Method for thawing frozen plasma, and method for producing plasma-derived products and blood coagulation factor VIII products.

Abstract

The goal is to realize a technology that allows for consistent control of cryoprecipitate yield in the production of plasma-derived products without causing a decrease in coagulation factor activity due to thermal denaturation. [Solution] The method for thawing frozen plasma according to the present disclosure includes a temperature adjustment step and a thawing step. In the temperature adjustment step, the surface temperature of the container containing the frozen plasma is raised from -20°C or below to -1.5°C or a temperature near -1.5°C or below. In the thawing step, after the temperature adjustment step, the frozen plasma is thawed at an ambient temperature of 2°C to 5°C to an average thawing rate of 40% to 80%.

Description

Technical Field

[0001] The present disclosure relates to a method for thawing frozen plasma. The present disclosure also relates to a method for producing a plasma fractionation preparation and a factor VIII coagulation preparation using a plasma component obtained by the method for thawing frozen plasma.

Background Art

[0002] Blood products are roughly classified into blood products for transfusion used for transfusion to patients and plasma fractionation preparations obtained by purifying plasma components. Plasma fractionation preparations are effectively utilized by manufacturing several types of preparations from the same raw material and providing the components required by patients as preparations.

[0003] The Japanese Red Cross Blood Center performs pre-storage leukocyte removal by passing donated blood through a leukocyte removal filter immediately after blood collection, and prepares fresh frozen plasma (Non-Patent Document 1). Fresh frozen plasma can be stored for 1 year after blood collection at -20°C or lower. When used, it is thawed at 30 to 37°C and used immediately after thawing. If it cannot be used immediately, it is stored at 2 to 6°C without being refrozen and used within 24 hours. In that case, a considerable amount of factor VIII contained therein is inactivated (about 30% in 24 hours). Also, since coagulation factors are considered to be inactivated to some extent during thawing, use after refreezing and rethawing is avoided (Non-Patent Document 1).

[0004] In addition, as a precaution during thawing when used for transfusion, the section on plasma preparations on the website of the Japanese Red Cross states that "if the temperature has not reached the thawing temperature, precipitation (cryoprecipitate) may occur and clog the filter. It is important to strictly control the temperature during thawing and thaw it completely. Also, if the thawing temperature is too high, protein thermal denaturation may make it unusable. Thawing at a high temperature causes a decrease in coagulation factor activity and the original transfusion effect cannot be obtained." (Non-Patent Document 2).

[0005] On the other hand, a standard procedure for preparing cryoprecipitate in-hospital is to thaw fresh frozen plasma by leaving it undisturbed in a refrigerated storage unit at 4°C (2-6°C) for 24 hours (up to a maximum of 30 hours) without stacking (Non-Patent Literature 3).

[0006] In the manufacture of plasma-derived products, blood coagulation factor VIII (Factor VIII) and other components are produced from cryoprecipitate. Therefore, temperature control is required according to the purpose, so as to allow precipitation (cryopropitate) during melting and to prevent a decrease in coagulation factor activity due to thermal denaturation.

[0007] To address the above issues, there is a report (Non-Patent Literature 4) that investigated temperature control during thawing of frozen plasma bags in order to adjust the fibrinogen content recovered in cryoprecipitate. Compared to the case where the frozen plasma bag was removed from the storage temperature of -30°C and immediately thawed in a constant temperature bath at 4±2°C, the fibrinogen content in the cryoprecipitate increased to 214% when the frozen plasma bag was removed from -30°C, left to stand in a low-temperature room at 4°C for 16 hours, and then thawed in a constant temperature bath at 4±2°C. Furthermore, in studies intended for the purification of blood coagulation factor VIII (factor VIII), three thawing procedures were investigated, with and without prior standing in a low-temperature room at -2°C for 18 hours after removing the frozen plasma bag from the storage temperature of -30°C. Condition A: Place the frozen plasma bag in a plastic container with the lid open after pre-standing (bag temperature: -5°C to -2°C). Condition B: The frozen plasma bag is placed in a plastic container covered with a sheet after being allowed to stand beforehand (bag temperature is -15°C to -10°C). Condition C: No prior standing. Compared to condition A, the fibrinogen content in cryoprecipitate decreased to 66% under condition B, and compared to condition A, it decreased to 40% under condition C. On the other hand, the yield of factor VIII after the manufacturing process was not affected among the three conditions A to C.

