A method for inactivating viruses in blood plasma by ultraviolet irradiation

Abstract

This invention provides a method for inactivating viruses in plasma using ultraviolet irradiation. The method involves adding 10–18.76 g / L of amino acids to the plasma as an inactivating protective agent, followed by inactivation under ultraviolet irradiation. This method effectively inactivates viruses in plasma while reducing the destruction of effective protein components in the plasma and minimizing the loss of coagulation factors. Furthermore, the residual amino acids in the inactivated plasma are harmless to the human body, providing safe plasma products that can be rapidly used in clinical settings.

Description

Technical Field

[0001] This invention relates to the field of virus inactivation technology for blood products, and particularly to a method for inactivating viruses in plasma by ultraviolet irradiation. Background Technology

[0002] Clinical practice has confirmed that many viruses can be transmitted through blood transfusions, causing post-transfusion viral infectious diseases. High-titer plasma containing antibodies against multiple viruses serves as the starting material for specific immunoglobulins and coagulation factors, and it primarily originates from state-approved single-donor plasma collection stations. The safety of raw plasma in my country is strictly controlled. Although each plasma sample undergoes rigorous testing procedures for viruses and other pathogens before collection, the unknown nature of viruses and limitations in testing methods mean that the absolute safety of raw plasma cannot be guaranteed.

[0003] Currently, the main methods for inactivating plasma viruses include: S / D inactivation, methylene blue photochemical treatment, psoralen photochemical treatment, and ultraviolet irradiation. S / D inactivation is mainly for frozen mixed plasma from multiple samples, using 1% TNBP and 1% Triton X-100 incubated at 30°C for 4 hours to inactivate lipid-enveloped viruses. Methylene blue photochemical treatment is a commonly used method for treating single-bag plasma, using 1μM methylene blue and 45000 lux of white fluorescent light (51) for 1 hour, or using a low-pressure sodium lamp at 200J / cm2 for 20 minutes. This method can inactivate lipid-enveloped viruses, and there is no method to completely remove methylene blue (bacterial mutagen). In order to avoid side reactions during the implementation of psoralen photochemical treatment, it needs to be carried out under anaerobic conditions, which has high environmental requirements. The principle behind ultraviolet (UV) irradiation inactivating viruses in plasma is as follows: UV radiation acts on the DNA and RNA of viruses and bacteria, causing thymine to dimerize with thymine, thymine to cytidine, and cytosine to cytosine on the DNA chain. On RNA, it induces the formation of hydrates and uridine dimers. Therefore, when bacteria or viruses are irradiated with UV radiation, their nucleoproteins and DNA strongly absorb the irradiation energy, causing DNA chain breaks and disrupting the cross-linking of nucleic acids and proteins, leading to the death of the virus or bacteria. UV radiation can be divided into three bands: Band A (320–380 nm, UVA), Band B (290–320 nm, UVB), and Band C (190–290 nm, UVC). When most bacteria and viruses receive a cumulative UV dose of 20 mJ, their inactivation rate can reach over 99%. Ultraviolet light at 253.7 nm can effectively destroy the genetic material (DNA or RNA) of microorganisms, preventing bacteria and viruses from replicating and transcribing their genetic material, thus killing bacterial pathogens and eliminating sources of infection. However, while killing bacteria and viruses, ultraviolet light also disrupts the hydrogen bonds in plasma protein molecules, affecting the integrity of the protein's secondary structure and leading to protein denaturation, inactivation, and decreased titer. All of the above plasma virus inactivation methods suffer from varying degrees of chemical reagent residue after inactivation or incomplete virus inactivation.

