WO1998009660A1 - Viral inactivation of biological fluids with biomolecule activity retention - Google Patents

Abstract

The invention involves methods for inactivating virus in biological fluid preparations such as IgG and products produced thereby. The method involves maintaining the biological fluid preparations such as IgG at an elevated pH for sufficient period of time and under conditions whereby the preparation is rendered free of active virus while maintaining the biological activity of the IgG.

Description

V1RAL INACTIVATION OF BIOLOGICAL FLUIDS WITH BIOMOLECULE ACTIVITY RETENTION

Background of the Invention 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.

Perhaps the most vexing problem with eliminating viral contamination from a biological isolate is that those conditions which tend to eradicate numerous species of virus also tend to destroy the desired biological activity of the biological isolate. For example, incubation of a preparation containing a biological isolate at an elevated temperature (60°C for ten hours or 100°C for one hour) usually results in successful viral destruction. Such elevated temperature, however, also can cause denaturation, aggregation and destruction of proteins such as immunoglobulins. Likewise, ultraviolet irradiation will inactivate virus, but again can cause denaturation, aggregation or destruction of proteins such as immunoglobulins. For these reasons, heat and radiation typically are used in connection with the sterilization of non- biological materials such as medical equipment and research tools.

Chemical treatment of biological isolates has been proposed to destroy virus while retaining the desired biological activity of the biological isolate. Solvent-detergent mixtures have been found useful for inactivating enveloped viruses such as Human Immunodeficiency Virus (HIV) or Hepatitis C Virus (HCV). For example, a popular solvent-detergent mixture includes tri(n-butyl)phosphate in combination with a detergent such as Octoxynol 9™, Tween- 80™, Sodium Cholate, Triton X-45™, and Triton X-100™, 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. In addition, the solvent-detergents must be removed prior to final formulation, resulting in additional processing steps that can be time consuming, expensive, and result in yield losses of IgG. Biological isolates also have been treated with soluble alcohols (C₄-C _Q) at low pH (4.0-7.0) to destroy virus as described in US Patent 5.071,650 to Dove et. al. Alcohols, however, tend to denature proteins.

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. Typically, 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. It is known in the art that most protein is somewhat unstable at pHs substantially outside of neutral (physiological) pH.

The '650 patent, however, is consistent with those prior art methodologies for eliminating viral contamination where pH is varied outside of a neutral range. In particular, the prior art suggests that if pH is to be varied outside of a neutral range, then low pH is desirable. For example, US Patent 4,939,176 teaches a method of inactivating enveloped viruses present in purified biologically active protein products, including IgG. The virus is eliminated by contacting the preparation with caprylic acid "under conditions sufficient to substantially reduce the activity of the virus without adversely affecting the amount of biological activity of the proteins". Temperature, pH and other variable are adjusted to make sure that the caprylic acid is present, in non-ionized form, at a specified concentration. Although the ' 176 patent suggests that the caprylic acid will inactivate virus over a broad range of pH (4-10), the preferred range is 4.5 to 8.5. This presumably is the range over which most proteins are stable. The specific examples involving immunoglobulin are carried out at a pH of between 4.8 and 8.0. The ' 176 patent, therefore, tends to teach carrying out the procedure in a neutral to slightly acidic range. Another patent, US 4,762,714, deals directly with the use of pH to inactivate Retrovirus (non- enveloped virus) in a preparation of immunoglobulin. The isolated immunoglobulin is incubated at a pH equal to or less than about 4.25 at a temperature of about 20°C for at least three days. 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. For decades, viruses have been classified in part by their relative susceptibility to pH. For example, Herpes Simplex type I and II virus have been distinguished based on their relative susceptibility to inactivation at elevated pH conditions. Likewise, 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. This literature about classification of virus based on susceptibility to inactivation by pH, however, is confusing in that different buffers, media and the purity levels of viral preparation were used by different investigators, which confounds reaching conclusions about the absolute pH conditions sufficient to inactivate virus. Perhaps this is because the purpose was not to measure the absolute pH conditions for sufficient viral inactivation, but rather was to measure the relative susceptibility to pH of two different viruses exposed to the same conditions.

