CA2450730A1 - Methods for sterilizing preparations of monoclonal immunoglobulins - Google Patents

Abstract

Methods are disclosed for sterilizing preparation of monoclonal immunoglobulins to reduce the level of active biological contaminants such a viruses, bacteria, yeasts, molds, mycoplasmas, prions and parasites.

Description

METHODS FOR STERILIZING PREPARATIONS OF MONOCLONAL
IMMUNOGLOBULINS
Field of the Invention The present invention relates to methods for sterilizing preparations of monoclonal immunoglobulins to reduce the level of active biological contaminants therein, such as viruses, bacteria, yeasts, molds, mycoplasmas, prions and/or parasites.
Background of the Invention Antibodies are produced by organisms in response to exposure to foreign substances that the body perceives as a threat. Antibodies, or as they are collectively known, immunoglobulins (2g), are proteins secreted by cells of the immune system known as B-cells or plasma cells. The structure of immunoglobulins is complex, but is well characterized. In brief, each immunoglobulin consists of a complex of protein chains known as the heavy and light chains. Each heavy chain is linked to a single light chain via disulfide bonds. The resulting complex is in turn linked by additional disulfide bonds to an identical heavy-light chain complex. This basic unit can be assembled by the cell into several specialized forms by varying the structure and number of heavy chains.
Different heavy chain structures produce differing molecules, known as "classes" of immunoglobulins. These classes may also have different numbers of the basic units described above.
The production of these various physical forms of the immunoglobulin molecule occurs in a sequential manner. During this process, the specificity of the molecule for a single molecule or antigen remains unchanged. This is because the changes described above all occur to the portion of the immunoglobulin molecule that is not involved in determining the specificity of the particular immunoglobulin molecule. This "hypervariable region" is subject to an unusually high degree of recombination events during B-cell maturation. These recombination events cease prior to the production of the first immunoglobulin molecule by the cell. The.result is that from a relatively small number of variable region genes, the body generates a large number of potential immunoglobulin molecules of differing specificities.
Once a B-cell encounters a molecule to which its own immunoglobulin molecule binds (an "antigen"), and upon receiving signals from other cells in the immune system, the B-cell first multiplies into a large number of identical cells, (collectively referred to as a clone) and then differentiates into an immunoglobulin-secreting plasma cell. In this way, the extremely large number of potential immunoglobulin molecules that might be manufactured is limited to only those molecules that recognize antigens to which the body must respond.
The vast array of immunoglobulin specificities that are produced results in an ongoing protection for the body against infection from those organisms that the body has made immunoglobulins against in the past. Taken in sum, the result is that the immunoglobulins contained in the plasma from a single donor may have millions of useful immunoglobulin specificities. A preparation of immunoglobulins from plasma is thus referred to as a polyclonal immunoglobulin preparation, since it contains the immunoglobulin molecules produced by all of the plasma cell clones in the body.
Polyclonal immunoglobulins are particularly useful for treating human disease in which the ability to produce Ig is absent or impaired. Since all plasma cell clones are affected, a mixture of all the immunoglobulin specificities found in the plasma is needed to correct the deficiency. In contrast, when an extreme degree of specificity is required, or when a single defined therapeutic goal is sought, polyclonal immunoglobulins are not the best solution. Instead, an immunoglobulin preparation consisting of the immunoglobulin molecules produced by a single clone with the desired specificity is the most precise and predictable solution. Such a preparation is known as a monoclonal immunoglobulin.
Monoclonal immunoglobulin have many differences as compared to polyclonal immunoglobulins. Their monospecificity makes them very precise when used as detection reagents. As therapeutics, they are free of confounding or dangerous side effects that arise from polyclonal immunoglobulin preparations, such as the introduction of immunoglobulins of unwanted specificities being introduced into the patient. Their physical characteristics may also be different. Since each monoclonal immunoglobulin has a unique and unvarying structure, its potential for stability, degradation, aggregation, temperature sensitivity and other~characteristics are unique and unchanging. Once a suitable monoclonal immunoglobulin has been chosen for production, its characteristics will not change, and it thus can be manufactured with great consistency and assurance of its performance and storage characteristics.
The ability to tailor production volumes to product requirements also makes monoclonal immunoglobulin a highly desirable alternative to polyclonal immunoglobulins.
Monoclonal immunoglobulin preparations are made in one of three general fashions. The first involves production in a cell culture system, the second uses an animal as a temporary bioreactor for monoclonal immunoglobulin production, and the third involves inserting the gene for a desired monoclonal immunoglobulin into an animal in such a manner as to induce continuous production of the monoclonal immunoglobulin into a fluid or tissue of the animal so that it can be continuously harvested (transgenic production).
Each of these methods may result in contamination of the product by pathogens. In the first method, the cells producing the monoclonal immunoglobulin may harbour undetected viruses that can be produced in the culture system.
Contamination of the culture system by bacteria, yeast or mold may also occur.
Both of the remaining methods involve the use of an animal to either serve as a host for the monoclonal immunoglobulin-producing cells or as a bioreactor to manufacture the monoclonal immunoglobulin product itself.
Obviously, these products face the risk of contamination by pathogens infecting or harboured by the host animal. Such pathogens include, viruses, bacteria, yeasts, molds, mycoplasmas, and parasites, among others.
Consequently, it is of utmost importance that any biologically active contaminant in the monoclonal immunoglobulin product be inactivated before the product is used. This is especially critical when the product is to be administered directly to a patient. This is also critical for various monoclonal immunoglobulin products which are prepared in media which contain various types of plasma and which may be subject to mycoplasma or other viral contaminants.
Previously, most procedures have involved methods that screen or test products for a particular contaminant rather than removal or inactivation of the contaminant from the product. Products that test positive for a contaminant are merely not used. Examples of screening procedures include the testing for a particular virus in human blood from blood donors. Such procedures, however, are not always reliable and are not able to detect the presence of viruses in very low numbers. This reduces the value or certainty of the test in view of the consequences associated with a false negative result. False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the product is contaminated.
In conducting experiments to determine the ability of l0 technologies to inactivate viruses, the actual viruses of concern are seldom utilized. This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal. In their place, model viruses of the same family and class are used.
In general, it is acknowledged that the most difficult viruses to inactivate are those with an outer shell made up of proteins, and that among these, the most difficult to inactivate are those of the smallest size. This has been shown to be true for gamma irradiation and most other forms of radiation as these viruses diminutive size is a consequence of their small genome. The magnitude of direct effects of radiation upon a molecule are directly proportional to the size of the molecule, that is the larger the target molecule, the greater the effect. As a corollary, it has been shown for gamma-irradiation that the smaller the viral genome, the higher the radiation dose required to inactive it.
Among the viruses of concern for~both human and animal-derived biologics, the smallest viruses of concern belong to the family of Parvoviruses and the slightly larger protein-coated Hepatitis virus. In humans, the Parvovirus B19, and Hepatitis A are the agents of concern. In porcine-derived products and tissues, the smallest corresponding virus is Porcine Parvovirus. Since this virus is harmless to humans, it is frequently chosen as a model virus for the human B19 Parvovirus and Hepatitis A. The demonstration of inactivation of this model parvovirus is considered adequate proof that the method employed will kill human B19 virus and Hepatitis A, and by extension, that it will also kill the larger and less hardy viruses such as HIV, CMV, Hepatitis B and C and others.
More recent efforts have focused on methods to remove or inactivate contaminants in the products. Such methods include heat treating, filtration and the addition of chemical inactivants or sensitizers to the product. Heat treatment requires that the product be heated to approximately 60°C for about 70 hours which can be damaging to sensitive products.
Heat inactivation can destroy 50% or more of the biological activity of the product. Filtration involves filtering the product in order to physically remove contaminants.
Unfortunately this method may also remove products that have a high molecular weight. Further, in certain cases small viruses may not be removed by the filter because of the larger molecular structure of the product. The procedure of chemical sensitization involves the addition of noxious agents which bind to the DNA/RNA of the virus and which are activated either by UV or radiation to produce reactive intermediates and/or free radicals which bind to the DNA/RNA or break the chemical bonds in the backbone of the DNA/RNA of the virus or crosslink or complex it in such a way that the virus can no longer replicate. This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and can not be administered to a patient.
Irradiating a product with gamma radiation is another method of sterilizing a product. Gamma radiation is effective in destroying viruses and bacteria when given in high total doses (Keathly et al., "Is There Life After Irradiation? Part 2," BioPharm July-August, 1993, and Leitman, Use of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease," Transfusion Science 10:219-239 (1989)). The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, protein and protein-containing products. In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman). U.S. Patent No. 4,620,908 discloses that protein products must be frozen prior to irradiation in order to maintain the viability of the protein product. This patent concludes that "[i]f the gamma irradiation were applied while the protein material was at, for example, ambient temperature, the material would be also completely destroyed, that is the activity of the material would be rendered so low as to be virtually ineffective". This would apply as well to monoclonal immunoglobulins which are, of course, proteins. Unfortunately, many sensitive biologicals, such as monoclonal antibodies (Mab), would lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.
In view of the difficulties discussed above, there remains a need for methods of sterilizing monoclonal immunoglobulins that are effective for reducing the level of active biological contaminants without an adverse effect on the monoclonal immunoglobulins.
Summary of the Invention Accordingly, it is an object of the present invention to provide methods of sterilizing preparations of monoclonal immunoglobulins by reducing the level of active biological _g_ contaminants without adversely affecting the monoclonal immunoglobulins. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized. and attained by the compositions and methods particularly pointed out in the written description and claims hereof .
l0 In accordance with these and other objects, a first embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect the preparation of monoclonal immunoglobulins from radiation; and (ii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins.
A second embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) adding to a preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect the preparation of monoclonal. immunoglobulins from radiation; and (ii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins.
A third embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation _g_ comprising: (i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect the preparation of monoclonal immunoglobulins from radiation; (ii) adding to the preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect the preparation of monoclonal immunoglobulins from radiation; and (iii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins. According to this embodiment, steps (i) and (ii) may be reversed.
The invention also provides a composition comprising at least one monoclonal immunoglobulin and a least one stabilizer selected from the group consisting of: ascorbic acid or a salt or ester thereof; glutathione; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; uric acid or a salt or ester thereof; methionine; histidine; N-acetyl cysteine; the dipeptide glycine-glycine; diosmin; silymarin; a mixture of ascorbic acid, or a salt or ester thereof, and uric acid, or a salt or ester thereof; a mixture bf ascorbic acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; a mixture of ascorbic acid, or a salt or ester thereof, uric acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; and a mixture of uric acid, or a salt or ester thereof and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, said at least one stabilizer being present in an amount effective to preserve said monoclonal immunoglobulin for its intended use following sterilization of the composition with radiation.
Brief Description of the Drawings Figures 1 and 2 are graphs showing the protective effects of certain stabilizers on lyophilized anti-insulin monoclonal immunoglobulin exposed to 45 kGy of low dose gamma irradiation.
Figures 3A-3C are graphs showing t-he protective effects of certain stabilizers on lyophilized anti-insulin monoclonal immunoglobulin exposed to 45 kGy of low dose gamma irradiation.
Figure 4 & 5 are graphs showing the protective effects of primary lyophilizing and secondary lyophilizing on' the sensitivity of a monoclonal immunoglobulin.
l0 Figures 6-11 are graphs showing the protective effect of certain stabilizers on the activity of lyophilized anti insulin monoclonal immunoglobulin.
Figure 12 is a graph showing the protective effect of stabilizers on the activity of lyophilized anti-insulin monoclonal immunoglobulin when the sample was irradiated at a high dose rate (30 kGy/hr).
Figure 13 is a graph showing the effect of a stabilizer on IgM activity after irradiation with gamma radiation.
Figure 14 and 15 are graphs showing the effect of stabilizers on immobilized anti-insulin monoclonal immunoglobulin after irradiation with gamma radiation.
Figures 16A, 16B, 17A, and 17B are graphs showing the effect of ascorbate at varying concentrations on immobilized anti-insulin monoclonal immunoglobulin after irradiation with gamma radiation.
Figures 18A-18H are graphs showing the effect of stabilizers on lyophilized anti-insulin monoclonal immunoglobulin supplemented with human serum albumin or sucrose, after irradiation with gamma radiation.
Figures 19A-19F are graphs showing the effect of stabilizers on lyophilized anti-insulin monoclonal immunoglobulin supplemented with bovine serum albumin, after irradiation with gamma radiation.
Figures 20A and 20B are graphs showing the effect of various doses of gamma radiation on anti-insulin monoclonal immunoglobulin, with and without ascorbate.
Figures 21A and 21B are graphs showing the effect of low pH (4.5) on the stabilizing effect of L-ascorbic acid on monoclonal immunoglobulin irradiated to 45 kGy with gamma radiation.
Figure 22 is a graph showing the level of viral inactivation by irradiation with gamma radiation of an anti-insulin monoclonal immunoglobulin contaminated with porcine parvovirus (PPV).
Figures 23A and 23B are~graphs showing the effect of the presence or absence of sodium ascorbate on the level of activity retention achieved when irradiating monoclonal immunoglobulins in both liquid and lyophilized forms with e-beam radiation.
Detailed Description of the Preferred Embodiments A. Defini tioas Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as is commonly understood by one of ordinary skill ~in the relevant art. All patents and publications mentioned herein are expressly incorporated by reference.

