[go: up one dir, main page]

WO1998009660A1 - Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules - Google Patents

Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules Download PDF

Info

Publication number
WO1998009660A1
WO1998009660A1 PCT/US1996/014373 US9614373W WO9809660A1 WO 1998009660 A1 WO1998009660 A1 WO 1998009660A1 US 9614373 W US9614373 W US 9614373W WO 9809660 A1 WO9809660 A1 WO 9809660A1
Authority
WO
WIPO (PCT)
Prior art keywords
preparation
vims
igg
maintained
virus
Prior art date
Application number
PCT/US1996/014373
Other languages
English (en)
Inventor
Frank J. Riske
Fred Rothstein
James E. Woiszwillo
Original Assignee
Epic Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epic Therapeutics, Inc. filed Critical Epic Therapeutics, Inc.
Priority to PCT/US1996/014373 priority Critical patent/WO1998009660A1/fr
Publication of WO1998009660A1 publication Critical patent/WO1998009660A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A61L2/0088—Liquid substances

Definitions

  • Biological fluids including blood, may be contaminated with virus, bacteria and other pathogens such as prions. Such contamination poses a great health risk to patients who might receive an isolate from the biological fluid for a therapeutic purpose. Inadvertent contamination of such isolates with pathogens can cause severe illness and, in some instances, death. For example, contaminated blood products administered to patients have resulted in transmissions to those patients of the human immunodeficiency virus, resulting in death from acquired immunodeficiency syndrome.
  • the immunoglobulin IgG is the principle circulating antibody providing humoral immunity from infection and disease. Purified preparations of IgG derived from blood and other sources are used for therapeutic purposes, diagnostic purposes and scientific research purposes. There is a pressing need for a simple procedure to eliminate viral and other pathogenic contamination from blood products, including purified IgG products.
  • a popular solvent-detergent mixture includes tri(n-butyl)phosphate in combination with a detergent such as Octoxynol 9TM, Tween- 80TM, Sodium Cholate, Triton X-45TM, and Triton X-100TM, as described by Pique et al., Vox Sang 63:251-256 (1992); Piet et al., Transfusion, 30:591-598 (1990); Horowitz et al., Transfusion, 25:216-219 (1985); and Horowitz et al., Blood, 79:826-831 (1992).
  • An important disadvantage to the use of a solvent-detergent mixture is that this method fails to inactivate non- enveloped virus.
  • the '650 patent represents, to some extent, a departure from the typical prior art teaching respecting methods for eliminating viral contamination while maintaining biological activity.
  • the published literature suggests carrying out the viral inactivation methods in a narrow pH range, usually between 6 and 8, presumably to make sure that the biological activity of the desired material is not lost as a result of varying the pH outside of that range.
  • most protein is somewhat unstable at pHs substantially outside of neutral (physiological) pH.
  • the immunoglobulin is said to retain its biological activity.
  • the ability of elevated pH to kill virus and inactivate prions is well documented, but not as a methodology for eliminating virus from a biologically labile isolate such as a protein.
  • viruses have been classified in part by their relative susceptibility to pH.
  • Herpes Simplex type I and II virus have been distinguished based on their relative susceptibility to inactivation at elevated pH conditions.
  • Reovirus and Rotavirus have been distinguished from one another on this basis.
  • Picamaviridae are separated into the acid sensitive Rhino and Aphthoviruses and the acid resistant Enteroviruses.
  • the prior art relating to rendering a preparation of IgG free of virus would appear to teach away from simply using elevated pH because (1) the prior art references discuss pH but do not indicate that elevating pH is the solution to the problem; (2) the prior art indicates that chemical agents other than pH are essential to eliminate virus; (3) the prior art actually teaches using a low pH, instead of a high pH, to eliminate a certain type of virus from a preparation of IgG; and (4) the prior art suggests, if anything, that IgG is stable only at neutral or low pH.
  • the invention involves the discovery that viruses, even robust viruses, can be inactivated in a biological fluid preparation by elevating pH, without destroying the desired native biological activity in the biological fluid preparation.
