WO2000018969A1 - Procede selectif d'inactivation de virus dans des compositions biologiques - Google Patents

Procede selectif d'inactivation de virus dans des compositions biologiques Download PDF

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Publication number
WO2000018969A1
WO2000018969A1 PCT/US1999/021245 US9921245W WO0018969A1 WO 2000018969 A1 WO2000018969 A1 WO 2000018969A1 US 9921245 W US9921245 W US 9921245W WO 0018969 A1 WO0018969 A1 WO 0018969A1
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preparation
composition
ethyleneimine oligomer
solution
washing
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PCT/US1999/021245
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English (en)
Inventor
Clark Mcwhorter Edson
Andrei A. Purmal
Samuel K. Ackerman
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Pentose Pharmaceuticals, Inc.
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Priority to JP2000572416A priority Critical patent/JP2002525131A/ja
Priority to CA002345230A priority patent/CA2345230A1/fr
Priority to AU64970/99A priority patent/AU6497099A/en
Priority to EP99952914A priority patent/EP1115889A4/fr
Publication of WO2000018969A1 publication Critical patent/WO2000018969A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2796/00—Viruses not covered by groups C12N2710/00 - C12N2795/00

Definitions

  • This invention relates to methods and compositions for the selective inactivation of animal viruses in biological compositions, such as blood.
  • blood transfusion Following traumatic injury (or during surgery), an organism may require a blood transfusion to prevent death due to blood loss. In humans and certain domesticated animals, blood transfusion has enabled the survival of injured individuals who would otherwise have died from blood loss.
  • Blood proteins include antibodies, complement proteins, and proteins involved in the blood clotting cascade.
  • each of the different types of blood cells plays a unique role in maintaining the health of the organism.
  • Red blood cells for instance, are essential for the transport of oxygen and carbon dioxide gases to and from the cells of a multicellular organism.
  • Another type of blood cell, a platelet is involved in initiating blood clotting; thrombocytopenia patients have a platelet deficiency and are prone to bleeding disorders.
  • One caveat in using blood transfusions is the danger of transmitting blood-borne viruses from donor blood to a recipient.
  • viral diseases e.g., hepatitis A, B, and C, acquired immunodeficiency syndrome, and cytomegalovirus infections
  • Screening donor blood for viral markers can help reduce the transmission of viruses to recipients, but many screening methods are directed to only a few discrete viruses and are therefore incomplete or less than 100% sensitive.
  • a number of agents have been developed that are capable of inactivating viruses found in blood, as well as viruses found in other biological compositions, such as mammalian and hybridoma cell lines, products of cell lines, milk, colostrum, urine, and sperm.
  • ethyleneimine monomer and ethyleneimine oligomers are very effective viral inactivating agents. These agents are themselves toxic, and must therefore be rendered non-toxic before a product, such as blood or milk proteins, may be used clinically.
  • a viral inactivating compound such as ethyleneimine dimer, is added to a biological composition to inactivate infectious viruses that might be present in the composition.
  • a quenching agent is then added to inactivate the ethyleneimine dimer that remains after viral inactivation has taken place.
  • the end result is a biological composition that is relatively free of infectious viruses, but that is contaminated with quenched inactivating agent and with quenching agent.
  • the present invention features a method for inactivating viruses in purified mammalian a-nucleated cell preparations, and a method for removing a virus inactivating agent from a treated biological composition by washing with an inert solution.
  • the invention features a method of inactivating animal viruses in a preparation of purified mammalian a-nucleated cells that includes contacting the preparation with a selective ethyleneimine oligomer inactivating agent under viral inactivating conditions for a period of time sufficient to inactivate at least some of the animal viruses in the preparation.
  • the cells are red blood cells or platelets, and the ethyleneimine oligomer is PEN102.
  • the animal viruses in the preparation are inactivated, or preferably at least 98% of the animal viruses in the preparation are inactivated.
  • the mammalian cell is from a mammal that is a human, a non-human primate, a domesticated mammal, or an endangered mammal.
  • the method further includes washing the contacted preparation with a solution that does not quench the ethyleneimine oligomer, where the washing reduces the concentration of the ethyleneimine oligomer in the washed preparation.
  • the concentration of the ethyleneimine oligomer in the washed preparation is at or below a concentration of the ethyleneimine oligomer that is toxic.
  • the solution is sterile unbuffered saline and the washing is manual.
  • the washing includes includes the steps of: (i) layering the contacted preparation on a mesh having pores with diameters smaller than the diameters of the a-nucleated cells; and (ii) continuously flowing the solution that does not quench the ethyleneimine oligomer over the contacted preparation.
  • the washing includes the steps of: (i) adding a volume of the solution that is at least three times the volume of the preparation; and (ii) removing the solution from the preparation. Preferably, the washing is repeated at least two times. In another embodiment of the first aspect, the washing is automated.
