JP2013075838A - Method of producing purified hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of efficiently producing a purified hemoglobin that can be used as a raw material of pharmaceuticals, such as an artificial oxygen carrier, especially the method capable of industrial mass production.SOLUTION: In the method of producing the purified hemoglobin, when the purified hemoglobin is obtained from an erythrocyte hemolysis liquid by a purification process including ultrafiltration, the ultrafiltration is executed with an ultrafiltration membrane having filter specifications of an albumin blocking rate of 50% or less and a γ-globulin blocking rate of 98% or more.

Description

  The present invention relates to a method for producing purified hemoglobin, and particularly to a production method that can be industrially implemented.

When hemoglobin in blood is processed into a blood product and used, the collected blood is hemolyzed to obtain erythrocyte hemolyzed blood and then purified. Red blood cells separated from blood can be lysed by osmotic shock, but in recent years, a common hemolysis technique is an organic solvent (solvent) called a solvent detergent (SD treatment) method and a surfactant (detergent). ) And hemolysis that also serves as virus inactivation. After hemolysis by this SD method, the surfactant and organic solvent are removed with a synthetic adsorbent to obtain red blood cell hemolysate.
In addition, when hemoglobin is processed into blood products and administered to humans for therapeutic purposes, it is necessary to guarantee the sterility and virus inactivation of the product. Purification including virus inactivation treatment is also guaranteed.

Various types of virus inactivation techniques are known. Among them, for purification of hemoglobin, technologies other than virus inactivation by energy that are restricted in application due to fear of hemoglobin alteration, specifically chemical viruses. Inactivation treatment and physical virus removal treatment can be applied. The chemical virus inactivation treatment is typically an SD treatment method using the above biocompatible organic solvent and a surfactant.
The physical virus removal treatment is typically size exclusion, and is nanofiltration in which the virus is removed by filtration with a very fine pore size that can remove the virus (referred to as a virus removal membrane).

  In the production of typical purified hemoglobin at present, erythrocyte lysate obtained by SD treatment of erythrocytes is adsorbed with a synthetic adsorbent and then treated with an ultrafiltration membrane having a fractional molecular weight of 100,000. Purify by removing unnecessary substances such as solvents and surfactants used in SD treatment, blood group substances such as stroma and sialic acid, removing viruses by nanofiltration, and concentrating to a certain concentration Hemoglobin can be obtained (see Patent Document 1).

  In these steps, the ultrafiltration treatment using the ultrafiltration membrane having a fractional molecular weight of 100,000 is the most important production efficiency in terms of filtration volume, treatment time, hemoglobin recovery rate, and the like. As a method for increasing the recovery rate of hemoglobin in this step, the ultrafiltration device is made into a circulation type, and a diluted solution such as a weak alkaline phosphate buffer or sodium hydrogen carbonate solution is added to the primary side, and diafiltrate is added. Techniques for hydration and hydroconcentration are known.

International Publication No. 2006/003926

Hemoglobin is a valuable resource and it is desirable to recover it in high yield. However, in the ultrafiltration step described above, in order to increase the recovery rate of hemoglobin, it is necessary to prepare a large amount of diluent and a large storage tank is required. Furthermore, a large capital investment is required because the hemoglobin concentration decreases and the capacity increases due to the ultrafiltration treatment. For example, when ultrafiltration membrane treatment is performed on 100 kg of erythrocyte hemolyzed blood having a hemoglobin concentration of 0.2 g / g by a hydroconcentration operation method using 100 kg of a diluent, 600 kg of hemoglobin is recovered to obtain a 75% hemoglobin recovery amount. Filtration with diluent is required.
Thus, a great deal of labor and a very long time are required to achieve a high recovery rate of hemoglobin.

  An object of the present invention is to provide a method for producing purified hemoglobin which can be industrially implemented and can recover hemoglobin in which virus inactivation and sterility are guaranteed in high yield from blood.