Some conclusions about viral inactivation at elevated pH can be drawn from the literature. Very high pH (> O.IM NaOH, pH >13 in water) is suggested as useful in sterilizing a nonbiological material such as a surface or a column, for example, wiping down a counter top or washing a chromatographic column and the like to assist in rendering that surface virus (and prion) free. For example, Grun et al., "Viral Removal/Inactivation by Purification of Biopharmaceuticals, BioPharm Volume 5, No. 9, Page 22-30", involves a discussion of viral removal/inactivation which occurs during the process of purification of biopharmaceuticals. Studies were conducted to show that virus was inactivated at very low pH (3.0 to 4.0) and at very high pH (13.0 to 14.0). The authors conclude with respect to pH that because "sodium hydroxide is generally used to sanitize chromatography columns and is not considered to be part of the purification process, reduction factors resulting from virus inactivation by sodium hydroxide cannot be added to the overall clearance data". "Such data do, however, provide some assurance that viral contaminants are not accumulating on the column resins creating the potential for leaching into the product during a later purification cycle." Furthermore, NaOH at this pH also removes proteins, lipids and other components that are tightly bound to column resins by denaturation and fragmentation of these materials. The use of elevated pH, thus, has not been known as a treatment for a biological isolate.

Perhaps this is, in part, because of the confusion created by the above-mentioned literature leaving unclear which viruses under which conditions will be inactivated. More certainly, it is because elevated pH is known to destroy the biological activity of proteins.

In summary, the prior art established that those of ordinary skill in the art did not consider elevated pH to be the solution to rendering an IgG fraction free of active virus. Numerous references teach various methodologies specifically adapted to render a fraction of IgG free of virus; none suggests using elevated pH to achieve this. For example, US Patent 4,939.176 teaches that one should destroy enveloped virus in an IgG preparation by treatment with caprylic acid. US Patent 4,814,277 teaches that one should eliminate virus from an IgG preparation by treatment with neutral hydrolases. US Patent 4,762,714 teaches that one should use low pH to eliminate retroviruses. When taken as a whole, 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.

Summary of the Invention 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. Surprisingly, it has been determined that 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. It specifically has been found with respect to IgG that 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.. According to one aspect of the invention, 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. More preferably 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. Most preferably, 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.

According to another aspect of the invention, a product obtainable by any one of the foregoing processes is provided. In one embodiment, 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.

According to another aspect of the invention, a method is provided 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. In most embodiments, the pH is maintained at between 10.0 and 12.0. In one important embodiment, 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. In particular, it has been shown that a Herpesvirus, a Togavirus, a Parvovirus. and a Picomavirus can be inactivated according to the methods set forth above. According to still another aspect of the invention, a product obtainable by the foregoing process is provided. In particular, 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.

According to yet another aspect of the invention, a method is provided 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.

These and other aspects of the invention are described in great detail below.

Detailed Description of the Invention 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. Thus, 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. Secondly, it was unexpected that robust viruses such as a Togaviruses and a Parvoviruses would be eliminated at the pHs employed according to the invention. Thirdly, it was unexpected that the desired biological activity of material such as proteins would survive the pH conditions employed to inactivate virus. In fact, in connection with the susceptibility of prions to elevated pH, it had been postulated that prions were inactivated because proteins were being denatured. Fourthly, it was unexpected that a protein such as IgG would aggregate over an extremely narrow range of elevated pH, making critical the pH employed in inactivating virus in a biological preparation containing IgG. The method thus is selective for viral inactivation. leaving other selected biomolecules such as IgG substantially active.

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. Thus, as described herein, 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. The term biological fluid preparation, as defined herein, includes suspensions or solutions of biological molecules that have been suspended or dissolved in nonbiological fluids such as buffers, chemical solutions and water. For example, 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.