[0004] Amino acids are not only the structural units of proteins, but also play a crucial role in maintaining protein structure and function. For example, glycine, also known as α-aminoacetic acid, is a polar, uncharged amino acid and a non-essential amino acid, containing one amino group and one carboxyl group. Due to its small molecular size, it readily serves as a flexible linker in proteins, facilitating the stabilization of native protein structures and improving the stability of protein formulations. Arginine, a polar, positively charged amphoteric amino acid with a guanidinium group at one end, can form multiple hydrogen bonds and is widely used as a general solvent additive for protein refolding, aggregation inhibition, and column chromatography. Arginine has the ability to inhibit protein aggregation without compromising protein stability and is commonly used as a protein stabilizer. Arginine hydrochloride, as a protein aggregation inhibitor in the production of biopharmaceuticals, can be used as a stabilizer for protein drugs in liquid and freeze-dried formulations. Summary of the Invention

[0005] In view of this, the present invention aims to propose a method for inactivating viruses in plasma by ultraviolet irradiation. By adding amino acids that are harmless to the human body to the plasma, various viruses present in plasma proteins can be effectively inactivated, while maintaining the integrity of the coagulation factor structure during the inactivation process. This provides an effective and specific solution for the safe production of blood products and the safe injection of plasma.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A method for inactivating viruses in plasma by ultraviolet irradiation involves adding 10–18.76 g / L of amino acids to the plasma as an inactivating protective agent, followed by inactivation under ultraviolet irradiation.

[0008] Furthermore, the intensity of the ultraviolet irradiation is 160–180 J / m². 2 .

[0009] Furthermore, the duration of the ultraviolet irradiation is 30–60 seconds.

[0010] Furthermore, the amino acid is selected from at least one of glycine, arginine hydrochloride, and arginine.

[0011] Furthermore, the amino acid is arginine hydrochloride.

[0012] Furthermore, the method for inactivating viruses in plasma by ultraviolet irradiation includes the following steps:

[0013] S1. Mix one or more portions of plasma at 2–8°C;

[0014] S2. Measure the volume of plasma and perform ultrafiltration using a filter plate with a pore size of 1 μm at a temperature of 25±2℃ to obtain plasma filtrate;

[0015] S3. Add amino acids to the plasma filtrate, and make the final mass fraction of amino acids in the plasma filtrate 10-18.76 g / L;

[0016] S4. Measure the absorption factor of the plasma filtrate at 254 nm using an ultraviolet absorption spectrophotometer, and determine the dose range of ultraviolet irradiation based on the measured absorption factor.

[0017] Furthermore, the plasma is derived from any one of the following: human, pig, dog, rat, cow, or horse plasma.

[0018] Plasma can treat various coagulation factor deficiency diseases and is also suitable for certain immunodeficiency patients. Given the important role of plasma, it is necessary to ensure that the plasma is virus-free and that the activity of coagulation factors in the plasma is maintained. Amino acids, as non-specific stabilizers of proteins, can play a certain protective role for plasma proteins during ultraviolet (UV) inactivation. The structure, charge distribution, and ability to form multiple hydrogen bonds of amino acids can protect the hydrogen bonds of protein molecules in plasma, maintaining the integrity of protein structure during inactivation. The applicant has discovered that by adding a certain amount of amino acids during UV irradiation, the amino acids act as inactivation protectants, not only achieving the effect of UV inactivation of viruses but also effectively protecting coagulation factors in plasma from UV damage. The UV irradiation method of this invention for inactivating viruses in plasma can remove lipid-enveloped or non-lipid-enveloped viruses. The pathogenic characteristic of SARS-CoV-2 is that it has an envelope; therefore, this invention can also be used for the inactivation of SARS-CoV-2, providing a new approach for the preparation of high-titer SARS-CoV-2 plasma.

[0019] Compared with existing technologies, the method for inactivating viruses in plasma by ultraviolet irradiation according to the present invention has the following advantages:

[0020] (1) By using ultraviolet light to inactivate viruses and amino acids as inactivation protectants, the virus in plasma is effectively inactivated while the destruction of effective protein components in plasma proteins is reduced, ensuring that coagulation factors in plasma are not lost. Furthermore, the amino acids remaining in the plasma after inactivation are harmless to the human body, providing safe plasma products that can be quickly used in clinical practice.

