APPARATUS AND METHOD FOR INACTIVATIVING VIRUSES AND PATHOGENS IN CONVALESCENT PLASMA UNITS FROM RECOVERED COVID-19 PATIENTS

Abstract

The novel coronavirus COVID-19 has caused a worldwide pandemic of enormous proportions resulting in significant levels of morbidity and mortality, tremendous pressures on the healthcare system, personal freedoms and society, and an unprecedented impact on the economies of the United States and the world. There are still significant unknowns about this very contagious and deadly virus, and these unknowns are coupled with no natural immunity. A promising therapeutic strategy is the utilization/transfusion of convalescent plasma from recovered COVID-19 patients. There are, however, risks involved in such transfusions from residual virus and other adventitious viruses and bacteria. These risks can be minimized by the pathogen clearance of convalescent plasma units in a hospital setting. There is an immediate need for the rapid pathogen inactivation/clearance of convalescent plasma units from recovered COVID-19 patients.

The present invention is a physical pathogen reduction and inactivation apparatus and method for controlling or eliminating transfusion-transmittable infections in convalescent plasma from recovered COVID-19 donors. The invention inactivates both nonenveloped and enveloped viruses as well as pathogenic bacteria and parasites in units of human plasma, while retaining the potency of natural biologically-active proteinaceous products in the pathogen-reduced plasma. The invention uses critical, near-critical or supercritical fluids for viral and pathogen reduction of units of donor blood plasma in blood bags. The apparatus is in the form of a transportable mobile unit, where it can be used in hospitals, blood banks, and medical facilities.

Claims

1. A treatment for COVID-19 patients using convalescent plasma which is pathogen reduced by SuperFluids which are supercritical, near-critical and critical fluids with or without small molar quantities of polar cosolvents.

2. The treatment of claim 1 wherein the SuperFluids are nitrous oxide (N.sub.2O) and carbon dioxide (CO.sub.2).

3. The treatment of claim 2 wherein the ratio of N.sub.2O to CO.sub.2 ranges from 90% to 100% N.sub.2O, and from 10% to 0% CO.sub.2.

4. The treatment of claim 3 wherein the ratio of N.sub.2O to CO.sub.2 99% N.sub.2O to 1% CO.sub.2

5. The treatment of claim 2 wherein the SuperFluids are at a pressure of 2,000 to 5,000 psig and a temperature of 20° C. to 50° C.

6. The treatment of claim 6 wherein the SuperFluids are at a pressure of 2,500 to 3,500 psig and a temperature of 35 to 40° C.

7. The treatment of claim 6 wherein the SuperFluids are at a pressure of 3,000 psig and a temperature of 37° C.

8. A method of treating for COVID-19 patients using convalescent plasma which is pathogen reduced by SuperFluids which are supercritical, near-critical and critical fluids with or without small molar quantities of polar cosolvents.

9. The method of claim 8 wherein the SuperFluids are nitrous oxide (N.sub.2O) and carbon dioxide (CO.sub.2).

10. The method of claim 9 wherein the ratio of N.sub.2O to CO.sub.2 ranges from 90% to 100% N.sub.2O, and from 10% to 0% CO.sub.2.

11. The method of claim 10 wherein the ratio of N.sub.2O to CO.sub.2 99% N.sub.2O to 1% CO.sub.2

12. The method of claim 9 wherein the SuperFluids are at a pressure of 2,000 to 5,000 psig and a temperature of 20° C. to 50° C.

13. The method of claim 12 wherein the SuperFluids are at a pressure of 2,500 to 3,500 psig and a temperature of 35 to 40° C.

14. The method of claim 13 wherein the SuperFluids are at a pressure of 3,000 psig and a temperature of 37° C.

15. An apparatus for making multiple units of pathogen-reduced COVID-19 convalescent plasma which is pathogen reduced by SuperFluids which are supercritical, near-critical and critical fluids with or without small molar quantities of polar cosolvents.

16. The apparatus of claim 15 which comprises: (a) a pressure vessel containing plasma in a sample bag surrounded by a hydraulic fluid; (b) a pump for increasing or decreasing the volume or pressure of the hydraulic fluids surrounding the sample bag; (c) a pressure vessel containing SuperFluids in a product bag surrounded by a hydraulic fluid; (d) a pump for increasing or decreasing the volume or pressure of the hydraulic fluids surrounding the product bag; (e) a pump for introducing a SuperFluids into the product bag; (f) a pump for introducing a second SuperFluids into the product bag; (g) chillers for maintaining the SuperFluids in a liquid state; (h) heaters for maintain the temperature of the hydraulic fluids in the pressure vessels; (i) connecting lines to move fluids from the sample bag to the product bag; (j) a back-pressure regulator to contain and release pressure in the apparatus; (k) controllers for managing volumes, pressures and temperatures; and (l) a rotating carousel for processing several plasma units sequentially, once a plasma bag is processed, the plasma bag will be disengaged from the SFS feed and pressurizing source, and the carousel will be rotated, advancing the CFI-processed plasma bag for automatic removal. Subsequently, a new plasma bag will be rotated into place for processing.

