifugal washing is performed using centrifugation.
- cryopreservation of erythrocyte layer after centrifugal washing The erythrocyte liquid which is the precipitate layer after the centrifugal washing is stored frozen and prepared for the next erythrocyte membrane removal purification step.
- the temperature for cryopreservation is ⁇ 60 to ⁇ 85 ° C., preferably ⁇ 70 to ⁇ 85 ° C. It is not necessary to add a protective solution for preventing hemolysis, such as glycerin.
- Example 1 Red blood cell concentrate-LR “Nichika” (derived from 200 mL of human blood, expiration date of use: 40 to 70 days) in 4 units, washing solution A (0.67 w / w% sodium bicarbonate, 0.43 w / w%) Add sodium chloride) to make 1kg, and mix.
- washing solution A (0.67 w / w% sodium bicarbonate, 0.43 w / w%) Add sodium chloride
- Using a centrifuge manufactured by BECKMAN COULTER, model: Avanti J-HC, rotor model: JS-3.4A), centrifuge at 3000 rpm for 12 minutes at a temperature of 4 ° C.
- the washing solution A is added to the erythrocyte layer obtained by removing the supernatant and mixed to make a total volume of 1 liter. Thereafter, the same centrifugal supernatant removal operation as described above is repeated twice. After the final centrifugation supernatant removal operation, the obtained red blood cell layer is directly put into a 1-liter separation bag and stored frozen at ⁇ 80 ° C.
- -Potassium hexacyanoferrate (III) solution Dissolve 5 g of potassium hexacyanoferrate (III) in water to make 100 mL. Sample solution preparation Weigh 0.2 mL of the sample to be measured, add 0.4 mL of water, and stir. Weigh 0.2 mL of this solution, add 10 mL of phosphate buffer and stir. Using a centrifuge (manufactured by Hitachi Koki Co., Ltd., model: Hitachi Multipurpose Centrifuge CF16RX, rotor model number: T5SS), centrifuge at 3000 rpm for 30 minutes at a temperature of 4 ° C. The supernatant after centrifugation is used as a sample solution.
- a centrifuge manufactured by Hitachi Koki Co., Ltd., model: Hitachi Multipurpose Centrifuge CF16RX, rotor model number: T5SS
- sample solution A 3 mL of sample solution is put into two cells, and it is set as “sample solution A” and “sample solution B”.
- 30 microliters of water is added to sample solution A and stirred, and the absorbance A1 at a wavelength of 630 nm is measured using water as a control.
- 30 microliters of potassium cyanide solution is added and stirred, and after standing for 2 minutes, the absorbance A2 is measured.
- 30 microliters of potassium hexacyanoferrate (III) solution was added to sample solution B and stirred, and after standing for 3 minutes, the absorbance B1 was measured, and immediately after measurement, 30 microliters of potassium cyanide solution was added and stirred.
- hemoglobin metation rate (%) [(A1-A2) / (B1-B2)] x 100
- the hemoglobin methalation rate measured as described above was 1.3%.
- Example 2 A production intermediate product was prepared in the same manner as in Example 1 except that Washing Solution B (1.29 w / w% sodium bicarbonate, final concentration of sodium bicarbonate when mixed with erythrocyte solution was about 154 mM) was used. did. The pH of the mixture after thawing was 7.6. Methelation rate was 1% or less. (Comparative example)
- the expired erythrocyte concentrate is washed and cryopreserved in the same manner as in Example, except that physiological saline is used in place of the sodium bicarbonate aqueous solution as the centrifugal washing solution. Further, the cryopreserved washed erythrocytes are thawed in the same manner as in the examples, and the pH of the mixture after thawing and the hemoglobin metrification rate are measured in the same manner as in the examples. The pH was 6.3, and the hemoglobin metation rate was 9.3%. Compared with the Examples, the hemoglobin quantification clearly progressed.
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Abstract
An issue is to prevent a rise in a methemoglobinizing rate of a blend after thawing, in a freeze-preservation method after removing plasma, leucocytes, and blood platelets from red-blood concentrate when extracting to purify hemoglobin material of an artificial oxygen carrier of a hemoglobin base from red-blood concentrate. Disclosed is a hemoglobin material intermediate product used in artificial oxygen carrier manufacturing of a hemoglobin base, characterized in that pH is 7.1 - 7.5 in a thawed solution after freeze-preservation of red blood cells attained by cleaning to remove plasma, leucocytes and blood platelets using an isotonic cleaning solution that contains a pH adjuster, from red-blood concentrate.
Description
本発明はヘモグロビンベースの人工酸素運搬体の製造に用いるヘモグロビン原料中間生成物に関する。
The present invention relates to a hemoglobin raw material intermediate product used in the production of a hemoglobin-based artificial oxygen carrier.
出血治療又は梗塞治療等に用いられる人工酸素運搬体として、ヘモグロビンベースのものが、様々に検討されて来た。ヘモグロビンベースのものとしては、非カプセル型（化学修飾ヘモグロビンなど）とカプセル型（ヘモグロビン含有リポソームなど）のものがある。ヘモグロビン原料は、血液を遠心分離等の処理によって、赤血球を分離した後、赤血球膜を除去し、ヘモグロビンを抽出精製する方法が知られている（例えば特許文献１、特許文献２参照。）
また、ヘモグロビンのメト化を抑制するために還元剤を含有するものが知られている。（例えば、特許文献３参照） Various artificial oxygen carriers used for bleeding treatment or infarction treatment have been studied. As the hemoglobin-based one, there are a non-capsule type (chemically modified hemoglobin and the like) and a capsule type (hemoglobin-containing liposome and the like). As the hemoglobin raw material, a method is known in which blood cells are separated by red blood cells by a process such as centrifugation, and then the red blood cell membrane is removed and hemoglobin is extracted and purified (see, for example, Patent Document 1 and Patent Document 2).
In addition, those containing a reducing agent in order to suppress the methemoglobin formation of hemoglobin are known. (For example, see Patent Document 3)
また、ヘモグロビンのメト化を抑制するために還元剤を含有するものが知られている。（例えば、特許文献３参照） Various artificial oxygen carriers used for bleeding treatment or infarction treatment have been studied. As the hemoglobin-based one, there are a non-capsule type (chemically modified hemoglobin and the like) and a capsule type (hemoglobin-containing liposome and the like). As the hemoglobin raw material, a method is known in which blood cells are separated by red blood cells by a process such as centrifugation, and then the red blood cell membrane is removed and hemoglobin is extracted and purified (see, for example, Patent Document 1 and Patent Document 2).