[0008] Furthermore, Patent Documents 1 to 9 disclose technology related to thawing devices for thawing frozen plasma bags. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2001-218817 [Patent Document 2] Japanese Patent Publication No. 2007-061245 [Patent Document 3] Japanese Patent Publication No. 2009-050316 [Patent Document 4] Japanese Patent Publication No. 2013-252204 [Patent Document 5] Japanese Patent Publication No. 2016-019614 [Patent Document 6] Japanese Patent Publication No. 2017-164221 [Patent Document 7] Japanese Patent Publication No. 2018-061719 [Patent Document 8] Japanese Patent Publication No. 2023-068504 [Patent Document 9] International Publication No. 2023 / 190337 [Non-patent literature]

[0010] [Non-Patent Document 1] Japanese Society of Thrombosis and Hemostasis Glossary "Fresh frozen plasma (FFP)" Created on 2015 / 02 / 17 / Updated on 2024 / 09 / 30 (https: / / jsth.medical-words.jp / words / word-478 / ). [Non-Patent Document 2] Japanese Red Cross Society website, Pharmaceutical Information, Transfusion Procedure, Plasma Products, "1. Thawing Method of Plasma Products" (https: / / www.jrc.or.jp / mr / transfusion / procedure / plasma / ). [Non-Patent Document 3] Cryoprecipitate in-hospital preparation protocol. Journal of the Japanese Society of Transfusion Medicine and Cell Therapy, Vol. 62, No. 6, 664-672, 2016. [Non-Patent Document 4] Modulation of fibrinogen content in cryoprecipitate by temperature manipulation during plasma processing. TRANSFUSION Vol.32, 755-759, 1992. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] As mentioned above, in the manufacture of plasma-derived products, blood coagulation factor VIII (Factor VIII) and other components are produced from cryoprecipitate. Therefore, temperature control is required to allow precipitation (cryopricipitate) during the thawing of fresh frozen plasma bags, while preventing a decrease in coagulation factor activity due to thermal denaturation, and to maintain a consistent yield of cryoprecipitate.

[0012] To date, no technology has been reported for the manufacture of plasma-derived products that can control the yield of cryoprecipitate without causing a decrease in coagulation factor activity due to thermal denaturation. The technologies described in Patent Documents 1 to 9 all focus on the safe and hygienic thawing of frozen plasma bags without using hot water by immersion in a constant temperature bath. No studies have been conducted on thawing conditions for frozen plasma bags with consideration for the manufacture and yield of plasma-derived products.

[0013] One aspect of this disclosure aims to realize a technology that can control the yield of cryoprecipitate to a constant level in the manufacture of plasma-derived products without causing a decrease in coagulation factor activity due to thermal denaturation. [Means for solving the problem]

[0014] The inventors of the present invention have conducted intensive studies to solve the above problems. As a result, it has been found that the retention rate of factor VIII activity (titer) in plasma is higher as the temperature is lower, and inactivation at -0.8°C or higher is remarkable. Then, by paying attention to this phenomenon and setting the thawing conditions of frozen plasma, it has been found that denaturation of coagulation factor activity due to heat is not caused, and the yield of cryoprecipitate can be controlled constantly, leading to the completion of the present invention.

[0015] A method for thawing frozen plasma according to one aspect of the present disclosure is a temperature adjustment step of raising the surface temperature of a container storing frozen plasma from -20°C or lower to a temperature in the vicinity of -1.5°C or -1.5°C or lower, and a thawing step of thawing the frozen plasma at an outside air temperature of 2°C or higher and 5°C or lower until the average thawing rate reaches 40% or higher and 80% or lower including a method for thawing frozen plasma.

[0016] A method for producing a plasma fractionation preparation according to one aspect of the present disclosure is a temperature adjustment step of raising the surface temperature of a container storing frozen plasma from -20°C or lower to a temperature in the vicinity of -1.5°C or -1.5°C or lower, a thawing step of thawing the frozen plasma at an outside air temperature of 2°C or higher and 5°C or lower until the average thawing rate reaches 40% or higher and 80% or lower after the temperature adjustment step, and a production step of pooling the plasma components obtained in the thawing step as raw plasma and producing a plasma fractionation preparation from the raw plasma including a method for producing a plasma fractionation preparation.

