WO2002056824A2

WO2002056824A2 - Pressure cycling inactivation of pathogens in biological materials used for therapeutics or vaccines

Info

Publication number: WO2002056824A2
Application number: PCT/US2001/041623
Authority: WO
Inventors: Robert A. Hess; Mark M. Manak; Sandra K. Dusing
Original assignee: Boston Biomedica, Inc.
Priority date: 2000-08-10
Filing date: 2001-08-08
Publication date: 2002-07-25
Also published as: WO2002056824A3; AU2002246493A1

Abstract

The invention is based on the discovery that viruses, bacteria or fungi present in biological materials can be inactivated without substantial loss of protein function, using high pressure at temperatures above or below room temperature (e.g., below 0 °C or above 30 °C). The new methods can be used to decontaminate recombinant protein solutions, serum, plasma, and other biological materials, which can be contaminated with viruses such as parvoviruses (e.g., human parvovirus, porcine parvovirus, and murine parvovirus) and retroviruses such as human immunodeficiency virus (HIV), herpesviruses (e.g., herpes simplex virus, HSV), flaviviruses (e.g., hepatitis C virus, HCV), reoviruses, and picornaviruses,bacteria (e.g., Bacillus, Enterococcus, Staphylococcus, Listeria, Pseudomonas) or fungi (e.g., Candida).

Description

PRESSURE CYCLING INACTIVATION OF PATHOGENS IN BIOLOGICAL MATERIALS USED FOR THERAPEUTICS OR VACCINES

FIELD OF THE INVENTION This invention relates to decontamination methods, and more particularly to high pressure cycling methods for inactivating viruses or bacteria in biological materials used for therapeutics or vaccines.

BACKGROUND OF THE INVENTION

As described in co-pending application PCT/US99/13461 , incorporated herein by reference in its entirety, a need exists for effective, rapid, and efficient methods for decontaminating recombinant protein solutions, serum, blood products, and other biological materials while retaining desired therapeutic or antigenic properties.

SUMMARY OF THE INVENTION

The invention is based upon the discovery that pathogens such as viruses, bacteria, or fungi present in biological materials can be inactivated without substantial loss of protein function, using high pressure at temperatures above or below room temperature (e.g., below 0°C or above 30°C). The new methods can be used to decontaminate recombinant protein solutions, serum, plasma and plasma derivatives, preparations derived from cell culture, human tissue, or animal tissue, and other biological materials, which can be contaminated with viruses such as parvoviruses, retroviruses including the human immunodeficiency virus (HIV), herpesviruses, flaviviruses including the hepatitis C virus, reoviruses, and picornaviruses; bacteria such as Bacillus, Enterococcus, Listeria, Pseudomonas, or Staphylococcus; and fungi such as Candida; as well as other viruses, bacteria, fungus, and other pathogens, such as those listed in PCT/US99/13461 and references cited therein, all of which are incorporated herein by reference in their entirety.

In one embodiment, the invention features a method for decontaminating a mixture that contains factor VIII. The method includes the steps of adjusting the temperature of the mixture to -20°C or lower (e.g., -20 to -40°C, or lower, such as -20, -25, -30, -35, or -40°C, or intermediate ranges); and exposing the temperature- adjusted mixture to an elevated pressure between 5,000 psi and 50,000 psi (e.g., 5,000, 10,000, 20,000, 30,000, 40,000, or 50,000, or intermediate ranges), to obtain a decontaminated mixture.

In another embodiment, the invention features a method for decontaminating a mixture that contains natural or recombinant proteins (e.g., those used for therapeutics or vaccines). The method features the step of exposing the mixture to an elevated pressure between 5,000 psi and 100,000 to obtain a decontaminated mixture. The method can also include the step of adjusting the temperature of the mixture prior to the exposing step (e.g., to a temperature of 25°C or higher, such as 25 to 40°C, 40 to 60°C, 60 to 80°C, or higher; or to a temperature of 25°C or lower, such as 15 to 25°C, 0 to 15°C, or 0 to -40°C or lower; or intermediate ranges).

Still another embodiment of the invention features a method for decontaminating a mixture contaminated with a parvovirus. The method includes the steps of adjusting the temperature of the mixture to 25°C or higher (e.g., 25 to 40°C, 40 to 60°C, 60 to 80°C, or higher; or other intermediate ranges such as 30 to 80°C), and exposing the temperature-adjusted mixture to an elevated pressure between 50,000 psi and 100,000 psi (e.g., 70,000 to 90,000 psi) to obtain a decontaminated mixture.

Yet another embodiment of the invention features a method for decontaminating a mixture contaminated with a virus, bacteria, or fungus. The method features the step of exposing the mixture to an elevated pressure between 5,000 psi and 100,000 psi (e.g., 5,000 to 25,000 psi, 25,000 to 50,000 psi, 50,000 to 80,000 psi, or 80,000 to 100,000, or other intermediate ranges appropriate for the proteins of interest) to obtain a decontaminated mixture. The method can also include the step of adjusting the temperature of the mixture prior to the exposing step (e.g., to a temperature of 25°C or higher, such as 25 to 40°C, 40 to 60°C, 60 to 80°C, or higher; or to a temperature of 25°C or lower, such as 15 to 25°C, 0 to 15°C, or 0 to - 40°C or lower; or intermediate ranges).

In any of the embodiments of the invention, the exposing step can include cycling pressure between atmospheric pressure and said elevated pressure 1 to 300 times (e.g., 5 to 100 times, 10 to 60 times, e.g., 20, 30, 40, or 50 times). The elevated pressure can be maintained, for example, on average, for less than about 60 seconds (e.g., <30 or <15 seconds) in each of the 1 to 300 cycles.

The mixture to be decontaminated can be, for example, a recombinant protein solution, blood plasma or a plasma derivative, or material derived from cell culture, human tissue, or animal tissue. The mixture can be, for example, contaminated with one or more viruses (e.g., a retrovirus (e.g., HIV), a herpesvirus, a reovirus, a flavivirus, a parvovirus, or a picornavirus), bacteria, or fungi.

The invention provides a number of advantages, including the avoidance of the use of chemical antiviral agents and scalability. Also, the application of pressure allows inactivation procedures to be carried out at temperatures significantly lower than are required by heat-only viral inactivation procedures. As a result, protein activity can partially or fully endure the new virus activation methods. The new methods can also inactivate viruses that are resistant to traditional thermal or chemical viral inactivation methods. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a plot of logs of virus inactivation vs. temperature for the inactivation of a parvovirus using a static pressure of 80,000 psi (-■-) or pulsed pressure of 80,000 psi (-♦-). FIG. 2 is a plot of fNIII activity (% relative to a non-pressure treated control) as a function of treatment pressure of human blood plasma.

FIG. 3 is a plot of fNIII activity (% relative to a non-pressure treated control) as a function of pressure (-♦-, 80,000 psi; -■-, 50,000 psi) and time at elevated pressure.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have shown that viruses can be inactivated in a biological sample such as a recombinant protein solution by application of cycled pressure. Thus, for example, virus titer can be reduced by at least several logs by treating a sample with 1 to 100 cycles of 5,000 to 100,000 psi (where pressure is maintained in each cycle for 5 to 300 seconds), at temperatures below about 0°C or above about 25°C. A more than 8-fold reduction in parvovirus titer was achieved by treating a contaminated solution with sixty cycles between atmospheric pressure and 80,000 psi at 60°C. Gram positive and gram negative bacteria can be inactivated by 5- to 8-fold following 10 cycles of treatment at 50,000 psi at -5°C. Sample volumes can be of any size. The activity of relatively stable proteins, which can withstand cycled pressures between 50,000 psi and 80,000 psi, or even higher pressures such as 100,000, can be expected to remain high after inactivation of parvoviruses and most other viruses.

Applicants have also shown that more than 70% of the activity of factor VIII. an important blood protein that is particularly unstable under various standard conditions for virus inactivation, is retained after treatment with three cycles between atmospheric pressure and 50,000 psi at either -20°C or -40°C. As indicated in Example 15 of co-pending application PCT/US99/13461, incorporated by reference in its entirety, HIV-1 and HSV-1 titers were each reduced by 6 logs by application of 5 pressure cycles (each cycle including 60 seconds at 50,000 psi and 60 seconds at atmospheric pressure) at -10°C.

Appropriate conditions for inactivating pathogens while retaining biological activities of proteins have to take into consideration the stability of the individual protein under each of the PCT conditions and the corresponding level of inactivation for each pathogen desired. Many bacteria, including Pseudomonas, E. coli, and Bacillus, and many enveloped viruses, such as retroviruses and herpesviruses. are readily inactivated upon pressurization, even under static conditions at room temperature. Some bacteria, such as Enterococcus, and non-enveloped viruses, such as MS2 and parvoviruses, are particularly resistant to inactivation and require more rigorous treatment conditions. Conditions that include either temperatures below 0°C or above 50°C, higher pressures (>50,000 psi), and pressure cycling conditions provide effective inactivation of these more resistant pathogens (see Examples). Specifically, conditions have been found that can inactivate parvovirus by 4 logs (at -35°C ) or by up to 8 logs (at 60°C) at cycles of 80,000 psi. The harsher conditions, however, can also lead to inactivation of some classes of proteins (such as several of the multimeric proteins), while other proteins are still quite stable under these conditions.

Monomeric proteins, such as alkaline phosphatase, amylase, lipase, and albumin, many recombinant proteins, as well as many of the immunoglobulins (IgA, IgG, IgM) are quite stable under pressures exceeding 50,000 psi, and at elevated temperatures. Other proteins which should also be stable under these conditions include many of the coagulation factors such as Factors VII, IX, and XIII, von Willebrand's factor, fibrin, fibrinogen, Protein C, CI inhibitor, Bovine Serum Albumin, viral subunits, growth hormones and various small peptides. ft will be possible, therefore, to achieve very efficient inactivation of viruses (>6 logs, including parvovirus) while retaining these activities.

Another class of proteins, such as, Lactose Dehydrogenase, ALT, and Factor VIII, are multimeric proteins that are particularly susceptible to inactivation using physical means such as temperature and pressure. In this patent application, wc provide examples of conditions needed to retain Factor VIII activity and still inactivate viruses. However, the more gentle conditions for preservation of this biological activity are not as effective in inactivating parvovirus. Nevertheless, more than 4 logs inactivation of these viruses can be achieved while still retaining >80% of Factor VIII activity, by exposing these proteins to cycled pressure of less than about 50,000 psi (e.g., in the range of about 30,000 psi to about 50,000). Other multimeric proteins, such as AST, GGT, and creatine kinase are intermediate in stability to pressures.

Inactivation of viruses and bacteria can be achieved under conditions that retain at least some activity of even the more labile proteins, such as Factor VIII. More rigorous inactivation conditions can be applied to preparations where more stable proteins are found, and up to 8 logs of inactivation of parvovirus can be achieved as shown in Example 1.

Conditions recited in the following example for inactivating parvovirus are expected to be even more effective in inactivating a large range of bacteria and viruses, particularly non-enveloped viruses. Similarly, conditions that preserved

Factor VIII activity will also preserve the protein activity of the more stable proteins.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 : Effects of Temperature on the Inactivation of a Parvovirus

Porcine parvovirus (PPV) was selected as representative of viruses that contaminate biological materials of interest. PPV is a small, non-enveloped virus that is highly resistant to thermal and chemical viral inactivation procedures. Conditions resulting in inactivation of PPV can also cause inactivation of most endogenous viral contaminants such as HIN HSN HCN and human B 19 parvovirus as well as most potential exogenous viral contaminants such as murine parvovirus.

A recombinant protein solution at 37°C was spiked with PPV at a ratio of 1 part virus in 100 parts solution. The spiked solution was loaded in 250 μl volumes into cut-off 1 cc disposable plastic syringes fitted at each end with a rubber syringe plunger seal and heat-sealed in polyethylene bags containing sylotherm oil (FTS

Kinetics) as a pressurization medium. Sylotherm oil can be used interchangeably with other pressurization media such as isopropanol, 70% ethanol, or 50 or 60% ethylene glycol. Samples were stored at -20°C after preparation. A pressure chamber was equilibrated to -40°C, -30°C, -10°C, +2°C, +30°C, and +60°C in a series of experiments. For each experimental temperature, a sample was removed from -20°C storage and immediately placed in the pressure chamber, where it was allowed to equilibrate to the chamber temperature for ten minutes. For the first sample of each series, 80,000 psi static pressure was applied to the sample for 30 minutes. For the second sample of the series, the sample was exposed to sixty cycles of pressure, where each cycle consisted of 30 seconds at 80,000 psi and 60 seconds at atmospheric pressure. Upon completion of the pressure treatment, the samples were stored immediately at -20°C until an endpoint assays were performed. For the viral endpoint assay, the quantity of PPV in each sample was determined by in vitro assay on PK13 cells in a 96-well plate format. Virus infection was scored on the basis of viral cytopathology and staining with a fluorescein- conjugated antibody directed against PPV. Fluorescence staining results were used to determine the tissue culture infective dose₅₀ (TCID50) titer. The viral inactivation results of this experiment are indicated in Fig. 1 by the symbol (-■-) for static pressure, and by the symbol (-♦-) for cycled pressure. The data indicate that static pressure treatment was less effective than cycled pressure treatment at most temperatures evaluated. The data also indicate that cycled pressure treatment was most effective at the lowest (i.e., -40°C and -30°C) and highest (e.g., +60°C) temperatures. Pressure cycling at +60°C provided more than an 8-log reduction in viral titer, while cycling at -30°C afforded about a 4.5 log reduction.

Protein activity was relatively unaffected by both the pressure cycling and static pressure treatment.

Example 2 - Retention of Factor VIII Activity at -40°C

Plasma samples in 0.25 ml volumes were loaded into cut-off 1 cc disposable plastic syringes fitted at each end with a rubber syringe plunger seal and heat-sealed in polyethylene bags containing 50% ethylene glycol. After sample preparation, the plasma was stored at -70°C until exposed to cycled pressure. Each sample was equilibrated to the processing temperature of -40°C for a period of 20 minutes prior to exposure to were exposed to pressure cycling treatment PCT. This treatment consisted of 50 cycles of 30 seconds at elevated pressure and 60 seconds at atmospheric pressure.

Factor VIII activity was measured by chromogenic assay (ACTICHROME® VIILC, American Diagnostica Inc.) modified for a microwell plate format.

As shown in FIG. 2, plasma treated at -40°C with fifty 30 second pulses at 80,000 psi retained less than 20% fNIII activity relative to a control plasma sample maintained at atmospheric pressure and -40°C, whereas plasma treated with 50,000 psi pressure retained approximately 70% fNIII activity.

Example 3 - Retention of Factor VIII Activity at -20°C

Plasma samples in 0.25 ml volumes were loaded into cut-off 1 cc disposable plastic syringes fitted at each end with a rubber syringe plunger seal and heat-sealed in polyethylene bags containing 50% ethylene glycol After sample preparation, the plasma was stored at -70°C until exposed to cycled pressure. Each sample was equilibrated to the processing temperature of -20°C for a period of 20 minutes prior to exposure to three cycles of either 50,000 psi or 80,000 psi of pressure for periods of time from 15 - 240 seconds.

For samples treated at 50,000 psi, Factor VIII (fNIII) was measured by chromogenic assay (ACTICHROME® VIILC, American Diagnostica Inc.) modified for a microwell plate format.

As shown in Fig. 3, plasma treated at -20°C, with three pulses of 15 seconds at 80,000 psi and 60 seconds at atmospheric pressure retained less than 30% fNIII activity relative to a control plasma sample maintained at atmospheric pressure, whereas samples treated with 30 second pulses of 50,000 psi pressure pulses retained greater than 70% of fNIII activity. These results demonstrated that short pulses of pressure at equal to or less than 50,000 psi were most effective in maintaining fVIII activity. Example 4 — Low Temperature Inactivation of Bacteria