  • a container containing the contacted preparation is in a machine that performs the following steps under sterile conditions: (i) pumping the preparation out of the container; (ii) diluting the preparation with the solution that does not quench the ethyleneimine oligomer; (iii) removing the solution from the preparation, wherein the solution is discarded; and (iv) pumping the preparation back into the container.
  • the machine performs steps (ii) and (iii) at least two times.
  • the method further includes quenching the contacted preparation with a quenching agent.
  • the quenching agent may be soluble or may be immobilized on a solid-phase support.
  • the invention features a method for selectively inactivating animal viruses in a biological composition that includes the steps of: (a) contacting the composition with a selective ethyleneimine oligomer inactivating agent under viral inactivating conditions for a period of time sufficient to inactivate at least some of the animal viruses in the composition; and (b) washing the composition with a solution that does not quench an ethyleneimine oligomer, wherein the washing reduces the amount of the ethyleneimine oligomer in the composition.
  • the concentration of the ethyleneimine oligomer in the washed composition is at or below a concentration of the ethyleneimine oligomer that is toxic.
  • the solution is sterile unbuffered saline and the washing step is automated.
  • a container containing the contacted composition in the washing step, is in a machine that performs the following steps under sterile conditions: (i) pumping the composition out of the container; (ii) diluting the composition with the solution that does not quench the ethyleneimine oligomer; (iii) removing the solution from the composition, wherein the solution is discarded; and (iv) pumping the composition back into the container.
  • the machine performs steps (ii) and (iii) at least two times.
  • the washing step includes: (i) adding a volume of the solution that is at least three times the volume of the composition; and (ii) removing the solution from the composition. Preferably, the washing step is repeated at least two times.
  • the composition includes a mammalian cell and the washing step includes: (i) layering the contacted composition on a mesh having pores with diameters smaller than the diameter of the mammalian cell; and (ii) continuously flowing the solution that does not quench the ethyleneimine oligomer over the contacted composition.
  • the mammalian cell may be an a-nucleated cell (e.g., a platelet or a red blood cell).
  • the composition is a cell-free composition and the washing step is manual.
  • purified is meant a preparation that contains, by volume, at least 50%), more preferably, at least 70%, more preferably at least 85%, even more preferably at least 95%, and most preferably, at least 98% of the indicated component.
  • a purified preparation of red blood cells contains at least 50% by volume red blood cells.
  • ethyleneimine oligomer is meant a compound having (1) an aziridino moiety or a halo-hydrocarbon-amine moiety, and, preferably, (2) two or more nitrogen atoms separated by hydrocarbon moieties. These compounds are also referred to as “inactivating agents,” or “selective inactivating agents.”
  • PEN 102 One preferred ethyleneimine oligomer of the invention is PEN 102, which has the following formula:
  • a second preferred ethyleneimine oligomer of the invention is PEN 103, which has the following formula:
  • An inactivating agent has "selectivity" for nucleic acids or “selectively' reacts with nucleic acids if the comparative rate of reaction of the inactivating agent with nucleic acids is greater than the rate of reaction with other biological molecules, e.g., proteins, carbohydrates or lipids.
  • nucleic acid is meant both DNA and RNA, both single and double stranded.
  • “Inactivating,” “inactivation,” or “inactivate,” when referring to nucleic acids means to substantially eliminate the template activity of DNA or RNA, for example, by destroying the ability to replicate, transcribe or translate a message.
  • the inhibition of translation of an RNA molecule can be determined by measuring the amount of protein encoded by a definitive amount of RNA produced in a suitable in vitro or in vivo translation system.
  • viruses the term means diminishing or eliminating the number of infectious viral particles measured as a decrease in the infectious titer or number of infectious virus particles per ml. Such a decrease in infectious virus particles is determined by assays well known to a person of ordinary skill in the art.
  • “Viral inactivating conditions” refers to the conditions under which the viral particles are incubated with the selective ethyleneimine oligomer inactivating agents of this invention, including, for example, time of treatment, pH, temperature, salt composition and concentration of selective inactivating agent so as to inactivate the viral genome to the desired extent.
  • Viral inactivating conditions are selected from the conditions for selective modification of nucleic acids described in U.S. Patent Application Serial No. 08/855,378, hereby incorporated by reference.
  • “inactivate at least some of the animal viruses” is meant that at least
  • the viruses in the treated preparation are inactivated, preferably at least 70%) of the viruses are inactivated, more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95%, still more preferably, at least 99%, and most preferably, 100% of the viruses in the treated preparation are inactivated.