  The inventors of the present invention have studied the production of purified hemoglobin, particularly focusing on the ultrafiltration membrane used in the ultrafiltration process, aiming at the efficiency of the ultrafiltration process. By making the ultrafiltration membrane a specific filter specification, specifically, it has been found that the yield is very effective even if the difference in the albumin inhibition rate is slight. The ultrafiltration membrane is available as an industrially manufactured commercial product, but from among these, a specific filter spec can be selected and used to implement the present invention and obtain its effects. .

Specifically, when using a conventional commercially available ultrafiltration membrane, the albumin rejection is 80%, but the theoretical value is calculated assuming that the molecular weights of albumin and hemoglobin are almost equal. Then, focusing on the possibility of equivalent recovery at about 100 kg with respect to the above-mentioned conventional required dilution amount (about 600 kg) by limiting to filter specifications with an albumin rejection of 50% or less.
Similarly, to obtain a 90% hemoglobin recovery rate, a filter with an albumin rejection rate of 80% requires filtration with a 1000 kg diluent, whereas a filter with an albumin rejection rate of 50% requires an equivalent recovery at approximately 300 kg. We also focused on the possibility of
When industrialization of the production of purified hemoglobin is considered, the amount of the diluted solution and the amount of the filtrate shown in the examples are further scaled up to 2 times and 3 times. Therefore, by selecting the optimum filter specification, it is possible to significantly reduce the capital investment cost, the ultrafiltration membrane treatment time, and the subsequent concentration time.

The theoretical value was calculated as follows.
As a precondition, it is assumed that 100 kg of the storage tank is completely filtered, and that hemoglobin (Hb) has permeated in accordance with the albumin blocking rate defined by the filter specification, and thereafter, the hydroconcentration with 100 kg of the diluent is repeated. In addition, it is assumed that the molecular weights of albumin (molecular weight: 67,000) and hemoglobin (molecular weight: 64,500) are almost the same, and the ultrafiltration treatment shows the same filterability.
For example, ultrafiltration membrane treatment is performed by a hydroconcentration operation method using 100 kg of diluted solution of 100 kg of erythrocyte hemolyzed blood having a hemoglobin concentration of 0.2 g / g. When 100 kg of the liquid storage tank is completely filtered, and then hydroconcentration with 100 kg of the diluent is repeated, the amount of diluent necessary for each recovery rate is as follows.

From the above
(1) In the case of a filter with an albumin rejection of 50% 75% (equivalent to 15 kg of Hb) Hb recovery necessary for collecting Hb: 100 kg
90% (equivalent to 18 kg of Hb) of Hb recovery required: 300 kg
(2) In the case of a filter with an albumin rejection rate of 80% Amount of diluent required for 75% (equivalent to 15 kg of Hb) of Hb recovery: 600 kg
90% (equivalent to 18 kg of Hb) of Hb recovery required: 1000 kg of diluent

Therefore, the present invention has been conceived in which an ultrafiltration membrane having a specific filter specification is used in the hemoglobin purification step.
That is, in the method for producing purified hemoglobin according to the present invention, when obtaining purified hemoglobin from erythrocyte lysate by a purification step including ultrafiltration, the ultrafiltration is performed with an albumin inhibition rate of 50% or less and a γ-globulin inhibition. The measurement is carried out with an ultrafiltration membrane having a filter specification of 98% or more. The molecular weight cut-off of the ultrafiltration membrane is preferably about 100,000.

  The erythrocyte lysate is preferably a chemical virus-inactivated erythrocyte lysate obtained by lysing erythrocytes by a solvent detergent method using an organic solvent and a surfactant.

  In the present invention, after the ultrafiltration, nanofiltration is usually performed with a filter having a pore size of about 15 to 70 nm.

In the method for producing purified hemoglobin according to the present invention, when the erythrocyte lysate is treated with an ultrafiltration membrane (usually a fractional molecular weight of 100,000), the filter specifications of the ultrafiltration membrane are limited, The amount of water added can be kept to a minimum, a filtrate with a higher hemoglobin concentration can be obtained, and a high hemoglobin recovery rate can be achieved. As a result, it is possible to significantly reduce manufacturing equipment investment costs for industrialization, ultrafiltration membrane treatment time, and subsequent concentration time.
When purifying erythrocyte hemolysate hemolyzed by the SD treatment method, the present invention uses the solvent and surfactant used together with unnecessary substances derived from blood such as stroma and blood group substances. Provided is an industrially feasible method for producing purified hemoglobin, which makes it possible to recover hemoglobin, which has been removed to a level acceptable by some biological system, from blood in a high yield.