  • pH can be elevated to a level just below that which will destroy the activity of many biological materials and maintained at that level for a sufficient period of time to destroy or otherwise inactivate pathogens such as viruses and prions.
  • IgG IgG
  • pH above a certain critical level causes an unacceptable loss of biological activity, whereas just below this level causes virtually no loss in biological activity, yet high inactivation of virus. It further was unexpected that even the most robust viruses appear sensitive to extended pH treatment below this critical level.
  • the invention therefore, provides a simple, rapid, cost-effective viral inactivation method. It also provides a method that is capable of inactivating both enveloped and non-enveloped viruses..
  • a method for treating an IgG preparation to render the preparation free of active virus is provided.
  • the preparation is maintained at a pH of between 10.0 and 11.6 for a sufficient period of time and under conditions such that the pH inactivates the virus in the preparation.
  • the IgG preparation is maintained at a pH of between 10.5 and 11.4, and ever more preferably between about 10.9 and 11.3.
  • the IgG preparation is maintained at a pH of between about 1 1.1 and 1 1.3.
  • the pH then is adjusted to between about 4.5 and 8.0. .
  • the foregoing methods can be carried out over a vast range of temperatures, although preferably the temperature is between about 4°C and 37°C. Most preferably the preparation is maintained at the desired pH at a temperature of between about 18°C and 27°C. The foregoing conditions are applied preferably for between about 4 hours and 7 days, and most preferably are applied for between 1 and 5 days.
  • the IgG preparation can be obtained from any source. In one important embodiment it is obtained from a body fluid. In particularly important embodiments, it is obtained from blood or ascites fluid.
  • the product is an IgG preparation rendered free of active virus by treating the preparation at a pH of between about 10.0 and 1 1.6 for a sufficient period of time and under conditions such that the pH inactivates the virus in the preparation.
  • Preferred conditions of treatment are as specified above.
  • a method for treating a biological fluid preparation containing a non-virus biological material, the biological material having a native biological activity, to render the preparation free of both an enveloped and a non-enveloped active virus.
  • the method involves maintaining the biological fluid preparation at pH conditions above about 10.0 for a sufficient period of time and under conditions such that the pH inactivates the enveloped and the non-enveloped virus in the preparation, wherein the pH is low enough whereby the native biological activity is substantially retained.
  • the pH is maintained at between 10.0 and 12.0.
  • the pH is maintained between 10.5 and 11.4 and preferably between 10.9 and 11.3.
  • the pH then is adjusted to between about 4.5 and 8.0.
  • the foregoing method can be carried over a broad range of temperatures, but preferably is carried out between about 4°C and 37°C. More preferably the method is carried out at a temperature of between 18°C and 27°C.
  • the foregoing treatment is carried out typically for between 4 hours and 7 days, and more typically between 1 and 5 days.
  • the foregoing method unexpectedly, is capable of inactivating both non-enveloped and enveloped viruses containing either DNA or RNA as the genetic material.
  • a Herpesvirus, a Togavirus, a Parvovirus. and a Picomavirus can be inactivated according to the methods set forth above.
  • a product obtainable by the foregoing process is provided.
  • the invention provides a product obtained by maintaining a biological fluid preparation containing a nonvirus biological material, the biological material having a native biological activity, at pH conditions above 10.0 for sufficient period of time and under conditions such that the pH destroys the enveloped and the non-enveloped virus in the preparation.
  • the pH is low enough whereby the native biological activity is substantially retained.
  • a method for treating a biological fluid preparation containing a non-virus biological material, the biological material having a native biological activity, to render the preparation free of active prions.
  • the method involves maintaining the biological fluid preparation at pH conditions above about 10.0 for a sufficient period of time and under conditions such that the pH inactivates the prions in the preparation, wherein the pH is low enough whereby the native biological activity is substantially retained.
  • the pH then is adjusted to between about 4.5 and 8.0. In most embodiments the pH is maintained between 10.0 and 12.0.