[0021] (2) The addition of amino acids can increase the light transmittance of plasma and improve the virus inactivation effect under the same irradiation dose. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. First, it should be noted that the data in the following experimental examples were obtained by the inventors through numerous experiments. Due to space limitations, only a portion of these data is shown in the specification, and those skilled in the art can understand and implement the present invention based on this data. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various modifications or alterations to the invention, and these modifications or alterations also fall within the scope of protection of this application.

[0023] This invention discloses a method for inactivating viruses in plasma using ultraviolet irradiation. The method involves adding 10–18.76 g / L of amino acids to the plasma as an inactivating protective agent, followed by inactivation under ultraviolet irradiation. This method utilizes ultraviolet irradiation to inactivate or remove viruses and other microorganisms that may be present in plasma proteins. Furthermore, the addition of amino acids reduces the damage to effective protein components in plasma proteins, protecting clotting factors and maximizing the plasma's utility while ensuring its safety. While simply irradiating plasma with ultraviolet light can inactivate viruses, it can damage hydrogen bonds in plasma protein molecules, affecting the integrity of the protein's secondary structure and thus preventing the plasma from exerting its beneficial effects.

[0024] Specifically, the method for inactivating viruses in plasma by ultraviolet irradiation according to the present invention includes the following steps:

[0025] S1. Mix one or more portions of plasma at 2–8°C;

[0026] S2. Measure the volume of plasma and filter it using a filter plate with a pore size of 1 μm at a temperature of 25±2℃ to obtain plasma filtrate;

[0027] S3. Add amino acids to the plasma filtrate, and make the final mass fraction of amino acids in the plasma filtrate 10-18.76 g / L;

[0028] S4. Measure the absorption factor of the plasma filtrate at 254 nm using an ultraviolet absorption spectrophotometer, and determine the dose range of ultraviolet irradiation based on the measured absorption factor.

[0029] Example 1

[0030] The main reagents, raw materials, and instruments used in the experiments of this invention are as follows:

[0031] Plasma: The experimental plasma was fresh frozen human plasma, purchased from Hualan Biological Engineering Co., Ltd.

[0032] Viruses: Sindbis virus, purchased from the American Type Culture Collection Center (ATCC); porcine parvovirus (ppv), purchased from the China Institute of Veterinary Drug Control.

[0033] Cells: Vero cells used for amplifying Sindbis virus were purchased from the American Type Culture Collection Center (ATCC); PK-15 cells used for amplifying porcine parvovirus were purchased from the China Institute of Veterinary Drug Control. The experimental viruses were amplified from cells infected with the working virus strain, and the viral titer must not be lower than 6.0 μg TCID⁻¹. 50 / 0.1ml. The viral titer used in the experiment must be pre-titrated before conducting the virus inactivation experiment. If the minimum usage conditions are not met, the virus must be amplified again.

[0034] Plasma virus inactivation device: developed by Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0035] Arginine hydrochloride: analytical grade, purchased from Tianjin Tianyao Co., Ltd.

[0036] All other reagents were of analytical grade, and all other instruments were standard domestic models.

[0037] Virus inactivation and detection in plasma

[0038] Preparation of contaminated plasma: Fresh frozen human plasma was thawed at 30°C. 300 mL of the thawed plasma was placed in a clean, covered container. Arginine hydrochloride was added in the specified ratio. The indicator virus solution and plasma were then mixed at a 1:9 ratio. Sindbis virus is an enveloped indicator virus, while porcine parvovirus is a non-enveloped indicator virus. To better illustrate the effectiveness of the ultraviolet irradiation method for inactivating plasma viruses according to this invention, representative Sindbis virus and porcine parvovirus were selected as representative indicator viruses for the experiment.

[0039] Preparation of untreated plasma: Thaw fresh frozen human plasma at 30°C, take 300 mL and place it in a clean container with a lid, then add arginine hydrochloride in the specified proportion.