Hereinafter, the present invention will be described more specifically.
In the process of recovering and purifying hemoglobin from blood, the present invention is a process in which virus inactivation treatment is performed with a combination of a solvent and a surfactant, and a combination of use of a synthetic adsorbent and ultrafiltration treatment is performed, that is, addition is performed. A solvent, a surfactant, and unnecessary substances such as stroma and blood group substances are adsorbed and removed by a synthetic adsorbent, and further subjected to ultrafiltration treatment with a pore size having a fractional molecular weight of about 100,000. Removing solvents, surfactants and unwanted substances such as stroma and blood group substances that could not be adsorbed and removed by the adsorbent to a level acceptable by the human or some biological system in which the hemoglobin is used, As a result, the efficiency of nanofiltration performed as a physical virus removal process is increased, and the sterilization process is further improved. The present invention relates to a method that guarantees virus inactivation and sterility by filtering with a filter, and enables purification of hemoglobin to be recovered in a high yield.

  A combination of a solvent and a surfactant used in an SD treatment method as a chemical inactivation treatment and hemolysis treatment of an enveloped virus, if it can be removed by a synthetic adsorbent treatment and an ultrafiltration operation. Any solvent and surfactant combination known in US Pat. In particular, trialkyl phosphates such as tri- (n-butyl) phosphate and the product known as Tween 80, as well as non-ionic oil soluble aqueous detergents such as the trade name Triton X-100, sodium cholate, and others in the art In combination, known solvent and surfactant combinations are preferably used. Further, the chemical virus inactivation treatment performed by purification of hemoglobin may be in a state where erythrocytes are pre-hemolyzed or not hemolyzed.

  As a chemical virus inactivation treatment in the present invention, a synthetic adsorbent that can be used as a means for removing a solvent and a surfactant added in blood is a commercially available Amberlite XAD series which is a copolymer of styrene or acrylic and divinylbenzene. (FPX-66, XAD-2000) is effective and the synthetic adsorbent removes most of the added solvent and surfactant. Furthermore, as the purification of hemoglobin, it becomes possible to remove unnecessary stroma and blood group substances.

Preparations treated with synthetic adsorbents are substances that are not necessary for purification of solvents, surfactants and hemoglobin that could not be removed by adsorption treatment with synthetic adsorbents, such as stroma and blood group substances. Is removed.
Furthermore, the ultrafiltration operation acts as a prefiltering process for nanofiltration, and a certain amount of virus can be removed. The amount of the synthetic adsorbent used and the processing time can be selected as desired by considering the removal effect and economic efficiency.
In addition, the amount of solvent-surfactant or stromal and blood group substances to be removed by the ultrafiltration treatment depends on the synthetic adsorbent treatment conditions such as the amount of synthetic adsorbent used in the synthetic adsorbent treatment performed in the previous step and the treatment time Determined by the balance of
Furthermore, the synthetic adsorbent has a strong resistance to alkalis and heat, and can be sterilized under warming conditions by immersion in an aqueous alkali solution before use, thus ensuring the pyrogen-free and sterility of the preparation. Further, sodium hydroxide is preferably used as the alkaline aqueous solution.

  If the preparation that has been subjected to chemical virus inactivation treatment is subjected to ultrafiltration as it is, the solvent, surfactant and stroma in the preparation may cause clogging of the filter, resulting in an increase in filtration time. This increases the membrane area of the expensive filter, requires frequent replacement of the filter, and leads to a decrease in yield. For this reason, the adsorption process by a synthetic adsorbent is usually performed before the ultrafiltration process.