  • Preferred method parameters are as detailed above.
  • the invention involves the discovery that both enveloped and non-enveloped virus can be eliminated from a biological fluid preparation if the biological fluid preparation is maintained long enough at an elevated pH, which pH, however, is low enough to permit the desired biological activity to be retained.
  • Several aspects of this discovery were unexpected. Firstly, it was unexpected that a broad range of both enveloped and non-enveloped viruses would be inactivated at the pHs employed according to the invention.
  • the conditions of the invention have been employed to inactivate an enveloped DNA virus, an enveloped RNA virus, a non-enveloped DNA virus and a non-enveloped RNA virus.
  • the invention in one important aspect, involves the inactivation of virus in biological fluids containing immunoglobulins.
  • the immunoglobulins (IgG, IgM, IgA, IgE, IgD) are found in the globulin fraction of vertebrate serum proteins. They constitute the circulating antibody population and provide the humoral immune response necessary to fight infection and disease. Purified immunoglobulins are necessary for scientific research, immunological protocols, and therapeutic uses, such as for passive immunization. Antibodies are also used as diagnostics, and can be used in vivo in targeting drugs and the like to specific tissues and organs. Immunoglobulins may be derived from mammals or may be created ex vivo.
  • Polyclonal antibodies are produced by injecting an animal, such as a mouse, rat or rabbit, or even a human, with an antigen, collecting blood or ascites, and isolating the immunoglobulin fraction that binds to the antigen, if desired, typically by passage of the immunoglobulin fraction through an affinity column to which the antigen has been immobilized.
  • Monoclonal antibodies can be created by fusing the normal antibody-producing lymphocyte to a myeloma cell line to form a hybridoma.
  • the "immortalized” fused cell causes the continuous production of the antibody of interest which is usually recovered from ascites fluid.
  • Monoclonal antibodies typically are isolated from the other proteins present in the ascites fluid before use in scientific research, in diagnostic kits or in therapeutic regimens.
  • Monoclonal antibodies also can be produced in vitro recombinantly, including via the use of phage.
  • Immunoglobulin prepared and/or isolated according to any of the foregoing methods may be treated according to the methods of the invention.
  • the method can be used to inactivate viruses present in any biological fluid of humans or other animals, including, but not limited to, whole blood, blood serum, blood plasma, urine, cerebrospinal fluid, lymph fluid, mucus, tears, saliva, secretions and ascites.
  • the methods of the invention also can be used to inactivate viruses in biological fluids such as fermentation fluid and tissue culture or cell culture fluids.
  • biological fluid preparation includes suspensions or solutions of biological molecules that have been suspended or dissolved in nonbiological fluids such as buffers, chemical solutions and water.
  • a solution containing an immunoglobulin that has been precipitated from a biological fluid preparation and resuspended in a buffer is specifically included within the definition of the term biological fluid preparation.
  • viruses in the biological fluid preparation can be inactivated either before isolation of the biomolecule of interest, after isolation of the biomolecule of interest, or even without further isolation of the biomolecule of interest. If the biomolecule is first isolated, then the isolate itself can be mixed with or, if a precipitate, resuspended in an alkaline buffer to ensure inactivation of virus that may contaminate the isolate.
  • the pH of the biological fluid preparation is adjusted to the range as described above with a basic compound in accordance with methods well known to those skilled in the art.
  • the pH of the biological fluid preparation may be adjusted at temperature of 4°C - 37°C by the addition of a sufficient amount of an alkaline or high pH compound in solid or liquid form.
  • the high pH compound can be in the form of a solution such as a sodium hydroxide (NaOH) solution, preferably a 0.02-1.0 N NaOH solution.
  • NaOH sodium hydroxide
  • KOH KOH.
  • Useful buffers for adjusting the pH of the biological fluid preparation include, but are not limited to, CAPS (3-cyclohexylamino-l propanesulfonic acid) carbonate buffers, borate buffers, phosphate buffers, glycine, sodium carbonate buffers and sodium bicarbonate/carbonate buffers. It is preferred that the pH be adjusted in a manner that reduces the likelihood of damage to the IgG by local pH effects that are above the critical range.