[0040] Inactivation process: In a biological laboratory, the inlet of a plasma virus inactivator is immersed in untreated plasma, and the outlet is led into a clean plasma collection container; the UVC light source is turned on (UVC intensity is 160-180 J / m²). 2 Once the light intensity stabilizes and the equipment reaches the set temperature of 20°C, turn on the peristaltic pump. Pump the contaminated plasma and uncontaminated plasma into the spiral quartz glass inactivation tubes respectively, ensuring that the plasma is completely irradiated to inactivate the virus throughout the process. The plasma is irradiated with ultraviolet light for 30–60 seconds.

[0041] Collect the inactivated plasma (i.e., virus-inactivated plasma) and take samples for testing. Collect the inactivated plasma (without virus) and take samples for testing.

[0042] Virus testing:

[0043] (1) The cells used for detection were prepared at a concentration of 0.5–1.5 × 10⁻⁶. 5 Add 100 μL of the solution to each well of a 96-well cell culture plate at a density of 1 / mL; incubate the cell culture plate at 37°C in a 5% CO2 incubator for 12–24 hours.

[0044] (2) The plasma samples after exposure to the virus, the virus-inactivated plasma samples, and the virus-positive control samples were serially diluted in a gradient of 10 times.

[0045] (3) Add 10 μL of the solution sequentially to columns 2 through 11 of the cell culture plate. -1 ~10 -10 Serially diluted virus was added to each well at 100 μL; columns 1 and 12 are normal cell controls;

[0046] (4) Incubate the culture plate at 37°C in a 5% CO2 incubator, observe the cell pathogenesis daily, and record the number of diseased cell wells until the cells in the cell control wells can no longer maintain normal morphology.

[0047] (5) Calculate the residual virus titer (TCID50) in each sample based on the number of lesion wells with cytopathic effect of 50% or more.

[0048] (6) According to the formula: Virus titer reduction value = zero-point control virus titer - virus titer after process treatment, calculate the virus titer reduction value of the infected plasma sample before and after virus inactivation.

[0049] Detection of coagulation factors: The activity of coagulation factors in plasma is detected using a coagulation analyzer.

[0050] Sample 1 consisted of 11 g / L of arginine hydrochloride added to contaminated plasma, mixed thoroughly, and then placed in an inactivation apparatus under a light intensity of 170 J / m². 2 The flow rate was 1.9 L / h, and the irradiation treatment lasted for 44.51 s.

[0051] Sample 2 consisted of 11 g / L arginine hydrochloride added to contaminated plasma, mixed thoroughly, and then placed in an inactivation apparatus under a light intensity of 180 J / m². 2 The flow rate was 1.9 L / h, and the irradiation treatment lasted for 44.51 s.

[0052] Sample 3 was prepared by adding 11 g / L of arginine hydrochloride to contaminated blood plasma, mixing thoroughly, and then placing it in an inactivation apparatus under a light intensity of 160 J / m². 2 The flow rate was 1.9 L / h, and the irradiation treatment lasted for 44.51 s.

[0053] The specific experimental results are shown in Tables 1 to 3.

[0054] Table 1. Results of residual virus titer detection in plasma treated with arginine hydrochloride before and after inactivation.

[0055]

[0056] Table 2. Results of coagulation factor titers before and after inactivation in Sample 2

[0057]

[0058] Table 3. Results of coagulation factor titers in uninactivated plasma (without arginine hydrochloride) before and after inactivation.