In addition, those containing a reducing agent in order to suppress the methemoglobin formation of hemoglobin are known. (For example, see Patent Document 3)
ヘモグロビン含有リポソーム型人工酸素運搬体の様なヘモグロビンベースの人工酸素運搬体に使用するヘモグロビン原料は、従来、ヒト由来の輸血用血液製剤である赤血球濃厚液から抽出精製する方法が多く検討されている。ヒト血液をヘモグロビン原料として用いる場合、血液製剤の有効利用の観点から、ヒトから採血された血液の使用期限（日本においては、採血後２１日）を過ぎた期限切れの赤血球濃厚液を用いることは、許容されうる。赤血球濃厚液から血漿、白血球、血小板を出来る限り、洗浄除去した後、一旦、凍結保存し、次の赤血球膜除去精製工程に備えるのが合理的な方法である。
Many hemoglobin raw materials used in hemoglobin-based artificial oxygen carriers such as hemoglobin-containing liposome-type artificial oxygen carriers have been studied by extracting and purifying from erythrocyte concentrates, which are blood products for blood transfusions derived from humans. . When using human blood as a hemoglobin raw material, from the viewpoint of effective use of blood products, using an expired erythrocyte concentrate that has passed the expiration date of blood collected from a human (21 days after blood collection in Japan) Be acceptable. It is a reasonable method to clean and remove plasma, white blood cells, and platelets from the concentrated red blood cell as much as possible, and then cryopreserve and prepare for the next red blood cell membrane removal and purification step.
洗浄され、凍結保存された濃縮赤血球液は解凍され、赤血球膜除去精製工程を経て、赤血球膜除去ヘモグロビンとなり、さらに、公知の方法により、リポソームでカプセル化されてヘモグロビン含有リポソーム懸濁液の様なヘモグロビンベース人工酸素運搬体となる。
The concentrated erythrocyte solution that has been washed and cryopreserved is thawed, passed through a erythrocyte membrane removal and purification step to become erythrocyte membrane-removed hemoglobin, and further encapsulated with liposomes by a known method, such as a hemoglobin-containing liposome suspension. It becomes a hemoglobin-based artificial oxygen carrier.
しかしながら、ヘモグロビンは酸化されてメトヘモグロビンとなると酸素運搬能を失うので、ヘモグロビンの抽出精製工程におけるヘモグロビンのメト化防止は非常に重要である。洗浄液により、赤血球濃厚液から、血漿、白血球、血小板を出来る限り洗浄除去し、濃縮した濃縮赤血球液を、一旦、凍結保存し次の工程に供するために解凍して使用する方法において、濃縮赤血球液中の赤血球は、工程における凍結及び解凍の操作の際に、大部分が溶血するので、溶血赤血球膜及び漏れ出したヘモグロビンの混合物となるが、洗浄液として生理食塩水を使用する従来の方法は、上記の凍結保存した濃縮赤血球液を解凍後、液中のヘモグロビンメト化率が上昇してしまうという問題が有った。これを防止することを目的とした還元剤を使用する方法は、その後の製造工程において、還元剤又は還元剤の分解物、代謝物等の除去が困難になるなどの問題があり、採用するのは難しかった。
However, when hemoglobin is oxidized to methemoglobin, the oxygen carrying ability is lost. Therefore, prevention of methemoglobin formation in the hemoglobin extraction and purification process is very important. In a method in which plasma, white blood cells, and platelets are washed and removed as much as possible from the concentrated red blood cell solution using a washing solution, and the concentrated concentrated red blood cell solution is frozen and stored and then thawed for use in the next step. Most of the erythrocytes in the process are hemolyzed during the freezing and thawing operations in the process, so it becomes a mixture of the hemolyzed erythrocyte membrane and the leaked hemoglobin, but the conventional method using physiological saline as a washing solution is: After thawing the above-contained concentrated erythrocyte liquid, there was a problem that the hemoglobin metholysis rate in the liquid increased. The method of using a reducing agent for the purpose of preventing this has a problem that it becomes difficult to remove the reducing agent or a decomposition product of the reducing agent, a metabolite, etc. in the subsequent manufacturing process, and is adopted. Was difficult.
そこで本発明は、ヘモグロビンベース人工酸素運搬体製造に供するために保存される、ヘモグロビン原料の製造中間生成物において、還元剤を添加物として使用せずに、凍結保存した原料の解凍時にヘモグロビン原料の中間生成物のヘモグロビンのメト化率上昇を防止する方法を提供することを目的とするものである。
Accordingly, the present invention provides an intermediate product of hemoglobin raw material stored for use in the production of a hemoglobin-based artificial oxygen carrier, and does not use a reducing agent as an additive. It is an object of the present invention to provide a method for preventing an increase in the intermediate product hemoglobin metration rate.
上記課題を解決するため、本発明は、ヘモグロビンベースの人工酸素運搬体のヘモグロビン原料を、輸血用血液製剤に代表される赤血球濃厚液から抽出精製する一連の製造工程において、前記赤血球濃厚液から、血漿、白血球、血小板を洗浄除去した後、一旦、凍結保存し、次の赤血球膜除去、精製工程に備える方法において使用するヘモグロビン原料中間体であって、上記の凍結保存後の解凍時のヘモグロビン原料の製造中間生成物におけるヘモグロビンのメト化率上昇を防止するものである。
尚、前記赤血球濃厚液から、血漿、白血球、血小板を洗浄除去するとは、試料中に実質的に血漿、白血球、血小板を含まないことを言い、具体的には、前記赤血球濃厚液に２倍量の洗浄液を添加し、撹拌後、２６３０Gで１２分間遠心し、上清を捨てる遠心洗浄操作を３回行って得た最終沈殿画分と同等程度の除去を言う。好ましくは、前記赤血球濃厚液に４倍量の洗浄液を添加し、撹拌後、２６３０Gで１２分間遠心し、上清を捨てる遠心洗浄操作を５回行って得た最終沈殿画分と同等程度の除去を言う。 In order to solve the above problems, the present invention provides a hemoglobin raw material for a hemoglobin-based artificial oxygen carrier, in a series of production steps for extracting and purifying the red blood cell concentrate represented by blood products for blood transfusion, from the red blood cell concentrate, After washing and removing plasma, leukocytes, and platelets, it is a hemoglobin raw material intermediate used in the method for preparing for the subsequent red blood cell membrane removal and purification step, and is the hemoglobin raw material at the time of thawing after the above cryopreservation This prevents an increase in the rate of hemoglobin methalation in the production intermediate product.
The removal of plasma, leukocytes, and platelets from the erythrocyte concentrate means that the sample does not substantially contain plasma, leukocytes, and platelets. Specifically, the amount of erythrocyte concentrate is doubled. After adding the washing solution, the mixture is stirred, centrifuged at 2630 G for 12 minutes, and the supernatant is discarded. Preferably, 4 times the amount of washing solution is added to the erythrocyte concentrate, and after stirring, centrifuged at 2630 G for 12 minutes, and the supernatant is discarded. Say.
尚、前記赤血球濃厚液から、血漿、白血球、血小板を洗浄除去するとは、試料中に実質的に血漿、白血球、血小板を含まないことを言い、具体的には、前記赤血球濃厚液に２倍量の洗浄液を添加し、撹拌後、２６３０Gで１２分間遠心し、上清を捨てる遠心洗浄操作を３回行って得た最終沈殿画分と同等程度の除去を言う。好ましくは、前記赤血球濃厚液に４倍量の洗浄液を添加し、撹拌後、２６３０Gで１２分間遠心し、上清を捨てる遠心洗浄操作を５回行って得た最終沈殿画分と同等程度の除去を言う。 In order to solve the above problems, the present invention provides a hemoglobin raw material for a hemoglobin-based artificial oxygen carrier, in a series of production steps for extracting and purifying the red blood cell concentrate represented by blood products for blood transfusion, from the red blood cell concentrate, After washing and removing plasma, leukocytes, and platelets, it is a hemoglobin raw material intermediate used in the method for preparing for the subsequent red blood cell membrane removal and purification step, and is the hemoglobin raw material at the time of thawing after the above cryopreservation This prevents an increase in the rate of hemoglobin methalation in the production intermediate product.