  • the number of viruses in a preparation may be measured by the number or titer of infectious viral particles per ml of preparation. Such a measurement may be accomplished by a variety of well known virus titer assays.
  • animal virus is meant a virus capable of infecting a cell from an animal.
  • Animal viruses may be DNA or RNA viruses, and may be enveloped or non-enveloped viruses or viroids.
  • animal viruses include, without limitation, poxviruses, herpes viruses, adenoviruses, papovaviruses, parvoviruses reoviruses, orbiviruses, picomaviruses, rotaviruses, alphaviruses, rubiviruses, influenza viruses, type A and B, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumo viruses, rhabdo viruses, lyssaviruses, orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhioviruse
  • biological composition is meant a composition containing cells or a composition containing one or more biological molecules, or a composition containing both cells and one or more biological molecules.
  • Cell-containing compositions include, for example, mammalian blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood plasma, platelet-rich plasma, semen, placental extracts, mammalian cell culture or culture medium, products of fermentation, and ascites fluid.
  • Biological compositions may also be cell-free, and contain at least one biological molecule.
  • biological molecule any class of organic molecule normally found in living organisms including, for example, nucleic acids, polypeptides, post- translationally modified proteins (e.g., glycoproteins), polysaccharides, and lipids.
  • Biological molecule-containing biological compositions include, for example, serum, blood cell proteins, blood plasma concentrate, blood plasma protein fractions, purified or partially purified blood proteins or other components, a supernatant or a precipitate from any fractionation of the plasma, purified or partially purified blood components (e.g., proteins or lipids), mammalian colostrum, milk, urine, saliva, a cell lysate, cryoprecipitate, cryosupernatant, or portion or derivative thereof, compositions containing proteins induced in blood cells, and compositions containing products produced in cell culture by normal or transformed cells (e.g., via recombinant DNA or monoclonal antibody technology).
  • an “a-nucleated cell” is meant a cell which, when mature, lack
  • a solution that does not quench an ethyleneimine oligomer is meant a solution that does not contain a quenching agent (e.g., a thiophosphate or a thiosulfate).
  • a quenching agent e.g., a thiophosphate or a thiosulfate.
  • a quenching agent when contacted with an ethyleneimine oligomer, renders the contacted ethyleneimine oligomer non-toxic.
  • Preferred solutions that are incapable of reacting with an ethyleneimine oligomer are unbuffered saline and water.
  • a “quenching agent” is meant a thiophosphate or a thiosulfate, or a compound containing a thiophosphate or a thiosulfate that, when contacted with an ethyleneimine oligomer, is capable of rendering the contacted ethyleneimine oligomer non-toxic.
  • domesticated mammal any non-human mammal that a human maintains. For example, cows, donkeys, mules, elephants, horses, llamas, camels, goats, sheep, reindeer, dogs, cats, pigs, ferrets, rabbits, mice, rats, hamsters, and guinea pigs are domesticated animals.
  • any mammal that a human keeps as a pet such as a pet skunk or a pet wolf, is also a domesticated mammal.
  • an endangered mammal is meant a mammal that is included on the Endangered Species List compiled by the U.S. Fish and Wildlife Service as of September 25, 1998.
  • red blood cell preparations are used to transfuse a recipient, without prior inactivation of viruses.
  • the methods and compositions of the present invention allow the inactivation of viruses in red blood cell preparations prior to their use in transfusions.
  • the inactivating agents described herein are selective for the nucleic acids that are a major component of viruses
  • viral nucleic acid can be selectively inactivated over the other molecules (e.g., proteins and lipids) present in the red blood cell preparation.
  • the invention features an in vitro method for removing an ethyleneimine oligomer from a biological composition following virus inactivation without using a quenching agent.
  • This method results in a biological composition that is relatively free not only of contaminating viruses, but also relatively free of quenched (i.e., non-toxic) ethyleneimine oligomer and unreacted quenching agent.
  • FIG. 1 is a schematic diagram showing the chemical reactions that take place during the post-column derivatization of PEN 102 or quenched PEN 102.
  • Fig. 2 is a schematic flow diagram showing the post-column reaction hardware used in the HPLC analysis of ethyleneimine oligomers.
  • Fig. 3 is the HPLC elution profile of PEN102.
  • Fig. 4 is a bar graph showing the distribution of PEN 102 between the red blood cell component and the plasma component of fresh baboon whole blood (spun hematocrit (HCT) of 38%) following treatment with 12 mM (1000 ⁇ g/ml; 0.1% v/v) PEN102 for 6 hours at 20°C.
  • HCT pun hematocrit
  • Fig. 5 is a bar graph showing the effectiveness of manual washing of PEN 102 from the red blood cell fraction of whole baboon blood (spun HCT 38%) following treatment with 12 mM PEN102 for 6 hours at 20°C.
  • the number of manual washes of the RBC fraction with nonbuffered saline (1:10 v/v) for 10 min. at 20°C is indicated, and residual PEN 102 in the RBC fraction is shown as a percentage.
  • Fig. 6 is a bar graph showing the effectiveness of manual washing of PEN 102 from the red blood cell fraction of whole baboon blood (SPUN HCT 38%) following treatment with 1000 ⁇ g/ml PEN102 for 6 hours at 20°C.