[0059]

[0060] Table 1 shows the residual virus titer of plasma treated with arginine hydrochloride before and after inactivation. Table 1 indicates that at an irradiation intensity of 160–180 J / m², the residual virus titer remained relatively stable. 2 Irradiation treatment for 30–60 seconds can effectively inactivate lipid-enveloped viruses such as Sinderby virus and non-lipid-enveloped viruses such as porcine parvovirus. Furthermore, under identical conditions, a light intensity of 160 J / m² is effective. 2 Sample 3 and light intensity of 180 J / m 2 Sample 2 showed no significant difference in inactivation effects against Sindbis virus and porcine parvovirus. Although the UV light intensity was reduced for Sample 3, it still achieved good virus inactivation. This is because UV irradiation, with the synergistic effect of amino acids, utilizes amino acids to inhibit plasma protein aggregation, effectively reducing the absorbance of plasma proteins and improving the efficiency of UV irradiation. Furthermore, by using amino acids to inhibit protein aggregation, more viruses are exposed, reducing protein shielding or encapsulation of the viruses, allowing more viruses to be exposed to UV irradiation. Therefore, when using the UV irradiation method of this invention to inactivate viruses in plasma, it not only protects clotting factors in the plasma but also reduces UV irradiation intensity and duration while ensuring the effectiveness of virus inactivation.

[0061] Table 2 shows the results of coagulation factor titers of sample 2 before and after UV irradiation inactivation. The UV irradiation intensity was 180 J / m². 2 The flow rate was 1.9 L / h, and the irradiation treatment time was 44.51 s. Table 2 shows that before and after UV irradiation, the decrease in various indicators of coagulation factors in plasma was relatively small, all within 30%, remaining within the normal range. This indicates that the addition of arginine hydrochloride during UV inactivation has a good protective effect on coagulation factors in plasma. When the UV light intensity was 160 J / m²... 2and 170J / m 2 At the same time, the coagulation factors in the plasma also remained within the normal range. Due to space limitations, this application only shows an ultraviolet light intensity of 180 J / m². 2 The result.

[0062] Table 3 shows the results of coagulation factor titers before and after inactivation in plasma without added arginine hydrochloride and virus. The ultraviolet light intensity was 180 J / m². 2 The flow rate was 1.9 L / h, and the irradiation treatment lasted for 44.51 s. As shown in Table 3, under the same conditions, due to the lack of the protective effect of arginine hydrochloride, the activity of coagulation factors in plasma was significantly reduced after ultraviolet irradiation. Except for factor IX, the titers of factors VIII and VII decreased by more than 30%.

[0063] Example 2

[0064] Comparative Example 1: After thawing frozen plasma at 30°C, the absorption factor of the original plasma was detected at 254 nm using a UV spectrophotometer.

[0065] Sample 1: After thawing frozen plasma at 30°C, the volume of plasma was measured. Ultrafiltration was performed using a filter plate with a pore size of 1 μm at a temperature of 25±2°C to obtain plasma filtrate. Arginine was added to the plasma filtrate to make the final mass fraction of arginine in the plasma filtrate 10.53 g / L. After mixing, the absorption factor of the plasma was detected at 254 nm using a UV spectrophotometer.

[0066] Sample 2: After thawing frozen plasma at 30°C, the volume of plasma was measured. Ultrafiltration was performed using a filter plate with a pore size of 1 μm at a temperature of 25±2°C to obtain plasma filtrate. Glycine was added to the plasma filtrate to make the final mass fraction of glycine in the plasma filtrate 10.53 g / L. After mixing, the absorption factor of the plasma was detected at 254 nm using a UV spectrophotometer.

[0067] Sample 3: After thawing frozen plasma at 30°C, the volume of plasma was measured. Ultrafiltration was performed using a filter plate with a pore size of 1 μm at a temperature of 25±2°C to obtain plasma filtrate. Glycine was added to the plasma filtrate to make the final mass fraction of glycine in the plasma filtrate 15 g / L. After mixing, the absorption factor of the plasma was detected at 254 nm using a UV spectrophotometer.

[0068] Sample 4: After thawing frozen plasma at 30°C, the volume of plasma was measured. Ultrafiltration was performed using a filter plate with a pore size of 1 μm at a temperature of 25±2°C to obtain plasma filtrate. Glycine was added to the plasma filtrate to make the final mass fraction of glycine in the plasma filtrate 18.7675 g / L. After mixing, the absorption factor of the plasma was detected at 254 nm using a UV spectrophotometer.