The removal of plasma, leukocytes, and platelets from the erythrocyte concentrate means that the sample does not substantially contain plasma, leukocytes, and platelets. Specifically, the amount of erythrocyte concentrate is doubled. After adding the washing solution, the mixture is stirred, centrifuged at 2630 G for 12 minutes, and the supernatant is discarded. Preferably, 4 times the amount of washing solution is added to the erythrocyte concentrate, and after stirring, centrifuged at 2630 G for 12 minutes, and the supernatant is discarded. Say.
（１）ヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料の中間生成物であって、赤血球濃厚液から、ｐH調整剤を含有する等張の洗浄液を用いて血漿、血小板、白血球を洗浄除去して得た赤血球液を、凍結保存した後解凍した液のpHが7.1～7.5である事を特徴とするヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料の製造中間生成物。
(1) An intermediate product of a hemoglobin raw material used in the production of a hemoglobin-based artificial oxygen carrier, washing and removing plasma, platelets and leukocytes from an erythrocyte concentrate using an isotonic washing solution containing a pH adjusting agent. The intermediate product of the hemoglobin raw material used for the production of a hemoglobin-based artificial oxygen carrier, wherein the pH of the solution obtained by freezing and thawing the erythrocyte solution obtained in the above is 7.1 to 7.5.
（２）前記洗浄液が８０ｍMから１５４ｍMの炭酸水素ナトリウムと前記洗浄液を等張とするために必要な濃度の他の塩を含むことを特徴とする上記（１）に記載のヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物。
(2) The hemoglobin-based artificial oxygen delivery according to (1) above, wherein the cleaning solution contains 80 mM to 154 mM sodium hydrogen carbonate and other salts required to make the cleaning solution isotonic. Hemoglobin raw material intermediate product used for body production.
（３）凍結保存前の前記赤血球液が、ヘマトクリット５５～６０％であることを特徴とする上記（１）または（２）に記載のヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物。
(3) The hemoglobin raw material intermediate product used for the production of a hemoglobin-based artificial oxygen carrier according to (1) or (2) above, wherein the red blood cell solution before cryopreservation is hematocrit 55-60% .
（４）前記解凍後の赤血球液中のヘモグロビンメト化率が3%以下である事を特徴とする（１）ないし（３）に記載のヘモグロビンベースの人工酸素運搬体製造用のヘモグロビン原料中間生成物。
(4) The hemoglobin raw material intermediate production for producing a hemoglobin-based artificial oxygen carrier according to any one of (1) to (3), wherein a hemoglobin methation rate in the erythrocyte fluid after thawing is 3% or less object.
（５）前記赤血球濃厚液が採血日より110日以内である事を特徴とする（１）ないし（４）に記載のヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物。
(5) The hemoglobin raw material intermediate product used for producing a hemoglobin-based artificial oxygen carrier according to (1) to (4), wherein the erythrocyte concentrate is within 110 days from the blood collection date.
（６）（１）ないし（５）に記載のヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物からヘモグロビンを抽出精製し、前記ヘモグロビンをリポソーム化して製造する事を特徴とするヘモグロビン含有リポソーム懸濁液。
(6) Hemoglobin-containing, characterized in that hemoglobin is extracted and purified from the hemoglobin raw material intermediate product used for the production of the hemoglobin-based artificial oxygen carrier according to (1) to (5), and the hemoglobin is produced into liposomes Liposome suspension.
本発明の、ヘモグロビンベースの人工酸素運搬体のヘモグロビン原料を赤血球濃厚液から抽出精製する時、赤血球濃厚液から血漿、白血球、血小板を洗浄除去した後、一旦、凍結保存し、次の赤血球膜除去、精製工程に備える方法において、解凍後の製造中間生成物のpHを7.1～7.７に設定する事により、解凍後の中間生成物のヘモグロビンのメト化率上昇を抑制することができる。
When the hemoglobin raw material of the hemoglobin-based artificial oxygen carrier of the present invention is extracted and purified from the erythrocyte concentrate, the plasma, leukocytes, and platelets are washed and removed from the erythrocyte concentrate, and then frozen and stored for the next erythrocyte membrane removal. In the method for preparing for the purification step, by setting the pH of the intermediate product after thawing to 7.1 to 7.7, it is possible to suppress an increase in the rate of hemoglobin in the intermediate product after thawing.
以下、本発明を具体的に説明する。
Hereinafter, the present invention will be specifically described.
＜赤血球濃厚液＞
本発明で用いる赤血球濃厚液は、輸血用血液製剤である赤血球濃厚液に代表されるが、血液保存液を混合したヒト血液から白血球、血小板及び血漿の大部分が除去された赤血球層に赤血球保存用添加液が混和されたものである。血液製剤の有効利用の観点より、前記赤血球濃厚液の使用期限を経過した(採血後21日間以上経過)ものを使用する。前記赤血球濃厚液から、ヘモグロビンベースの人工酸素運搬体のヘモグロビン原料を抽出精製する場合、採血日より110日以内、好ましくは90日、最適には70日以内のものを用いることが好ましい。これ以上の日数が、採血日より経過したものは、ヘモグロビン原料の抽出精製工程においてヘモグロビンの回収率が悪くなり、また、ヘモグロビンのメト化が進行してしまう傾向がある。 <Red blood cell concentrate>
The erythrocyte concentrate used in the present invention is typified by erythrocyte concentrate, which is a blood product for blood transfusion. However, erythrocyte storage is carried out in an erythrocyte layer from which most of leukocytes, platelets and plasma have been removed from human blood mixed with blood storage solution. The additive solution is mixed. From the viewpoint of effective use of blood products, those that have passed the expiration date of the erythrocyte concentrate (21 days or more after blood collection) are used. When the hemoglobin raw material of the hemoglobin-based artificial oxygen carrier is extracted and purified from the erythrocyte concentrate, it is preferable to use a hemoglobin source that is within 110 days, preferably 90 days, and optimally within 70 days from the blood collection date. If the number of days is longer than the blood collection date, the recovery rate of hemoglobin in the hemoglobin raw material extraction / purification process tends to be poor, and the hemoglobin tends to be methed.