  • the number of manual washes of the RBC fraction with nonbuffered saline (1 :10 v/v) for 10 min. at 20°C is indicated, and residual PEN102 in the RBC fraction is shown as ⁇ g of PEN102 per ml of RBC.
  • Fig. 7 is a bar graph showing the quenching of the ethyleneimine dimer PEN 102 (6mM) from human blood (plasma (gray bar) and red blood cells
  • ethyleneimine oligomer inactivating agents For example, most mature mammalian red blood cells, unlike those of other vertebrate animals, lack nuclei and, hence, lack nucleic acid. Thus, treatment of the cells with an ethyleneimine oligomer inactivating agent that inactivates nucleic acids allows for the selective inactivation of the nucleic acid of any viruses contaminating the red blood cell preparation, while leaving the red blood cells unaffected. Accordingly, as we demonstrate below, ethyleneimine oligomer-mediated inactivation of nucleic acids in red blood cell preparations does not affect the in vivo longevity of the cells.
  • the invention also provides a method for removing the ethyleneimine oligomer from the treated biological composition (e.g., blood), prior to use of the composition by repeatedly washing the composition with a solution that does not quench the ethyleneimine oligomer (e.g., sterile unbuffered saline).
  • a solution that does not quench the ethyleneimine oligomer e.g., sterile unbuffered saline.
  • the biological composition is a composition containing cells (e.g., sperm)
  • the treated cells may be washed by repeated steps of resuspension in a solution that does not quench an ethyleneimine oligomer and isolating the cells by centrifugation.
  • the treated milk proteins may be, for example, diluted with a solution that does not quench an ethyleneimine oligomer, and then dialyzed to remove the ethyleneimine oligomer.
  • the method of the present invention allows the generation of a biological composition free of both virus and quenching agent.
  • the ethyleneimine oligomer inactivating agents of the present invention are compounds having (1) an aziridino moiety or a halo-hydrocarbon-amine moiety, and, preferably, (2) two or more nitrogen atoms separated by hydrocarbon moieties.
  • an ethyleneimine oligomer may have one of the following five formulas: (1) CH 2
  • Ethyleneimine oligomers can also be substituted so long as this does not eliminate the essential property of the ethyleneimine (i.e., the inactivation of nucleic acids).
  • the ethyleneimine oligomers are substituted with halogens and have the general formula ⁇ -Hal-(CH 2 -CH 2 -NH) n H.
  • the "n" is an integer between 2-5, inclusive.
  • the ethyleneimine oligomer inactivating agents of the present invention also include both aziridino compounds and halo-hydrocarbon-amine compounds.
  • the aziridino compounds have the formula:
  • each of R,, R 2 , R 3 , R 4 , R 6 , R 7 , and R 8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R l5 R 2 , R 3 , R 4 , R 6 , R 7 , and R 8 cannot all be H;
  • R 5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive;
  • X is a pharmaceutically acceptable counter-ion; and
  • n is an integer between 2 and 10, inclusive.
  • the halo-hydrocarbon-amine compounds can have the formula ⁇ -X,- [R r N + (R 2 , R 3 )-] n R 4 (X 2 ) n > where X, is Cl or Br; R j is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R 2 , R 3 , and
  • R 4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R 2 , R 3 , and R 4 cannot all be H when R, contains 2 carbon atoms;
  • X 2 is a pharmaceutically acceptable counter-ion; and
  • n is an integer between 2 and 10, inclusive.
  • these compounds can have the formula ⁇ -X,-CH 2 CH 2 - N + H ⁇ HR,- ⁇ , R 4 )-] n R 5 (X 2 " ) n+ i > where X ! is Cl or Br; each of R l9 R 3 , R 4 , and R 5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R 2 is a divalent hydrocarbon moiety having 3 or 4 carbon atoms; X 2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
  • These compounds can be prepared by the aziridine-initiated oligomerization of an halo-hydrocarbon- amino compound, followed by conversion of the aziridino group to the corresponding halide compound.
  • red blood cells e.g., lymphocytes, neutrophils, and platelets
  • biological molecules e.g., clotting factors and complement
  • whole blood may be separated into the following components: (1) the red blood cell (RBC) portion (which includes a small portion of the white blood cells) and (2) the plasma (which also includes a small portion of the white blood cells).
  • RBC red blood cell
  • Apheresis System (commercially available form Haemonetics Corp., Braintree, MA) may be used to isolate red blood cells from whole blood. It should be noted that this system may also be used to separate other a-nucleated cells (e.g., platelet cells) from whole blood.
  • a-nucleated cells e.g., platelet cells
  • ethyleneimine oligomers are useful compounds for the selective inactivation of viral nucleic acids, their inherent alkylating abilities may render them toxic to most nucleated cells. Thus, prior to introduction of the treated red blood cells into the recipient animal, it is desirable to remove the ethyleneimine oligomer from the cells, or at least reduce the concentration of the ethyleneimine oligomer to a level that is non-toxic. Of course, if the cells or biological molecules are to be used in vivo, the washing step, in addition to the ethyleneimine oligomer treatment step, must be conducted under sterile conditions.
  • PEN 102 the toxic level for the ethyleneimine oligomer used in the studies described below, PEN 102, is greater than 1 ⁇ g/ml (i.e., a concentration of 1 ⁇ g/ml PEN 102 or less is non-toxic).
  • the toxic levels of other ethyleneimine oligomers may be readily assessed using the murine lymphoma mutagenesis assay.