[0017] A method for producing a factor VIII preparation for blood coagulation according to one aspect of the present disclosure is a temperature adjustment step of raising the surface temperature of a container storing frozen plasma from -20°C or lower to a temperature in the vicinity of -1.5°C or -1.5°C or lower, a thawing step of thawing the frozen plasma at an outside air temperature of 2°C or higher and 5°C or lower until the average thawing rate reaches 40% or higher and 80% or lower after the temperature adjustment step, A purification step is performed in which the plasma components obtained in the melting step are pooled to form raw plasma, and blood coagulation factor VIII is purified from the cryoprecipitate precipitated in the raw plasma, and A formulation process for formulating the purified blood coagulation factor VIII obtained in the purification process, This is a method for producing a blood coagulation factor VIII preparation, which includes [the specified ingredient]. [Effects of the Invention]

[0018] According to one aspect of this disclosure, in the manufacture of plasma-derived products, the yield of cryoprecipitate can be controlled to a constant level without causing a decrease in coagulation factor activity due to thermal denaturation. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows the changes in factor VIII activity over time in plasma (whole blood and apheresis). [Figure 2] This figure shows the change in the surface temperature of a bag when the ambient temperature is gradually increased: 30 hours at -3°C, 20 hours at -0.8°C, and 14 hours at 2°C. [Figure 3] This figure shows the change in the surface temperature of the bag when the ambient temperature is -3°C. [Figure 4] This figure shows the change in the surface temperature of the bag when the ambient temperature is -1.5℃. [Figure 5] This figure shows the change in the surface temperature of the bag when the ambient temperature is -0.8℃. [Figure 6] This figure shows the change in the surface temperature of the bag when the ambient temperature is 2°C. [Modes for carrying out the invention]

[0020] [Method for thawing frozen plasma] A method for thawing frozen plasma according to one aspect of this disclosure is: A temperature adjustment step in which the surface temperature of the container containing the frozen plasma is raised from below -20°C to a temperature near -1.5°C or below -1.5°C, and The process includes a thawing step, after the temperature adjustment step, in which the frozen plasma is thawed at an ambient temperature of 2°C to 5°C to an average thawing rate of 40% to 80%.

[0021] [Temperature adjustment process] In the temperature adjustment step, the surface temperature of the container containing the frozen plasma is raised from -20°C or below to -1.5°C or a temperature near -1.5°C. In this specification, "temperature near -1.5°C or below" refers to the temperature range of variation when raising the surface temperature of the container with a target temperature of -1.5°C or below, for example, between -2.2°C and -1.6°C.

[0022] The aforementioned temperature control step suppresses the decrease in coagulation activity of the frozen plasma thawed in the subsequent thawing step, thereby enabling consistent control of the cryoprecipitate yield.

[0023] Examples of frozen plasma include plasma obtained by filtering the blood of mammals such as humans through a leukocyte-removing filter and freezing it at -20°C or below (for example, -20°C, -30°C, -40°C, or -50°C or below). Examples of frozen plasma include fresh frozen plasma (FFP). The blood may be obtained by whole blood collection or by component collection.

[0024] A method for thawing frozen plasma according to one aspect of this disclosure involves thawing frozen plasma stored in a container. Examples of such containers include blood bags and blood bottles. Blood bags are preferred as such containers because they are commonly used in medical settings.

[0025] Examples of means for raising the surface temperature of a container storing frozen plasma from below -20°C to -1.5°C or a temperature near -1.5°C include controlling the ambient temperature. In this specification, "ambient temperature" refers to the temperature outside the container (outside temperature).

[0026] When raising the surface temperature of the container by setting the ambient temperature, it is preferable to raise the ambient temperature in at least two stages (for example, two, three, four, five, or more stages) and more preferable to raise the ambient temperature in at least three stages, in order to further suppress the inactivation of factor VIII activity in plasma.

[0027] An example of a temperature adjustment process with at least two stages based on the setting of the outside temperature is shown below.