As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise.
As used herein, the term "immunoglobulin" is used synonymously with the term "antibody", and encompasses all classes of immunoglobulins including, without limitation, IgG, IgM, IgA, IgD and IgE and all subclasses of immunoglobulins such as the IgG subclasses IgGl, IgG2, IgG3, and IgG4 found in or produced by cells or animals including humans. The team "immunoglobulin" encompasses both membrane immunoglobulins and secreted immunoglobulins. Membrane immunoglobulins are transmembrane proteins of B cells, and act as the B cells' antigen receptor. Secreted immunoglobulins are structurally identical to their membrane counterparts except that they lack the traps-membrane region of amino acids at the C-terminus of membrane immunoglobulins. Secreted immunoglobulins are present in extracellular fluids and secretions.
The term "immunoglobulin" also encompasses fragments of immunoglobulins including, without limitation, fragments F(ab')2, Fab', Fab, Fc, Facb, pFc', and Fd, as well as immunoglobulin derivatives and metabolites as are known in the art. Metabolites of immunoglobulin are products resulting from the metabolism of immunoglobulins by a living organism. A wide variety of derivatives of immunoglobulins as are known in the art may be prepared by known methods, which typically involve breaking peptide or disulfide bonds in the immunoglobulin.
Immunoglobulins may also be derivatized to include modified or synthetic or unnatural amino acids. Derivatives of immunoglobulins also include immunoglobulins conjugated to a moiety such as a toxin (e.g. diphtheria toxin, ricin), a labelling molecule (e. g. fluorescin, Texas Red), a radioactive atom or molecule (e. g. last) for therapeutic or diagnostic use, an enzyme (e. g. avidin, horseradish peroxidase, alkaline phosphatase), et cetera. Immunoglobulins may include post-translational modifications such as phosphorylation, glyocsylation, myristilation, prenylation, ADP-ribosylation, methylation, acetylation, hydroxylation, carboxylation, and oxidation-reduction, or may be cationized or anionized to alter the overall charge of the immunoglobulin.
The term "monoclonal immunoglobulins" refers to homogeneous immunoglobulins produced by a single clone, and are to be contrasted with polyclonal immunoglobulins. Monoclonal immunoglobulins are usually made from hybridomas, which are prepared by fusing immunized mouse or rat spleen cells with a non-secretor myeloma using polyethylene glycol (PEG). The fusion mixture is plated out in HAT medium, containing hypoxanthine, aminopterin and thymidine. Aminopterin blocks a.
metabolic pathway which can be bypassed if hypoxanthine and thymidine are present. The myeloma cells lack this bypass and consequently die in the HAT medium. Spleen cells die naturally in culture after one or two weeks, but fused cells survive because they have the immortality of the myeloma and the metabolic bypass of the spleen cells. Some of the fused cells secrete antibody, and the supernatants are tested in a specific assay. Wells which produce the desired antibody are then cloned. Monoclonal immunoglobulins may also be produced by various techniques familiar to one skilled in the art of genetic engineering that cause the inducible or constitutive expression of a single set of endogenous genes that code for a single immunoglobulin, or that involve the addition of such a set or portion of a set, of genes into a cell or organism in such a manner as to result in either the inducible or 3o constitutive expression of the resulting single set of genes that code for a single immunoglobulin.
Monoclonal immunoglobulins may be produced using the above-described techniques in combination with in vitro cell culture, in vivo cell culture (for example as an ascites culture) or by the techniques of transgenesis. Transgenesis involves the insertion of one or more genes into a recipient organism. The recipient organism then produces the protein product of these genes, either constitutively or following induction, and either incorporates them or secretes the into a tissue (including eggs) or fluid (including blood, sweat, milk or urine). The resulting tissue or fluid is then harvested and the desired monoclonal immunoglobulin is purified from it.
As used herein, the term "preparation of monoclonal immunoglobulins" encompasses, without limitation: (1) compositions consisting solely of monoclonal immunoglobulins (such as monoclonal immunoglobulins of a single specificity or combinations of monoclonal immunoglobulins of different specificities and/or different classes) and which may contain impurities (including the naturally-occuring components of a tissue or fluid into which the monoclonal antibody was produced by transgenic means); (2) compositions comprising monoclonal immunoglobulins, pharmaceutically acceptable diluents, carriers, adjuvants, liposomes and other therapeutic agents, et cetera, as are known in the art; (3) partially-purified in-process intermediate preparations of monoclonal immunoglobulins; and (4) articles containing monoclonal immunoglobulins or having monoclonal immunoglobulins immobilized upon them or otherwise disposed thereon. Preferred "preparations of monoclonal immunoglobulins" are discussed below.
In therapeutic applications, monoclonal immunoglobulins are typically combined with buffer or salt solutions as are known in the art. The monoclonal immunoglobulin may also be combined with another therapeutic agent. For instance, anti-platelet monoclonal immunoglobulins which prevent platelets from aggregating are being evaluated for long-term therapy for the prevention of formation of thrombi. In this application, the anti-platelet monoclonal immunoglobulin is administered together with aspirin. The monoclonal immunoglobulin and the other therapeutic agent may be co-packaged for administration by, for example, intravenous injection. This may take the form of simple inclusion into a single external package, or they may be provided in a single container. An example of a more advanced form of packaging would be the use of monoclonal immunoglobulins in liposome preparations. Typically, the immunoglobulin is embedded a.n at least the outer layer of the liposome where it can act as a targeting agent by binding to structures on or in the desired cells or tissues. The drug, which is contained within the liposome, is then released at this specific site, providing a more concentrated drug therapy with a larger therapeutic index than achievable by ordinary systemic therapy.
For therapeutic use, the monoclonal immunoglobulins are typically provided in liquid, frozen, or freeze-dried (lyophilized) form packaged under nitrogen or vacuum. The monoclonal immunoglobulins are usually then reconstituted with sterile water (if required, or thawed as needed) and administered by an appropriate route, such as an intramuscular injection or intravenous injection either directly or following placement in an IV bag. In accordance with the invention, the preparation of monoclonal immunoglobulins may be irradiated at any stage, including but not limited to as a raw material, a purified or partially-purified in-process intermediate, in bulk or in individual or multi-dose packaging, before or after packaging, after dilution for administration, or in the IV bag or other delivery vehicle itself. The irradiation may be carried out at any convenient temperature that does not have a deleterious effect upon the preparation, and which may be above or below the freezing or eutectic point of the preparation.