  Ultrafiltration performed in the present invention is also referred to as cross-flow filtration or tangential flow filtration (TFF), and unlike dead-end filtration, which is a method performed by filtration sterilization or the like, the liquid is allowed to flow in parallel to the filtration membrane surface to remove dirt. This is a method of filtering while removing. In other words, the filtration membrane cassette used for cross-flow filtration has a structure that is laminated in a flat membrane shape, and the treatment liquid flows between the flat membrane and the flat membrane continuously in parallel, and the treatment flows in parallel with the flat membrane. This is a method in which particles deposited on the flat membrane surface are washed away by the flow of the liquid to prevent the formation of a gel layer due to the accumulation of particles and perform stable filtration.

As a material for the filtration membrane, generally, regenerated cellulose such as cellulose acetate and synthetic polymer such as polyethersulfone are preferably used.
In addition, filter membranes with a molecular weight cut and a pore size suitable for the purpose are used, but the exact pore size is not to mention the substances that must be maintained in the liquid preparation and the solvent-surfactant to be removed. Should be determined by the size of the substance that does not need to be maintained in some virus and liquid preparations. In particular, when purifying hemoglobin from blood, the pore size having a fractional molecular weight of about 100,000 for the purpose of removing stroma and blood group substances and for the purpose of increasing the efficiency of nanofiltration treatment as a physical virus inactivation treatment. Is suitable.

Furthermore, the present inventors have made extensive studies on the relationship between the filter specification of an ultrafiltration membrane having a fractional molecular weight of about 100,000, the hemoglobin recovery rate and the removal target substance removal rate, and albumin defined as the filter specification. (Molecular weight: 67,000) Correlation was found between the rejection rate, the hemoglobin recovery rate, and the removal target substance removal rate. According to the present invention, an ultrafiltration membrane having an albumin blocking rate of 50% or less is selected and used. As a result, as shown in the above-mentioned example, a diluted solution with a case of using an albumin blocking rate of 80% is used. The difference between the volume and the filtrate volume will be further doubled and tripled on the industrial scale, and the required liquid volume difference will also be 2 according to the albumin rejection rate value that is the filter spec. A difference of 3 times or 3 times will occur.
In addition, as another filter specification of the ultrafiltration membrane, the γ-globulin blocking rate is preferably 98% or more.

  Further, in the cross flow filtration system, the operation with the membrane area corresponding to the amount of the processing liquid can be performed by controlling the effective filtration area of the filtration membrane cassette attached to the filtration device. That is, it is possible to cope with a scale from a small capacity to a large capacity by controlling the number of filtration membrane cassettes to be mounted on the filtration device according to the amount of the processing liquid.

  Furthermore, since the in-line sterilization is possible when the cross-flow filtration apparatus is used in the present invention, it is possible to guarantee the pyrogen-free and sterility of the preparation as in the case of the synthetic adsorbent treatment. As a method of in-line sterilization, high-temperature steam or an alkaline aqueous solution heated to about 50 ° C. is generally circulated in the apparatus. Further, sodium hydroxide is preferably used as the alkaline aqueous solution.

  Cross-flow filtration is a batch system that repeats the process of returning to a specified amount of circulating fluid with a dispersion medium when filtration is performed without controlling the amount of necessary substances circulating in the cross-flow filtration device. In addition, there is a diafiltration method in which the amount of the dispersion medium supplied to the circulating fluid is controlled in accordance with the amount of the permeating liquid and the amount of the circulating fluid is maintained at a specified amount. .

  The dispersion medium is not limited as long as it is a solvent that can stably disperse and dissolve necessary substances. Further, the presence or absence of components such as an osmotic pressure adjusting agent and a pH adjusting agent in the dispersion medium is not limited at all as long as it does not cause an action of deteriorating and destroying the cross flow membrane.

  The resulting preparation is then subjected to a physical virus removal treatment by nanofiltration treatment. As a filter used for nanofiltration, a hollow fiber microporous membrane, a PVDF membrane or the like generally formed of regenerated cellulose is used. Since virus removal by nanofiltration is mainly due to the mechanism of multi-sieve effect, ie physical removal of virus particles, the exact pore size must be removed by the substance that must be maintained in the preparation and by size exclusion Should be determined according to the size of the virus.

