JPS6025411B2 - Manufacturing method of hemoglobin solution - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides hemolysis of human red blood cells, quantitative separation of stromal fat, reduction of potassium content, as well as sterile filtration of the solution, which is stable in storage without the risk of hepatitis. and a method for producing a hemoglobin solution having a content of stabilized 2,3-diphosphoglycerate which is insoluble.

Commercially available so-called "blood substitutes" have the following advantages with respect to blood storage. [1} When using,
No need for blood type testing at all.

■ Longer term is possible.

{3' There is no risk of hepatitis.

The biggest drawback of commercially available blood substitutes is that they cannot replace many of the special functions of blood, such as the oxygen-carrying action of red blood cells.

Isotonic human hemoglobin solutions have existed as blood substitutes, especially for several years in preclinical trials.
Since it is clear that a critical factor in the occurrence of shock is the lack of oxygen supply, such drugs would be worth pursuing, especially for shock therapy. Attempts have already been made to use aqueous emulsions of fluorocarbon compounds as oxygen carriers, especially for the infusion of separated organs. However, since there is no reasonable method of removing these substances,
Even today, there are still insurmountable obstacles to intravenous injection into the body. This is because such foreign substances that cannot be physically decomposed are inevitably stored in tissues. On the other hand, free hemoglobin is a normal urine component, and depending on each case, a portion is reabsorbed and left to natural metabolism.

The safety of known hemoglobin agents against hepatitis is not considered to be a problem. However, for clinical use, safety is naturally of great importance. This is because contagiousness is not eliminated when donated blood is used as a starting material. Methods are already known in which a blood-killed physiologically compatible hemoglobin solution can be prepared from human red blood cells by treatment that reduces the potassium content and removes the stromal lipids released by hemolysis. However, during testing of this known hemoglobin solution, shortcomings were revealed that have prevented its clinical use to date. In particular, kidney damage was observed. Other known hemoglobin solutions do not cause kidney damage, but their production involves, inter alia, a dialysis process, which removes phosphorus, which plays an important role in physiologically important enzyme affinity. Acid esters, i.e. 2.3-
Diphosphoglycerate is quantitatively removed. The starting material containing red blood cells is treated with a dilute solution of 3-propiolactone at a temperature range of 5 to 15 qo for less than 25 minutes, and the amount of 8-propiolactone incorporated in the dilute solution is the same as that of red blood cells. 4 to 12 days per liter of precipitation, then using a neutral washing solution or using a slightly alkaline solution to wash off the excess 8-bropiolactone and its reaction products, and then A red blood cell lysate produced by stirring red blood cells in a cytolytic solvent such as distilled water is treated with an enteric ion exchange resin in the H+ form until the pH value is reduced to 5.0 to 5.5; After removing the resin and precipitated stromal mass by centrifugation, adjusting the hemoglobin concentration to a desired level,
It is characterized by adding additives for adjusting isotonicity and for pH value and ferroheme stabilization, followed by aseptic handling.

This combination of critical processing steps results in a product whose usage dose eliminates the risk of hepatitis.

Treatment with an 8-propiolactone solution is preferably carried out using a solution of 6 to 16 times of 8-propiolactone per 1.5 minutes of red blood cell precipitation of 1.2 to 3.2 hours/100'. It will be done.

The remaining 8-bropiolactone in the red blood cell supernatant was then reduced to 20 to 10% by washing with a centrifuge four times.
wc%. At this time, a solution containing common salt or sodium bicarbonate is preferably used as the washing liquid. Other preferred bran washes include tribasic sodium phosphate solution and sodium carbonate solution. These purified solutions are further diluted to 1/50 during subsequent hemolysis. Only at these concentrations does 8-propiolactone come into contact with hemoglobin and other internal components of the cell. Subsequently, it can be fully reacted. Distilled water is used as the preferred diluting solvent.

However, it is also possible to add physiologically suitable electrolyte solutions, such as saline solutions, glucose solutions or bicarbonate solutions. Quite surprisingly, it has been found that during the treatment carried out in this way, the enzyme system responsible for the hydrolysis of 2,3-diphosphoglystate is clearly inhibited. Excluding the 8-propiolactone treatment step, the product obtained by the process according to the invention contains about twice as much free phosphate after storage for 3 months compared to the product according to the process according to the invention. .