It will be understood by those skilled in the art that 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. For example, 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. Another useful alkaline solution is 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. For example, the compound used to decrease the pH to neutral or below could be H₃PO₄, preferably 0.1 - 0.5N. Other useful compounds include citric acid, glycine HC1, and H₂SO₄.

The pH-adjusted biological fluid preparation is incubated within a temperature range that is nondestructive to the immunoglobulin. Although 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. It also is possible that 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. It is important to note that although other agents capable of inactivating viruses can be included when the biological fluid preparation is maintained at the pH conditions of the invention, it is the action of the elevated pH to inactivate the active virus present, at levels acceptable for maintaining the desired biological activity in the biological fluid preparation, that forms the basis of the invention. Thus, the methods of the invention can be carried out in the absence of any of the materials known in the prior art for inactivating viruses. This is an important aspect of the invention, in that the materials typically added in the prior art need not be added or removed after viral inactivation according to the methods of the invention. Thus, the biological fluid preparation can be free of β-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. We have demonstrated 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:

VIRAL INACTIVATION PROGRAM VIRUSES EMPLOYED IN VALIDATING

Encephalomyo- Picornaviridae Non-enveloped RNA Hepatitis A Yes carditis (EMC)

Polio Type 1 No

Hepatitis A Picornaviridae Non-enveloped RNA Yes

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,

Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae and the Unnamed Family for African Swine Fever Virus. Of course, it is understood that with further research, 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. In listing a viral family, it is understood that the members of the viral family are included in the list of viral agents that can be inactivated by the present invention. For example, the Family Herpesviridae includes the following:

Sub-family Alphaherpesviridae (Herpes Simplex-like Viruses) Genus Simplexvirus

Human Pathogens: Herpes Simplex Vims 1 and 2, Cercophithecine Herpesvirus 1 (B-vims)

Animal Pathogens: Infectious Bovine Rhontracheitis Vims, Bovine Mammillitis Virus, Cercopithecine Herpesvirus 1 (B-Virus)

Genus Varicellavirus

Human Pathogens: Varicella-Zoster Vims

Animal Pathogens: Pseudorabies Vims, Equine Rhinopneumonitis, Coital Eanthema Viruses

Sub-family Betaherpesviridae (The Cytomegaloviruses) Genus Cytomegalovirus

Human Cytomegalovirus Genus Muromegalovirus Murine Cytomegalovims

Sub-family Lymphocryptovirus (Epstein-Barr-like Vimses)

Genus Lymphocryptovirus (Epstein-Barr-like Vimses) Human Pathogen: Epstein-Barr Vims Animal Pathogens: Baboon Herpesvirus, Pongine (chimpanzee) Herpesvirus

Most preferably, the present method is useful for the inactivation of human pathogens such as HIV, Hepatitis A (HAV), Hepatitis B (HBV), Hepatitis C (HCV), Human B19 (Parvovirus), Herpes Simplex Vims (HSV) Types 1 and 2, and Poliovimses. 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. The capsid contains four stmctural proteins and is quite stable to acid and heat treatment. 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.

Furthermore, many types of bacteria will be inactivated by the methods of the invention. In general, 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. Thus the invention pertains to eliminating pathogens from biological fluid preparations.

It is believed that 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.

Certain of the foregoing and other features of the invention will described further below in the examples.

Examples Vimses Studied

Five vimses, Bovine Parvovirus (BPV), Bovine Viral Diarrhea vims (BVD), Porcine Pseudorabies Vims (PPV) Murine Encephalomyocarditis (EMC) and Hepatitis A were separately spiked into purified gammaglobulin solutions, the pH adjusted to between pH 1 1.0 and pH 1 1.1, and the solutions incubated for up to 120 hours at room temperature. The solutions were then assayed at various time intervals for viral inactivation. Spiked immunoglobulin solutions without pH adjustment were used as controls. Bovine Viral Diarrhea vims (BVD, strain KY-22) is a 40-70 nm, enveloped. RNA- containing vims. Recent studies of the viral genome and physical characteristics of Hepatitis C vims (HCV, formerly known as non-A and non-B hepatitis) have shown it to be a member of the Flavivims family, most closely related to the Pestivims genus. Because HCV cannot be propagated in vitro, and there are no animal models available for HCV infection other than chimpanzees, BVD has been used as a model for HCV in process validation studies.