  • the pH-adjusted biological fluid preparation is incubated, with or without stirring, for a sufficient amount of time to inactivate virus without denaturation of the immunoglobulin present in the fluid. Although some viruses are totally inactivated in a period of one hour or less at the pH ranges set forth above, the more robust viruses require longer exposure to the pH conditions. Typically the exposure will be between four hours and seven days and more typically at least twenty-four hours and not more than five days.
  • the pH of the incubation mixture at 4°C - 37°C typically is readjusted to a neutral or acidic pH after completion of the viral inactivation method and prior to formulation, use or storage.
  • the compound used to decrease the pH to neutral or below could be H 3 PO 4 , preferably 0.1 - 0.5N.
  • Other useful compounds include citric acid, glycine HC1, and H 2 SO 4 .
  • the pH-adjusted biological fluid preparation is incubated within a temperature range that is nondestructive to the immunoglobulin.
  • elevated temperature may perhaps accelerate viral inactivation, it also can result in denaturation, alteration, or aggregation of the desired biological material. Therefore, according to preferred embodiments, the pH conditions are maintained at a temperature between 4°C and 37°C and more preferably between about 18°C and 27°C. This latter range is particularly desirable for proteins such as immunoglobulins, the structure of which is unaffected in this temperature range.
  • the nature of the buffer and its concentration can affect the stability of a protein such as an immunoglobulin at elevated pH.
  • a buffer could directly or indirectly affect viral inactivation when employing the methods of the invention to inactivate virus.
  • Examples of useful buffers and the effects of the buffer on immunoglobulin are detailed below. It is expected that other parameters can influence the stability of the immunoglobulin and the degree of viral inactivation. Materials which stabilize the immunoglobulin to the effects of elevated pH may permit even slightly higher pHs to be employed. Likewise, other agents capable of causing viral inactivation may be included in the mixture when the biological fluid preparation is subjected to the elevated pH conditions of the invention. It is expected that all of the foregoing parameters are interrelated, and that varying one will influence another.
  • the biological fluid preparation can be free of ⁇ -propiolactone, free of soluble alcohols, free of solvent-detergents, free of caprylic acid, free of hydrolases, free of protein stabilizers and the like.
  • the present method can be used to inactivate viruses having a wide range of structural, physiochemical and replicative characteristics, including both enveloped and non-enveloped, double-stranded and single-stranded DNA and RNA (positive and negative strand) viruses of humans, plants, bacteria and animals, including insects.
  • viruses having a wide range of structural, physiochemical and replicative characteristics, including both enveloped and non-enveloped, double-stranded and single-stranded DNA and RNA (positive and negative strand) viruses of humans, plants, bacteria and animals, including insects.
  • viral kill of an enveloped DNA virus, an enveloped RNA virus, a non-enveloped DNA virus, and a non- enveloped RNA virus We have demonstrated, in particular, viral kill using the methods of the invention of Herpesvirus, Togavirus, Parvovirus, and Picomavirus.
  • the foregoing include Hepatitis A and models for Hepatitis C, and Parvo B19, which are all blood borne viruses of importance in humans:
  • EMC Encephalomyo- Picornaviridae Non-enveloped RNA Hepatitis A Yes carditis
  • Viral inactivation by the present method should inactivate most, if not all, of the 61 families of viruses recognized by the ICTV, including bacteriophages, insect viruses, such as Bacculoviruses, and plant viruses.
  • the human and animal viruses that may be inactivated by this method are the viruses found in the following taxonomic families: Picornaviridae, Calciviridae, Togaviridae, Flaviviridae, Coronaviridae, Rhabodoviridae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Hepadnaviridae, Parvoviridae,
  • viruses may be classified or reclassified into other families, such as known but presently unclassified viruses and such as the viral agents that cause the spongiform encephalopathies.
  • Family Herpesviridae includes the following:
  • Alphaherpesviridae (Herpes Simplex-like Viruses) Genus Simplexvirus