[0028] A first step involves maintaining an ambient temperature of -3.0°C or higher and 0.0°C or lower (preferably -2.5°C or higher and -0.5°C or lower, more preferably -1.8°C or higher and -1.2°C or lower) for 1 hour or more and 20 hours or less; and, The second step involves maintaining an ambient temperature of -2.3°C to 0.8°C (preferably -1.8°C to 0.3°C, more preferably -1.0°C to -0.6°C) for 1 hour to 20 hours; A temperature control process, including a temperature adjustment process.

[0029] The first step involves maintaining an ambient temperature of -4.5°C or higher and -1.5°C or lower (preferably -4.0°C or higher and -2.0°C or lower, more preferably -3.5°C or higher and -2.5°C or lower) for 1 hour or more and 30 hours or less; A second step involves maintaining an ambient temperature of -3.0°C or higher and 0.0°C or lower (preferably -2.5°C or higher and -0.5°C or lower, more preferably -1.8°C or higher and -1.2°C or lower) for 1 hour or more and 20 hours or less; and, The third step involves maintaining an ambient temperature of -2.3°C to 0.8°C (preferably -1.8°C to 0.3°C, more preferably -1.0°C to -0.6°C) for 1 hour to 20 hours; A temperature control process, including a temperature adjustment process.

[0030] The first step involves maintaining an ambient temperature of -7.0°C to -3.5°C (preferably -6.5°C to -4.0°C, more preferably -5.5°C to -4.5°C) for 1 hour to 30 hours; The second stage involves maintaining an ambient temperature of -4.5°C or higher and -1.5°C or lower (preferably -4.0°C or higher and -2.0°C or lower, more preferably -3.5°C or higher and -2.5°C or lower) for 1 hour or more and 30 hours or less; A third step involves maintaining an ambient temperature of -3.0°C or higher and 0.0°C or lower (preferably -2.5°C or higher and -0.5°C or lower, more preferably -1.8°C or higher and -1.2°C or lower) for 1 hour or more and 20 hours or less; and, The fourth step involves maintaining an ambient temperature of -2.3°C to 0.8°C (preferably -1.8°C to 0.3°C, more preferably -1.0°C to -0.6°C) for 1 hour to 20 hours; A temperature control process, including a temperature adjustment process.

[0031] [Melting process] In the thawing step, after the temperature adjustment step, the frozen plasma is thawed at an ambient temperature of 2°C to 5°C (for example, 2°C, 3°C, 4°C, or 5°C) to an average thawing rate of 40% to 80%. The thawing step yields the thawed plasma components.

[0032] The average melting rate can be determined by separating the contents (plasma) stored in the container into liquid and solid components, and calculating the ratio of the liquid weight to the total weight of the contents in the container.

[0033] In order to prevent a temperature rise in the plasma components thawed in the process after the thawing step and to improve the quality of the raw plasma described later, the average thawing rate of frozen plasma in the thawing step is 40% to 80%, preferably 50% to 70%, and more preferably 55% to 65%.

[0034] To prevent a rapid rise in the temperature of the frozen plasma, the ambient temperature during the thawing process is preferably 1.5°C to 5.5°C, more preferably 2°C to 5°C, and even more preferably 2°C to 4°C.

[0035] To prevent a rapid rise in the temperature of the frozen plasma, the thawing process time is preferably between 1 hour and 30 hours, more preferably between 2 hours and 20 hours, and even more preferably between 5 hours and 10 hours.

[0036] A preferred thawing process involves thawing the frozen plasma at an ambient temperature of 2°C to 5°C to an average thawing rate of 40% to 80%.

[0037] [Method for manufacturing plasma-derived products] A method for producing a plasma-derived product according to one aspect of the present disclosure includes a manufacturing step of pooling plasma components obtained by the method for thawing frozen plasma to obtain raw plasma, and producing a plasma-derived product from the raw plasma.

[0038] Examples of plasma-derived products include albumin preparations, immunoglobulin preparations, and blood coagulation factor preparations. Examples of blood coagulation factor preparations include blood coagulation factor VIII preparations, blood coagulation factor IX preparations, and fibrinogen preparations. The method for producing the plasma-derived product is suitable for the production of blood coagulation factor preparations, and more suitable for the production of blood coagulation factor VIII preparations, in that it can suppress the inactivation of factor VIII activity in the plasma during the thawing of frozen plasma.

[0039] Completely thawed raw plasma can be obtained by recovering the plasma components from the container containing the plasma components obtained by the aforementioned frozen plasma thawing method, pooling the plasma components, etc.

[0040] The production of plasma-derived products from raw plasma can be carried out using known methods. For example, it can be produced by precipitating and fractionating the desired component using Cohn's cold ethanol fractionation method.

[0041] In order to further suppress the inactivation of factor VIII activity in the plasma, it is preferable to raise the temperature of the frozen plasma by raising the ambient temperature in at least two stages (preferably at least three stages) during the temperature adjustment step of the frozen plasma thawing method.

[0042] [Method for manufacturing blood coagulation factor VIII preparations] A method for producing a blood coagulation factor VIII preparation according to one aspect of the present disclosure includes a purification step of pooling plasma components obtained by the method of thawing frozen plasma to obtain raw material plasma, purifying blood coagulation factor VIII from cryoprecipitate precipitated in the raw material plasma, and a formulation step of preparing the purified blood coagulation factor VIII obtained in the purification step.

[0043] In order to further suppress the inactivation of factor VIII activity in the plasma, it is preferable to raise the temperature of the frozen plasma by raising the ambient temperature in at least two stages (preferably at least three stages) during the temperature adjustment step of the frozen plasma thawing method.

[0044] (purification process) In the purification step, the plasma components obtained by the thawing method of the frozen plasma are pooled to form raw plasma, and blood coagulation factor VIII is purified from the cryoprecipitate precipitated in the raw plasma. It is preferable to further purify the purified blood coagulation factor VIII by ion exchange chromatography or the like, as this yields a blood coagulation factor VIII preparation with high purity.

[0045] (Formulation process) In the formulation step, the purified blood coagulation factor VIII obtained in the purification step is formulated. For example, additives used in the manufacture of blood coagulation factor preparations (e.g., sugars, salts, surfactants, pH adjusters, etc.) can be added to the formulation.

[0046] Prior to the formulation process, it is preferable to remove the virus from the purified blood coagulation factor VIII by filtering it through a virus removal membrane. Furthermore, prior to the final formulation, it is preferable to perform a virus inactivation treatment by heating the purified blood coagulation factor VIII.

[0047] A blood coagulation factor VIII preparation manufactured by the method for manufacturing the blood coagulation factor VIII preparation described above is also included in one aspect of this disclosure. By administering the blood coagulation factor VIII preparation to a subject, the blood coagulation factor VIII in the subject's plasma can be supplemented, and bleeding in the subject can be suppressed. The blood coagulation factor VIII preparation can be used to treat hemophilia, such as hemophilia A.

[0048] The amount of purified blood coagulation factor VIII in a blood coagulation factor VIII preparation may be, for example, 250 to 1000 international units.

[0049] The dosage form of the blood coagulation factor VIII preparation may be, for example, liquid, powder (lyophilized powder, dried powder), capsule, tablet, or frozen.

[0050] The route of administration of factor VIII blood coagulation preparations may be, for example, intravenous or subcutaneous. The method of administration of factor VIII blood coagulation preparations may be, for example, by syringe or by a needle-free device.

[0051] Target populations for administration of blood coagulation factor VIII preparations include, for example, humans and non-human animals, more specifically, vertebrates such as birds and mammals. Mammals include laboratory animals such as mice, rats, rabbits, guinea pigs, rhesus monkeys, crab-eating macaques, and primates other than humans; companion animals (pets) such as dogs and cats; livestock such as pigs, cattle, goats, sheep, and horses; or humans.

[0052] The dosage and frequency of administration of blood coagulation factor VIII preparations can be appropriately selected according to the severity of symptoms, age, sex, weight, and administration method.

[0053] 〔summary〕 A method for thawing frozen plasma according to Embodiment 1 of the present disclosure includes a temperature adjustment step of raising the surface temperature of a container storing frozen plasma from -20°C or below to -1.5°C or a temperature near -1.5°C, and a thawing step of thawing the frozen plasma after the temperature adjustment step at an ambient temperature of 2°C to 5°C to an average thawing rate of 40% to 80%.

[0054] The method for thawing frozen plasma according to Embodiment 2 of this disclosure is such that, in Embodiment 1, the container may be a blood bag.

[0055] In the method for thawing frozen plasma according to embodiment 3 of this disclosure, in embodiment 1 or 2, the ambient temperature may be raised in at least two stages during the temperature adjustment step.

[0056] A method for producing a plasma-derived product according to aspect 4 of the present disclosure includes a temperature adjustment step of raising the surface temperature of a container storing frozen plasma from -20°C or below to -1.5°C or a temperature near -1.5°C; a thawing step of thawing the frozen plasma after the temperature adjustment step at an ambient temperature of 2°C to 5°C to an average thawing rate of 40% to 80%; and a manufacturing step of pooling the plasma components obtained in the thawing step to use raw plasma, and producing a plasma-derived product from the raw plasma.

[0057] In the method for producing a plasma-derived product according to Embodiment 5 of this disclosure, in Embodiment 4, the ambient temperature may be raised in at least two stages during the temperature adjustment step.

[0058] A method for producing a blood coagulation factor VIII preparation according to aspect 6 of the present disclosure includes: a temperature adjustment step of raising the surface temperature of a container storing frozen plasma from -20°C or below to -1.5°C or a temperature near -1.5°C; a thawing step of thawing the frozen plasma after the temperature adjustment step at an ambient temperature of 2°C to 5°C to an average thawing rate of 40% to 80%; a purification step of pooling the plasma components obtained in the thawing step to obtain raw material plasma and purifying blood coagulation factor VIII from cryoprecipitate precipitated in the raw material plasma; and a formulation step of preparing the purified blood coagulation factor VIII obtained in the purification step.

[0059] In the method for producing a blood coagulation factor VIII preparation according to aspect 7 of this disclosure, in aspect 6, the ambient temperature may be raised in at least two stages during the temperature adjustment step.

[0060] The embodiments of the present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to the following embodiments, and various forms are possible in terms of details. Furthermore, the present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included in the technical scope of the present invention. In addition, all references cited herein are incorporated by reference. [Examples]

[0061] [Example 1] Stability of Factor VIII activity To investigate the effect of the thawing process temperature on factor VIII activity, tubes (5 mL) containing plasma (whole blood and apheresis) were placed in a constant temperature bath, and the changes in factor VIII activity over time were observed. The results are shown in Figure 1. The temperatures shown in Figure 1 represent the set temperature of the constant temperature bath. The vertical axis of Figure 1 shows the titer retention rate, with the factor VIII activity (titer retention rate) of the plasma immediately before being placed in the constant temperature bath set to 100%.

[0062] As shown in Figure 1, the retention rate of activity was higher at lower temperatures, and inactivation was particularly pronounced above -0.8°C. Therefore, it was considered effective in retaining factor VIII activity to control the temperature increase so that the temperature of the frozen plasma as a container surface temperature is lower than -0.8°C, i.e., in this study, -1.5°C or a temperature close to -1.5°C. Furthermore, it was considered that thawing conditions that can improve the yield of factor VIII activity can be set by shortening the subsequent thawing time at higher temperatures.

[0063] [Example 2] Investigation of the time required for the melting process using a simulated bag (a bag containing plasma) In the manufacture of plasma-derived products, pooled plasma, collected from a large number of individuals, is used as the starting material, meaning that thousands to tens of thousands of donated plasma bags are used in a single batch. When proceeding with the thawing process of such a large quantity of frozen plasma bags, the surface temperature of the containers will vary to some extent from bag to bag. Therefore, when raising the surface temperature of the containers to -1.5°C or a temperature near -1.5°C, if the ambient temperature is raised by 1 degree Celsius between 2°C and 5°C, the temperature may rise to above -1.5°C. Thus, it is preferable to proceed with the thawing process by gradually raising the ambient temperature until the target surface temperature of the containers is reached.

[0064] We investigated the target temperature and required time at the end of each step in the process of gradually increasing the temperature.

[0065] First, we examined the changes in the surface temperature of the bag when the temperature was gradually increased. Figure 2 shows the changes in the surface temperature of the bag when frozen plasma was removed from a storage temperature of -20°C or lower and the temperature was gradually increased at an ambient temperature of -3°C for 30 hours, -0.8°C for 20 hours, and 2°C for 14 hours. As indicated by the arrows, there were periods of little temperature change (6-29 hours and 38-50 hours from the start of thawing), and it was considered that shortening these periods would be effective in purifying and obtaining factor VIII.