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 (EMC) 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.

Preparation of Purified Gammaglobulin Solution Purified immunoglobulin was produced by the Cohn-Oncley alcohol fractionation process (Cohn et al., J Am Chem. Soc. (1946) 68:459-475, Oncley et al., J Am. Chem. Soc. (1949) 71 :541-550) or by a purification process developed by Middlesex Sciences that does not include alcohol fractionation (described below).

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 T₀ 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_comroι 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. At 24h, 48h, 72h, 96h and 120h, a 5ml aliquot was removed, adjusted to pH 6.5 - 8.0 and frozen immediately at or below -70°C.

Thus, the following samples were generated: T₀: pH 10.95 - 11.1 Incubation T₂₄: pH 10.95 - 1 1.1 Incubation T₇₂: pH 10.95 - 11.1 Incubation

T₉₆: pH 10.95 - 1 1.1 Incubation T₁₂₀: pH 10.95 - 1 1.1 Incubation T₁₂₀: pH 6.5 - 8.0 Incubation

Vims Titration

Samples were quick thawed by placing the -70°C material in a 37°C water bath. Samples were tested undiluted and at ten-fold dilutions in triplicate on the appropriate indicator cell line. For Bovine Parvovims and BVD, Bovine Turbinate (BT) cells at 30-50% confluence in 60mm Petri dishes were washed once with Balanced Salts Solution (BSS) and then received 0.2ml of either the test sample, positive controls (known amounts of vims in media), or negative controls (no vims into Dulbecco's Modified Eagle's Medium and 10% horse semm). After 30 - 90 minutes of incubation at 36°C ± 2°C the test material was removed and the plates overlayed with agar and allowed to solidify. Plates were then placed in a humidified CO₂ 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 and Hepatitis A vims were treated in a similar manner, but were assayed for plaque formation on Vero cells (EMC), PK-13 cells (Pseudorabies), and FRhK / JP cells (Hepatitis A).

The limit of sensitivity for this assay is <1.67 PFU/ml based on the following calculation: <1 plaque / 3 dishes / 0.2ml per dish = <1.67 PFU/ml. 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

The results are shown in Tables 1 through 5. 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 T₀due 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.

BVD Table 1 : Bovine Viral Diarrhea (BVD) Vims Titers after Incubation at pH 10.95-1 1.1

"Vims brought to non-detectable levels. As shown in Table 1, pH 10.95 - 1 1.1 incubation reduced BVD titer in 5% human IgG by >6.03 logs between 72 and 96h (vims brought to non-detectible levels).

BPV Table 2. Bovine Parvovims (BPV) 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 T₀ and T_{con o]} samples (vims in IgG) showed a 1.6 and 1.89 log_I0 reduction respectively, in relation to a T₀ 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 Table 3. Encephalomyocarditis (EMC) Titers after Incubation at pH 10.95 - 1 1.1.

^s 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.

* Vims unaffected by incubation in buffer for 120h. ^a 1 PFU detected in 3 plates at a 10-1 dilution (0.2 ml/plate). ^b 0 PFU detected in undiluted samples on 3 plates (0.2 ml/plate)

Undiluted samples interfered with plaquing efficiency; therefore, values are reported at 10-1 dilution.

EMC was reduced to non-detectible levels (>5.74 logs) between 48 and 72h incubation at pH 10.95 - 11.1. Porcine Pseudorabies Virus

Table 4. Porcine Pseudorabies Vims Titers after Incubation at pH 10.95 - 11.1.

, n mal titer difference between these samples indicating that IgG has no effect on vims. Data suggest that the vims losses some infectivity (-2.5 log) after prolonged incubation (120h). However, complete inactivation in treated samples occurred -48 hours. ^a 1 PFU detected in 3 plates (0.2 ml/plate). ^b 0 PFU detected in 3 plates (0.2 ml/plate)

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.