  • Herpes Simplex Vims 1 and 2 Cercophithecine Herpesvirus 1 (B-vims)
  • Betaherpesviridae The Cytomegaloviruses Genus Cytomegalovirus
  • Genus Lymphocryptovirus (Epstein-Barr-like Vimses) Human Pathogen: Epstein-Barr Vims Animal Pathogens: Baboon Herpesvirus, Pongine (chimpanzee) Herpesvirus
  • HIV is an enveloped, single positive strand RNA vims having a size of 80-1 10 nm in diameter and is classified as a retrovirus.
  • HAV is a non-enveloped, single-stranded RNA vims having a size of approximately 27 nm and having positive polarity with a 5' terminal protein and a 3' poly(A) tail.
  • HBV is an enveloped, circular, partially single-stranded DNA vims having a size of approximately 42-47 nm.
  • HCV also know as non-A, non-B hepatitis vims, is an enveloped, single positive strand RNA vims having a size of approximately 30-60 nm in diameter.
  • HSV is a large, enveloped, linear, double-strand DNA vims having a size of approximately 150-250 nm.
  • Poliovimses are non-enveloped, single-stranded RNA vimses having a virion diameter of approximately 27-30 nm.
  • Parvovirus is a non-enveloped, linear, single-stranded DNA vims having a size of approximately 20-25 nm and high resistance to physiochemical reagents. It is well known that a Parvovirus is a highly persistent vims. It is also expected that prions are inactivated by the methods of the invention.
  • the pH range tolerated by most microorganisms extends over 3 to 4 units, but rapid growth may be confined to 1 unit or less.
  • Pathogens such as Pneumococcus, Neisseria, and Bruce lla have a more restricted range than E. coli which cannot withstand a pH much above 8.
  • the invention pertains to eliminating pathogens from biological fluid preparations.
  • the methods of the invention can be applied to molecules other than immunoglobulins. It is particularly useful in connection with proteins having a native biological activity (in the organism in which the protein is found in nature) that depends upon the overall configuration of the protein and intramolecular bonding. Thus, the invention is useful in connection with rendering preparations of albumin vims free. It likewise is useful in connection with rending preparations of Antithrombin III, Factor VIII, human growth hormone, insulin and other blood derived (or recombinantly produced) proteins vims free.
  • Biomolecules in the biological fluid that preferably retain a native activity after exposure to the viral inactivation methods described herein also include, but are not limited to, nucleic acids, and carbohydrates. It will be understood by those skilled in the art that the biomolecule could also be a targeted molecule such as a pharmaceutical drug.
  • Bovine Parvovirus BPV
  • Bovine Viral Diarrhea vims BBV
  • Porcine Pseudorabies Vims PV
  • Murine Encephalomyocarditis EMC
  • Hepatitis A EMC
  • Bovine Viral Diarrhea vims BBV, strain KY-22
  • RNA- containing vims RNA- containing vims.
  • HCV Hepatitis C vims
  • Bovine Parvovims (BPV, Dubovi strain) is an 18-26 nm, non-enveloped DNA- containing vims of the Parvoviridae family. Human parvovims B- 19 is a concern for human blood-derived products but cannot be propogated in vitro. Therefore, the animal Parvoviruses (porcine, bovine, canine) are used as models for the process validation studies. Parvovims also represents a class of vimses that is very difficult to inactivate.
  • Murine Encephalomyocarditis Vims is a member of the Picornaviridae family which includes Poliovims and Hepatitis A.
  • the vims is approximately 20-30 nm in diameter and consists of a single molecule of RNA covered by an icosahedral shell.
  • EMC is often used as a model vims for Polio and Hepatitis A, and all three vimses are extremely resistant to low pH.
  • Porcine Pseudorabies Vims is a large ( ⁇ 200nm), complex enveloped vims of the family Herpesviridae, containing linear double stranded DNA.
  • the family includes the human pathogens Herpes Simplex 1 and 2, Varicella zoster Vims, Epstein-Barr Vims, and Cytomegalovims.
  • Hepatitis A is a non-enveloped, single-stranded RNA vims having a size of approximately 27 nm and having positive polarity with a 5' terminal protein and a 3' poly(A) tail.
  • the capsid contains four structural proteins and is quite stable to acid and heat treatment.
  • Viral Inactivation The viral stock (supplied and stored at -70°C) was quick thawed in a 37°C ⁇ 2°C water bath and then immediately added with stirring to 10% of the volume of either a buffer solution or to a solution containing purified human gamma globulin. The material was stirred for a few minutes and then a 5ml sample was removed, adjusted to between pH 6.5 and 8.0 and then immediately frozen at -70°C. This represented the T 0 sample. A second 5ml sample was treated as above but then stored at 24°C ⁇ 2°C for time periods of up to 120h. This sample was a control to measure the stability of the vims in the test material over time, in the absence of high pH treatment (T comrol ).