17. The apparatus of claim 16 wherein the hydraulic fluid is oil or water.

18. The apparatus of claim 17 wherein the hydraulic fluid is water.

19. The apparatus of claim 16 wherein the sample and product bags are multiport plastic bags.

20. The apparatus of claim 19 wherein the multiport plastic bags are made of polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), perfluoroalkoxy alkanes (PFA) or fluorinated ethylene propylene (FEP).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows a TEM (Transmission Electron Microscopy) photomicrograph of coronavirus;

[0025] FIG. 2 shows before-and-after TEM (Transmission Electron Microscopy) photomicrographs of normal viral activity before CFI and after CFI disruption and inactivation of bacteriophage D-6 virus;

[0026] FIG. 3 shows before-and-after SEM (Scanning Electron Microscopy) photomicrographs of normal viral activity before CFI and after CFI disruption and inactivation of yeast (Saccharomyces cerevisiae);

[0027] FIG. 4 is a schematic illustration of the CFIU bench-top unit process flow diagram; and

[0028] FIG. 5 an illustration of the CFIU bench-top unit, showing the side elevation and frame.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are lipid-enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases, the largest among known RNA viruses. The diameter of the virus particles is around 120 nm.

[0030] The envelope of the virus in electron micrographs appears as a distinct pair of electron dense shells, as shown in FIG. 1 with well-defined spikes. Infection begins when the virus enters the host organism and the spike protein attaches to its complementary host cell receptor. After attachment, a protease of the host cell cleaves and activates the receptor-attached spike protein. Depending on the host cell protease available, cleavage and activation allows cell entry through endocytosis or direct fusion of the viral envelop with the host membrane.

[0031] The CFI (Critical Fluid Inactivation) technology of the present invention has the capability to physically disrupt viral particles. FIG. 2 shows transmission electron microscopic (TEM) images of stains of bacteriophage virus <D-6 before and after CFI treatment. FIG. 3 shows before-and-after SEM (Scanning Electron Microscopy) photomicrographs of normal viral activity before CFI and after CFI disruption and inactivation of yeast (Saccharomyces cerevisiae). FIGS. 2 and 3 illustrate the ability of critical fluid inactivation (CFI) to inactivate enveloped viruses and a variety of other tough microorganisms. Also, like the SD technique developed by the New York Blood Center, CFI inactivates enveloped viruses by a lipid solubilization mechanism, dissolving away the protective lipid coat.

[0032] HCV 229E is able to grow in human cell lines such as MRC-5 and produces CPE consisting of rounding and sloughing of cells. MRC5 (ATCC CCL-171) is a human lung fibroblastic cell line obtained from a normal 14-week-old male fetus. It supports the replication of a number of respiratory viruses including human coronaviruses. MRC-5 cells, 80-90% confluent, will be infected at a relatively high multiple-of-infection (MOI of 0.1 to 0.2) and the virus will be harvested 24-48 hours post infection before CPE is visible. HCV OC43 shows no cross reactivity with HCV strain 229E, and is able to grow in human cell lines such as HCT-8 (ATCC CCL-244) and produces CPE consisting of vacuolation and sloughing of cells. Mouse hepatitis virus strain MHV-A59, a mouse coronavirus, will be grown in NCTC clone 1469, a mouse liver cell line, in which it produces CPE consisting of syncytia, rounding and sloughing of cells. Human coronavirus SARS-CoV-2 will be grown in human cell lines such as Vero (ATCC CCL-81) which produces CPE consisting of rounding and detachment of cells.

[0033] The virus stocks generated above were titrated in 96 well plates by our standard TCID.sub.50 procedure on their respective host cells. Briefly, confluent monolayers of the host cells will be infected with serial log dilutions of the virus in replicates of 8. CPE will be monitored for 5-10 days and the number of wells showing CPE will be used to calculate the TCID.sub.50 by the Karber method. The duration of the assay that gives the highest titers will be optimized initially. Additionally, virus titrations will also be performed by qPCR of viral nucleic acids and ELISA and/or lateral flow assays for viral antigens in the culture supernatants in the TCID.sub.50 assay.