  • the goal of the washing step is to reduce the concentration of the ethyleneimine oligomer in the desired treated biological composition to a level at or below that determined to be non-toxic.
  • One method to remove ethyleneimine oligomer compounds from a treated red blood cell preparation is to subject the cells to repeated washings with nonbuffered sterile saline (i.e., sterile 0.9% NaCl). Following each washing step, a sample of the biological composition being treated (and washed) may be tested for the presence of the ethyleneimine oligomer at a concentration higher than that previously determined to be toxic. If the concentration is found to be at a toxic level, at the very minimum, at least one additional washing step should be performed prior to the in vivo use of the treated composition. As a safety measure, once a non-toxic level of an ethyleneimine oligomer is achieved, an additional washing step is preferably performed prior to the in vivo use of the treated composition.
  • red blood cells following treatment of whole blood
  • the treated whole blood is diluted with approximately 3X volume of sterile 0.9% NaCl (i.e., 15 ml saline is added to 5 ml blood).
  • the packed RBC volume is resuspended in approximately 9X volume of sterile 0.9% NaCl, and allowed to mix (under gentle mechanical agitation) for 10 minutes at 22°C.
  • RBC component is then isolated by centrifugation, and the washing step with 9X volume of sterile saline is repeated until the concentration of the ethyleneimine oligomer in the RBC component is at or below the concentration determined to be non-toxic (as determined, for example, using the mouse lymphoma mutagenicity assay described below) .
  • isolated platelets are treated with a concentration of ethyleneimine oligomer for an amount of time and at an incubation temperature sufficient to inactivate at least some of the viruses in the platelet preparation.
  • the platelets are next repeatedly washed in at least 4X volume of sterile saline solution until the concentration of the ethyleneimine oligomer is at or below the concentration determined to be non-toxic.
  • the cells may be collected in mesh having pores of a diameter smaller than the cells' diameter, and then washed under a continuous flow of a solution that does not quench an ethyleneimine oligomer for a period of time sufficient to lower the concentration of the ethyleneimine oligomer in the cells to a non-toxic level.
  • the ethyleneimine oligomer-treated cells may collected in a mesh bag having pores of a diameter smaller than the cells' diameter. The bag may then be repeatedly dipped in a solution that does not quench the ethyleneimine oligomer until the concentration of the ethyleneimine oligomer in the cells is reduced to a non-toxic level.
  • washing steps described in the following examples are manual washings under sterile conditions, it will be understood that automated washing may be employed to free a biological composition from an ethyleneimine oligomer.
  • a machine may be designed to wash ethyleneimine oligomer treated cells under sterile conditions.
  • purified platelets may be treated with ethyleneimine oligomer under viral inactivating conditions for a period of time sufficient to inactivate at least some of the viruses in the platelet preparation.
  • This treatment step may be performed by combining the platelets with the ethyleneimine oligomer in a sterile container, such as a sterile plastic bag.
  • the bag may then be attached to the machine such that machine can, under sterile conditions, pump the cells out of the bag (and, additionally rinse the bag with sterile 0.9% NaCl).
  • the machine may then dilute the platelets with sterile saline, gently mix the platelets for a desired time at a desired temperature, collect the platelets by centrifugation, discard the "used” sterile 0.9% NaCl, and add "fresh” 0.9% NaCl, and repeat the mixing- centrifugation-discarding process for a desired number of times. After the final collection of the platelets by centrifugation, the platelets may be resuspended in "fresh” 0.9%) NaCl, or in another desired solution (e.g., blood), and returned to the original container. Platelets thus virally inactivated and washed may be used immediately, stored, or frozen as desired.
  • ethyleneimine oligomers that render them able to inactivate viral nucleic acids also enable them to damage and/or induce mutations in the genomic DNA of mammalian cells.
  • a ethyleneimine oligomer-treated biological composition may be used in vivo (or in vitro, in, for example, the case of treated sperm for in vitro fertilization)
  • the mouse lymphoma mutagenicity assay is one method to determine the toxicity concentration level of a particular ethyleneimine oligomer.
  • This assay uses a murine lymphoma cell which is heterozygous at the thymidine kinase (TK) locus (i.e., TK +/-) grown in the presence of the toxin, 5- trifluorothymidine (TFT), to screen different concentrations of the particular ethyleneimine oligomer.
  • TK +/- cells and TK -/- cells are viable in normal culture media; however, in the presence of TFT, only the TK -/- cells will grow because the TK +/- cells will incorporate the toxic TFT into their DNA.
  • TK+/- murine lymphoma cells are exposed to a toxic concentration of an ethyleneimine oligomer, they may undergo a single-step forward mutation to a TK-/- genotype, enabling them to grow in the presence of TFT.
  • a concentration of an ethyleneimine oligomer which does not result in the growth of TK +/- murine lymphoma cells in the presence of 5- trifluorothymidine (TFT) is non-toxic.
  • mutagenicity test we used below to screen the toxicity level of the ethyleneimine oligomer PEN102 was performed by Convance Laboratories Inc. (Vienna, VA). L5178Y murine lymphoma cells (which are heterozygous at the
  • TK locus grown in the presence of 5-trifluorothymidine (TFT) were exposed to different concentrations of PEN 102.
  • TFT 5-trifluorothymidine
  • a concentration of 1 ⁇ g/ml PEN 102 did not allow the growth (i.e., the forward mutation) of L5178Y TK +/- cells in the presence of 5-trifluorothymidine (TFT).
  • a concentration of less than or equal to 1 ⁇ g/ml PEN102 is non- toxic
  • a PEN102-treated biological composition e.g., a treated RBC preparation
  • a PEN102-treated biological composition e.g., a treated RBC preparation
  • Ethyleneimine oligomers are routinely analyzed by cation exchange HPLC. Because ethyleneimine oligomers do not contain a chromophore, the following method uses post-column derivatization which reacts only with primary amino groups, thereby eliminating complications of multiple reaction sites (ethyleneimine oligomers contain only one primary amino group) or interfering compounds.
  • a schematic of the reactions that take place during HPLC of PEN102 and quenched PEN102 is shown on Fig. 1. The method has been determined to be linear within the range of 10-230 ng of PEN 102. a. Starting materials for HPLC analysis
  • FIG. 2 A schematic diagram of the HPLC post column reaction hardware is shown on Fig. 2.
  • Jasco FP-920 intelligent Fluorescent detector (Jansco Inc., Easton, MD) Pickering Laboratories PCX 3100 post-column reaction module Alkion cation-exchange column 4 x 150 mm; Pickering Laboratories Catalog No. 9410917
  • OPA o-pthalaldehyde
  • This method uses cationic exchange chromatography with post column derivatization of ethyleneimine oligomer primary amino groups and fluorescent detection.
  • the HPLC elution profile of PEN 102 is shown on Fig. 3. 1. Prepare samples in potassium phosphate eluent, pH 6. Dilute 1 ⁇ l
  • PEN 102 at a final concentration of 1: 8 x 10 4 with the eluent.
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • the desired blood component e.g., red blood cells
  • virally inactivated plasma may be used to purify virus-free plasma proteins, such as blood clotting factors or albumin.
  • Table I shows the results of the RBC biochemistry tests.
  • the blood was separated into a plasma fraction and a red blood cell (RBC) fraction.
  • RBC red blood cell
  • the concentration of PEN102 in the plasma and RBC fractions was determined using the HPLC analysis described above. The concentration of PEN 102 in the plasma fraction was found to be
  • Eighty ml of treated baboon blood was divided in 6 portions (about 13 ml each) in 50 ml sterile tubes.
  • the average volume of RBC in each portion was about 5 ml.
  • Unbuffered sterile saline i.e., 0.9% NaCl in water
  • the RBC fraction was separated by centrifugation at 2000 rpm (1248 x g) for 5 min. at room temperature, and the diluted plasma fraction removed.
  • the RBC fraction was next subjected to a washing cycle: To the remaining RBC (about 5 ml in each tube), a new portion of unbuffered sterile saline (about 4.5 ml) was added. The tubes were incubated with gentle agitation at room temperature for 10 min. At the end of the incubation, RBC fraction was separated by centrifugation. After each cycle, a small aliquot of the RBC suspension was removed to determine the concentration of PEN 102 by HPLC (see method above). The washing cycle was repeated four times. After the fourth cycle, the six tubes of RBC were combined together and unbuffered sterile saline added to give a final hematocrit (HCT) of 46%.
  • HCT hematocrit
  • Fig. 5 The effectiveness of the washing procedure is shown on Fig. 5, which demonstrates a rapid reduction in the percentage of PEN102 in the RBC fraction.
  • concentration of PEN102 remaining in the RBC fraction (in ⁇ g/ml of RBC) after each washing cycle is shown on Fig. 6.
  • the data in Fig. 6 demonstrates that the residual concentration of PEN102 in the RBC after 4 washings was lower than the safety level for PEN 102, 1 ⁇ g/ml, which was determined by mutagenicity testing on mouse lymphoma cells (performed by Covance, Vienna, VA).
  • PEN 102 treated RBC from baboon 214 Five ml portions of PEN 102 treated RBC from baboon 214 were labeled with either Cr or biotin using standard labeling protocols (see, e.g., Valeri et al., Transfusion 24: 105-108, 1984).
  • standard labeling protocols see, e.g., Valeri et al., Transfusion 24: 105-108, 1984.
  • to label RBCs with 51 Cr approximately 20-30 ml of RBCs were incubated for 30 min. at 37°C with 0.5 ⁇ Ci 51 Cr (disodium chromate) per ml of blood (commercially available from, for example, Dupont/NEN, Boston, MA), and then washed to remove the unincorporated 51 Cr-label.
  • the RBCs were biotin-labeled using the Biotin-X- NHS kit, commercially available from Calbiochem (San Diego, CA). The 51 Cr- labeled RBCs and biotin-labeled RBCs were then combined and infused back into baboon 214.
  • Table II shows the in vivo survival time of the PEN102 treated, washed, and stored RBC compared to the in vivo survival time of normal RBCs that had been removed from baboon 214, labeled, and re-infused.
  • the half-life (T50) of the RBCs is shown in days.
  • a quenching agent either soluble (i.e., Na-thiosulfate) or solid- phase.
  • soluble i.e., Na-thiosulfate
  • solid- phase i.e., Na-thiosulfate
  • the red blood cell (RBC) and plasma fraction of the blood were separated by centrifugation (10,000 rpm, 5 min.), and the RBCs were opened by adding 9 volumes of water.
  • the concentration of PEN 102 was determined in RBC and in the plasma fraction of the blood by HPLC (Fig. 7).
  • both sodium thiosulfate and solid phase-bound thiophosphate groups were capable of quenching PEN102.
  • the plasma quenched with sodium thiosulfate contained only 6.8 ⁇ g/ml PEN102
  • the solid phase quencher containing thiophosphate groups was even more effective.
  • the plasma quenched with this system contained only 1.5 ⁇ g/ml PEN102, and the red blood cells contained only 0.9 ⁇ g/ml PEN102 after 2 hours.
  • incubation of PEN 102 treated red blood cells with the solid- phase quencher for two hours lowered the concentration of PEN 102 in the red blood cells to a non-toxic level.
  • Blood is collect from a dog and divided into two portions. The first portion is incubated with an amount of ethyleneimine oligomer sufficient to inactivate at least some of the animal viruses in the portion. Following incubation, the red blood cells from this treated portion are isolated, washed four times with sterile unbuffered saline, as described above, and then frozen in glycerol for two weeks at -70°C.
  • the second portion is incubated for 6 hours at 22°C, but without any addition of ethyleneimine oligomer.
  • the red blood cells are isolated and washed four times with sterile saline, and then frozen in glycerol for two weeks at -70°C.
  • the cells are thawed, and cells in the first portion (i.e., the ethyleneimine oligomer treated portion) are labeled with 51 Cr while the cells in the second portion (i.e., the untreated portion) are labeled with biotin.
  • the cells are then combined and re-infused to the donor dog.
  • the survival time of the 51 Cr labeled red blood cells is compared to the survival time of the biotin-labeled cells, and no significant difference is predicted to be found between the two.
  • ethyleneimine oligomer treatment does not affect the survival time of canine red blood cells in vivo.
  • the purified platelets are divided into two equal portions, one of which (i.e., the PEN 102 treated portion) is treated for 6 hours at room temperature with 2.1 ml of 240 mM PEN102 in 0.25 M NaH 2 P0 4 (20X stock solution of PEN 102 is prepared immediately before treatment) with the final calculated total concentration of PEN 102 in the platelet-containing solution being 12 mM (1000 ⁇ g/ml; 0.1 % v/v).
  • the second portion i.e., the untreated portion
  • the two portions of platelets are next separated by centrifugation, and the supernatant removed.
  • the pelleted cells are next resuspended in sterile unbuffered saline, incubated at room temperature on a rocker for 10 min., and re-pelleted by centrifugation. After four cycles of this resuspension re- pelleting, the platelets are frozen in glycerol for two weeks at -70°C.
  • both the treated and the untreated portions of platelets are thawed and labeled with 51 Cr, as described above.
  • 51 Cr labeled PEN 102 treated platelets are transfused into one recipient baboon and the 51 Cr labeled untreated platelets are transfused into a second recipient baboon. Both of the recipient baboons are the same gender and roughly the same age.
  • the T50 of the PEN102 treated platelets is predicted to be approximately equal to that of the untreated platelets.
  • Blood donated by a human blood donor is incubated with an amount of ethyleneimine oligomer sufficient to inactivate at least some of the animal viruses in the blood.
  • the ethyleneimine oligomer treated blood is separated into three components: (1) the red blood cells (RBCs), (2) the platelets, and (3) the plasma.
  • the plasma and platelets are frozen in glycerol and stored at -70°C.
  • the isolated RBCs are spread onto a mesh having pores with diameters that are smaller than the diameter of a human RBC, and rinsed with a continuous flow of sterile unbuffered saline until the concentration of ethyleneimine oligomer in the cells is at or below the concentration determined to be non-toxic in the mouse lymphoma forward mutation assay described above.
  • the washed cells are next stored for 18 hours at 4°C.
  • the sample of cells is then divided into two. The first of the two portions of washed, treated, and stored RBCs are subjected to testing for a series of biochemical parameters. No significant changes in the biochemical characteristics of the washed, treated RBC are predicted to be seen as compared to a sample of washed, untreated
  • the second of the two portions of washed, treated, and stored RBCs are labeled with biotin, and re-infused back into the same human donor. Tracking of the biotin-labelled cells demonstrates that the ethyleneimine oligomer treated cells survive in vivo for a length of time comparable that of untreated biotin- labeled red blood cells that are similarly isolated (i.e., using the MCS®+ Apheresis System), washed, and stored.
  • a first sample of bovine urine is collected and incubated with an amount of an ethyleneimine oligomer sufficient to inactivate at least some of the viruses in the urine.
  • a second sample of bovine urine is collected and subjected to the same incubation conditions as the first sample, but in the absence of any ethyleneimine oligomer.
  • Both urine samples are next greatly diluted with sterile water until the concentration of the ethyleneimine oligomer is below that determined to be toxic in a mouse lymphoma mutagenicity assay (such as that described above and commercially performed by Covance, Vienna, VA).
  • the urine from both samples is then subjected to protein purification techniques to isolate the desired urine protein (e.g., the Tamm-Horsfall glycoprotein).
  • Standard protein purification techniques include HPLC, and described in general technique laboratory manuals (see, e.g., Scopes, R. K., Protein Purification: Principles and Practice, ed. C. R. Cantor, Spring- Verlag Inc., New York, NY, 1982; Coligan, J.E., Current Protocols in Protein Science, John Wiley & Sons, New York, NY, 1996).
  • a comparable of amount of the desired urine protein is predicted to be purified from both the ethyleneimine oligomer treated urine and the untreated urine.