8 according to the present invention, as proven by direct measurements.
- By treatment with propiolactone, the 2,31-diphosphoglycerate component in the hemoglobin solution is stabilized at a value of about 0.4 mmol per 0.8 mmol of hemoglobin over a period of more than 3 months. Without this stabilization, the salt of 2,3-diphosphoglycerate would be
per batch, 0 to 0.25mMol. change between. A particularly suitable solution for washing pigs according to the invention is 3.8%
of sodium bicarbonate solution or 1.6% sodium chloride solution. When diluting, especially the volume of red blood cells
．． It is diluted by fugaku. A particularly preferred cation exchange resin is an acidic polystyrene sulfonate ion exchange resin, especially Dowex 50 WX (trade name:
There is a resin commercially available as (manufactured by mjcal). This resin has an exchange capacity of 4 to 5 mval with a dry weight of 9. Other suitable ion exchange resins include:
For example, Amberlite IR-120 (product name RO
HM & HAAS), o-Karb225 (trade name, manufactured by PERMUTIT), Lewatit SIO0 (trade name, manufactured by FARBENFAB RIKEN BAYER), etc. are commercially available. According to the method of the present invention, the potassium component is reduced by first cation-exchanging K+ into H+, followed by adding distilled water and centrifuging.

It is advantageous to carry out this in such a way that approximately 6.3 hours of hemoglobin are present per 100 secretions. The stable pH value is preferably 7.4 at the end of the treatment. One preferred tonicity adjusting agent is glucose, with sodium acetate and sodium bicarbonate also being suitable. However, in combination with other known isotonicity adjusting agents such as sodium chloride, mannitol, sorbitol, or any combination thereof, physiologically acceptable amounts of calcium chloride, magnesium chloride, sodium lactate, etc. are all equally suitable for this purpose. In accordance with this invention, a hemoglobin-enriched product can be created by freezing and partially rethawing a prepared hemoglobin solution;
By repeating the concentration process, it is possible to achieve a hemoglobin concentration of 1.5%, that is, the same concentration as the hemoglobin concentration in blood.

The relative viscosity of such a solution is approximately 1.8 at 370 even in the normal blood count range. The use of sterile flasks and blood transfer equipment provides a variant containing the enhanced active substance in a closed system. The final adjustment of isotonicity is carried out so that the obtained superconcentrate does not thereby become hypertonic.
This is preferably carried out after concentration. The following examples illustrate the method of this invention.

The products obtained in the following examples have already been tested in mice, rats,
It has been tested on rabbits, dogs, pigs and humans and has been found to have good compatibility and transport effectiveness. EXAMPLE 500 volumes of blood from each of 16 blood donors were collected, the red blood cell precipitate was separated from the blood, combined and diluted with two volumes of 1.6% sodium chloride solution, freshly distilled P- Propiolactone was added for 6 days at 1000 ml and heated at 10-120 ml for 15 minutes.

This was subjected to refrigerated centrifugation for 2 minutes, the supernatant was removed by suction, and the red blood cells were precipitated 4 times, each time at a concentration of 1.6%.
Wash on a centrifuge using approximately 1.1 parts of sodium chloride solution, followed by 5 parts of sterile deionized distilled water.
2 added to it. After lighting for 5 minutes, add 50 liters of high purity (PA quality) cation exchange resin Dowex 50.
0 was added and 1N sodium hydroxide solution was added until the stromal lipids precipitated clearly, and the pH value was adjusted to 5.0.
And so.

After that, it is inactivated at pH 5.5, and after 1 minute, it becomes 20,000 ml.
centrifuged. Approximately 6 to 7
Approximately 5% hemoglobin solution was collected. glucose 4
After adding 0.0 ml, adjust the pH using sodium hydroxide solution.
The value was adjusted to 7.4 and the solution was further centrifuged as described above and filtered through a cellulose-asbestos filter (Bad
EK manufactured by Seitz-Werke in Kreuznach
Bonito mold was filtered using SI). Good results can also be obtained using filters of the following types: St.
Fil from Gallen's Filtrox-Werke
trox-Stem filter, Firma Pal
FilterKerzen's "U1tipore"
Type - Both are cellulose asbestos thin layer filters or cellulose-acetate thin layer filters.
These are respectively GSUP type filters (product name MLLL
Manufactured by mORE, registered trademark Millipore) and S
ar■ri female type SMII07 filter (product name S
ARTORIUS-WERKE). The oxygen binding curve of the product prepared in this example is 1
After storage for 2 months at 0q○, 1 at pH 7.4.
9bi orr. showed the correctness of P5.

If converted to the pH value in red blood cells of 7.2, the hemoglobin P50 value of the above product is 2-feed on. corresponds to Fresh red blood cell P5q direct'ma2orr. It is. This oxygen partial pressure is such that the hemoglobin product is 50% of its maximum oxygen binding capacity.
The value is saturated to 0%. Example 2 The same procedure as in Example 1 was carried out, except that 8-propiolactone was added to the red blood cell suspension.