本発明で用いる赤血球濃厚液は、輸血用血液製剤である赤血球濃厚液に代表されるが、血液保存液を混合したヒト血液から白血球、血小板及び血漿の大部分が除去された赤血球層に赤血球保存用添加液が混和されたものである。血液製剤の有効利用の観点より、前記赤血球濃厚液の使用期限を経過した(採血後21日間以上経過)ものを使用する。前記赤血球濃厚液から、ヘモグロビンベースの人工酸素運搬体のヘモグロビン原料を抽出精製する場合、採血日より110日以内、好ましくは90日、最適には70日以内のものを用いることが好ましい。これ以上の日数が、採血日より経過したものは、ヘモグロビン原料の抽出精製工程においてヘモグロビンの回収率が悪くなり、また、ヘモグロビンのメト化が進行してしまう傾向がある。 <Red blood cell concentrate>
The erythrocyte concentrate used in the present invention is typified by erythrocyte concentrate, which is a blood product for blood transfusion. However, erythrocyte storage is carried out in an erythrocyte layer from which most of leukocytes, platelets and plasma have been removed from human blood mixed with blood storage solution. The additive solution is mixed. From the viewpoint of effective use of blood products, those that have passed the expiration date of the erythrocyte concentrate (21 days or more after blood collection) are used. When the hemoglobin raw material of the hemoglobin-based artificial oxygen carrier is extracted and purified from the erythrocyte concentrate, it is preferable to use a hemoglobin source that is within 110 days, preferably 90 days, and optimally within 70 days from the blood collection date. If the number of days is longer than the blood collection date, the recovery rate of hemoglobin in the hemoglobin raw material extraction / purification process tends to be poor, and the hemoglobin tends to be methed.
＜赤血球濃厚液の洗浄＞
赤血球の洗浄液としては、従来、生理食塩水が使用されているが、本発明においては、赤血球濃厚液を本発明の洗浄液を用いて洗浄して得た赤血球液を凍結保存後に解凍した混合物のpHを7.1～7.７に、好ましくは7.3～7.5に調整される洗浄液である。具体的には、炭酸水素ナトリウムなどのｐH調整剤として含有するものであり、炭酸水素ナトリウムであれば８０ｍMから１５４ｍM含有し、洗浄液を等張とするために必要な濃度の塩化ナトリウムなどの他の塩を含むものである。 <Washing of erythrocyte concentrate>
Conventionally, physiological saline is used as the erythrocyte washing solution, but in the present invention, the pH of the mixture obtained by thawing the erythrocyte solution obtained by washing the erythrocyte concentrated solution with the washing solution of the present invention after cryopreservation. Is a cleaning solution adjusted to 7.1 to 7.7, preferably 7.3 to 7.5. Specifically, it is contained as a pH adjusting agent such as sodium hydrogen carbonate. In the case of sodium hydrogen carbonate, it contains 80 mM to 154 mM, and other concentrations such as sodium chloride required to make the washing solution isotonic. It contains salt.
赤血球の洗浄液としては、従来、生理食塩水が使用されているが、本発明においては、赤血球濃厚液を本発明の洗浄液を用いて洗浄して得た赤血球液を凍結保存後に解凍した混合物のpHを7.1～7.７に、好ましくは7.3～7.5に調整される洗浄液である。具体的には、炭酸水素ナトリウムなどのｐH調整剤として含有するものであり、炭酸水素ナトリウムであれば８０ｍMから１５４ｍM含有し、洗浄液を等張とするために必要な濃度の塩化ナトリウムなどの他の塩を含むものである。 <Washing of erythrocyte concentrate>
Conventionally, physiological saline is used as the erythrocyte washing solution, but in the present invention, the pH of the mixture obtained by thawing the erythrocyte solution obtained by washing the erythrocyte concentrated solution with the washing solution of the present invention after cryopreservation. Is a cleaning solution adjusted to 7.1 to 7.7, preferably 7.3 to 7.5. Specifically, it is contained as a pH adjusting agent such as sodium hydrogen carbonate. In the case of sodium hydrogen carbonate, it contains 80 mM to 154 mM, and other concentrations such as sodium chloride required to make the washing solution isotonic. It contains salt.
従来使用されて来た生理食塩水はpH緩衝能がなく、pHが不安定で、外的環境によりpHは4.5～8.0の範囲を変動してしまう。我々は、pHが低い場合は、赤血球濃厚液を洗浄後に、所定期間凍結保存した後、解凍した赤血球液のpHも低くなり、それと関連して、ヘモグロビンのメト化が進行することを見出した。この問題を解決する為に、凍結保存し解凍した赤血球のpHを調整する必要が有る事が鋭意検討の結果、判明した。前記pH範囲がこれ以下であると、解凍操作後のヘモグロビンのメト化率が上昇してしまい、また、pH範囲がこれ以上であるとヘモグロビンが変性してしまう可能性がある。解凍操作後の赤血球液（実際には、赤血球が溶血し、溶血赤血球膜と漏出ヘモグロビンの混合物となっている）のpHをこの範囲に収める為には、洗浄液のpHを調整する必要が有るのである。
Physiological saline solution that has been used in the past has no pH buffering ability, has an unstable pH, and the pH varies between 4.5 and 8.0 depending on the external environment. We have found that when the pH is low, the erythrocyte concentrate is washed and then frozen and stored for a predetermined period of time, and then the pH of the thawed erythrocyte solution is also lowered. In order to solve this problem, as a result of intensive studies, it was found that it was necessary to adjust the pH of erythrocytes that had been frozen and thawed. If the pH range is below this range, the hemoglobin metation rate after the thawing operation increases, and if the pH range is above this range, the hemoglobin may be denatured. In order to keep the pH of the erythrocyte fluid after thawing (actually, the erythrocyte is hemolyzed and becomes a mixture of hemolyzed erythrocyte membrane and leaked hemoglobin) within this range, it is necessary to adjust the pH of the washing solution. is there.
従来から使用して来た生理食塩水を使うと、既述の様に、解凍時の混合物のpHが低くなり過ぎ、解凍時の混合物のpHが低くなるのと関連して、ヘモグロビンのメト化率が上昇してしまう。この為、炭酸水素ナトリウム水溶液などのよりpHの高い洗浄液を使用して、融解操作後のヘモグロビン原料の製造中間物のpHが低くなり過ぎない様にする。即ち、使用期限の経過した赤血球濃厚液に炭酸水素ナトリウム水溶液を添加し、遠心分離して、白血球、血小板、血漿を出来る限り除去し、pHを弱アルカリ性に保った状態にて赤血球層を回収し、凍結保存する。遠心分離を利用して洗浄する遠心洗浄は、前記赤血球濃厚液に２倍から４倍量の洗浄液を添加し、撹拌後、2630Gの条件で遠心し、上清を捨てる遠心洗浄操作を３回から５回行って実施する。上清を捨てて得られ凍結保存に供される赤血球液のヘマトクリットは５５％から６０％であることが好ましい。
When using normal saline, the pH of the mixture at the time of thawing becomes too low and the pH of the mixture at the time of thawing becomes low, as described above. The rate will rise. For this reason, a washing solution having a higher pH such as an aqueous sodium hydrogen carbonate solution is used so that the pH of the production intermediate of the hemoglobin raw material after the melting operation does not become too low. That is, an aqueous solution of sodium bicarbonate was added to the erythrocyte concentrate after the expiration date and centrifuged to remove leukocytes, platelets and plasma as much as possible, and the erythrocyte layer was collected with the pH kept weakly alkaline. Store frozen. Centrifugal washing is performed using centrifugation. The washing solution is added to the erythrocyte concentrate 2 to 4 to 4 times in volume, stirred, centrifuged at 2630G, and the supernatant is discarded from 3 times. Perform 5 times. The hematocrit of the erythrocyte liquid obtained by discarding the supernatant and subjected to cryopreservation is preferably 55% to 60%.