  • the T comro ⁇ samples were removed from the water bath after up to 120h of incubation and frozen at or below -70°C.
  • the remaining spiked starting material was then adjusted to pH 1 1.0 - 11.1 with 0.2 N NaOH and incubated for 120 hours at room temperature.
  • a 5ml aliquot was removed, adjusted to pH 6.5 - 8.0 and frozen immediately at or below -70°C.
  • T 96 pH 10.95 - 1 1.1 Incubation
  • T 120 pH 10.95 - 1 1.1 Incubation
  • T 120 pH 6.5 - 8.0 Incubation
  • test material was removed and the plates overlayed with agar and allowed to solidify. Plates were then placed in a humidified CO 2 incubator and examined daily until plaques formed in the positive control cultures. Cultures were then overlaid with agarose containing neutral red to enhance plaque visualization and read when the dye penetrated to the cell layer and the plaques were fully formed.
  • Murine Encephalomyocarditis EMC
  • Porcine Pseudorabies EMC
  • Hepatitis A FRhK / JP cells
  • the test material (without viral spike) was also tested on the indicator cells for the presence of toxicity (without viral spike) and for inhibition of viral plaquing at low vims titer (40 PFU/ml).
  • the viral inactivation values were calculated based on the dilution of the test material where inhibition of viral plaquing or toxicity were absent.
  • Example 1 Inactivation of Virus by pH 11
  • Viral titers are express as Plaque Forming Units (PFU) per ml. Titer was reported as ⁇ 1.67 x 10° PFU/ml when no vims was detected. The Adjusted Titer reflects the dilution of the sample in relation to T 0 due to the addition of base in raising the pH to 10.95 - 1 1.1 and acid in returning it to pH 5.0 - 8.0 upon completion of the procedure.
  • PFU Plaque Forming Units
  • BVD Table 1 Bovine Viral Diarrhea (BVD) Vims Titers after Incubation at pH 10.95-1 1.1
  • BPV titers were reduced by >4.15 logs in 5% IgG after 48h of treatment. However, there was interaction (possibly neutralization) between the vims and IgG in the absence of high pH treatment. Both the T 0 and T con o] samples (vims in IgG) showed a 1.6 and 1.89 log I0 reduction respectively, in relation to a T 0 sample where vims was spiked into buffer. Also in our plaquing control, only 6 plaques out of 40 PFU were counted in a 10 "2 dilution of IgG. This data is shown in Table 2.
  • EMC Encephalomyocarditis
  • vims 1.66 log reduction after incubation of vims in 5% IgG after 120h. However, vims is totally inactivated (>5.74 logs) between 48 and 72h.
  • EMC was reduced to non-detectible levels (>5.74 logs) between 48 and 72h incubation at pH 10.95 - 11.1.
  • Log !0 reduction values were calculated by comparing Log, 0 PFU/ml of the treated samples to the TO sample. Adjusted titer reflects the volume expansion of the sample due to base and acid used during treatment. Porcine Pseudorabies titer was reduced by > 6.70 logs (complete inactivation) between 48h and 72h of incubation at pH 10.95-11.1.
  • Hepatitis A Table 5 Hepatitis A Vims Titers after Incubation at pH 10.95 - 11.1.
  • Hepatitis A titer was reduced by 2.4 logs following 120h at pH 10.95 - 11.0.
  • Hepatitis A vims was spiked into purified human IgG (5% protein). This material was purified from a single unit of commercial frozen plasma which did not exhibit a titer to Hepatitis A antigen.
  • the human IgG fraction was purified from the plasma using a modification of a process developed at Middlesex Sciences for purifying human IgG from Cohn Fraction II & III. This method does not employ alcohol fractionation. Briefly, the plasma was quick thawed in a 37°C water bath, filtered through a 0.8/0.2u depth filter and Triton X-100 was added with stirring to bring the final concentration to 2%.
  • the human IgG in the material was then precipitated from the plasma with 2 volumes of a polymer solution containing 20% PEG 3350 mw and 20% polyvinylpropylene (PVP) 40,000 mw.
  • the mixture was adjusted to a pH of 6.2, stirred for 30 min. and then the precipitate containing IgG was separated from the aqueous phase by centrifugation at 5000g for 30 minutes.
  • the liquid resulting was poured off and the precipitate was suspended in 25mM sodium phosphate buffer adjusted to pH 4.3, passed through a charcoal and nominal filter (0.2u), adjusted to pH 6.0, and then loaded on a DEAE- Sepharose Fast Flow column.
  • the flow through of the column was diluted with one volume of 0.2M carbonate-bicarbonate buffer, pH 10 and then loaded on a Q-Sepharose Fast Flow column.