Bacterial stocks including Bacillus cereus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were diluted in plasma and placed in polyethylene tubes in approximately 280 ml volumes and stored at -76°C prior to treatment with static or cycled pressure after a four minute pre-equilibration to the -5°C processing temperature. PCT treatment was performed at 20,000; 30,000; 40,000; and 50,000 psi using 10 cycles of two minute duration. Static pressure of 30,000 and 50,000 psi was applied for an equivalent period of time to additional plasma samples containing bacteria. Viable bacterial cell counts were determined by dilution and plating on appropriate culture medium. The application of pulses of 50,000 psi pressure resulted in a 4 - 8 log reduction of culturable viable bacteria counts for the strains of bacteria tested. Application of static pressure resulted in lesser degree of bacteria inactivation with levels ranging from less than 1 log with Enterococcus to six logs with Pseudomonas. Bacterial stocks including Bacillus cereus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were diluted in plasma and placed in polyethylene tubes in approximately 280 μl volumes and stored at -76°C prior to treatment with static or cycled pressure after a four minute pre-equilibration to the -5°C processing temperature. PCT treatment was performed at 20,000; 30,000; 40,000; and 50,000 psi using 10 cycles of two minute duration. Static pressure of 30,000 and 50,000 psi was applied for an equivalent period of time to additional plasma samples containing bacteria. Viable bacterial cell counts were determined by dilution and plating on appropriate culture medium. The application of pulses of 50,000 psi pressure resulted in a 4 to 8 log reduction of culturable viable bacteria counts for the strains of bacteria tested. Application of static pressure resulted in a lesser degree of bacterial inactivation, with levels ranging from less than 1 log with Enterococcus to six logs with Pseudomonas. The results are shown in Table 1. Attorney Docket No 07985-028 WO 1

Table 1. Inactivation of bacteria by PCT treatment

* Results are presented as colony forming units of bacteria per milliliter (CFU/ml)

Example 5 — High Temperature Inactivation of Bacteria

The experiment of Example 4 will be repeated, equilibrating the samples to temperatures in the range of -5°C to 60°C.

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A method for decontaminating a mixture that contains factor VIII, the method comprising:

(a) adjusting the temperature of the mixture to -20°C or lower; and (b) exposing the temperature-adjusted mixture to an elevated pressure between 5,000 psi and 50,000 psi to obtain a decontaminated mixture.

2. The method of claim 1 , wherein the mixture is contaminated with a virus.

3. The method of claim 1, wherein the mixture is contaminated with bacteria.

4. The method of claim 1, wherein the mixture is contaminated with fungi.

5. The method of claim 1, wherein the exposing step comprises cycling pressure between atmospheric pressure and said elevated pressure 1 to 300 times.

6. The method of claim 5, wherein said elevated pressure is maintained, on average, for less than about 60 seconds in each of the 1 to 300 cycles.

7. The method of claim 1, wherein the temperature is adjusted to between about -35°C and about -20°C in step (a).

8. The method of claim 1, wherein the mixture is a recombinant protein solution.

9. The method of claim 1, wherein the mixture comprises blood plasma or plasma derivatives.

10. The method of claim 1, wherein the mixture comprises material derived from cell culture, human tissue, or animal tissue.

1 1. A method for decontaminating a mixture that contains natural or recombinant proteins, the method comprising exposing the mixture to an elevated pressure between 5,000 psi and 100,000 psi to obtain a decontaminated mixture.

12. The method of claim 11, further comprising adjusting the temperature of the mixture to 25°C or higher prior to the exposing step.

13. The method of claim 11 , further comprising adjusting the temperature of the mixture to 25°C or lower prior to the exposing step.

14. A method for decontaminating a mixture contaminated with a parvovirus, the method comprising: (a) adjusting the temperature of the mixture to 25°C or higher; and

(b) exposing the temperature-adjusted mixture to an elevated pressure between 50,000 psi and 100,000 psi to obtain a decontaminated mixture.

15. The method of claim 14, wherein the temperature of the mixture is adjusted to between about 30°C and about 80°C in step (a).

16. The method of claim 14, wherein the elevated pressure is between about 70,000 psi and about 90,000 psi.

17. The method of claim 14, wherein the exposing step comprises cycling pressure between atmospheric pressure and said elevated pressure 1 to 300 times.

18. The method of claim 17, wherein said elevated pressure is maintained, on average, for less than about 60 seconds in each of the 1 to 300 cycles.

19. A method for decontaminating a mixture contaminated with a virus, bacteria, or fungus, the method comprising exposing the mixture to an elevated pressure between 5,000 psi and 100,000 psi to obtain a decontaminated mixture.

20. The method of claim 19, wherein the exposing step comprises cycling pressure between atmospheric pressure and said elevated pressure 1 to 300 times.

21. The method of claim 19, further comprising adjusting the temperature of the mixture to 25°C or higher prior to the exposing step.

22. The method of claim 19, further comprising adjusting the temperature of the mixture to 25°C or lower prior to the exposing step.