[0069] Sample 5: After thawing frozen plasma at 30°C, the volume of plasma was measured. Ultrafiltration was performed using a filter plate with a pore size of 1 μm at a temperature of 25±2°C to obtain plasma filtrate. Arginine hydrochloride was added to the plasma filtrate to make the final mass fraction of arginine hydrochloride in the plasma filtrate 10.53 g / L. After mixing, the absorption factor of the plasma was detected at 254 nm using a UV spectrophotometer.

[0070] Sample 6: Frozen plasma was thawed at 30°C, and its volume was measured. Ultrafiltration was performed at 25±2°C using a 1μm pore size filter to obtain plasma filtrate. Arginine and glycine were added to the plasma filtrate, ensuring a final mass fraction of 10 g / L for arginine and 3 g / L for glycine. After mixing, the absorbance factor of the plasma was measured at 254 nm using a UV spectrophotometer.

[0071] The specific test results are shown in Table 4.

[0072] Table 4. Results of absorption factors of different amino acids as plasma inactivation protectants

[0073] combination Types of amino acids Amino acid concentration (g / L) Absorption factor Compare with Example 1 / 0 28.1 Sample 1 Arginine 10.53 27.3 Sample 2 glycine 10.53 22.8 Sample 3 glycine 15 26.4 Sample 4 glycine 18.7675 26.4 Sample 5 Arginine hydrochloride 10.53 21.2 Sample 6 Arginine + Glycine 10g / LArg, 3g / LGly 26.2

[0074] Table 4 shows the absorption factor results of different amino acids as plasma inactivation protectants. As can be seen from Table 4, compared with other amino acids, adding arginine hydrochloride to plasma as a plasma inactivation protectant can significantly reduce the plasma absorption factor and increase the inactivation efficiency of the inactivator. The reduction in plasma absorption factor improves the efficiency of ultraviolet irradiation and enhances the transmittance of ultraviolet light through plasma, thus significantly synergistically enhancing the inactivation effect of ultraviolet light on viruses. The decrease in viral titer is related to the intensity and duration of ultraviolet irradiation. The absorption factor of the plasma filtrate was measured at 254 nm using an ultraviolet absorption spectrophotometer, and the dosage range of ultraviolet irradiation was determined based on the measured absorption factor.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for inactivating viruses in blood plasma by ultraviolet irradiation, characterized in that, 10–18.76 g / L of amino acids were added to the plasma as an inactivating protective agent, followed by inactivation under ultraviolet irradiation at an intensity of 160–180 J / m². 2 .

2. The method for inactivating viruses in plasma by ultraviolet irradiation according to claim 1, characterized in that, The duration of ultraviolet irradiation is 30-60 seconds.

3. The method for inactivating viruses in plasma by ultraviolet irradiation according to claim 1, characterized in that, The amino acid is selected from at least one of glycine, arginine hydrochloride, and arginine.

4. The method for inactivating viruses in plasma by ultraviolet irradiation according to claim 3, characterized in that, The amino acid in question is arginine hydrochloride.

5. The method for inactivating viruses in plasma by ultraviolet irradiation according to claim 1, characterized in that, The method includes the following steps: S1. Mix one or more portions of plasma at 2-8°C; S2. Measure the volume of plasma and perform ultrafiltration using a filter plate with a pore size of 1 μm at a temperature of 25±2℃ to obtain plasma filtrate; S3. Add amino acids to the plasma filtrate, and make the final mass fraction of amino acids in the plasma filtrate 10~18.76g / L; S4. Measure the absorption factor of the plasma filtrate at 254 nm using an ultraviolet absorption spectrophotometer, and determine the dose range of ultraviolet irradiation based on the measured absorption factor.

6. The method for inactivating viruses in plasma by ultraviolet irradiation according to claim 1, characterized in that, The plasma is derived from any one of the following: human, pig, dog, rat, cow, or horse plasma.

Images