  In the process of purifying hemoglobin from blood in the present invention, Ultipor VF DV20 manufactured by Pall and Viresolve NFP manufactured by Millipore were effectively used.

  One of the critical parameters of nanofiltration is the amount of impurities contained in the preparation. In the present invention, a solvent-surfactant added as a chemical virus inactivation treatment, and stroma and blood group substances are added. It was removed by a combination of the use of a synthetic adsorbent and an ultrafiltration operation, thereby improving the efficiency of nanofiltration treatment, that is, reducing the treatment time and improving the yield.

  The resulting preparation is then concentrated by dehydrating excess water by ultrafiltration. In the present invention, the ultrafiltration performed in the concentration operation is the same system as the ultrafiltration described above as the operation for removing the solvent-surfactant and stroma and blood group substances, that is, cross-flow filtration, tangential flow filtration (TFF) ) Is used.

  Filter membranes with molecular weight cuts and pore sizes depending on their purpose are used, but the exact pore size depends on the size of the material that must be maintained in the liquid preparation and the material that need not be maintained in the liquid preparation. Should be determined. In particular, when concentrating hemoglobin, a pore size having a fractional molecular weight of about 10,000 to 30,000 is suitable. According to the present invention, the concentration of 45 w / w% as the hemoglobin concentration was made possible.

  The resulting preparation is then filtered through a sterilizing filter having a pore size of 0.2 μm for sterilization purposes. As the membrane material of the filter used for the sterilization filter, regenerated cellulose such as cellulose acetate, polyethersulfone, PVDF and the like are used. In the process of purifying hemoglobin from blood in the present invention, the hemoglobin concentration is 45 w / w%, and filtration is possible with very good filtration characteristics even though the viscosity is very high.

The present invention will be described in more detail with reference to the following examples, but these are not intended to limit the present invention.
Example 1
An appropriate amount of physiological saline was added to a concentrated erythrocyte preparation from which platelets, leukocytes, and plasma components were removed from human whole blood, and the lower phase was recovered after centrifugation to obtain 25 kg of washed erythrocytes.
25 kg of virus inactivated SD solution (TNBP, TritonX-containing aqueous solution) is added to 25 kg of the washed erythrocytes, and the virus and inactivation-treated hemolysate having a solvent and surfactant concentration of 0.3% TNBP-0.2% TritonX-100 50 kg of treatment liquid was obtained.
The virus-inactivated hemolysis solution was circulated in a column packed with 10 L of synthetic adsorbent Amberlite XAD series (FPX-66) resin.

Thereafter, a filter membrane having a filter specification of albumin blocking rate ≦ 44% and γ-globulin blocking rate ≧ 99.7% (material: polyethersulfone, pore size: fractional molecular weight 100,000, effective filtration area: 4.2 m ² , A permeate was obtained by performing ultrafiltration with a cross-flow filtration apparatus (Zaltocon 2 plus, manufactured by Sartorius) equipped with Sartorius.
This ultrafiltration treatment is a series of operations in which 30 kg of filtrate is first recovered, and then 10 kg of aqueous sodium bicarbonate solution prepared to 20 mM as a dispersion medium is added to 20 kg of the remaining liquid, and 10 kg of permeate is recovered by filtration. Was repeated 15 times to obtain about 180 kg of ultrafiltration treatment liquid.

Subsequently, the ultrafiltration treatment solution was subjected to nanofiltration treatment with a virus removal filter Viresolve NFP Opticap Capsule (Millipore) under the condition of 0.2 MPa.
About the total amount of the treatment liquid obtained, a cross flow filtration apparatus (Zaltocon 2 plus, equipped with a filtration membrane (material: polyethersulfone, pore size: fractional molecular weight 30,000, effective filtration area: 1.2 m ² , manufactured by Sartorius) Concentration was carried out by Sartorius) to obtain a preparation with a hemoglobin concentration of 42 w / w%.
The obtained hemoglobin preparation solution was about 7.5 kg, and the hemoglobin yield calculated from the ratio of the hemoglobin preparation solution hemoglobin amount to the hemoglobin amount in the prepared washed erythrocytes was 54.3%.