重要なことは、洗浄された赤血球液を凍結保存しさらに解凍するという操作において、ｐH調整剤を含有する洗浄液を用いた場合であっても、ｐHの低下が発生するので、そのｐHの低下を考慮し、洗浄液のｐH調整剤の濃度を調整することである。ｐH調整剤として炭酸水素ナトリウムのほか、酢酸ナトリウム、水酸化ナトリウム、炭酸ナトリウム、リン酸三ナトリウム、リン酸水素二ナトリウム、リン酸水素二カリウムなどを用いることができる。これらのｐH調整剤を含有する生理的食塩水などの緩衝能を有する洗浄液に用いて、洗浄液のpHを弱アルカリ性に維持する事も可能であり、この場合も解凍後の混合物のpHを低くなり過ぎない様に出来るが、炭酸水素ナトリウム水溶液は洗浄液調整の簡便さ及び安全性の観点より好ましい。
What is important is that, in the operation of storing frozen erythrocytes in a frozen state and further thawing, even when a washing solution containing a pH adjusting agent is used, a drop in pH occurs. In consideration, the concentration of the pH adjusting agent in the cleaning liquid is adjusted. As a pH adjuster, sodium acetate, sodium acetate, sodium hydroxide, sodium carbonate, trisodium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and the like can be used. It is possible to maintain the pH of the washing solution to be weakly alkaline by using it in a washing solution having a buffering capacity such as physiological saline containing these pH adjusting agents. In this case, the pH of the mixture after thawing is lowered. However, an aqueous sodium hydrogen carbonate solution is preferable from the viewpoints of easy cleaning liquid adjustment and safety.
＜遠心洗浄後の赤血球層の凍結保存＞
前記の遠心洗浄後の沈殿層である赤血球液を凍結保存して、次の赤血球膜除去精製工程に備える。凍結保存の温度は－60～－85度Cであり、好ましくは－70～－85度Cである。グリセリン等の溶血防止の為の保護液は添加する必要はない。 <Cryopreservation of erythrocyte layer after centrifugal washing>
The erythrocyte liquid which is the precipitate layer after the centrifugal washing is stored frozen and prepared for the next erythrocyte membrane removal purification step. The temperature for cryopreservation is −60 to −85 ° C., preferably −70 to −85 ° C. It is not necessary to add a protective solution for preventing hemolysis, such as glycerin.
前記の遠心洗浄後の沈殿層である赤血球液を凍結保存して、次の赤血球膜除去精製工程に備える。凍結保存の温度は－60～－85度Cであり、好ましくは－70～－85度Cである。グリセリン等の溶血防止の為の保護液は添加する必要はない。 <Cryopreservation of erythrocyte layer after centrifugal washing>
The erythrocyte liquid which is the precipitate layer after the centrifugal washing is stored frozen and prepared for the next erythrocyte membrane removal purification step. The temperature for cryopreservation is −60 to −85 ° C., preferably −70 to −85 ° C. It is not necessary to add a protective solution for preventing hemolysis, such as glycerin.
＜凍結保存後の解凍＞
所定期間凍結保存された赤血球液は室温にて解凍され、解凍後の赤血球液は血液保冷庫にて所定期間保存し、赤血球膜除去精製工程に供える。遠心洗浄後の赤血球液の凍結保存期間はウィンドウピリオド（ウィルスに感染してから検査で検出出来る様になるまでの空白期間）対応のためのルックバック（遡及調査）の観点より6ケ月間以上が好ましい。解凍後の中間物は血液保冷庫にて2～8度Cで保存し、ヘモグロビンのメト化防止の観点より、3日以内、好ましくは1日以内に、次の赤血球膜除去精製工程に移る。 <Thaw after freezing storage>
The erythrocyte liquid frozen and stored for a predetermined period is thawed at room temperature, and the erythrocyte liquid after thawing is stored in a blood cool box for a predetermined period of time and used for the erythrocyte membrane removal purification step. The cryopreservation period of the erythrocyte fluid after centrifugal washing is more than 6 months from the viewpoint of a lookback (retrospective survey) for window period (blank period from virus infection until detection becomes possible). preferable. After thawing, the intermediate is stored at 2 to 8 ° C. in a blood cooler, and is transferred to the next erythrocyte membrane removal and purification step within 3 days, preferably within 1 day, from the viewpoint of preventing hemoglobin methation.
所定期間凍結保存された赤血球液は室温にて解凍され、解凍後の赤血球液は血液保冷庫にて所定期間保存し、赤血球膜除去精製工程に供える。遠心洗浄後の赤血球液の凍結保存期間はウィンドウピリオド（ウィルスに感染してから検査で検出出来る様になるまでの空白期間）対応のためのルックバック（遡及調査）の観点より6ケ月間以上が好ましい。解凍後の中間物は血液保冷庫にて2～8度Cで保存し、ヘモグロビンのメト化防止の観点より、3日以内、好ましくは1日以内に、次の赤血球膜除去精製工程に移る。 <Thaw after freezing storage>
The erythrocyte liquid frozen and stored for a predetermined period is thawed at room temperature, and the erythrocyte liquid after thawing is stored in a blood cool box for a predetermined period of time and used for the erythrocyte membrane removal purification step. The cryopreservation period of the erythrocyte fluid after centrifugal washing is more than 6 months from the viewpoint of a lookback (retrospective survey) for window period (blank period from virus infection until detection becomes possible). preferable. After thawing, the intermediate is stored at 2 to 8 ° C. in a blood cooler, and is transferred to the next erythrocyte membrane removal and purification step within 3 days, preferably within 1 day, from the viewpoint of preventing hemoglobin methation.
＜解凍後の混合液のpH測定＞
解凍後の液は、凍結及び解凍の操作の際に、赤血球の大部分が溶血するので、溶血赤血球膜及び漏れ出したヘモグロビン及び炭酸水素ナトリウム水溶液の混合物となる。この混合物検体3mLを取り、22～25度Cの範囲内で、電極を検体に浸漬させて、約4分間緩やかに攪拌し、1分間静置した後にpH測定を行う。 <Measurement of pH of the mixture after thawing>
Since most of the red blood cells are hemolyzed during the freezing and thawing operations, the thawed solution becomes a mixture of the hemolyzed red blood cell membrane and the leaked hemoglobin and aqueous sodium hydrogen carbonate solution. Take 3 mL of this mixed sample, immerse the electrode in the sample within the range of 22 to 25 ° C., gently stir for about 4 minutes, and let stand for 1 minute, and then measure the pH.
解凍後の液は、凍結及び解凍の操作の際に、赤血球の大部分が溶血するので、溶血赤血球膜及び漏れ出したヘモグロビン及び炭酸水素ナトリウム水溶液の混合物となる。この混合物検体3mLを取り、22～25度Cの範囲内で、電極を検体に浸漬させて、約4分間緩やかに攪拌し、1分間静置した後にpH測定を行う。 <Measurement of pH of the mixture after thawing>
Since most of the red blood cells are hemolyzed during the freezing and thawing operations, the thawed solution becomes a mixture of the hemolyzed red blood cell membrane and the leaked hemoglobin and aqueous sodium hydrogen carbonate solution. Take 3 mL of this mixed sample, immerse the electrode in the sample within the range of 22 to 25 ° C., gently stir for about 4 minutes, and let stand for 1 minute, and then measure the pH.