$ Vims unaffected by IgG.

Hepatitis A titer was reduced by 2.4 logs following 120h at pH 10.95 - 11.0. Preparation of Purified Human IgG from Plasma Deficient in Titer to Hepatitus A Virus

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.

Conclusion from the Viral Studies In conclusion, 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).

Stability of Gamma Globulin The integrity of human gamma globulin to pH 1 1.0 incubation was determined by accessing the stmcturai and functional activity of treated human IgG. Purified human gamma globulin (-5-10% protein), obtained from commercial sources either as a finished product or a pre-formulation bulk product was used in the studies. Material was buffer exchanged into 5mM phosphate buffer, pH 5.0 and then adjusted with the same buffer to 1-5% protein as determined spectrophotometrically (E^l% ₂₈₀ = 13.8). 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. Human IgG was analyzed pre and post high pH treatment by HPLC for stmcturai integrity as well as in several assays for assessing functional activity such as 1) EIA for Rubeolla, Rubella, Pneumonia, Diphtheria; 2) Cytotoxicity assay for Diphtheria; and 3) Plaque neutralization assays (Polio and Measles). These studies (Examples 2- 7) show that pH 11.0 treated gamma globulin is functionally active.

Example 2: The Stability of Gammaglobulin Incubated at pH 11

Triplicate samples (10 ml) of purified human gamma globulin from the Massachusetts State Lab (Jamaica Plain, MA) at 5% protein and 24°C ± 2°C were raised to pH 11.0 ± 0.1 with 0.2N NaOH under stirring and then treated for up to 120 hours. The preparations were maintained at pH 11.0 ± 0.1 by adjustment with 0.2N NaOH or 0.5N H₃PO₄ Following treatment the pH was readjusted to pH 5.0, and the samples were filtered, and analyzed (40 ul) by gei permeation HPLC. Table 6. Percent of Gammaglobulin Monomer. Dimer and Oligomer during pH 1 1 Incubation

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

The pH of an IgG solution (5%) was raised with 0.2 NaOH. under stirring, and material (10ml) was removed at pH 11.0 ±0.05, 11.3, ±0.05, 11.6, ±0.05 and 12.0, ±0.05 and then placed in separate vessels. Samples were removed from each vessels at 24 hour intervals, the pH was adjusted, and samples analyzed as described in Example 2.

Table 7. Percent of Monomer, Dimer and Oligomer during pH 11 to pH 12 Incubation

nt = not tested ^* material precipitated (ppt)

Based on the foregoing results, it was concluded that incubation at pH 11.0 caused only minimal formation of polymeric IgG. However, when the pH was raised to 11.3 and 11.6, significant formation of polymeric IgG was observed at all time points. The material precipitated almost immediately upon adjustment to pH 12.0 suggesting rapid irreversible denaturation.

Example 4. Effect of Buffer Composition on Gammaglobulin Stability

5 Example 4a. Phosphate Concentration

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 H₃PO₄. 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

10 as described in the previous examples.

Table 8. Effect of Phosphate Concentration on Monomer, Dimer, and Oligomers at pH 1 1. Sample Monomer Dimer Oligomer / .

(%)

⁰ 5% protein loss versus 4.4mM phosphate

The study shows that although the amount of oligomers was modest in all phosphate 35 conditions examined, the oligomers increased (approximately 2-fold) as the concentration of phosphate ion increased from 4.4mM to 55mM. The anion effect appears to plateau at between 43-55mM phosphate. The percentage of oligomers was similar in dH₂0 and 4.4mM phosphate but there was about 5% less protein in the former as determined spectrophotometrically (E^l% _2g0 = 13.8). Table 8 shows that low molarity phosphate buffer is preferable for maintaining IgG integrity. Example 4b. Carbonate-Sulfate Buffer Versus Phosphate Buffer

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 9. Comparison of Phosphate and Carbonate Buffer on Monomers, Dimers. and Oligomers at pH 11.