Thereafter, filtration sterilization was performed by sterilization filter sartopore 2 (material: polyethersulfone, effective filtration area 0.45 m ² ) having a pore diameter of 0.2 μm for sterilization.
The SD removal effect (TNBP, Triton X-100 quantification) and stromal removal effect (phosphatidylserine quantification) were analyzed by the above treatment. The analysis results are shown in Table 1.

As shown in Table 1, about 99.999% of the solvent TNBP in the finally obtained hemoglobin solution was removed with respect to the added amount. Moreover, surfactant TritonX-100 was removed below the limit of quantification.
In stromal analysis, phosphatidylserine was removed to 1.0 μg / g.
In addition, by operating in the order of synthetic adsorbent treatment and ultrafiltration treatment, stable treatment was possible in terms of time and yield in ultrafiltration treatment and nanofiltration.

(Comparative Example 1)
Washed erythrocytes as in Example 1 except that the ultrafiltration treatment in Example 1 was carried out in place of a filter membrane with a filter specification of albumin blocking rate ≦ 62% and γ-globulin blocking rate ≧ 99.7%. Were subjected to the same virus inactivation treatment, column circulation treatment, nanofiltration treatment after ultrafiltration treatment, and crossflow filtration to obtain a hemoglobin preparation having a hemoglobin concentration of 42 w / w%.

  The obtained hemoglobin preparation solution was about 2.8 kg, and the hemoglobin yield calculated from the ratio of the hemoglobin preparation solution hemoglobin amount to the hemoglobin amount in the prepared washed erythrocytes was 18.2%.

The hemoglobin preparation solution was sterilized by filtration using a 0.2 μm pore size sterilizing filter as in Example 1.
The SD removal effect (TNBP, quantification of TritonX-100) and the stromal removal effect (phosphatidylserine quantification) were analyzed in the same manner as in Example 1. The analysis results are shown in Table 1.
As shown in Table 1, about 99.997% of the solvent TNBP in the finally obtained hemoglobin solution was removed with respect to the added amount. Moreover, surfactant TritonX-100 was removed below the limit of quantification. Moreover, the phosphatidylserine which performed the stromal analysis was removed by 0.6 microgram / g. In addition, by operating in the order of synthetic adsorbent treatment and ultrafiltration treatment, stable treatment was possible in terms of time and yield in ultrafiltration treatment and nanofiltration.
However, the hemoglobin yield calculated from the ratio of the amount of hemoglobin obtained to the amount of hemoglobin in the prepared washed erythrocytes was 18.2%.

  When the hemoglobin yield calculated from the ratio of the hemoglobin amount obtained with respect to the hemoglobin amount in the prepared washed red blood cells in Example 1 and Comparative Example 1 was compared, it was 18.2% in Comparative Example 1. On the other hand, in Example 1, it is 54.3%, and there is a correlation between the albumin rejection rate defined as the filter specification of the ultrafiltration membrane and the hemoglobin recovery rate, and a filter having an albumin rejection rate of 50% or less may be used. It was revealed that the recovery was excellent.

Claims (3)

  When obtaining purified hemoglobin from erythrocyte lysate by a purification step including ultrafiltration, the ultrafiltration is performed by ultrafiltration according to a filter specification having an albumin blocking rate of 50% or less and a γ-globulin blocking rate of 98% or more. A method for producing purified hemoglobin, which is carried out with a membrane.   The method for producing purified hemoglobin according to claim 1, wherein the erythrocyte lysate is a chemically virus-inactivated erythrocyte lysate obtained by lysing erythrocytes by a solvent detergent method using an organic solvent and a surfactant. .   The method for producing purified hemoglobin according to claim 1, wherein nanofiltration is performed with a filter having a pore diameter of 15 to 70 nm after the ultrafiltration.