[0034] CFI experiments are conducted in a CFIU prototype, as shown in FIG. 4, which is a bench-top unit, designed to be easily transportable to the point of use, such as a hospital serology laboratory. The center of the CFIU prototype is a 316 stainless-steel (SS) high-pressure cylinder (9.5 cm OD×33.5 cm L) with an internal volume of approx. 800 mL containing a plasma bag on a Luer-Lok attached to a ⅛″ 316 SS high-pressure tubing rated at 8,500 psig. This tubing passes through a top flange and cap, and is secured in place with ⅛″ Swagelok fittings. The cap is secured to the vessel with 8¾″ screws torqued to 60 ft-lb. The vessel is rated for 5,000 psig at 22° C. with a hydrostatic test pressure of 6,500 psig (High Pressure Equipment Company, Erie, Pa., USA). The vessel is equipped with a pressure relief valve (PRV) set at 3,000 psig, the planned operating pressure of the prototype. The vessel is wrapped with copper heating coils originating from a circulation heater in order to maintain the vessel at operating temperature, usually ˜ 40° C.

[0035] Plasma is first introduced into the plasma bag via a 50 mL syringe attached to the ⅛″ SS tubing outside of the cylinder via a second Luer-Lok. Alternatively, as planned for commercial units, the plasma bag with plasma is connected to the first Luer-Lok and the vessel sealed before proceeding to the next operational step. After the plasma is introduced, valve V-9 is closed. All three high-pressure Isco syringe pumps are then zeroed. Warm water (˜40° C.) is then introduced into the vessel via water syringe pump C by opening V-8 and V-12 connected to the water syringe pump C. Fresh water is resupplied to the water syringe pump C via valve V-11. After coming to operating temperature, the system is pressurized to operating pressure.

[0036] The plasma is then kept at operating pressure and temperature for a specific residence time of minutes to an hour. After the designated residence time has been achieved, the plasma bag is decompressed by first opening valve V-10 and releasing pressure through the back-pressure regulator, BPR-1. The effluent SFS, now a gas, is bubbled through a liquid trap containing 10% Chlorox and then vented through a HEPA filter to the atmosphere. Simultaneous to the decompression of the plasma bag, the pressure outside of the plasma bag is reduced by running pump C in reverse. The treated plasma is then recovered.

[0037] FIG. 5 shows a design for a beta-site CIFU unit for treating COVID-19 convalescent plasma in a hospital setting. The design in FIG. 5 has the capability of processing several plasma units sequentially, using a rotating carousel design. One operation procedure is performed on each plasma unit in sequence. Once a plasma bag is processed, the plasma bag will be disengaged from the SFS feed and pressurizing source, and the carousel will be rotated, advancing the CFI-processed plasma bag for automatic removal. Subsequently, a new plasma bag will be rotated into place for processing.

[0038] The detailed description set forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not limited in scope by the specific embodiments herein disclosed. The embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

EXAMPLES

Example 1: Single-Stage CFIU™ Inactivation of Human Coronavirus-229E (HCV-229E) Virus Using 1% CO.SUB.2 .in a SFS Mixture N.SUB.2.O:CO.SUB.2.::99:1 (CFIU-II-180)

[0039] In this single-stage CFIU-II-180 experiment, FBS was used instead of human plasma to avoid neutralization of human viruses by potential antibodies in donor plasma. The sample bag was loaded with 80 mL of SFS (N.sub.2O:CO.sub.2::99:1 at ˜2,250 psig and 40° C.) followed by −60 mL of FBS sample.

[0040] The virus titration results are listed in Table 2. The spike control showed a titer of 4.94 log TCID.sub.50/mL, and the 4° C. control had a titer of 3.87 log TCID.sub.50/mL, consistent with spiking the plasma with the virus stock at a 1:10 ratio. The Virus Reduction Factor (VRF) obtained for the CFI bag product before degassing was 1.61 log TCID.sub.50.

TABLE-US-00001 TABLE 2 CFIU-II-180 - HCV-229E Virus Titration Results (6 days Post Infection) CFIU-II-180 Dilution Number of Titer (log VRF (log Sample (3.sup.u) wells +/8 TCID.sub.50/mL) TCID.sub.50) 4° C. Plasma 3 8 3.87 0.00 Control 4 7 5 2 8 1 3 8 3.87 0.00 t&T Plasma 4 5 Control 5 2 6 3 CFIU Bag 0 6 2.26 1.61 Product 1 8 2 1 3 0 Spike Control 5 8 4.94 N/A 6 7 7 4 8 1
Inactivation levels can be increased by increasing the number of transfers of plasma to SFS or stages as demonstrated in U.S. Provisional Patent Application No. 63/090,707.