Various preparations of monoclonal immunoglobulins are available for therapeutic use, as set forth in Table 1.
Table 1: Therapeutic Monoclonal Immunoglobulin Products Physical Name mAb Name TargetIndication Company Status State*

Metastatic LicensedFD

Anti- Breast HerceptinTrastuzumabHER2 Cancer Genentech, Anti- B Cell non- L

Rituxan Rituximab CD20 Hodgkin's Genentech, I

Anti- ClinicalFD

Xolair OmalizumabIgE asthma Genetech, Trials Relapsed Metastatic Anti- Breast Clinical Anti-'iIEGFVEGF Cancer Genetech, Trials Anti- Severe ClinicalFD

Xanelim Anti-CDllaCDlla Psoriasis Genetech, Trials Anti- ClinicalFD

LDP-02 Anti-a4b7 a4b7 IBD Genetech, Trials Anti-HER2 r Clinical 2C4 Solid TumorsGenetech, Trials I

Crohn's ClinicalFD

Remicade InfliximabcA2 Disease Centacor Trials Gleevec CML Novartis C

Berlex/

I~ Campath~ Alemtuzu~ ~ CLL ~ Millennium Source for above Table: A practical guide to ELISA, D.M.
Kemeny, Pergamon Press *; FD - Freeze-Dried (Lyophylized), L - Liquid, I - Immobilized on a surface, C - Capsule (Oral Dosing) containing dried material _17_ Preparations of monoclonal immunoglobulins also find application as research tools and a vast number are used for diagnostic purposes, particularly in blood work. Typical research tools and diagnostic tests involving monoclonal immunoglobulins include enzyme-linked imunnosorbent assays (ELISA), radioimmunoassays (RIA), magnetic-bead-based assays and separation kits, and fluorescent activated cell analysis and/or sorting, among others. It is desirable to sterilize such diagnostic tools, both to preserve them, as well as for safety, as they are handled by lab technicians. Preparations of monoclonal immunoglobulins used for diagnostic purposes often comprise a solid support, such as a dipstick or plastic plate, having the monoclonal immunoglobulins immobilized on or in it by covalent chemistry, drying, or other known means. In accordance with the invention, an entire commercial package or kit, containing the solid support with monoclonal immunoglobulins affixed thereto, instructions, containers of reagents (for instance a reference sample for generating a standard curve), et cetera, may be sterilized in accordance with the invention. Table 2 sets forth a number of commercial diagnostic monoclonal immunoglobulin products.

Table 2:
Diagnostic Monoclonal Immunoglobulin Products Physical Name mAb NameTarget IndicationCompany Status State*

i CMV IgG ELISA Sigma FD , IgM ELISA Sigma FD

HSV Type FD/L

and 2 (IgG) ELISA Sigma Rubella FD/L

IgG

(Indirect ELISA Sigma Rubella FD

IgG

(Capture) ELISA Sigma Lyme FD

Disease (Indirect ELISA Sigma Mumps FD

(Indirect) ELISA ~ Sigma Pregnancy Test Kits J&J

FAGS FD/L

analysis (diagnosti BD.

c imaging) Coulter Sources for above Table: A practical guide to ELISA, D.M.
Kemeny, Pergamon Press *: FD - Freeze-Dried (Lyophylized), L - Liquid, I - Immobilized on a surface, C - Capsule (Oral. Dosing) containing dried material As used herein, the term "sterilize" is intended to mean a reduction in the level of at least one active biological 9_ contaminant found in the preparation of monoclonal immunoglobulins being treated according to the present invention.
As used herein, the term "biological contaminant" is intended to mean a contaminant that, upon direct or indirect contact with a preparation of monoclonal immunoglobulins, may have a deleterious effect on a preparation of monoclonal immunoglobulins or upon a recipient thereof. Such biological contaminants include the various viruses, bacteria and parasites known to those of skill in the art to generally be found in or infect preparation of monoclonal immunoglobulins.
Examples of biological contaminants include, but are not limited to, the following: viruses, such as human immuno-deficiency viruses and other retroviruses, herpes viruses, parvoviruses, filoviruses, circoviruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses (including hepatitis B and hepatitis C), pox viruses, toga viruses, Epstein-Barr virus and parvoviruses; bacteria, such as Escherichia, Bacillus, Campylobacter, Streptococcus and Staphylococcus; parasites, such as Trypanosoma and malarial parasites, including Plasmodium species; yeasts; molds; mycoplasmas; and prions. As used herein, the term "active biological contaminant" is intended to mean a biological contaminant that is capable of causing the deleterious effect.
2S As used herein, the term "a biologically compatible solution" is intended to mean a solution to which monoclonal immunoglobulins may be exposed, such as by being suspended or dissolved therein, and remain viable, i.e., retain their essential biological and physiological characteristics.
As used herein, the term "a biologically compatible buffered solution" is intended to mean a biologically compatible solution having a pH and osmotic properties (e. g, tonicity, osmolality and/or oncotic pressure) suitable for maintaining the integrity of monoclonal immunoglobulins.
Suitable biologically compatible buffered solutions typically have a pH between 4 and 8.5 and are isotonic or only moderately hypotonic or hypertonic. Biologically compatible buffered solutions are known and readily available to those of skill in the art.
As used herein, the term "stabilizer" is intended to mean a compound or material that reduces any damage to the preparation of monoclonal immunoglobulins being irradiated to a level that is insufficient to preclude the~safe and effective use of the preparation of monoclonal immunoglobulins.
Illustrative examples of stabilizers include, but are not limited to, the following: antioxidants, such as ascorbic acid.
and tocopherol; and free radical scavengers, such as ethanol.
Preferred examples of stabilizers include, but are not limited to, the following: fatty acids, including 6,8-dimercapto-octanoic acid (lipoic acid) and its derivatives and analogues (alpha, beta, dihydro, bisno and tetranor lipoic acid), 2o thioctic acid, 6,8-dimercapto-oct'anoic acid, dihydrolopoate (DL-6,8-dithioloctanoic acid methyl ester), lipoamide, bisonor methyl ester and tatranor-dihydrolipoic acid, furan fatty acids, oleic and linoleic and palmitic acids and their salts and derivatives; flavonoids, phenylpropaniods, and flavenols, such as quercetin, rutin and its derivatives, apigenin, aminoflavone, catechin, hesperidin and, naringin; carotenes, including beta-carotene; Co-Q10; xanthophylls; polyhydric alcohols, such as glycerol, mannitol; sugars, such as xylose, glucose, ribose, mannose, fructose and trehalose; amino acids, such as histidine, N-acetylcysteine (NAC), glutamic acid, trypt'ophan, sodium carpryl N-acetyl tryptophan and methionine;
azides, such as sodium azide; enzymes, such as Superoxide Dismutase (SOD) and Catalase; uric acid and its derivatives, such as 1,3-dimethyluric acid and dimethylthiourea;
allopurinol; thiols, such as glutathione and reduced glutathione and cysteine; trace elements, such as selenium;
vitamins, such as vitamin A, vitamin C (including its derivatives and salts such as sodium ascorbate and palmitoyl ascorbic acid) and vitamin E (and its derivatives and salts such as tocopherol acetate and alpha-tocotrienol); chromanol-alpha-C6; 6-hydroxy-2,5,7,8-tetramethylchroma-2 carboxylic acid (Trolox) and derivatives; extraneous proteins, such as gelatin and albumin; tris-3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186); citiolone; puercetin; chrysin; dimethyl sulfoxide (DMSO);
piperazine diethanesulfonic acid (PIPES); imidazole;
methoxypsoralen (MOPS); 1,2-dithiane-4,5-diol; reducing substances, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT); cholesterol; probucol; indole derivatives; thimerosal; lazaroid and tirilazad mesylate;
proanthenols; proanthocyanidins; ammonium sulfate; Pegorgotein (PEG-SOD); N-text-butyl-alpha-phenylnitrone (PBN); 4-nydroxy-2,2,6,6-Tetramethylpiperidin-1-oxyl (Tempol); mixtures of ascorbate, urate and Trolox C (Asc/urate/Trolox C); proteins, peptides and dipeptides; the Glycine homodipeptide glycine-glycine (Gly-Gly); reduced glutathione; diosmin; pupurogalin;
gallic acid and its derivatives including but not limited to propyl gallat;, sodium formaldehyde sulfoxylate and silymarin.
As used herein, the term ~~residual solvent content°
is intended to mean the amount or proportion of freely-available liquid in the preparation of monoclonal immunoglobulins. Freely-available liquid means that liquid, such as water or an organic solvent (e. g. ethanol, isopropanol, polyethylene glycol, etc.), present in the preparation of monoclonal immunoglobulins that is not bound to or complexed with one or more of the non-liquid components of the preparation of monoclonal immunoglobulins. Freely-available liquid includes intracellular water. The residual solvent contents related as water referenced herein refer to levels determined by the FDA approved, modified Karl Fischer method (Meyer and Boyd, Analytical Chem., 31, 215-219, 1959; May, et al., J. Biol. Standardization, 10, 249-259, 1982; Centers for Biologics Evaluation and Research, FDA, Docket No. 89D-0140, .83-93; 1990). Quantitation of the residual levels of other solvents may be determined by means well known in the art, depending upon which solvent is employed. The proportion of residual solvent to solute may also be considered to be a reflection of the concentration of the solute within the solvent. When so expressed, the greater the concentration of the solute, the lower the amount of residual solvent.
As used herein, the term "sensitizer" is intended to mean a substance that selectively targets viral, bacterial, prion and/or parasitic contaminants, rendering them more sensitive to inactivation by radiation, therefore permitting the use of a lower rate or dose of radiation and/or a shorter time of irradiation than in the absence of the sensitizer.
Illustrative examples of suitable~sensitizers include, but are not limited to, the following: psoralen and its derivatives and analogs (including 3-carboethoxy psoralens); angelicins, khellins and coumarins which contain a halogen substituent and a water solubilization moiety, such as quaternary ammonium ion or phosphonium ion; nucleic acid binding compounds; brominated hematoporphyrin; phthalocyanines; purpurins; porphorins;
halogenated or metal atom-substituted derivatives of dihematoporphyrin esters, hematoporphyrin derivatives, benzoporphyrin derivatives, hydrodibenzoporphyrin dimaleimade, 3o hydrodibenzoporphyrin, dicyano disulfone, tetracarbethoxy hydrodibenzoporphyrin, and tetracarbethoxy hydrodibenzo-porphyrin dipropionamide; doxorubicin and daunomyci.n, which may be modified with halogens or metal atoms; netropsin; BD