[0066] Therefore, regarding the setting temperature for the melting process, we set the target container surface temperature to -2°C, which is a temperature close to -1.5°C or lower. We then investigated the necessary time to achieve the target value (-2°C) in three stages: ambient temperature -3°C (target bag surface temperature at the end of the process is around -4.5°C; first melting process), ambient temperature -1.5°C (target bag surface temperature at the end of the process is around -3°C; second melting process), and ambient temperature -0.8°C (target bag surface temperature at the end of the process is around -2°C; third melting process).

[0067] Figure 3 shows the change in the surface temperature of the bag when the ambient temperature is -3°C. Figure 4 shows the change in the surface temperature of the bag when the ambient temperature is -1.5°C. Figure 5 shows the change in the surface temperature of the bag when the ambient temperature is -0.8°C. Figure 6 shows the change in the surface temperature of the bag when the ambient temperature is 2°C. In Figures 3 to 6, the vertical axis represents the surface temperature of the bag, and the horizontal axis represents the elapsed time (minutes) after the ambient temperature was set.

[0068] As a result, it was confirmed that the bag surface temperature increased efficiently in 2 hours during the first melting process, reaching an average value of -4.5°C (Figure 3). Therefore, a time of 2 hours for the first melting process was considered optimal.

[0069] Regarding the second melting process, the average temperature of the bag surface rose to around -3°C after 2 hours from the start of the process. However, the minimum bag surface temperature in the center of the bag storage container (basket) reached -3°C after 3 hours from the start of the process. Therefore, a duration of 3 hours was considered optimal for the second melting process (Figure 4).

[0070] Regarding the third melting step, the average temperature of the bag surface rose to -2°C after 4 hours from the start of the process, and the maximum value of the bag surface temperature was -1.5°C or lower. Therefore, a duration of 4 hours for the third melting step was considered optimal (Figure 5).

[0071] [Example 3] Investigation of the time required for a 2°C process using a simulated bag (a bag containing plasma) Based on the results of the first to third melting processes, we used a simulated bag to investigate the duration of the 2°C process based on the bag surface temperature and melting state during the 2°C process.

[0072] In the manufacture of plasma-derived products, pooled plasma, collected from a large number of individuals, is used as the starting material. However, thousands to tens of thousands of donated plasmas are used in a single batch. When thawing such a large quantity of frozen plasma bags, it is difficult to match the thawing rate, and a distribution of thawing rates exists. The thawing rate can be determined by separating the contents of the bag into liquid and solid and calculating the ratio of the liquid weight to the total weight of the contents. In an example where frozen plasma bags are thawed at 2°C, an analysis of the thawing rate and distribution ratio under conditions that do not hinder plasma recovery from the bag yields the results shown in Table 1. It was confirmed that the guideline for the thawing rate is 60%, and that approximately 85% falls within the range of 40% to 80%.

[0073] [Table 1]

[0074] Based on the above, we investigated the changes in bag surface temperature and melting state during the 2°C process after the first to third melting processes using 50 simulated bags. The 50 bags were arranged in a basket of 6 bags x 7 to 10 bags (vertical x horizontal) and placed in a constant temperature room. The 10 bags on the outer perimeter were designated as the outer bag, and the 10 bags in the center were designated as the inner bag, and the melting rate of each bag was measured.

[0075] Starting at approximately -2°C, the bag surface temperature rose for the first 5 hours, reaching an average temperature of -1°C, after which the temperature change leveled off (Figure 6). Regarding the thawing state, after 5 hours the outside / inside of the bag was 30% / 20%, after 6 hours 50% / 30%, after 7 hours 70% / 50%, and after 8 hours 80% / 60%, with the average thawing rate reaching approximately 60% after 7 hours. The bag thawing rate and distribution ratio after 7 hours are shown in Table 2. Approximately 78% fell within the 40% to 80% range, and no difference was observed compared to the case of thawing a large amount of frozen plasma bags. Therefore, a time of 7 hours for the 2°C process was considered optimal.

[0076] [Table 2]

[0077] Based on the above, suitable frozen plasma bag thawing conditions for temperature and time control (process time reduction) that can suppress the inactivation of factor VIII activity in plasma include the following: "First thawing step: ambient temperature -3°C, 2 hours," "Second thawing step: ambient temperature -1.5°C, 3 hours," and "Third thawing step: ambient temperature -0.8°C, 4 hours," until the container surface temperature reaches -1.5°C or a nearby temperature of -1.5°C or lower, followed by a "2°C step, 7 hours."

[0078] [Example 4] Comparison of manufacturing results under melting conditions and cold melting conditions of the present invention The table below shows the production results on a 3,000L scale using raw plasma for the cold-thawing conditions at 2°C (control) and the preferred thawing conditions for the frozen plasma bag described above. In the examples, the yield of cryoprecipitate (cryo) was less variable, and both the specific activity and activity yield of factor VIII were improved compared to the control.

[0079] [Table 3] [Industrial applicability]

[0080] This disclosure can be used in the manufacture of plasma-derived products.

Claims

1. The surface temperature of the container holding the frozen plasma is changed from below -20°C to between -2.2°C and -1.5°C. A surface temperature adjustment process that raises the temperature until it reaches a certain point, and After the surface temperature adjustment step, the frozen plasma is subjected to an ambient temperature of 2°C to 5°C with an average thawing rate of 40%. A melting process that melts the material down to 80% or less. Includes, In the surface temperature adjustment process, the ambient temperature is adjusted between -7.0°C and 0.8°C in at least two steps. To raise, Methods for thawing frozen plasma.

2. The method for thawing frozen plasma according to claim 1, wherein the container is a blood bag.

3. In the aforementioned surface temperature adjustment step, The first stage involves maintaining an outside temperature of -3.0°C or higher and 0.0°C or lower for a period of 1 hour or more and 20 hours or less; and, After the first stage, the temperature is between -2.3°C and 0.8°C, and is higher than the ambient temperature in the first stage. This includes at least a second stage, which involves maintaining the outside temperature at a level of 1 hour to 20 hours. A method for thawing frozen plasma according to claim 1 or 2.

4. The surface temperature of the container holding the frozen plasma is changed from below -20°C to between -2.2°C and -1.5°C. A surface temperature adjustment process that raises the temperature until it reaches a certain point. After the surface temperature adjustment step, the frozen plasma is subjected to an ambient temperature of 2°C to 5°C with an average thawing rate of 40%. A melting process that melts the material to 80% or less, and A manufacturing process in which the plasma components obtained in the melting step are pooled to form raw plasma, and a plasma-derived product is manufactured from the said raw plasma, Includes, In the surface temperature adjustment process, the ambient temperature is adjusted between -7.0°C and 0.8°C in at least two steps. To raise, A method for producing plasma-derived products.

5. In the aforementioned surface temperature adjustment step, The first stage involves maintaining an outside temperature of -3.0°C or higher and 0.0°C or lower for a period of 1 hour or more and 20 hours or less; and, After the first stage, the temperature is between -2.3°C and 0.8°C, and is higher than the ambient temperature in the first stage. This includes at least a second stage, which involves maintaining the outside temperature at a level of 1 hour to 20 hours. A method for producing a plasma-derived product according to claim 4.

6. The surface temperature of the container holding the frozen plasma is changed from below -20°C to between -2.2°C and -1.5°C. A surface temperature adjustment process that raises the temperature until it reaches a certain point. After the surface temperature adjustment step, a thawing step is performed in which the frozen plasma is thawed at an ambient temperature of 2°C to 5°C until the average thawing rate is 40% to 80%. A purification step is performed in which the plasma components obtained in the melting step are pooled to form raw plasma, and blood coagulation factor VIII is purified from the cryoprecipitate precipitated in the raw plasma, and A formulation process for formulating the purified blood coagulation factor VIII obtained in the purification process, Includes, In the surface temperature adjustment process, the ambient temperature is adjusted between -7.0°C and 0.8°C in at least two steps. To raise, A method for manufacturing a blood coagulation factor VIII preparation.

7. In the aforementioned surface temperature adjustment step, The first stage involves maintaining an outside temperature of -3.0°C or higher and 0.0°C or lower for a period of 1 hour or more and 20 hours or less; and, After the first stage, the temperature is between -2.3°C and 0.8°C, and is higher than the ambient temperature in the first stage. This includes at least a second stage, which involves maintaining the outside temperature at a level of 1 hour to 20 hours. A method for producing a blood coagulation factor VIII preparation according to claim 6.

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