Conclusions from this study were that 2mM phosphate buffer conditions were superior to 20mM phosphate buffer, and both phosphate buffer conditions were superior to carbonate-sulfate buffer at either concentration, in terms of reducing or lowering the amount of oligomer. Furthermore, fragments were apparent in the carbonate-sulfate buffer, and not in phosphate buffer. Therefore, phosphate buffer was superior to the carbonate-sulfate buffer in maintaining protein integrity. Over the course of the experiment, with increasing time, the oligomer and fragment formation increased relatively slightly. Example 4c. Effect of NaCl on Gammaglobulin Integrity

Human IgG (-2% protein) was treated (96h) and analyzed as described previously. The material was in phosphate buffer (10 or 50mM) in the presence or absence of NaCl (lOOmM) prior to pH to 11.0 treatment. 5 Table 10. Comparison of Phosphate Buffer with and without NaCl on Monomers, Dimers and Oligomers at pH 1 1.

This experiment shows that increasing buffer ionic strength (increasing NaCl) 5 modestly increases the amount of oligomers. The absence of NaCl is preferable for maintaining

IgG integrity.

Example 5: Effect of Protein Concentration on Gammaglobulin Stability upon Incubation at pH 11.0

The purpose of this study was to assess the effect of protein concentration on the 0 formation of oligomers and fragments in a purified gammaglobulin solution. A commercial gammaglobulin solution (Gamimune™, 10% protein in glycine) was obtained from Bayer

Corp., formerly Miles Laboratories, (Elkhart, Ind.). A second gammaglobulin solution was obtained from the Massachusetts State Biological Laboratories [MSL, (Jamaica Plain, MA)], which had an initial protein concentration of 5%. The Bayer solution (60 ml) was diafiltered 5 against 10 volumes of 20mM phosphate buffer, pH 5.5. The solution was diluted 1 :10 witii deionized water for a final buffer concentration of 2mM phosphate and then concentrated to 5% protein. Samples with different protein concentrations were made by diluting the Bayer and

MSL solutions (5%) with 2mM phosphate to obtain 2.5% and 1% solutions. The pH of all samples was raised to pH 1 1.0 using 0.2N NaOH as described previously. The incubation was 0 stopped at 24, 48, 72, 96, 120 and 144 hours by adjusting the solution to pH 5.0-6.0 by addition of 0.5N H₃PO₄. Protein concentration was determined spectrophotometrically (E'^% ₂₈₀ = 13.8) and then samples were chromatographed by gel filtration HPLC for assessment of oligomers

(Table 1 1). The Control sample was not subjected to high pH treatment. Activity of gammaglobulin was determined by its reactivity to measles antigen. The measle assay is an ELISA kit from the Sigma Chemical Company (St. Louis, MO).

Table 1 1 : Activity and Stability of MSL and Bayer IVIG Treated at pH 1 1 at 5%, 2.5% and 1% protein

This study demonstrates that the bioactivity of the antibodies were maintained during high pH treatment from 24 to 144 hours. The activity of the treated samples was essentially equal to the untreated material.

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. In 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 Britain, CT); and Clostridium tetani (Tetanus), and Corynebacterium diphtheriae (Diphtheria), (both from Massachusetts State Lab, Jamaica Plain, MA). ELISA Tests Human gammaglobulin (Massachusetts State Lab, Jamaica Plain, MA) at 5% protein and in 5mM phosphate buffer was treated at high pH as described, previously. Samples (triplicates) were drawn at 72, 96 and 120 hours, concentrated to about 5% protein in a Centriprep 10K unit (Amicon Corp, MA), sterile filtered (0.2m) and then shipped on ice to the testing sites.

Table 12 ELISA Functional Activity of Gammaglobulin treated at pH 1 1

tn c σ d H C H m en x m m

H *ci°>

3 2₈0 13.8 c m *For Specific activity the ± value represents the sample standard deviation from the mean +%T₀ = Specific Activity Sample/Specific Activity T₀ x 100

A comparison of the functional activity of the treated samples to the untreated control (T₀) can be made based on the % T₀ 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. Functional Activity of Human Gammaglobulin Following Viral Inactivation Conditions (pH 11): Additional Functional Tests

Samples were tested at the Massachusetts State Lab (Jamaica Plain, MA) for 1) Diphtheria Antitoxin neutralization; 2) Plaque neutralization of Measles and Polio Vims; and 3) in a Fluorescent test for antibody to Varicella-Zoster Vims (Chicken Pox). Table 13. Functional Activity of Gammaglobulin treated at pH 11.

^* All samples pass these potency tests for commercial release of product

⁺ For plaque neutralization the ± value represents the sample standard deviation from the mean. The results are reported in absorbency units/ml (AU/ml) for Diphtheria vims

(neutralization of toxin effects on Vero cells), titers for Polio and Measles, and the titer of gammaglobulin (greatest dilution of material) at which Varicella-Zoster vims was not detected by immunofluorescene. 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 (T₀) samples show the same Diphtheria neutralization value and similar titers for the three vimses that were examined.

Claims

1. A method for treating an IgG preparation to render the preparation free of active vims comprising, maintaining the IgG 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 active vims in the preparation, and then adjusting the pH to between about 4.5 and 8.0.

2. A method of Claim 1 , wherein the IgG preparation is maintained at a pH of between about 10.5 and 11.4.

3. The method of Claim 1, wherein the IgG preparation is maintained at a pH of between about 10.9 and 1 1.3.

4. The method of Claim 1 , wherein the IgG preparation is maintained at a pH of between about 1 1.1 and 1 1.3.

5. The method of Claims 1 , 2, 3 or 4, wherein the IgG preparation is simultaneously maintained at a temperature of between 4 °C and 37 °C.

6. The method of Claim 5, wherein the IgG preparation is simultaneously maintained at a temperature of between about 18°C and 27°C.

7. The method of Claim 6, wherein the IgG preparation is maintained at said pH for at least 24 hours.

8. The method of Claim 5 wherein the preparation is a fraction obtained from a body fluid.

9. The method of Claim 5 wherein the preparation is a fraction obtained from blood.

10. A product obtainable by the process of any one of Claims 1, 2, 3, 4, 5, 6, 7, 8 and 9.

1 1. A method for treating a biological fluid preparation containing a non-vims biological material, the biological material having a native biological activity, to render the preparation free of an enveloped and a nonenveloped vims, comprising: maintaining the preparation at pH conditions above about 10.0 for a sufficient period of 5 time and under conditions such that the pH inactivates the enveloped and nonenveloped vims in the preparation, wherein the pH is low enough whereby the native biological activity is substantially retained; and, then adjusting the pH of the preparation to between about 6.0 and 8.0.

10 12. The method of claim 1 1 wherein the pH is maintained for a sufficient period of time to inactivate an enveloped DNA vims, an enveloped RNA vims, a nonenveloped DNA vims and a nonenveloped RNA vims.

13. The method of Claim 11 wherein the pH is maintained for a sufficient period of time and 15 under conditions sufficient to inactivate a herpesvirus, a togavirus, a parvovims and a picomavims.

14. The method of Claim 13, wherein the pH of the preparation is maintained between 10.0 and 12.0.

20

15. The method of Claim 13, wherein the pH of the preparation is maintained between 10.5 and 1 1.4.

16. The method of Claim 13, wherein the pH of the preparation is maintained between 10.9 25 and 11.3.

17. The method of Claims 1 1, 12, 13, 14, 15 or 16, wherein the preparation is maintained at a temperature of between 4^CC and 37 °C.

30 18. The method of Claim 17, wherein the preparation is maintained between 18 °C and 27°C.

SUBSTITUTE SHEET RULE 25

19. The method of Claim 18, wherein the preparation is maintained at said pH for at least 24 hours.

20. A product obtainable by the process of any one of Claims 11, 12, 13, 14, 15, 16, 17, 18 and 19.