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Abstract

L'invention porte sur un procédé d'inactivation de virus d'animaux dans une préparation de cellules purifiées de mammifères qui arrivées à maturité n'ont pas de noyau (par exemple des globules rouges ou des plaquettes), consistant à mettre en contact la préparation avec un agent inactivateur sélectif, oligomère d'éthylèneimine, dans des conditions d'inactivation pendant un temps suffisant pour inactiver au moins certains virus de la préparation. L'invention porte également sur un procédé d'extraction, après le processus d'inactivation, des virus dudit agent inactivateur de la composition biologique traitée, sans adjonction d'un agent d'extinction.
PCT/US1999/021245 1998-09-25 1999-09-22 Procede selectif d'inactivation de virus dans des compositions biologiques WO2000018969A1 (fr)

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JP2000572416A JP2002525131A (ja) 1998-09-25 1999-09-22 生物学的組成物においてウイルスを選択的に不活化する方法
CA002345230A CA2345230A1 (fr) 1998-09-25 1999-09-22 Procede selectif d'inactivation de virus dans des compositions biologiques
AU64970/99A AU6497099A (en) 1998-09-25 1999-09-22 Methods to selectively inactivate viruses in biological compositions
EP99952914A EP1115889A4 (fr) 1998-09-25 1999-09-22 Procede selectif d'inactivation de virus dans des compositions biologiques

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002067956A2 (fr) * 2001-01-22 2002-09-06 V.I. Technologies, Inc. Procede de purification d'une composition biologique
US6617101B1 (en) 1999-01-25 2003-09-09 V. I. TECHNOLOGIES, Inc. Lipophilic quenching of viral inactivating agents
US6617100B2 (en) 1998-09-25 2003-09-09 V.I. Technologies, Inc. Solid phase quenching systems
US6720136B2 (en) 1998-09-25 2004-04-13 V. I Technologies, Inc. Solid phase quenching systems
WO2005042060A2 (fr) * 2003-10-06 2005-05-12 V.I. Technologies, Inc. Procede et composition permettant de traiter un echantillon biologique
CN101012455B (zh) * 2005-11-14 2011-05-18 大连珍奥药业有限公司 一种生化物质的灭活方法、一种心肌肽素的制备方法及其用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005021363A1 (de) * 2005-05-04 2006-11-16 Basf Ag Biozide Beschichtungen

Citations (1)

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WO1997007674A1 (fr) * 1995-08-29 1997-03-06 Pentose Pharmaceuticals, Inc. Procedes et compositions de modification selective d'acides nucleiques

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Publication number Priority date Publication date Assignee Title
WO1997007674A1 (fr) * 1995-08-29 1997-03-06 Pentose Pharmaceuticals, Inc. Procedes et compositions de modification selective d'acides nucleiques

Non-Patent Citations (4)

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Title
BUDOWSKY ET AL.: "Principles of Selective Inactivation of the Viral Genome: Dependence of the Rate of Viral RNA Modification on the Number of Protonizable Groups in Ethyleneimine Oligomers", VACCINE RESEARCH, vol. 5, no. 1, 1996, pages 29 - 39, XP002926907 *
DATABASE WPI Derwent World Patents Index; AN 1992-413169/50 *
GABLER F.R.: "Cell Processing Using Cross-Flow Filtration", DEVELOPMENTS IN INDUSTRIAL MICROBIOLOGY, vol. 25, 1984, pages 381 - 396, XP002926909 *
WAGNER ET AL.: "Approaches to the Reduction of Viral Infectivity in Cellular Blood Components and Single-Donor Plasma", TRANSFUSION MEDICINE REVIEWS, vol. 5, no. 1, January 1991 (1991-01-01), pages 18 - 32, XP002926908 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6617100B2 (en) 1998-09-25 2003-09-09 V.I. Technologies, Inc. Solid phase quenching systems
US6720136B2 (en) 1998-09-25 2004-04-13 V. I Technologies, Inc. Solid phase quenching systems
US6617101B1 (en) 1999-01-25 2003-09-09 V. I. TECHNOLOGIES, Inc. Lipophilic quenching of viral inactivating agents
WO2002067956A2 (fr) * 2001-01-22 2002-09-06 V.I. Technologies, Inc. Procede de purification d'une composition biologique
WO2002067956A3 (fr) * 2001-01-22 2003-11-13 Vi Technologies Inc Procede de purification d'une composition biologique
WO2005042060A2 (fr) * 2003-10-06 2005-05-12 V.I. Technologies, Inc. Procede et composition permettant de traiter un echantillon biologique
WO2005042060A3 (fr) * 2003-10-06 2005-09-01 Vi Technologies Inc Procede et composition permettant de traiter un echantillon biologique
CN101012455B (zh) * 2005-11-14 2011-05-18 大连珍奥药业有限公司 一种生化物质的灭活方法、一种心肌肽素的制备方法及其用途

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JP2002525131A (ja) 2002-08-13

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