peptide, S2 peptide; S-303 (ALE compound); dyes, such as hypericin, methylene blue, eosin, fluoresceins (and their derivatives), flavins, merocyanine 540; photoactive compounds, such as bergapten; and SE peptide.
As used herein, the term "radiation" is intended to mean radiation of sufficient energy to sterilize at least some component of the irradiated monoclonal immunoglobulins. Types of radiation include, but are not limited to, the following:
(i) corpuscular (streams of subatomic particles such as neutrons, electrons, and/or protons); and (ii) electromagnetic (originating in a varying electromagnetic field, such as radio waves, visible (both mono and polychromatic) and invisible Light, ultraviolet radiation, x-radiation, and gamma rays and mixtures thereof). Such radiation is often described as either 25 ionizing (capable of producing ions in irradiated materials) radiation such as gamma rays and non-ionizing radiation such as visible light. The sources of such radiation may vary, however in general the specific source is of little material difference as long as sufficient radiation is given in an appropriate time and at an appropriate rate to effect sterilization. In practice, gamma radiation is usually produced by isotopes of Cobalt or Cesium, while X-rays are produced by machines that emit X-radiation, and electrons are often used to sterilize materials in a method known as "e-beam" irradiation that involves their production via a machine.
B. Particularly Preferred Embodiments A first preferred embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) reducing the residual solvent content of the preparation of monoclonal immunoglobulins to a level effective to protect the preparation of monoclonal immunoglobulins from radiation; and (ii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins.
~ A second embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that .is sensitive to radiation comprising: (i) adding to a preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect the preparation of monoclonal immunoglobulins from radiation; and (ii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins.
A third embodiment of the present invention is directed to a method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect the preparation of monoclonal immunoglobulins from radiation; (ii) adding to the preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect the preparation of monoclonal immunoglobulins from radiation; and (iii) irradiating the preparation of monoclonal immunoglobulins with radiation at an effective rate for a time effective to sterilize the preparation of monoclonal immunoglobulins. The order of steps (i) and (ii) may, of course, be reversed as desired.
According to the methods of the present invention, the residual solvent content of the preparation of monoclonal immunoglobulins may be reduced prior to irradiation of the preparation of monoclonal immunoglobulins with radiation. The residual solvent content is reduced to a level that is effective to protect the preparation of monoclonal immuno-globulins from the radiation. Suitable levels of residual solvent content may vary depending upon the nature and characteristics of the particular preparation of monoclonal immunoglobulins being irradiated and can be determined empirically by one skilled in the art. Preferably, when the solvent is water, the residual solvent content is less than about 10%, more preferably less than about 2.0%, more preferably less than about 1.0%, even more preferably less than about 0.5% and most preferably less than about 0.2%.
According to the methods of the present invention, the monoclonal antibody to be sterilized may be immobilized upon a solid surf ace by a means familiar to one skilled in the art.
According to the methods of the present invention, the rate of irradiation may be optimized to produce the most advantageous combination of product recovery and time required to complete the operation. Both low {<3 kGy/hour) and high {>3 kGy/hour) rates may be achieved by the appropriate application of the methods described herein.
While not wishing to be bound by any theory of operability, it is believed that the reduction in residual solvent content reduces the degrees of freedom of the preparation of monoclonal immunoglobulins and thereby protects it from the effects of the radiation. Similar results might therefore be achieved by lowering the temperature of the preparation of monoclonal immunoglobulins below its eutectic point or below its freezing point, or by vitrification to likewise reduce the degrees of freedom of the preparation of monoclonal immunoglobulins. These results may permit the use of a higher rate of irradation than might otherwise be acceptable. Thus the inventions described herein may be carried out at any temperature that does not result in damage to the monoclonal immunoglobulin.
The residual solvent content of the preparation of monoclonal immunoglobulins may be reduced by any of the methods and techniques known to those skilled in the art for reducing solvent from a preparation of monoclonal immunoglobulins. Such methods may include, but are not limited to, evaporation, concentration, centrifugal concentration, vitrification and spray-drying. A particularly preferred method for reducing the residual solvent content of a preparation of monoclonal immunoglobulins is lyophilization. According to a particularly . preferred embodiment of the present invention, a preparation of monoclonal immunoglobulins which has been lyophilized is stored under vacuum or an inert atmosphere (preferably a noble gas, such as helium or argon, more preferably a higher molecular weight noble gas, and most preferably argon) prior to irradation.
The radiation employed in the present invention may be any radiation effective for the inactivation of one or more biological contaminants of the preparation of monoclonal immunoglobulins being treated. The radiation may be corpuscular, including e-beam radiation. Preferably the radiation is electromagnetic radiation, including visible light, W light and mixtures of various wavelengths of electromagnetic radiation and a particularly preferred form of radiation is gamma radiation.
According to the methods of the present invention, the preparation of monoclonal immunoglobulins is irradiated with the radiation at a rate effective for the inactivation of one or more biological contaminants of the preparation of monoclonal immunoglobulins. Suitable rates of irradiation may vary depending upon the particular form of radiation and the nature and characteristics of the particular preparation of monoclonal immunoglobulins being irradiated and the particular biological contaminants being inactivated. Suitable rates of irradiation can be determined empirically by one skilled in the art. Preferably, the rate of irradiation is constant for the duration of the sterilization procedure. When this is impractical, a variable or discontinuous irradiation may be utilized.
according to a particularly preferred embodiment of the present invention, the rate of irradiation is not more than about 3.0 kGy/hour, more preferably between about 0.1 kGy/hr.
and 3.0 kGy/hr, even more preferably between about 0.25 kGy/hr and 2.0 kGy/hour, still even more preferably between about 0.5 kGy/hr and 1.5 kGy/hr and most preferably between about 0.5 kGy/hr and 1.0 kGy/hr.
According to another particularly preferred embodiment of the present invention, the rate of irradiation is at least about 3.0 kGy/hr., more preferably at least about 6 kGy/hr., even more preferably at least about 16 kGy/hr., and even more preferably at least about 30 kGy/hr and most preferably at least about 45 kGy/hr or greater.
The preparation of monoclonal immunoglobulins is irradiated with the radiation for a time effective for the inactivation of one or more biological contaminants of the preparation of monoclonal immunoglobulins. Combined with irradiation rate, the appropriate irradiation time results in the appropriate dose of irradiation being applied to the monoclonal immunoglobilin. Suitable ionization times may vary depending upon the particular form and rate of radiation and the nature and characteristics of the particular preparation of monoclonal immunoglobulins being irradiated and the particular biological contaminants being inactivated. Suitable irradiation times can be determined empirically by one skilled in the art.
Optionally, an effective amount of at least one sensitizing compound may be added to the monoclonal immunoglobulin prior to irradiation to enhance the anti-microbial effect of the irradiation, while employing the methods described herein to minimize the deleterious effects of irradiation upon the monoclonal immunoglobulin. Suitable sensitizers are known to those skilled in the art.
According to methods of the present invention, the irradiation of the preparation of monoclonal immunoglobulins may occur at any temperature which is not deleterious to the preparation of monoclonal immunoglobulins being treated.
According to a preferred embodiment, the preparation of monoclonal immunoglobulins is irradiated at ambient temperature. According to an alternate preferred embodiment, the preparation of monoclonal immunoglobulins is irradiated at reduced temperature, preferably at or below the freezing or eutectic point of the preparation of monoclonal immunoglobulins.
In order to avoid aggregation of the monoclonal immunoglobulins, the preparation of monoclonal immunoglobulins may have a pH of less than 7, preferably less than 6, more preferably less than 5, even more preferably less than 4, and most preferably less than 3.
It will be appreciated that combination of the several methods described herein may be employed to further minimize undesirable effects upon the monoclonal immunoglobulin caused by irradiation, while maintaining adequate effectiveness of the anti-microbial properties of the irradiation process.

Examples The following examples are illustrative, but not limiting, of the present invention. Other suitable modifications and adaptations are of the variety normally encountered by those skilled in the art and are fully within the spirit and scope of the present invention. Unless otherwise noted, all irradiation was accomplished using~a 6°Co source.
Example 1 In this experiment the protective effects of certain stabilizers were evaluated using lyophilized anti-insulin monoclonal immunoglobulin exposed to 45 kGy of low dose gamma irradiation. The stabilizers tested were: sodium ascorbate, methionine, and lipoic acid.
Method In 2 ml glass vials, a 0.5 ml total volume was lyophilized containing 50~.g anti-insulin monoclonal immunoglobulin, 5 mg bovine serum~albumin (1%) and either no stabilizer or 50mM of the stabilizer of interest. The samples were stoppered under vacuum. Samples were irradiated with gamma radiation (45 kGy total dose, dose rate 1.83 kGy/hr, temperature 4°C) and then reconstituted with water.
Immunoglobulin binding activity of independent duplicate samples was determined by a standard ELISA protocol:
96-well microtitre plates were coated overnight with 2.5ug/ml insulin antigen. Three-fold serial dilutions of anti-insulin monoclonal antibody samples starting at 5ug/ml were used. Goat anti-mouse Ig conjungated to phosphatase used at 50 ng/ml.
Sigma 104 alkaline phosphatase substrate was used at 1 mg/ml in DEA buffer. Binding activity was determined by absorbance at 405-620nm.
Relative protection was determined by estimating the shift in the titration curve (i.e. concentration of immuno-globulin needed to observe the same amount of binding) of the irradiated sample compared to an unirradiated sample at approximately 50% of the maximum absorbance signal for the unirradiated sample.
Results Lyophilized samples containing no stabilizer retained 50% of immunoglobulin avidity following irradiation with 45 kGy gamma irradiation. This is in contrast to previous results in which 45 kGy of gamma radiation destroyed essentially all the activity of immunoglubulin when it was irrradiated in solution.
Thus, it is apparent that the reduction in. residual water content by lyophilizing afforded significant protection on its own to the monoclonal immunoglobulin.
The addition of sodium ,ascorbate provided full recovery of activity after irradiation of the sample. Both methionine and lipoic acid provided significant recovery of activity (76-83%) of activity after irradiation as compared to the unirradiated sample. The results are shown in Figures 1 and 2. Similar results (65% recovery of activity) were also seen for pupurogalin (data not shown).
Example 2 In this experiment, the protective effects of certain stabilizers were evaluated using lyophilized anti-insulin monoclonal immunoglobulin exposed to 45 kGy of low dose gamma irradiation. The stabilizers tested were: sodium ascorbate, N-acetyl cysteine, glutathione and mixtures of urate/trolox and ascorbate/urate/trolox.
Me thod Tn 3 ml glass vials, a 1.0 ml total volume was lyophilized containing 100ug anti-insulin monoclonal immunoglobulin, 10 mg~bovine serum albumin (1%) and either no stabilizer or the stabilizer of interest. The samples were stoppered under vacuum, Samples were irradiated with gamma radiation (45 kGy total dose, dose rate 1.83 kGy/hr, temperature 4°C) and then reconstituted with 1.0 ml water.
Immunoglobulin binding activity of independent duplicate samples was determined by a standard ELISA protocol:
Maxisorb plates were coated overnight with 2.5ug/ml insulin antigen. Three-fold serial dilutions of anti-insulin monoclonal immunoglobulin samples starting at 5ug/ml were used.
Goat anti-mouse Ig conjugated to phosphatase was used at 50 ng/ml. Binding activity was determined by absorbance at 405-620nm.
Relative protection was determined using a parallel line analysis software package (PLA 1.2 from Stegmann Systemberatung).
Resu1 is Lyophilized samples containing no stabilizer retained 70°s of immunoglobulin avidity following irradiation with 45 kGy gamma irradiation. This is in contrast to previous results in which 45 kGy of gamma radiation destroyed essentially all the activity of immunoglubulin when it was irrradiated in solution.
Thus, it is apparent that the reduction in residual water content by lyophilizing afforded significant protection on its own protein.

The presence of sodium ascorbate increased recovery by 20%, i.e. such that there is 90% avidity recovered after irradiation. The remaining stabilizers resulted in recovery of 77-84% of avidity. The results are shown in Figures 3A-3C.
Example 3 In this experiment, the protective effects of primary lyophilizing (which leaves a relatively "high moisture" content in the product) and the combination of both primary and secondary lyophilizing (which results in a product with relatively "low moisture") on the radiation sensitivity of a monoclonal immunoglobulin were determined.
Methods ~In 3 ml glass vials, 1.0 ml total volume was lyophilized (using either only primary or a combination of both Z5 primary and secondary drying) containing 100ug anti-insulin monoclonal immunoglobulin, 10 mg bovine serum albumin (1%) and either no stabilizer or 100 mM of sodium ascorbate. The samples were stoppered under vacuum. Samples were irradiated with gamma radiation (45 kGy total dose, dose rate between 2.03 and 2.13 kGy/hr, temperature 4°C) and then reconstituted with 1.0 ml water.
Immunoglobulin binding activity of independent duplicate samples was determined by a standard ELISA protocol:
Maxisorb plates were coated overnight with 2.5ug/ml insulin antigen. Three-fold serial dilutions of anti-insulin mAb samples starting at 5ug/ml were used. Goat anti-mouse Ig conjugated to phosphatase was used at 50 ng/ml. Binding activity was determined by absorbance at 405-620nm.

lZesul is In the absence of a stabilizer, there was better recovery of the anti-insulin immunoglobulin after irradiation from the samples that had undergone the secondary "low moisture" drying cycle, i.e. a lower total moisture content in the absence of a stabilizer improved recovery.
In the presence of the stabilizer, however, there was very good recovery of antibody activity after 45 kGy irradiation, irrespective of whether the sample had undergone only the primary "high moisture" drying cycle or had also undergone the secondary "low moisture" drying cycle.
The results of this experiment are shown in Figures 4 and S.
Example 4 In this experiment, the protective effect of certain stabilizers on the activity of lyophilized anti-insulin monoclonal immunoglobulin was determined. The stabilizers tested were; sodium ascorbate; trolo.x/urate/ ascorbate mixtures; and N-acetyl cysteine.
2 0 Me thods Anti-insulin monoclonal immunoglobulin supplemented with 1% of human serum albumin (and, optionally, 5% sucrose) was lyophilized, stoppered under vacuum, and irradiated (total dose 45 kGy; dose rate between 1.83 and 1.88 kGy/hr).
Immunoglobulin binding activity was determined using the standard EZISA protocol described above.

Resu1 is Irradiation of lyophilized anti-insulin immuno-globulin supplemented with 1% HSA to a dose of 45 kGy resulted in an average loss of avidity of about 33%. The addition of the following stabilizers significantly improved recovery: 2omM
sodium ascorbate (100% recovery); 200~ZM trolox/l.5mM uratei20 mM ascorbate (87%) recovery); 20 mM N-acetyl cysteine (82%
recovery The addition of 5% sucrose to the lyophilized immunoglobulin containing 1% HSA resulted in an average loss of avidity of about 30% when irradiated to a dose of 45 kGy. The addition of the following stabilizers significantly improved recovery: 20mM sodium ascorbate (88% recovery); 200uM
trolox/l.5mM urate/20 mM ascorbate (84%) recovery); 20 mM N-acetyl cysteine (72% recovery).
The results of these experiments are shown in Figures 6-11.
Example 5 In this experiment, the protective effect of stabilizers (ascorbate) on the activity of lyophilized anti-.insulin monoclonal immunoglobulin was determined when the sample was irradiated at a high dose rate (30 kGy/hr).
Methods Anti-insulin monoclonal immunoglobulin was lyophilized and irradiated at a rate of 30 kGy/hr (total dose 45 kGy). Immunoglobulin binding activity was determined using the standard EhISA protocol described above.

Resu1 is Irradiation of lyophilized anti-insulin immunoglobulin to a dose of 45 kGy resulted in an average loss of activity of about 32%, The addition of 20mM sodium ascorbate provided 85% recovery of avidity compared to an unirradiated sample. The results are shown in Figure 12.
Examgle 6 In this experiment, an IgM monoclonal immunoglobulin specific for marine IgG3 was irradiated at a low dose rate in l0 the presence or absence of a stabilizer.
Method Liquid rat anti-marine IgG3 monoclonal IgM (in a PBS
buffer with 10 mM sodium azide; concentration of antibody was 666 ng/~1) was irradiated at a rate of 1.8 kGy/hr to a total dose of either 10 kGy or 45 kGy, Samples either contained no stabilizer or a stabilizer mixture containing 20 mM citrate, 300 uM urate and 200 mM ascorbate.
Immunoglobulin activity was analyzed by standard LISA protocol using marine IgG3 as the coating antigen and a phosphatase-conjugated anti-rat IgM detection antibody.
Resu3 is Liquid samples containing no stabilizer lost all functional immunoglobulin activity following irradiation with either lOkGy or 45 kGy gamma irradiation. The presence of a stabilizer mixture, however, provided full recovery of activity following irradiation with 10 kGy gamma radiation and 88%
recovery of activity following irradiation with 45 kGy gamma radiation. The results of this experiment are shown graphically in Figure 13.

Example 7 In this experiment, the protective effects of certain stabilizers were evaluated using immobilized anti-human insulin monoclonal immunoglobulin exposed to 45 kGy of low dose-rate gamma irradiation. The stabilizers tested were: sodium ascorbate, reduced glutathione, sodium formaldehyde sulfoxylate, and polypropylene glycol.
Me thod Two plates were coated with 100 ul/well of freshly prepared 2 ug/ml anti-insulin immunoglobulin in coating buffer overnight at 4°C. The plates were washed briefly three times with PBS. A two-fold dilution series of each stabilizer in PBS
was prepared. 100 u1 of a selected stabiliser solution was added to each well. The plates were covered tightly with a cap mat. One plate was irradiated at 1,92 kGy/hr for a total of 45 kGy at 4°C. The control plate received 0 kGy and was stored at 4°C.
Immunoglobulin binding activity was determined by a standard ELISA protocol. The plate wells were emptied and were washed four times with a full volume of PBS . A full volume of blocking buffer (approximately 380 u1) was added to all wells and incubated for two hours at 37°C. All wells were washed four times with TBST (TBS pH 7.4 with 0.05% TWEEN 20). One hundred u1 of 50 ng/ml biotin-labelled insulin in binding buffer was added to each well. The plates were covered with a plate sealer and incubated at 37°C while shaking (LabLine titer plate shaker set at 3) for 1.5 hours. The plates were then washed four times with TEST. One hundred u1 of 0.5 ug/ml phosphatase labelled Streptavidin (stock diluted 1:1000 in binding buffer) was added to each well. The plates were covered with a plate sealer and incubated at 37°C for one hour with shaking. The plates were then washed four times with TBST. One hundred u1 of 1 mg/ml Sigma 104 phosphatase substrate in DEA buffer was added to each well. The plates were then incubated at 37°C with shaking. Absorbance was determined at 405nm-628nm at 5 minute intervals.
Resu1 is As shown in Figures 14 and 15, sodium ascorbate exhibited a dose-dependent protective effect. Samples containing between 31-250 mM of sodium ascorbate exhibited 73~
81% greater retained activity.
l0 Samples containing glutathione exhibited approximately 25% greater retention of monoclonal immunoglobulin activity, that was dose dependent up to a glutathione concentration of about 31 mM.
Samples treated with sodium formaldehyde sulfoxylate exhib7.ted approximately 50% greater retained activity than control samples at a stabilizer concentration of 31 mM.
All three forms of polypropylene glycol (i.e.
polypropylene P400 (Fluka 81350);~polypropylene P1200 (Fluka 81370); and polypropylene P2000 (Fluka 81380)) exhibited a protective effect. Samples treated with polypropylene glycol exhibited approximately 50-60% increased retention of activity relative to control samples.
Example 8 In this experiment, the optimal concentration of sodium ascorbate to protect immobilized anti-insulin monoclonal immunoglobulins from 45 kGy of gamma irradiation was determined. Tt was also determined whether the presence of 1.5 mM uric acid has any effect on the stabilizing nature of ascorbate of immobilized monoclonal immunoglobulin exposed to 45 kGy gamma irradiation.

Method Two plates were coated overnight at 4°C with 100 u1 of 2.5 ug/ml anti-insulin monoclonal immunoglobulin in coating buffer. The coating solution was discarded and the wells washed two times with PBS. Twenty-five u1 of 4X ascorbate solution was added to appropriate wells. Seventy-five ~1 of water was added to the orate-free wells (rows a-d). Twenty five ~.1 of water was added to the orate containing wells (rows e-h). Fifteen u1 of 3 mM orate was added to the orate containing wells (rows e-h). The plates were covered with a 96-well cap mat. One plate was irradiated with gamma radiation at 1.9 kGy/hr for a total of 45 kGy at 4°C. The other plate was stored at 4°C as a travel control.
Immunoglobulin binding activity was determined by a standard ELISA protocol as follows. The well contents were removed; and the wells washed twice with a full volume of. PBS.
Non-specific binding sites were blocked by adding a full volume of blocking buffer (approximately 380 u1) to all wells and incubated for two hours at 37°C. All wells were washed three times with TBST. One hundred u1 of 10 ng/ml insulin-biotin in binding buffer was added to each well (stock diluted 1:100,000 in binding buffer). The plates were covered with a plate sealer and incubated at 37°C with shaking (LabLine titer plate shaker set at three) for one hour. The plates were washed with TBST for four sets of two washes each set, usually leaving five minutes between each set. One hundred u1 of 25 ng/ml phosphatase-labelled Streptavidin (stock diluted 1:20,000 in binding buffer) was added to each well. Plates were covered with a plate sealer and incubated at 37°C for one hour with shaking. Each plate was washed with TBST for four sets of two washes each set, usually leaving approximately five minutes between each set. One hundred u1 of 1 ng/ml Sigma 104 phosphatase substrate in DEA buffer was added to each well.

The plates were incubated at ambient temperature with nutation.
Absorbance was determined at 405nM-620nM.
Resu1 is It was determined that the optimal concentration of sodium ascorbate necessary to provide maximal protection of immobilized anti-insulin monoclonal immunoglobulins in an aqueous environment (in the absence of uric acid) is approximately 150 mM. Approximately 50% recovery of the anti~-insulin binding activity was achieved at a concentration of approximately 150 mM ascorbate. The addition of 1.5 mM uric acid resulted in a slight left shift in the ascorbate dose curve (~5 mM) and appeared to cause maximal recovery of activity to be achieved at a lower concentration of ascorbate (~3o mM). Figure 16A shows the complete data set, and Figure 16B is an expansion of the critical region of the data used to determine these values.
Example 9 In this experiment, the~optimal concentration of sodium ascorbate to protect immobilized monoclonal immunoglobulin from 45 kGy gamma irradiation was determined.
The experiment also determined whether the presence of 2.25 mM
of uric acid effects the stabilizing effect of ascorbate.
Method Two plates were coated overnight at 4°C with 1.00 uz of 2.5 ug/ml anti-insulin monoclonal immunoglobulin in coating buff er. The coating solution was discarded and the wells washed twice with PBS. Twenty-five u1 of 4X ascorbate solution was added to appropriate wells. Seventy-five u1 of water was added to the urate-free wells (rows a-d). Seventy-five u1 of 3 mM urate stock was added to the urate-containing wells (rows e-h)(f.c. - 2.25 mM). The plates were covered with a 96-well cap mat. One plate was irradiated with gamma radiation at 1.9 kGy/hr for a total of 45 kGy at 4°C. The other plate was stored at 4°C as a travel control.
Monoclonal immunoglobulin binding activity was determined as in Example 8.
Results As illustrated in Figures 17A and 17B, the optimal concentration of sodium ascorbate necessary to provide maximum protection of immobilized anti-insulin monoclonal immunoglobulin in an aqueous environment (in the absence of uric acid) was determined to be approximately 70 mM. This contrasted with Example 8, which showed the optimal concentration of ascorbate to be approximately 150 mM.
Approximately 100% recovery of anti-insulin binding activity was achieved in this example as opposed to approximately 50%
recovery in Example 8. The addition of uric acid (2.25 mM) again resulted in a slight left shift of the ascorbate dose curve (~5 mM) and appeared to cause maximum recovery of activity to be achieved at a lower concentration of ascorbate ~(~25 mM). It was found that there is a biphasic nature to the irradiated samples without uric acid. Recovery improved significantly between 0-20 mM ascorbate, levelled off from 20 50 mM ascorbate, and then went up again until maximum recovery was observed at approximately 70 mM ascorbate.
Example 10 In this experiment, the protective effect of various stabilizers on gamma irradiated freeze-dried anti-insulin monoclonal immunoglobulin supplemented with 1% human serum albumin (HSA) and 5% sucrose was evaluated. The stabilizers tested were: ascorbate (20mM); a mixture of trolox(200mM), urate (l.SuM) , and ascorbate (20mM) ; n-acetyl-1-cysteine (20mM) ;
reduced glutathione(2omM); and the dipeptide, Gly-Gly(2omM).
Me thod Samples were freeze-dried for approximately 64 hours and stoppered under vacuum and sealed with an aluminum, crimped seal. Samples were irradiated at a dose rate of 1.83-1.88 kGy/hr to a total dose of 45.1-46.2 kGy at 4°C.
Monoclonal immunoglobulin activity was determined by a standard EhISA protocol. Maxisorp plates were coated with human recombinant insulin at 2.5 ug/ml overnight at 4°C. The plate was blocked with 200 ~1 of blocking buffer (PBS, pH 7.4, 2% BSA) for two hours at 37°C and then washed six times with wash buffer (TBS, pH 7, 0.05% TWEEN 20). Samples were re-suspended in 500 u1 of high purity water (100 ng/ul), diluted to 5 ug/ml in a 300 u1 U-bottomed plate coated for either overnight or two hours with blocking buffer. Serial 3-fold dilutions were performed, with a final concentration of 0.0022~ug1ml. Plates were incubated for one hour at 37°C with agitation and then washed six times with a wash buffer.
Phosphatase-labelled goat anti-mouse IgG (H+L) was diluted to 50 ng/ml in binding buffer and 100 u1 was added to each well.
The plate was incubated for one hour at 37°C with agitation and washed six times with wash buffers. One hundred u1 of Sigma-104 substrate (1 mg/ml in DEA buffer) was added to each well and reacted at room temperature. The plate was read on a Multiskan MCC/340 at 40SnM with the 620nM absorbance subtracted.

Results As shown in Figures 18A-18H, freeze-dried anti-insulin monoclonal immunoglobulin, supplemented with 1% HSA, gamma irradiated to 45 kGy resulted in an average loss in . activity of 1.5 fold (average loss in avidity of 33%) .
Samples irradiated to 45 kGy in the presence of stabilizers gave varying results:
20 mM ascorbate = 100% recovery 200uM trolox, 1.5 mM urate, 20 mM ascorbate = ~87% recovery l0 20 mM, n-acetyl-1-cysteine = ~82% recovery 20 mM reduced glutathione = ~76% recovery 20 mM Gly-Gly = 100% recovery Adding 5% sucrose to freeze-dried anti-insulin monoclonal immur~oglobulin containing 1% HSA resulted in an average recovery of 70% of the activity in the sample irradiated to 45 kGy (average loss in activity of approximately 1.5 fold or approximately 30% loss ir_ avidity) .
The samples that radiated to 45 kGy in the presence of the aforementioned stabilizers had reduced activities upon addition of 5% sucrose:
2o mM ascorbate = ~88% recovery 200 uM trolox, 1.5 mM urate, 20 mM ascorbate = ~84% recovery 20 mM n-acetyl-1-cysteine = ~72% recovery 20 mM reduced glutathione = ~69% recovery 20 mM gly-gly = ~79% recovery Similar results have been obtained upon the addition of 2omM ascorbate, 20mM Gly-Gly or the addition of 20mM of both ascorbate and Gly-Gly to another monoclonal IgG preparation of different specificity (anti-Ig Lambda Light Chain).
Example 11 In this experiment, the protective effect of ascorbate(Asc,20mM), ascorbate(20mM)/urate(l.5mM)/trolox(200uM) cocktail(AUT), n-acetyl-cysteine(neutral form: NAC-n, acidic form: NAC-a, both at 20mM), Gly-Gly(20mM), reduced glutathione (GSH, 20mM) , diosmin (39. 3 uM) and silymarin (246 uM) on lyophilized anti-insulin monoclonal irnmunoglobulin was evaluated.
Me thod In 3 ml glass vials, 1.0 ml total volume containing 100 ~,~g anti-insulin monoclonal immunoglobulin; with 10 mg BSA
(1%) and either no stabilizer or the stabilizer of interest was lyophilized. Samples were irradiated with gamma radiation (45 kGy total dose, dose rate 1.83 kGy/hr, temperature 4°C) and~then reconstituted with 1 ml of water. Karl Fischer moisture analysis was performed on the quadruplicate samples that did not contain immunoglobulin.
Immunoglobulin binding activity of independent duplicate samples was determined by a standard ELISA protocol:
Maxisorp plates were coated overnight with 2.5 ug/ml insulin antigen. Three-fold serial dilutions of anti-insulin monoclonal immunoglobulin samples starting at 5 ug/ml were used. Goat anti-mouse phosphatase conjugate was used at 50 mg/ml. Relative potency values of irradiated samples compared to their corresponding unirradiated sample were calculated using the parallel line analysis software package (PLA 1.2 from Stegmann Systemberatung). Mass spectroscopy analysis was performed by M-scan, Inc. of WestchesterePennsylvania.
Results As illustrated in Figures 19A-19F, irradiation of lyophilized anti-insulin monoclonal immunoglobulin in the presence of 1% bovine serum albumin resulted in the loss of approximately 30% avidity (relative to unirradiated samples) of the immunoglobulin for its immobilized antigen. The addition of ascorbate alone improved the recovery by 20%, such that there was approximately 90% avidity recovered after irradiation. The addition of ascorbate/urate/trolox cocktail, the dipeptide Gly-Gly, neutral n-acetyl-cysteine, reduced glutathione, or silymarin resulted in recovery of 77-84%
avidity.
Similar results have been obtained upan the addition of 200mM ascorbate, 200mM Gly-Gly or the addition of 200mM of both ascorbate and Gly-Gly to two other monoclonal IgG
preparations of different specificity (anti-Ig Lambda Light Chain and anti-IgGl).
2 0 Exzmple 12 In this experiment, the stability of anti-insulin monoclonal immunoglobulin irradiated in the liquid form in the presence or absence of ascorbate was evaluated.
Me thod Anti-insulin monoclonal immunoglobulin was diluted to 1 mg/ml and irradiated at 4°C in the presence or absence of 200 mM ascorbate to a total dose of 0, 15, or 45 kGy of gamma radiation.

Immunoglobulin binding activity of independent duplicate samples was determined by a standard direct EZISA
protocol generally as described in the previous example.
Results As shown in Figures 20A and 20B, the addition of 200 mM ascorbate resulted in recovery of 100% of immunoglobulin binding activity of samples irradiated with 15 kGy of radiation and recovery of 71.7% and 80.4% of the activity of samples radiated with 45 kGy of radiation compared to the in-house dilution control and the 0 kGy plus ascorbate control, respectively. As determined by polyacrylamide gel electro-phoresis, irradiation of the anti-insulin immunoglobulins and the absence of the stabilizer resulted in protein aggregation as evidenced by high molecular weight'bands on polyacrylamide gels. Additionally, a significant loss of material was apparent. The addition of 200 mM ascorbate had a protective effect on the immunoglobulins irradiated at 15 kGy and 45 kGy, as demonstrated by the recovery of an intact IgGl band and as well as heavy and light chain bands.
When duplicates of the 15 kGy plus 200 mM ascorbate samples were averaged, the antigen binding activity was not significantly different from that of the dilution control. In contrast, irradiating the samples containing ascorbate to 45 kGy resulted in an average 2- and 2,5- fold decrease in avidity when compared to the in-house dilution control and stock control, respectively. The SDS-PAGE analysis indicated that in the absence of ascorbate, irradiating the anti-insulin monoclonal immunoglobulins resulted in significant loss of material and a generation of high molecular weight aggregate.
The addition of 200 mM ascorbate prevented aggregate formation and resulted in recovery of approximately 80% and approximately 50% of the immunoglobulins following irradiation to 15 kGy and 45 kGy, respectively.
Example 13 This experiment was conducted to determine whether low pH (4.5) diminishes the stabilizing effect of L-ascorbic acid on monoclonal immunoglobulin irradiated to 45 kGy with gamma radiation.
Method An anti-human insulin monoclonal Ig (Anti-Human Insulin Monoclonal Immunoglobulin, Purified Clone #7F8;BioDesign International #E86102M, lot 7125000) was irradiated as a liquid at a rate of 1.774 kGy/hr (6°Co) in the presence and absence of 200 mM L-Ascorbic acid to a total dose of 45kGy, at pH 6.8 and 4.5. Following irradiation, the samples were assayed for their antigen-specif is binding capability in an ELISA assay using insulin-coated plates as targets. Structural analysis of the Ig was done via standard SDS-PAGE electrophoresis under both reduced and non-reduced conditions.
Results As illustrated in Figures 21A and 21B, the ELISA
functional assay results clearly show that recovery of the monoclonal immunoglobulin in the presence of ascorbate is not dependent on pH. The graphs for pH 6.8 and 4.5 are virtually superimposable. A slight loss of activity is seen at both pH
values upon the addition of ascorbic acid and again following irradiation, however the magnitude of this reduction is small in comparison to the complete loss of activity seen when irradiation takes place in the absence of ascorbate.

SDS-PAGE electrophoresis gels showed a complete destruction of the immunoglobulin at 45 kGy in the absence of ascorbic acid at both pH 3.8 and pH 4.5. The addition of 200 mM ascorbic acid maintained the same apparent structure upon irradiation. A pH of 4.5 may have inhibited aggregation.
These results indicate that, in the presence of 200 mM ascorbic acid, monoclonal Ig can be irradiated to at least 45 kGy while retaining structure and activity at both pH 6.7 and 4 . 5 .
Example 14 In this experiment the level of viral inactivation and monoclonal immunoglobulin activity retention in anti-insulin monoclonal immunoglobulin infected with porcine parvovirus (PPV) irradiated with 6°Co gamma radiation at an approximate rate of 1.8 kGy/hour at 4°C was evaluated.
Method PPV was utilized as a model virus for Human Parvovirus B19, a non-enveloped virus that is considered the most difficult virus of concern in human-sourced biologics and a close analog of the other members of the Parvovirus family that are also considered the most difficult viruses of concern in animal-sourced biologics.
A high titre PPV stock was spiked into a preparation of a monoclonal immunoglobulin directed against insulin. A
protectant, (sodium ascorbate) was added to some samples at a final concentration of 200 mM.
The samples to be irradiated were exposed to 6°Co radiation at an approximate rate of 1.8 kGy/hour at 4°C.

_g8_ After irradiation of some of the samples, aliquots of the spiked samples were taken and used to titre the amount of remaining infective virus particles. Briefly, the samples were assayed in a viral detection bioassay known as a Cytopathic Effect test (CPE). A cell line capable of being infected by the PPV virus and lysed by it (Porcine Kidney cells, also known as PK-13 cells) were added to 96-well assay plates to form a monolayer of approximately 70% confluence. Quadruplicate aliquots of the samples were added to the wells in a limiting-dilution series (5-fold dilutions). The plates were then incubated for 7-8 days and then examined to determine if viable cells remained in the wells. The resulting data was analysed using a Limiting-Dilution method as described by Karber to determine the viral titer which is shown in the accompanying Figure as LogloTCIDSO Titer per 0.1m1.
Resu1 is As shown in Table 4 below and in Figure 22, the application of gamma radiation effectively inactivated the virus in a dose-dependent manner. The addition of 200mM sodium ascorbate to the monoclonal immunoglobulin resulted in a significant reduction in the viral inactivation at lower doses, but at higher doses this effect was much smaller. The application of 45 kGy of gamma radiation to samples containing ascorbate resulted in greater than 4 logs of viral inactivation.

-~9-Table 4:
'y Radiation Ascorbate TCTDso Loglo Reduction Dose Titre/0.1m1 of Viral Titre 0 kGy - 7.075 " + 7.95 kGy - 5.4125 1.66 " + 3.575 4.3 7S

30 kGy - 3.4 3.675 " + 2.35 ~ 5.6 45 kGY - 2.525 4.55 " + 2.35 5.6 Example 15 This experiment was conducted to evaluate the level 5 of activity retention achieved when irradiating monoclonal immunoglobulins in both liquid and lyophilized forms with e-beam radiation in the presence or absence of sodium ascorbate.
Me thod 10 Anti-Insulin IgG1 was tested in liquid and after having been lyophilized. Samples were prepared both with and without 200 and 20mM sodium ascorbate in the liquid and lyophilized state respectively.
The samples to be irradiated (both with and without ascorbate) were exposed to e-beam irradiation at an approximate rate of 45 kGy/hour at 77-88 °F. The e-beam energy was 7 MeV and a total dose of approximately 45 kGy was given. Control samples consisted of unirradiated samples with and without ascorbate that traveled to and from the irradiation site, and a reference control sample that did not travel to the irradiation site.
During transport the samples were kept at 4 °C. The samples are shown in Table 5 below:

Table 5: Samples Sample Name Physical Ascorbate Travel to e-Beam State Irradiator Dose (kGy) Fresh Ig Liquid 0 .
L-I/0 Liquid ~ 0 L-I/45 " ~ 45~

L-I/A/ o " ~I ~I 0 L-I/A/45 " ~ ~ 45 Fresh Ig Lyophilized 0 fd-I/0 Lyophilized ~ 0 fd -I/45 " ~ 45 fd -I/A/0 " ~ '~ 0 fd -I/A/45 " ~ ~ 45 After irradiation the lyophilized samples were reconstituted with distilled water. All samples were then tested in an anti-human insulin ELISA assay as described in Example 10. Approximate measures of the recovery of antigen-binding activity were performed by hand as the concentration of Ig that produced approximately 50% of the maximum OD.

Results As seen Table 6 below, when in the liquid state the application of E-beam radiation completely inactivated the Ig.
In the presence of ascorbate, there was a clear recovery of activity, but the magnitude was limited.
The lyophilization of the Ig prior to irradiation had a greater effect upon the recovery of activity than the addition of ascorbate alone to the liquid. Approximately 50% of the antigen-binding activity was retained when ascorbate-free Ig was irradiated. The addition of 20mM ascorbate prior to lyophilization resulted in complete recovery of activity. The results of the ELISA are shown in Figures 23A and 23B.

Table 6: Approximate Antigen-Binding Activity Sample Name Physical e-Beam X50% of ~ % Recovery State Dose Max of Activity ~~GY~ OD4os-szo Fresh Ig Liquid 0 0.065 L-I/0 Liquid 0 0.12 100 L-I/45 " 45 co 0 L-I/A/0 " 0 0.18 100 L-I/A/45 " 45 1.8 10 Fresh Ig Lyophilized 0 0.17 'fd-I/0 Lyophilized 0 0.21 100 fd -I/45 " 45 0.45 50 fd -I/A/0 " 0 0.22 100 fd -I/A/45 " 45 0.21 100 Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations, and other parameters without departing from the scope of the invention or any embodiments thereof. All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Claims (98)

CLAIMS:

1. A method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation, said method comprising:
(i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect said preparation of monoclonal immunoglobulins from said radiation; and (ii) irradiating said preparation of monoclonal immunoglobulins with a suitable radiation at an effective rate for a time effective to sterilize said preparation of monoclonal immunoglobulins.

2. A method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation, said method comprising:
(i) adding to a preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect said preparation of monoclonal immunoglobulins from said radiation; and (ii) irradiating said preparation of monoclonal immunoglobulins with a suitable radiation at an effective rate for a time effective to sterilize said preparation of monoclonal immunoglobulins.

3. A method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation, said method comprising:
(i) adding to a preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect said preparation of monoclonal immunoglobulins from said radiation; and (ii) irradiating said preparation of monoclonal immunoglobulins with a suitable radiation at a low rate for a time effective to sterilize preparation of monoclonal immunoglobulins.

4. A method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation, said method comprising:
(i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect said preparation of monoclonal immunoglobulins from said radiation; and (ii) irradiating said preparation of monoclonal immunoglobulins with a suitable radiation at a low rate for a time effective to sterilize said preparation of monoclonal immunoglobulins.

5. A method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation, said method comprising:
(i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect said preparation of monoclonal immunoglobulins from said radiation;
(ii) adding to said preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect said preparation of monoclonal immunoglobulins from said radiation; and (iii) irradiating said preparation of monoclonal immunoglobulins with a suitable radiation at an effective rate for a time effective to sterilize said preparation of monoclonal immunoglobulins, wherein steps (i) and (ii) may be performed in inverse order.

6. A method for sterilizing a preparation of monoclonal immunoglobulins that is sensitive to radiation, said method comprising:
(i) reducing the residual solvent content of a preparation of monoclonal immunoglobulins to a level effective to protect said preparation of monoclonal immunoglobulins from said radiation;
(ii) adding to said preparation of monoclonal immunoglobulins at least one stabilizer in an amount effective to protect said preparation of monoclonal immunoglobulins from said radiation; and (iii) irradiating said preparation of monoclonal immunoglobulins with a suitable radiation at a low rate for a time effective to sterilize said preparation of monoclonal immunoglobulins, wherein steps (i) and (ii) may be performed in inverse order.