＜解凍後の混合物のヘモグロビンメト化率測定＞
解凍後の混合物を遠心分離操作により溶血赤血球膜を沈降させた上清を試料溶液として用い、ヘモグロビンメト化率を測定する。 <Measurement of hemoglobin metrification rate of the mixture after thawing>
Using the supernatant obtained by precipitating the hemolyzed erythrocyte membrane by centrifuging the thawed mixture as a sample solution, the hemoglobin methation rate is measured.
解凍後の混合物を遠心分離操作により溶血赤血球膜を沈降させた上清を試料溶液として用い、ヘモグロビンメト化率を測定する。 <Measurement of hemoglobin metrification rate of the mixture after thawing>
Using the supernatant obtained by precipitating the hemolyzed erythrocyte membrane by centrifuging the thawed mixture as a sample solution, the hemoglobin methation rate is measured.
本発明のヘモグロビン原料中間体を従来公知の人工酸素運搬体の製造方法（例えば特開２００９－２６３２６９など）により、ヘモグロビンベースの人工酸素運搬体を製造することができる。
A hemoglobin-based artificial oxygen carrier can be produced from the hemoglobin raw material intermediate of the present invention by a conventionally known method for producing an artificial oxygen carrier (for example, JP-A-2009-263269).
次に本発明の実施例により、具体的に説明するが、発明はこれらの実施例に限定されるものではない。
Next, examples of the present invention will be specifically described, but the present invention is not limited to these examples.
＜赤血球濃厚液の洗浄及び洗浄赤血球の凍結保存＞
＜実施例１＞赤血球濃厚液-LR「日赤」（ヒト血液200mL由来、使用期限経過４０～７０日）4単位に、洗浄液A（0.67w/w%濃度の炭酸水素ナトリウム、0.43w/w%塩化ナトリウム）を加えて、全量を1kgとして、混和する。遠心機（BECKMAN COULTER社製、型式：Avanti J-HC、ローター型番：JS-3.4A）を用いて、回転数3000rpm、12分間、温度4度Cで遠心処理を行なう。遠心処理後、上清を除去して得られた赤血球層に、洗浄液Aを加えて、全量1リットルとして、混和する。その後、前記と同様の遠心上清除去操作を2回繰り返す。最後の遠心上清除去操作後には、得られた赤血球層をそのまま1リットル容量の分離バッグに入れ、－80度Cで冷凍保存する。 <Washing of erythrocyte concentrate and cryopreservation of washed erythrocytes>
<Example 1> Red blood cell concentrate-LR “Nichika” (derived from 200 mL of human blood, expiration date of use: 40 to 70 days) in 4 units, washing solution A (0.67 w / w% sodium bicarbonate, 0.43 w / w%) Add sodium chloride) to make 1kg, and mix. Using a centrifuge (manufactured by BECKMAN COULTER, model: Avanti J-HC, rotor model: JS-3.4A), centrifuge at 3000 rpm for 12 minutes at a temperature of 4 ° C. After centrifugation, the washing solution A is added to the erythrocyte layer obtained by removing the supernatant and mixed to make a total volume of 1 liter. Thereafter, the same centrifugal supernatant removal operation as described above is repeated twice. After the final centrifugation supernatant removal operation, the obtained red blood cell layer is directly put into a 1-liter separation bag and stored frozen at −80 ° C.
＜実施例１＞赤血球濃厚液-LR「日赤」（ヒト血液200mL由来、使用期限経過４０～７０日）4単位に、洗浄液A（0.67w/w%濃度の炭酸水素ナトリウム、0.43w/w%塩化ナトリウム）を加えて、全量を1kgとして、混和する。遠心機（BECKMAN COULTER社製、型式：Avanti J-HC、ローター型番：JS-3.4A）を用いて、回転数3000rpm、12分間、温度4度Cで遠心処理を行なう。遠心処理後、上清を除去して得られた赤血球層に、洗浄液Aを加えて、全量1リットルとして、混和する。その後、前記と同様の遠心上清除去操作を2回繰り返す。最後の遠心上清除去操作後には、得られた赤血球層をそのまま1リットル容量の分離バッグに入れ、－80度Cで冷凍保存する。 <Washing of erythrocyte concentrate and cryopreservation of washed erythrocytes>
<Example 1> Red blood cell concentrate-LR “Nichika” (derived from 200 mL of human blood, expiration date of use: 40 to 70 days) in 4 units, washing solution A (0.67 w / w% sodium bicarbonate, 0.43 w / w%) Add sodium chloride) to make 1kg, and mix. Using a centrifuge (manufactured by BECKMAN COULTER, model: Avanti J-HC, rotor model: JS-3.4A), centrifuge at 3000 rpm for 12 minutes at a temperature of 4 ° C. After centrifugation, the washing solution A is added to the erythrocyte layer obtained by removing the supernatant and mixed to make a total volume of 1 liter. Thereafter, the same centrifugal supernatant removal operation as described above is repeated twice. After the final centrifugation supernatant removal operation, the obtained red blood cell layer is directly put into a 1-liter separation bag and stored frozen at −80 ° C.
＜凍結保存洗浄赤血球の解凍＞
遠心洗浄後の赤血球層の180日間の凍結保存後に、室温にて解凍し、解凍時の混合物のpH測定およびヘモグロビンメト化率の測定を行なう。 <Thawing cryopreserved washed erythrocytes>
After 180-day cryopreservation of the erythrocyte layer after centrifugal washing, it is thawed at room temperature, and the pH of the mixture at the time of thawing and the measurement of the hemoglobin methoconization rate are performed.
遠心洗浄後の赤血球層の180日間の凍結保存後に、室温にて解凍し、解凍時の混合物のpH測定およびヘモグロビンメト化率の測定を行なう。 <Thawing cryopreserved washed erythrocytes>
After 180-day cryopreservation of the erythrocyte layer after centrifugal washing, it is thawed at room temperature, and the pH of the mixture at the time of thawing and the measurement of the hemoglobin methoconization rate are performed.
＜解凍後の混合物のpH測定＞
解凍時の混合物を十分に室温に戻した後、pH測定器（ザルトリウス社製、型式PP-50）を用いて、22～25度Cの範囲内でpH測定を行う。ガラス管に検体を3mL取り、攪拌子を入れる。電極を検体に浸漬させて、約4分間、緩やかに攪拌し、1分間静置した後、pH値を読み取る。pHは7.3であった。 <Measurement of pH of the mixture after thawing>
The mixture at the time of thawing is fully returned to room temperature, and then the pH is measured within a range of 22 to 25 ° C. using a pH meter (manufactured by Sartorius, model PP-50). Take 3mL of the sample in a glass tube and put a stirring bar. Immerse the electrode in the sample, gently stir for about 4 minutes, let stand for 1 minute, and then read the pH value. The pH was 7.3.
解凍時の混合物を十分に室温に戻した後、pH測定器（ザルトリウス社製、型式PP-50）を用いて、22～25度Cの範囲内でpH測定を行う。ガラス管に検体を3mL取り、攪拌子を入れる。電極を検体に浸漬させて、約4分間、緩やかに攪拌し、1分間静置した後、pH値を読み取る。pHは7.3であった。 <Measurement of pH of the mixture after thawing>
The mixture at the time of thawing is fully returned to room temperature, and then the pH is measured within a range of 22 to 25 ° C. using a pH meter (manufactured by Sartorius, model PP-50). Take 3mL of the sample in a glass tube and put a stirring bar. Immerse the electrode in the sample, gently stir for about 4 minutes, let stand for 1 minute, and then read the pH value. The pH was 7.3.
＜解凍後の混合物のヘモグロビンメト化率測定＞
試液の調製
・リン酸緩衝液：リン酸二水素カリウム0.548gを水に溶かし200mLとする（A液）。また、リン酸水素二ナトリウム12水和物3.58gを水に溶かし500mLとする（B液）。A液：B液＝１：３（容量）の割合で混合し、pH 7.4に調整する。
・シアン化カリウム溶液：シアン化カリウム5gを水に溶かし100mLとする。
・ヘキサシアノ鉄（ＩＩＩ）酸カリウム溶液：ヘキサシアノ鉄（ＩＩＩ）酸カリウム5gを水に溶かして100mLとする。
試料溶液調製
測定検体0.2mLを量り、水0.4mLを加えて攪拌する。この液0.2mLを量り、リン酸緩衝液10mLを加えて攪拌する。遠心機（日立工機社製、型式：日立多用途小型遠心機CF16RX、ローター型番：T5SS）を用いて、回転数3000rpm、30分間、温度4度Cで遠心処理を行なう。遠心処理後の上清を試料溶液とする。
測定方法
試料溶液3mLを2本のセルに入れ、「試料溶液A」及び「試料溶液B」とする。試料溶液Aに水30マイクロリットルを加えて攪拌し、水を対照とし、波長630nmにおける吸光度A1を測定する。測定後、直ちにシアン化カリウム溶液30マイクロリットルを加えて攪拌し、2分間放置後吸光度A2を測定する。試料溶液Bにヘキサシアノ鉄（ＩＩＩ）酸カリウム溶液30マイクロリットルを加えて攪拌し、3分間放置後吸光度B1を測定し、測定後直ちにシアン化カリウム溶液30マイクロリットルを加えて攪拌し、2分間放置後、吸光度B2を測定する。測定により得られた測定値A1、A2、B1、B2を用い、次の計算式に従って検体のヘモグロビンメト化率を求める。
計算式：ヘモグロビンメト化率（%）＝〔(A1－A2) ／(B1－B2)〕ｘ100
以上により測定したヘモグロビンメト化率は1.3%であった。 <Measurement of hemoglobin metrification rate of the mixture after thawing>
Preparation of test solution ・ Phosphate buffer solution: Dissolve 0.548 g of potassium dihydrogen phosphate in water to make 200 mL (solution A). Dissolve 3.58 g of disodium hydrogen phosphate 12 hydrate in water to make 500 mL (Liquid B). Mix A liquid: B liquid = 1: 3 (volume) and adjust to pH 7.4.
-Potassium cyanide solution: Dissolve 5 g of potassium cyanide in water to make 100 mL.
-Potassium hexacyanoferrate (III) solution: Dissolve 5 g of potassium hexacyanoferrate (III) in water to make 100 mL.
Sample solution preparation Weigh 0.2 mL of the sample to be measured, add 0.4 mL of water, and stir. Weigh 0.2 mL of this solution, add 10 mL of phosphate buffer and stir. Using a centrifuge (manufactured by Hitachi Koki Co., Ltd., model: Hitachi Multipurpose Centrifuge CF16RX, rotor model number: T5SS), centrifuge at 3000 rpm for 30 minutes at a temperature of 4 ° C. The supernatant after centrifugation is used as a sample solution.
Measurement method 3 mL of sample solution is put into two cells, and it is set as “sample solution A” and “sample solution B”. 30 microliters of water is added to sample solution A and stirred, and the absorbance A1 at a wavelength of 630 nm is measured using water as a control. Immediately after the measurement, 30 microliters of potassium cyanide solution is added and stirred, and after standing for 2 minutes, the absorbance A2 is measured. 30 microliters of potassium hexacyanoferrate (III) solution was added to sample solution B and stirred, and after standing for 3 minutes, the absorbance B1 was measured, and immediately after measurement, 30 microliters of potassium cyanide solution was added and stirred. Measure absorbance B2. Using the measured values A1, A2, B1, and B2 obtained by the measurement, the hemoglobin metation rate of the specimen is obtained according to the following calculation formula.
Calculation formula: hemoglobin metrification rate (%) = [(A1-A2) / (B1-B2)] x 100
The hemoglobin methalation rate measured as described above was 1.3%.
試液の調製
・リン酸緩衝液：リン酸二水素カリウム0.548gを水に溶かし200mLとする（A液）。また、リン酸水素二ナトリウム12水和物3.58gを水に溶かし500mLとする（B液）。A液：B液＝１：３（容量）の割合で混合し、pH 7.4に調整する。
・シアン化カリウム溶液：シアン化カリウム5gを水に溶かし100mLとする。
・ヘキサシアノ鉄（ＩＩＩ）酸カリウム溶液：ヘキサシアノ鉄（ＩＩＩ）酸カリウム5gを水に溶かして100mLとする。
試料溶液調製
測定検体0.2mLを量り、水0.4mLを加えて攪拌する。この液0.2mLを量り、リン酸緩衝液10mLを加えて攪拌する。遠心機（日立工機社製、型式：日立多用途小型遠心機CF16RX、ローター型番：T5SS）を用いて、回転数3000rpm、30分間、温度4度Cで遠心処理を行なう。遠心処理後の上清を試料溶液とする。
測定方法
試料溶液3mLを2本のセルに入れ、「試料溶液A」及び「試料溶液B」とする。試料溶液Aに水30マイクロリットルを加えて攪拌し、水を対照とし、波長630nmにおける吸光度A1を測定する。測定後、直ちにシアン化カリウム溶液30マイクロリットルを加えて攪拌し、2分間放置後吸光度A2を測定する。試料溶液Bにヘキサシアノ鉄（ＩＩＩ）酸カリウム溶液30マイクロリットルを加えて攪拌し、3分間放置後吸光度B1を測定し、測定後直ちにシアン化カリウム溶液30マイクロリットルを加えて攪拌し、2分間放置後、吸光度B2を測定する。測定により得られた測定値A1、A2、B1、B2を用い、次の計算式に従って検体のヘモグロビンメト化率を求める。
計算式：ヘモグロビンメト化率（%）＝〔(A1－A2) ／(B1－B2)〕ｘ100
以上により測定したヘモグロビンメト化率は1.3%であった。 <Measurement of hemoglobin metrification rate of the mixture after thawing>
Preparation of test solution ・ Phosphate buffer solution: Dissolve 0.548 g of potassium dihydrogen phosphate in water to make 200 mL (solution A). Dissolve 3.58 g of disodium hydrogen phosphate 12 hydrate in water to make 500 mL (Liquid B). Mix A liquid: B liquid = 1: 3 (volume) and adjust to pH 7.4.
-Potassium cyanide solution: Dissolve 5 g of potassium cyanide in water to make 100 mL.
-Potassium hexacyanoferrate (III) solution: Dissolve 5 g of potassium hexacyanoferrate (III) in water to make 100 mL.
Sample solution preparation Weigh 0.2 mL of the sample to be measured, add 0.4 mL of water, and stir. Weigh 0.2 mL of this solution, add 10 mL of phosphate buffer and stir. Using a centrifuge (manufactured by Hitachi Koki Co., Ltd., model: Hitachi Multipurpose Centrifuge CF16RX, rotor model number: T5SS), centrifuge at 3000 rpm for 30 minutes at a temperature of 4 ° C. The supernatant after centrifugation is used as a sample solution.
Measurement method 3 mL of sample solution is put into two cells, and it is set as “sample solution A” and “sample solution B”. 30 microliters of water is added to sample solution A and stirred, and the absorbance A1 at a wavelength of 630 nm is measured using water as a control. Immediately after the measurement, 30 microliters of potassium cyanide solution is added and stirred, and after standing for 2 minutes, the absorbance A2 is measured. 30 microliters of potassium hexacyanoferrate (III) solution was added to sample solution B and stirred, and after standing for 3 minutes, the absorbance B1 was measured, and immediately after measurement, 30 microliters of potassium cyanide solution was added and stirred. Measure absorbance B2. Using the measured values A1, A2, B1, and B2 obtained by the measurement, the hemoglobin metation rate of the specimen is obtained according to the following calculation formula.
Calculation formula: hemoglobin metrification rate (%) = [(A1-A2) / (B1-B2)] x 100
The hemoglobin methalation rate measured as described above was 1.3%.
＜実施例２＞ 洗浄液B（1.29w/w%濃度の炭酸水素ナトリウム、赤血球液との混合時炭酸水素ナトリウム終濃度約154ｍM）を用いる以外は、実施例１と同様に製造中間生成物を作成した。解凍後の混合物のｐHは7.6であった。メト化率は1％以下であった。
（比較例） <Example 2> A production intermediate product was prepared in the same manner as in Example 1 except that Washing Solution B (1.29 w / w% sodium bicarbonate, final concentration of sodium bicarbonate when mixed with erythrocyte solution was about 154 mM) was used. did. The pH of the mixture after thawing was 7.6. Methelation rate was 1% or less.
(Comparative example)
（比較例） <Example 2> A production intermediate product was prepared in the same manner as in Example 1 except that Washing Solution B (1.29 w / w% sodium bicarbonate, final concentration of sodium bicarbonate when mixed with erythrocyte solution was about 154 mM) was used. did. The pH of the mixture after thawing was 7.6. Methelation rate was 1% or less.
(Comparative example)
遠心洗浄液として、炭酸水素ナトリウム水溶液の代わりに生理食塩水を使う以外は、実施例と同様の方法で、期限切れ赤血球濃厚液の洗浄及び凍結保存を行なう。また、実施例と同様の方法で、凍結保存洗浄赤血球の解凍を行い、実施例と同様の方法で、解凍後の混合物のpH測定及びヘモグロビンメト化率の測定を行う。pHは6.3であり、ヘモグロビンメト化率は9.3%であった。実施例と比較して、明らかにヘモグロビンのメト化が進行した。
The expired erythrocyte concentrate is washed and cryopreserved in the same manner as in Example, except that physiological saline is used in place of the sodium bicarbonate aqueous solution as the centrifugal washing solution. Further, the cryopreserved washed erythrocytes are thawed in the same manner as in the examples, and the pH of the mixture after thawing and the hemoglobin metrification rate are measured in the same manner as in the examples. The pH was 6.3, and the hemoglobin metation rate was 9.3%. Compared with the Examples, the hemoglobin quantification clearly progressed.
Claims (4)
- ヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料の中間生成物であって、赤血球濃厚液から、ｐH調整剤を含有する等張の洗浄液を用いて血漿、血小板、白血球を洗浄除去して得た赤血球液を、凍結保存した後解凍した液のpHが7.1～7.5である事を特徴とするヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物。 An intermediate product of a hemoglobin raw material used for the production of hemoglobin-based artificial oxygen carriers, obtained by washing and removing plasma, platelets, and leukocytes from an erythrocyte concentrate using an isotonic washing solution containing a pH adjusting agent A hemoglobin raw material intermediate product for use in the production of an artificial oxygen carrier based on hemoglobin, characterized in that the pH of a solution obtained by thawing erythrocytes after freezing is 7.1 to 7.5.
- 前記洗浄液が８０ｍMから１５４ｍMの炭酸水素ナトリウムと前記洗浄液を等張とするために必要な濃度の他の塩を含むことを特徴とする請求項１に記載のヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物。 2. The hemoglobin-based artificial oxygen carrier according to claim 1, wherein the cleaning solution contains 80 mM to 154 mM sodium hydrogen carbonate and another salt at a concentration necessary to make the cleaning solution isotonic. Intermediate product of hemoglobin raw material.
- 凍結保存前の前記赤血球液が、ヘマトクリット５５～６０％であることを特徴とする請求項１または２に記載のヘモグロビンベースの人工酸素運搬体製造に用いるヘモグロビン原料中間生成物。 3. The hemoglobin raw material intermediate product used for producing a hemoglobin-based artificial oxygen carrier according to claim 1 or 2, wherein the red blood cell solution before cryopreservation is hematocrit 55 to 60%.
- 前記解凍後の赤血球液中のヘモグロビンメト化率が3%以下である事を特徴とする請求項１ないし３に記載のヘモグロビンベースの人工酸素運搬体製造用のヘモグロビン原料中間生成物。 The hemoglobin raw material intermediate product for producing a hemoglobin-based artificial oxygen carrier according to any one of claims 1 to 3, wherein a hemoglobin metration rate in the erythrocyte fluid after thawing is 3% or less.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02178233A (en) * | 1988-12-28 | 1990-07-11 | Terumo Corp | Liposome containing hemoglobin and preparation thereof |
| JP2002520338A (en) * | 1998-07-10 | 2002-07-09 | バイオピュア コーポレーション | Hemoglobin purification method |
| WO2005063814A1 (en) * | 2003-12-26 | 2005-07-14 | Oxygenix Co., Ltd. | Process for purification of hemoglobin |
| JP2008120760A (en) * | 2006-11-15 | 2008-05-29 | Terumo Corp | Hemoglobin- and allosteric factor-containing liposome suspension and its preparation method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02178233A (en) * | 1988-12-28 | 1990-07-11 | Terumo Corp | Liposome containing hemoglobin and preparation thereof |
| JP2002520338A (en) * | 1998-07-10 | 2002-07-09 | バイオピュア コーポレーション | Hemoglobin purification method |
| WO2005063814A1 (en) * | 2003-12-26 | 2005-07-14 | Oxygenix Co., Ltd. | Process for purification of hemoglobin |
| JP2008120760A (en) * | 2006-11-15 | 2008-05-29 | Terumo Corp | Hemoglobin- and allosteric factor-containing liposome suspension and its preparation method |
Non-Patent Citations (1)
| Title |
|---|
| HESS JR ET AL.: "The effect of two additive solutions on the post thaw storage of RBCs.", TRANSFUSION., vol. 41, no. 7, July 2001 (2001-07-01), pages 923 - 927 * |
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