After centrifugation and suction filtration of the supernatant, the red blood cell precipitate was washed using approximately 1.1 volumes of each of the following solutions: '1' solution containing sodium bicarbonate at 1/2 m/day; 2} '1- and a solution containing sodium bicarbonate per 1 〆 and 1 per 1 as in {3}
A solution containing 6 hours of NaCI in a 1:1 ratio, {4} a solution containing 16 hours of NaCI per night. Next, the same procedure as in Example 1 was performed up to the step of separating the stroma. Before brood filtration, the pH value was adjusted once again to 7.0 using 1N NaOH and 33 g of glucose per night was added, followed by the addition of 25 g of sodium bicarbonate per night. The final pH value was then adjusted to 7.4 using 1N NaOH solution. Example 3
The procedure up to the separation of the stroma was carried out in the same manner as in Example 1 or 2.

The concentration of hemoglobin corresponding to blood was then concentrated to 15/100 by the following procedure: the hemoglobin solution was frozen in 50/100 increments in fixed 1000/100 increments, and then the sterile blood It is melted at room temperature in a flask mounted on top so that the dark red melt can be continuously removed from the retained white icicles through the perforation of the stopper with a removal device. By repeating this operation, a solution containing 150 hemoglobin was obtained. This solution was then processed according to Example 1 to obtain a diluted product.
The embodiments of the present invention are as follows.

'1' B-propiolactone from 6 to 16 days per 1.5 parts of red blood cell sediment 1.2 to 3.2 times per 100
2. The method according to claim 1, wherein the method is used as a solution.

'21 The manufacturing method according to Claim 1 or Embodiment '1', characterized in that the treatment with 8-propiolactone is carried out at 10 to 1 o.

(3) The production method according to any one of Claim 1 and Embodiments '1' to '2', characterized in that washing is carried out four times to separate excess 8-propiolactone.

■ Use of sodium bicarbonate solution and/or sodium chloride solution, in particular 3.8% sodium bicarbonate solution and/or 1.6% sodium chloride solution, for washing the rice bran after treatment with 8-propioolactone. The manufacturing method according to claim 1 and any one of embodiments 1 to {3}.

(2) The manufacturing method according to claim 1 and any one of {1} to '4} of the embodiments, characterized in that an H+ type polystyrene sulfonate resin is used as the cation exchange resin.

{6} Claim 1 and embodiments {1} to {5) characterized in that isotonicity is adjusted using glucose, sodium acetate or sodium hydrogen carbonate.
} The manufacturing method described in any of the above. {7- The manufacturing method according to any one of Claim 1 and Embodiments '1} to {6}, characterized in that the pH value of the final product is adjusted using a sodium hydroxide solution. (According to any one of Claim 1 and Embodiments 1 to 7, wherein the bacteria are filtered using an 81 cellulose-asbestos filter or a thin layer made of cellulose acetate.) Manufacturing method.

Claims (1)

[Claims] 1. A starting material containing red blood cells is treated with a dilute solution of β-propiolactone at a temperature range of 5 to 15°C for a period of less than 25 minutes, and the β-propiolactone formulated in the dilute solution is The amount of piolactone used is 4 to 12 g per liter of red blood cell sediment, and then a neutral washing solution or a slightly alkaline solution is used to remove excess β-propiolactone and its reaction product. The red blood cell lysate, produced by washing off and then stirring the red blood cells in a cytolytic solution such as distilled water, is incubated with H^+ until the pH value drops to 5.0-5.5. After treatment with a cation-exchange resin of the form and removal of the resin and precipitated stromal mass by centrifugation, the desired hemoglobin concentration is adjusted and additives for isotonicity adjustment and pH value and ferroheme stabilization are added. A method for producing a hemoglobin solution, which is characterized by adding additives and then sterile filtration. 2. The starting material containing red blood cells is treated with a dilute solution of β-propiolactone at a temperature range of 5 to 15°C for a period of less than 25 minutes, and the amount of β-propiolactone used in the dilute solution is determined to be the same as that of red blood cells. 4 to 12 g per liter of sediment and then using a neutral washing solution, or
Alternatively, a red blood cell lysate produced by washing off excess β-propiolactone and its reaction products using a slightly alkaline solution and then stirring the red blood cells in a cell-destroying solvent such as distilled water. was treated with enteric ion photoconversion resin in the H^+ form until the pH value decreased to 5.0-5.5, and after removing the resin and precipitated stromal mass by centrifugation, the thus obtained The resulting solution is frozen in the form of a rod, then thawed in a vertical position, separating the hemoglobin-rich melt that flows from the ice block, repeating this operation if necessary, and then adding to it an isotonic adjustment solution. A process for producing a hemoglobin solution, characterized in that additives and additives for pH value and ferroheme stabilization are added, followed by sterile filtration.