7. The method according to claim 1, wherein said solvent is water.

8. The method according to claim 4, wherein said solvent is water.

9. The method according to claim 5, wherein said solvent is water.

10. The method according to claim 6, wherein said solvent is water.

11. The method according to claim 7, wherein said residual water content is reduced by the addition of an organic solvent.

12. The method according to claim 8, wherein said residual water content is reduced by the addition of an organic solvent.

13. The method according to claim 9, wherein said residual water content is reduced by the addition of an organic solvent.

14. The method according to claim 10, wherein said residual water content is reduced by the addition of an organic solvent.

15. The method according to claim 1, wherein said solvent is an organic solvent.

16. The method according to claim 4, wherein said solvent is an organic solvent.

17. The method according to claim 5, wherein said solvent is an organic solvent.

18. The method according to claim 6, wherein said solvent is an organic solvent.

19. The method according to claim 1, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

20. The method according to claim 4, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

21. The method according to claim 5, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

22. The method according to claim 6, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

23. The method according to claim 7, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

24. The method according to claim 8, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

25. The method according to claim 9, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

26. The method according to claim 10, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

27. The method according to claim 11, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

28. The method according to claim 12, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

29. The method according to claim 13, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

30. The method according to claim 14, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

31. The method according to claim 15, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

32. The method according to claim 16, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

33. The method according to claim 17, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

34. The method according to claim 18, wherein said preparation of monoclonal immunoglobulins is suspended in an organic solvent following reduction of said residual solvent content.

35. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins are selected from the group consisting of: IgG and fragments, derivatives, and metabolites thereof; IgM and fragments, derivatives, and metabolites thereof; IgA and fragments, derivatives, and metabolites thereof; IgD and fragments, derivatives, and metabolites thereof; IgE and fragments, derivatives, and metabolites thereof; and mixtures thereof.

36. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins are IgG or fragments or derivatives or metabolites thereof.

37. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins are IgM or fragments or derivatives or metabolites thereof.

38. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins are IgA or fragments or derivatives or metabolites thereof.

39. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins are IgD or fragments or derivatives or metabolites thereof.

40. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins are IgE or fragments or derivatives or metabolites thereof.

41. The method according to any one of claims 1-6, wherein said monoclonal immunoglobulins comprise immunoglobulin fragments selected from the group consisting of F(ab')2, Fab', Fab, Fc, Facb, pFc', and Fd; or metabolites or derivatives thereof.

42. The method according to any one of claims 1-6, wherein said effective rate is not more than about 3.0 kGy/hour.

43. The method according to any one of claims 1-6, wherein said effective rate is not more than about 2.0 kGy/hr.

44. The method according to any one of claims 1-6, wherein said effective rate is not more than about 1.0 kGy/hr.

45. The method according to any one of claims 1-6, wherein said effective rate is not more than about 0.3 kGy/hr.

46. The method according to any one of claims 1, 2, or 5, wherein said effective rate is more than about 3.0 kGy/hour.

47. The method according to any one of claims 1, 2, or 5, wherein said effective rate is at least about 6.0 kGy/hour.

48. The method according to any one of claims 1, 2, or 5, wherein said effective rate is at least about 18.0 kGy/hour.

49. The method according to any one of claims 1, 2, or 5, wherein said effective rate is at least about 45 kGy/hour.

50, The method according to any one of claims 1-6, wherein said preparation of monoclonal immunoglobulins is maintained in a low oxygen atmosphere.

51. The method according to any one of claims 1-6, wherein said preparation of monoclonal immunoglobulins is maintained in an argon atmosphere.

52. The method according to any one of claims 1-6, wherein said preparation of immunoglobulins is maintained in a vacuum.

53. The method according to any one of claims 1, 4, 5 or 6, wherein said residual solvent content is reduced by a method selected from the group consisting of lyophilization, drying, concentration, addition of solute, evaporation, chemical extraction, spray-drying, and vitrification.

54. The method according to any one of claims 1, 4, 5 or 6, wherein said residual solvent content is less than about 10%.

55. The method according to any one of claims 1, 4, 5 or 6, wherein said residual solvent content is less than about 5%.

56. The method according to any one of claims 1, 4, 5 or 6, wherein said residual solvent content is less than about 2%.

57. The method according to any one of claims 1, 4, 5 or 6, wherein said residual solvent content is less than about 1%.

58. The method according to any one of claims 1, 4, 5 or 6, wherein said residual solvent content is less than about 0.5%.

59. The method according to any one of claims 1-6, wherein at least one sensitizer is added to said preparation of monoclonal immunoglobulins prior to said step of irradiating said preparation of monoclonal immunoglobulins.

60. The method according to,any one of claims 1-6, wherein said preparation of monoclonal immunoglobulins contains at least one prion as a biological contaminant.

61. The method according to any one of claims 1-6, wherein said preparation of monoclonal immunoglobulins contains at least one virus as a biological contaminant.

62. The method according to claim 1 or 4, wherein at least one stabilizer is added to said preparation of monoclonal immunoglobulins prior to said step of irradiating said preparation of monoclonal immunoglobulins.

63. The method according to any one of claims 2, 3, 5 or 6, wherein at least one of said stabilizers is an antioxidant.

64. The method according to any one of claims 2, 3, 5 or 6, wherein at least one of said stabilizers is a free radical scavenger.

65. The method according to any one of claims 2, 3, 5 or 6, wherein at least one of said stabilizers reduces damage due to reactive oxygen species.

66. The method according to any one of claims 2, 3, 5 or 6, wherein at least one of said stabilizers is selected from the group consisting of: ascorbic acid or a salt or ester thereof; glutathione; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; uric acid or a salt or ester thereof;
methionine; histidine; N-acetyl cysteine; lipoic acid; sodium formaldehyde sulfoxylate; gallic acid or a derivative thereof;
propyl gallate and mixtures of two or more of said stabilizers.

67. The method according to claim 66, wherein said mixtures of two or more of said stabilizers are selected from the group consisting of: mixtures of ascorbic acid, or a salt or ester thereof, and uric acid, or a salt or ester thereof;
mixtures of ascorbic acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; mixtures of ascorbic acid, or a salt or ester thereof, uric acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; and mixtures of uric acid, or a salt or ester thereof; lipoic acid; sodium formaldehyde sulfoxylate; gallic acid or a derivative thereof;
propyl gallate and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

68. The method according to any one of claims 2, 3, 5, or 6, at least one of said stabilizers comprises the dipeptide glycine-glycine.

69. The method according to any one of claims 2, 3, 5, or 6, wherein at least one of said stabilizers comprises diosmin.

70. The method according to any one of claims 2, 3, 5, or 6, wherein at least one of said stabilizers comprises silymarin.

71. The method according to any one of claims 1-6, wherein said radiation is corpuscular radiation or electromagnetic radiation, or a mixture thereof.

72. The method according to claim 71, wherein said electromagnetic radiation is selected from the group consisting of radio waves, microwaves, visible and invisible light, ultraviolet light, x-ray radiation, gamma radiation and combinations thereof.

73. The method according to any one of claims 1-6, wherein said radiation is gamma radiation.

74. The method according to any one of claims 1-6, wherein said radiation is e-beam radiation.

75. The method according to any one of claims 1-6, wherein said radiation is visible light.

76. The method according to any one of claims 1-6, wherein said radiation is ultraviolet light.

77. The method according to any one of claims 1-6, wherein said radiation is x-ray radiation.

78. The method according to any one of claims 1-6, wherein said radiation is polychromatic visible light.

79. The method according to any one of claims 1-6, wherein said radiation is a combination of one or more wavelengths of visible and ultraviolet light.

80. The method according to any one of claims 1-6, wherein said irradiation is conducted at ambient temperature.

81. The method according to any one of claims 1-6, wherein said irradiation is conducted at a temperature below ambient temperature.

82. The method according to any one of claims 1-6, wherein said irradiation is conducted below the freezing point of the monoclonal immunoglobulin.

83. The method according to any one of claims 1-6, wherein said irradiation is conducted below the eutectic point of the monoclonal immunoglobulin.

84. The method according to any one of claims 1-6, wherein the pH of said preparation of monoclonal immunoglobulins is less than 7.

85. The method according to any one of claims 1-6, wherein the pH of said preparation of monoclonal immunoglobulins is less than 6.

86. The method according to any one of claims 1-6, wherein the pH of said preparation of monoclonal immunoglobulins is less than 5.

87. The method according to any one of claims 1-6, wherein the pH of said preparation of monoclonal immunoglobulins is less than 4.

88. The method according to any one of claims 1-6, wherein the pH of said preparation of monoclonal immunoglobulins is less than 3.

89. A composition comprising at least one monoclonal immunoglobulin and a least one stabilizer selected from the group consisting of: ascorbic acid or a salt or ester thereof;

glutathione; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; uric acid or a salt or ester thereof; methionine;
histidine; N-acetyl cysteine; the dipeptide glycine-glycine;
diosmin; silymarin; lipoic acid; sodium formaldehyde sulfoxylate; gallic acid or a derivative thereof; propyl gallate; a mixture of ascorbic acid, or a salt or ester thereof, and uric acid, or a salt or ester thereof; a mixture of ascorbic acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; a mixture of ascorbic acid, or a salt or ester thereof, uric acid, or a salt or ester thereof, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; and a mixture of uric acid, or a salt or ester thereof and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, said at least one stabilizer being present in an amount effective to preserve said monoclonal immunoglobulin for its intended use following sterilization of the composition with radiation.

90. The composition according to claim 89 having a residual solvent content that is sufficiently low to preserve said monoclonal immunoglobulin, curing sterilization by irradiation, for its intended use following sterilization of the composition with radiation.

91. The composition according to claim 89, having a residual solvent content of less than about 10%.

92. The composition according to claim 89, having a residual solvent content of less than about 5%.

93. The composition according to claim 89, having a residual solvent content of less than about 2%.

94. The composition according to claim 89, having a residual solvent content of less than about 1%.

95. The composition according to claim 89, having a residual solvent content of less than about 0.5%.

96. The composition according to claim 89, having a pH
less than 7.

97. The composition according to claim 89, having a pH
less than 6.

96. The composition according to claim 89, having a pH
less than 5.

97. The composition according to claim 89, having a pH
less than 4.

98. The composition according to claim 89, having a pH
less than 3.