Example 2: Characterization of CFI Treated Human Plasma

[0041] Changes to the composition, characteristics and functionality of the components of CFI treated plasma are identified to determine if these treatments result in changes that would lead to actual or potential adverse events upon transfusion of the respective treated plasma to recipients. The changes from the following treatments will be monitored and compared with untreated plasma. We compare: (1) physicochemical characteristics including protein aggregation and clot strength assays; (2) protein profiles by denaturing and native electrophoresis (1-D and 2-D); (3) secondary modifications of proteins (glycosylation, acylation, phosphorylation); (4) functionality of coagulation factors by assays such as PT, APTT, TT and specific single factor assays for intrinsic and extrinsic pathways; (5) vWF assays by ELISA, collagen binding assay and RIPA assay; (6) quantification by ELISA of various proteins in the clotting cascade such as fibrinogen, etc.; and (7) biochemical assays by SMAC analysis, a panel of 24 clinical laboratory parameters.

Example 3: Evaluate Antibodies to SARS-CoV-2 for Biological Activity and Potency as Well as Other Key Plasma Transfusion Biological Properties by In Vitro Immunological Assays

[0042] We evaluate the biological and activity of convalescent COVI-19 plasma treated by CFI technology for pathogen inactivation by in vitro immunological and biochemical assays. We have previously demonstrated that CFI technology has little of no impact on the bioactivity and potency of immunoglobulins. The effects of CFI N.sub.2O on a hyper-immunoglobulin at different temperatures (22 to 40° C.) and pressures (0 to 278 bars) are listed in Table 2 and compared to controls at atmospheric pressure showing little or no change in physical and potency parameters tested. We have also demonstrated that antigenicity and immunogenicity of HIV is retained after CFI inactivation.

TABLE-US-00002 TABLE 2 Effect of CFI N2O at Different Pressures and Temperatures on a Hyperimmuneglobulin NO.sub.2 HPLC- Anti- Protein ELISA bars/° C. SEC (%) Complementary (mg/ml) MEP Abs  0/22 94.7 >1.74 18.14 379.5 278/22 95.2 >1.74 17.39 370.8  0/29 101.4 >1.83 18.27 349.7 208/29 92.7 >1.77 17.65 313.8  0/40 104.3 >1.81 18.00 351.4 278/40 99.7 >1.78 17.84 385.4

Example 4: Virus Neutralization Assay for Antibody Activity and Potency

[0043] Virus neutralization assays are performed using the widely used and accepted classical infectivity titration assays. This assay is considered as the definitive proof in vitro of the functionality of an antibody preparation. In this assay, serial dilutions of the plasma/antibody are incubated in the presence of 100 TCID.sub.50 of the virus at 37 C for 1 hour and then added to the host cell monolayers in replicates of 8 in 96 well plates. The cells are monitored for CPE for the required number of days needed for highest sensitivity and the neutralization titers are calculated by the Karber method as the dilution at which CPE is inhibited in 50% of the time. Alternately, qPCR for viral RNA is performed on the culture supernatants at earlier time points to shorten the duration of the assay using published methods. The neutralization titers of the CFI treated plasma preparations are compared with those of the untreated counterparts.

Example 5: ELISA for Preservation of Antibody Titers

[0044] The CFI treated plasma samples are tested for the preservation of antibody titers by ELISA against S glycoprotein of the virus. Although patients can develop antibodies against multiple viral antigens, the focus is on the antibodies to S glycoprotein since these antibodies have the ability to prevent the binding of the virus to the host cell receptor thereby eliminating infectivity of the virus. ELISAs are performed against both the full-length S glycoprotein and the Receptor Binding Domain (RBD) by published methods (Amanat et al, 2020). In addition to detection of IgG, we also test the samples using secondary antibodies to IgM and IgA and correlate the results with neutralization titers to determine the role that these antibody classes play in protection and confirm that the CFI treatment preserves these activities as well. Additional methods such as western blotting are used, if necessary. CFI treated convalescent COVID-19 plasma with an antibody integrity and potency at least 90% of untreated convalescent COVID-19 plasma is recommended.

[0045] Thus, various embodiments of the present technology been described, and said embodiments are capable of further modification and variation by those skilled in the art. Accordingly, it is intended that the examples and the description be intended for illustration purposes only and that the inventions set forth in the claims shall encompass variations and equivalents.