  • the IgG fraction, bound to the column was washed thoroughly with 0.1 M carbonate- bicarbonate buffer pH 10, containing 60mM sodium sulfate.
  • the eluate, containing purified IgG was concentrated to about 5% protein using a 50kd ultrafiltration membrane, diafiltered into 5mM phosphate buffer pH 5.0, sterile filtered and then stored at 4°C.
  • the high pH process shows substantial inactivation of a human pathogenic vims (Hepatitis A) and model vimses for human pathogens such as EMC (polio), Bovine Parvovims (Human B 19) and BVD (Hepatitis C).
  • EMC polio
  • Bovine Parvovims Human B 19
  • BVD Hepatitis C
  • the materials were filtered (0.2u) and the oligomers, dimers, and monomers determined by gel permeation HPLC (60cm Tosohaus TSK 3000 column) under isocratic conditions at 0.8 ml/min using 0.01 M sodium phosphate. 150mM NAC1, pH 7.4 as the mobile phase. Chromatograms were analyzed using software which determined the area under the curves for the oligomers, dimers, and monomers and reported each as the percent of total area.
  • the results show minimal oligomer formation over 120 hours of treatment, and minimal increase in oligomer formation (-2%) between 24 and 120 hours.
  • Example 3 Stability of Gammaglobulin Incubated at pH 11, 11.3, 11.6 and 12.0
  • Human IgG samples were adjusted with 0.2N NaOH to pH 11.0 and incubated as indicated below. The pH was maintained at 1 1.0 ⁇ 0.1 by the addition of 0.2N NaOH or 0.5N H 3 PO 4 .
  • Human IgG (6.4% protein) was tested at sodium phosphate buffer concentrations from 4.4mM to 55mM. Samples were removed at 24 hour intervals, adjusted and analyzed by HPLC
  • Human IgG (2% protein) was treated with high pH as described in Example 4A. Human IgG was either in; a) lOOmM carbonate, 45mm sulfate buffer; b) lOmM carbonate. 4.5mM sulfate buffer (0.1 carb); c) 20mM phosphate buffer; or d) 2mM phosphate, pH 5.5 (0.1 phosphate) prior to raising the pH to 1 1.0. Samples were collected and analyzed at 24 and 96 hours as described previously.
  • Table 1 1 Activity and Stability of MSL and Bayer IVIG Treated at pH 1 1 at 5%, 2.5% and 1% protein
  • Oligomers increased as the protein concentration increased from 1-5%. However, at 5% protein concentration, oligomers account for ⁇ 5% of the total protein in the MSL material and about 10% or less in the Bayer material.
  • Example 6 Functional Activity of Human Gammaglobulin Following Viral Inactivation Conditions (pH 11) Human gammaglobulin binds to a number of antigens due to our exposure to numerous microorganisms vimses, and other materials. The functional activity of human gamma globulin was assessed by testing gammaglobulin binding to several disease related antigens/organisms in several different assay formats.
  • Example 6 we examined pH 1 1 treated IgG in ELISA for the presence of functional antibody to Rubella Vims (German Measles), Rubeola Vims (Measles), and Pneumococcal bacteria (all three from BBI-North American Clinical Laboratories, New England, CT); and Clostridium tetani (Tetanus), and Corynebacterium diphtheriae (Diphtheria), (both from Massachusetts State Lab, Jamaica Plain, MA).
  • a comparison of the functional activity of the treated samples to the untreated control (T 0 ) can be made based on the % T 0 value. This value represents specific antigen binding remaining post treatment. Clearly, human gammaglobulin binding to these antigens is maintained following high pH treatment for 120 hours.
  • Example 7 adds further support to the results reported in Example 6; namely that the functional activity of human gammaglobulin is maintained following high pH treatment. Treated and untreated (T 0 ) samples show the same Diphtheria neutralization value and similar titers for the three vimses that were examined.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention porte sur des procédés d'inactivation des virus dans des préparations de liquides biologiques, par exemple de l'IGg et les différentes substances y étant produites. Le procédé consiste à maintenir la préparation de liquide biologique, par exemple de l'IGg, à un pH élevé pendant un temps suffisant et dans des conditions la rendant exempte de virus actifs tout en préservant l'activité biologique de l'IgG.
PCT/US1996/014373 1996-09-06 1996-09-06 Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules WO1998009660A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US1996/014373 WO1998009660A1 (fr) 1996-09-06 1996-09-06 Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1996/014373 WO1998009660A1 (fr) 1996-09-06 1996-09-06 Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules

Publications (1)

Publication Number Publication Date
WO1998009660A1 true WO1998009660A1 (fr) 1998-03-12

Family

ID=22255750

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/014373 WO1998009660A1 (fr) 1996-09-06 1996-09-06 Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules

Country Status (1)

Country Link
WO (1) WO1998009660A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5906915A (en) * 1990-11-07 1999-05-25 Baxter International Inc. Method for storing red cells using reduced citrate anticoagulant and a solution containing sodium, citrate, phosphate, adenine and mannitol
WO2000040703A1 (fr) * 1999-01-08 2000-07-13 National Blood Authority Procede d'inactivation d'un virus
US20200369747A1 (en) * 2017-08-25 2020-11-26 Ichnos Sciences SA. Methods of inactivating viral contaminants

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1498821A (en) * 1975-10-24 1978-01-25 Wellcome Found Vaccines
EP0124506A2 (fr) * 1983-05-02 1984-11-07 IMMUNO Aktiengesellschaft für chemisch-medizinische Produkte Procédé pour inactiver des pathogènes
US4814277A (en) * 1986-05-30 1989-03-21 Immuno Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method of inactivating reproductive filterable pathogens
EP0312839A2 (fr) * 1987-10-17 1989-04-26 Bayer Ag Procédé pour la préparation d'une substance provoquant une immunité non spécifique
WO1994022305A1 (fr) * 1993-04-01 1994-10-13 Unilever N.V. Compositions desinfectantes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1498821A (en) * 1975-10-24 1978-01-25 Wellcome Found Vaccines
EP0124506A2 (fr) * 1983-05-02 1984-11-07 IMMUNO Aktiengesellschaft für chemisch-medizinische Produkte Procédé pour inactiver des pathogènes
US4814277A (en) * 1986-05-30 1989-03-21 Immuno Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method of inactivating reproductive filterable pathogens
EP0312839A2 (fr) * 1987-10-17 1989-04-26 Bayer Ag Procédé pour la préparation d'une substance provoquant une immunité non spécifique
WO1994022305A1 (fr) * 1993-04-01 1994-10-13 Unilever N.V. Compositions desinfectantes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5906915A (en) * 1990-11-07 1999-05-25 Baxter International Inc. Method for storing red cells using reduced citrate anticoagulant and a solution containing sodium, citrate, phosphate, adenine and mannitol
WO2000040703A1 (fr) * 1999-01-08 2000-07-13 National Blood Authority Procede d'inactivation d'un virus
US20200369747A1 (en) * 2017-08-25 2020-11-26 Ichnos Sciences SA. Methods of inactivating viral contaminants
US12351602B2 (en) * 2017-08-25 2025-07-08 Ichnos Sciences SA. Methods of inactivating viral contaminants

Similar Documents

Publication Publication Date Title
JP4841018B2 (ja) 生物学的製品の最終滅菌法
Piet et al. The use of tri (n‐butyl) phosphate detergent mixtures to inactivate hepatitis viruses and human immunodeficiency virus in plasma and plasma's subsequent fractionation
CZ190292A3 (en) Virus inactivated blood product
JPS61275228A (ja) 治療用タンパク組成物中のビ−ルスの光動的不活性化
JPH06102627B2 (ja) 脂質含有ウイルスを含まない蛋白質含有組成物及びその製造方法
EP0050061A2 (fr) Procédé pour réduire les activités indésirables de produits biologiques et pharmaceutiques
Chandra et al. Effectiveness of alternative treatments for reducing potential viral contaminants from plasma-derived products
Uemura et al. Inactivation and elimination of viruses during preparation of human intravenous immunoglobulin
US5041537A (en) Method of preparing a high-purity, virus safe, biologically active transferrin preparation
Kempf et al. Pathogen inactivation and removal procedures used in the production of intravenous immunoglobulins
JP2506370B2 (ja) 増殖性濾過性病原体の不活化法
Lee et al. New methods for inactivation of lipid‐enveloped and nonenveloped viruses
JP2879427B2 (ja) ウイルスおよびバクテリア汚染物の熱的不活性化中の生物学的および製薬的生成物の安定化
Roberts et al. Virus reduction in an intravenous immunoglobulin by solvent/detergent treatment, ion-exchange chromatography and terminal low pH incubation
US6465170B2 (en) Method for depleting viral and molecular pathogens in a biological material
WO1998009660A1 (fr) Procedes d'inactivation des virus dans des preparations de liquides biologiques preservant l'activite des biomolecules
Morgenthaler Securing viral safety for plasma derivatives
RU2411959C2 (ru) Способ получения безопасных в вирусном отношении биологических текучих сред
Alonso et al. Viral inactivation of intramuscular immune serum globulins
SK175299A3 (en) Process for reducing the concentration of viral and molecular pathogens in a biological material
JP3735137B2 (ja) 生物学的活性なタンパク質の溶液における殺ウイルス剤としてのアゾールの使用
Stephan Inactivation of hepatitis viruses and HIV in plasma and plasma derivatives by treatment with b-propiolactone/UV irradiation
Poulsen et al. Elimination of viral infection risk from blood samples for allergy testing.
JPS59110627A (ja) Ｂ型及び非ａ非ｂ型肝炎の危険の無い生物学的生産物
Aghaie et al. Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998512597

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase