Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to an improved method and immune globulin formulation containing a non-ionic surface active agent to prolong the serum half-life and to alter the immunomodulatory effect of immune globulin.
• Immune globulins are proteins produced by lymphoreticular tissues. There are 6 known classes of immune globulin: IgG, IgM, IgA, IgD, IgE and secretory IgA. IgG (also known as gamma-globulin) is the most abundant and the most therapeutically relevant class of immune globulin. The primary function of immune globulins is to specifically recognize and bind antigens through reversible bonding thereby facilitating the immune systems ability to eliminate antigens.
• IgG is a glycoprotein of approximately 150,000 Daltons consisting of 2 "heavy” (gamma) chains and 2 "light” (kappa or gamma) chains held together by disulphide as well as weak covalent bonds.
• IgGl subclasses of IgGl, IgG2, IgG3 and IgG4 comprising about 70%, 15%, 10% and 5% of total IgG in normal human serum respectively. These subclasses possess minor antigenic differences among their "heavy" chains resulting in distinct biological actions.
• immune globulins There are principally two types of immune globulins that are available as therapeutic agents: standard immune serum globulin preparations for general use, and immune globulin preparations that recognize specific antigens for use in specific disorders.
• Commercial examples of products in the former category include numerous brands of intravenous immune globulin (Gamimune N ® by Bayer; Sandoglobulin ® by Sandoz; Gammar-IV ® by Armour) as well as intramuscular immune globulin (Gamastan ® by Cutter; Gammar ® by Armour).
• H-BIG hepatitis B immune globulin
• Hep-B-Gammagee ® by MSD
• HyperHep ® varicella zoster immune globulin
• VZIG varicella zoster immune globulin
• Rh immune globulin WinRho ® and WinRho SD ® by Cangene; HypRho- D ® by Miles; Gamulin Rh ® by Armour; RhoGAM ® by Ortho Diagnostics).
• immune globulins The primary therapeutic basis for immune globulins is passive immunity conferred to the recipient through the direct introduction of extraneous "ready-made” antibodies.
• the major clinical utilities of immune globulins are prophylaxis and /or treatment of antigen-associated disorders.
• Immune globulins may be prepared by isolation of natural immune globulins from mammalian serum. Immune globulins prepared using Cohn's cold ethanol fractionation method suffers from relatively low product yield and IgG purity. The resultant product contains significant amounts of aggregated immune globulin which combines with complement (also termed anticomplementary activity) and produces adverse reactions in recipients if given by intravenous injection or infusion (see Huchet, J. et al., Rev. Fr. Transfus. 13:231, 1970; Chown, B. et al., Can. Med. Assoc. J. 100:1021, 1969; Barandun, S. et al, Vox Sang. 7: 157-174, 1962).
• complement also termed anticomplementary activity
• these immune globulin preparations must be injected intramuscularly (therefore termed intramuscular immune globulin).
• Intramuscular injections are painful.
• Drug absorption and peak levels of immune globulin are slow, and approximately half of the injected dose is lost due to local proteolysis and incomplete absorption.
• Significant amounts of IgA and IgM are also present in Cohn-prepared intramuscular immune globulin preparations which can cause anaphylactic reactions in certain recipients.
• intravenous injection or infusion of immune globulin is the preferred route of drug administration in the clinical setting due to instant bioavailability and rapid onset of therapeutic protection.
• Intravenous immune globulin products differ from intramuscular products in two fundamental ways. First, an intravenous preparation must contain a significantly smaller amount of aggregated immune globulin molecules (over about 94% monomeric immune globulin) thereby causing fewer anticomplementary adverse reactions. Second, the IgG content and product purity of intravenous immune globulin products are significantly higher (over about 95% IgG content) than intramuscular products. The low level of contamination with IgA or IgM in intravenous immune globulin (less than about 40 ug/mL) is also associated with lower incidences of adverse events such as anaphylactic reactions especially in agammaglobulinemic recipients.
• 3,903,262 describes the reduction of intermolecular disulphide bonds of immune globulin and alkylation of the resultant sulfhydryl groups.
• Schura of Germany also developed an immune globulin for intravenous injection by adsorption of immune globulin onto hydroxy-ethyl starch.
• these approaches are often technically unfeasible for manufacturing or residual reactants in the final preparation have been shown to cause undesirable outcomes such as a reduction in serum half-life of the immune globulin and elicitation of immunogenic reactions in recipients.
• Further improved chromatographic techniques e.g.
• Immune globulin prepared by improved processes may be administered by intravenous injection or infusion as well as other parenteral routes.
• Cangene's WinRho ® and WinRho SD ® are produced using a proprietary anion exchange chromatographic process and are the only commercial anti-Rh 0 D immune globulin preparations that can be administered safely by intravenous injection or infusion to humans. This is due to their relatively higher IgG purity and monomeric protein content as well as lower IgA /IgM contamination.
• Monoclonal immune globulins can be produced using recombinant and hybridoma techniques (see Canadian Patent number 1,303,534; Canadian Patent number 1,303,533; European Patent Application 87302620.7 published as EP 239,400; European Patent Application 93102609.0 published as EP 557,897; Fletcher, A. and Thompson, A., Transfus. Med. Rev. 9: 314-326, 1995; Alting-Mees, M. et al., Strat. Mol. Biol. 3: 1-9, 1990; Huse, W.D. et al., Science 246: 1275-1281, 1989; Sastry, L. et al., Proc. Natl. Acad. Sci.
• binding partners or domains may also be constructed using recombinant DNA techniques to incorporate the variable regions of a gene encoding a specific antibody (see PCT Patent Application PCT/GB93/00605 published as WO 93/19172; PCT Patent Application PCT/GB93/02492 published as WO 94/13804; PCT Patent Application PCT/EP90/01964 published as WO 91/07492; Bird et al., Science 242: 423- 426, 1988).
• Immune globulin preparations suitable for parenteral injection commonly consist of an immune globulin distributed in a physiologically compatible medium.
• This medium may be sterile water for injection (WFI) with or without isotonic amounts of sodium chloride.
• WFI sterile water for injection
• the recommended diluent for reconstituting commercial intravenous immune globulins such as Iveegam ® , Gammagard ® , or, Venoglobulin ®
• Sandoglobulin ® is supplied with 0.9% (w/v) sodium chloride solution as diluent (see Gahart, B.L. & Nazareno, A.R., Intravenous Medications: a handbook for nurses and allied health professionals, p.
• WinRho SD ® is reconstituted in 0.9% sodium chloride solution for intravenous injection.
• the immune globulin product by Schura is formulated as a solution of 165 mEq/L sodium ion and 120 mEq/L chloride ion with a final pH of 6.7.
• the Miles' intravenous immune globulin preparation, Gammimune ® when constituted, has an osmolality of 278 mOsm/L and a pH of 4.0-4.5.
• 4,396,608 and 4,499,073 also disclose a low pH (3.5-5.0) and low ionic strength ( ⁇ _0.001) immune globulin formulation for intravenous injection.
• the globulin protein concentration in the above preparations ranges from 0.5% to 20%.
• Carbohydrates and their derivatives such as glucose, maltose or mannitol may be included in immune globulin formulations to adjust the tonicity of the preparation.
• maltose (10%) is included in Miles' intravenous immune globulin preparation, Gammimune ® , to achieve isotonicity.
• Sucrose (5%) is included in Sandoz's intravenous immune globulin preparation, Sandoglobulin ® and in Armour's intravenous immune globulin preparation, Gammar- IV ® .
• the commercial intravenous immune globulin preparation, Venoglobulin ® contains 50 mg/mL D-sorbitol.
• amino acids such as glycine or histidine may be added to improve storage stability of the protein.
• glycine 0.3M
• commercial immune globulin preparations such as American Red Cross' intravenous immune globulin preparations, Polygam ® and Polygam S/D ® ; the intramuscular varicella zoster immune globulin preparation by Massachusetts Public Health Biologic Laboratories; and the intramuscular anti-Rh 0 D immune globulin preparations by Armour and Miles.
• U.S. patents Nos. 4,396,608 and 4,597,966 describe the use of glycine and histidine to stabilize immune globulin formulations.
• EP 392,717 describes the use of mannitol and glycine to stabilize and prevent aggregation of immune globulin in formulation.
• the prolongation of storage shelf-life of immune globulin preparations may also be accomplished by the addition of preservatives including organic mercurial derivatives such as thimerosal.
• Surface active agents are compounds that can lower the surface tension of water. All surface active agents are amphipathic possessing a hydrophobic end (e.g. one or more hydrocarbon chain(s)) as well as a hydrophilic moiety (which may or may not be ionic). A surface active agent may be classified as anionic, cationic, or non-ionic depending on the nature of its hydrophilic moiety. Soaps with carboxylate or sulphonate groups carry net negative charges and are examples of anionic surface active agents. Benzalkonium, an N- benzyl quaternary ammonium chloride and an antibacterial agent, carries a net positive charge and is an example of a cationic surface active agent.
• a non-ionic surface active agent contains a neutral group such as a carbohydrate which can hydrogen-bond with water.
• Tween ® and Span ® are two types of non-ionic surface active agent.
• Agents such as Span ® are partial esters of common fatty acids and sugar alcohol anhydrides derived from sorbitol.
• Agents such as Tween ® are derivatives of Span ® products with polyoxyethylene chains attached to non-esterified hydroxyl groups. Their hydrophilic property is due to free hydroxyl and /or oxyalkylene groups, and their hydrophobic property is due to their long chain fatty acids.
• Span ® type surface active agents are sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), sorbitan monooleate (Span 80), and sorbitan trioleate (Span 85).
• Tween 80 A commonly used member of Tween ® type surface active agent, Tween 80, is also known as Polysorbate 80, sorbitan mono-9- octadecenoate poly(oxy-l,2-ethanediyl) derivative, polyoxyethylene sorbitan monooleate, polyethylene oxide sorbitan monooleate, sorethytan monooleate, Sorlate, Monitan or Olothorb.
• polyoxyethylene sorbitan surface active agents comprise polyoxyethylene sorbitan monolaurate (Tween 20 or 21), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate (Tween 60 or 61), polyoxyethylene sorbitan tristearate (Tween 65), and polyoxyethylene sorbitan trioleate (Tween 85).
• a commercially available preparation of granulocyte colony stimulating factor, Neupogen ® contains 0.004% Polysorbate 80 to improve storage stability.
• Turbersol ® is a sterile isotonic solution of Tuberculin in phosphate buffered saline containing 0.0005% Polysorbate 80 as a stabilizer.
• U.S. Patent No. 4,902,500 discloses immune globulin formulations with improved storage stability containing at least one polyoxypropylene-polyoxyethylene block polymer (Pluronic 68).
• U.S. Patent No. 5,151,266 teaches a method of treating antibodies with an anionic detergents such as sodium dodecylsulfate (sodium lauryl sulfate), cetyl ammonium sulfate, or taurocholic acid, to increase the solubility of the antibody and to reduce its reticuloendothelial uptake.
• an anionic detergents such as sodium dodecylsulfate (sodium lauryl sulfate), cetyl ammonium sulfate, or taurocholic acid
• U.S. Patent Nos. 4,371,520 and 4,379,086 describes the use of alkylene oxide polymers such as polyethylene glycol in the fractionation process for isolating immune globulin-rich fractions from plasma.
• U.S. Patent Nos. 4,276,283 and 5,132,406 describe the use of alkylene oxide polymers such as polyethylene glycol in a precipitation step for isolating and purifying immune globulin-rich fractions.
• the ability of surface active agents to alter the pharmacologic properties of drugs has been examined to a limited extent, but their usefulness in immune globulin formulations in a clinical setting has not been established. Jekunen, A.
• Ellis, A.G. et al. (Cancer Chemother. Pharmacol. 38: 81-87, 1996) describes the effects of two surface active agents, Cremophor EL and Tween 80, on the pharmacokinetics of a chemotherapeutic non-protein drug, etoposide, in an isolated perfused rat liver experimental model. Co- administration of either surface active agent decreased the elimination half-life of etoposide. Masters, J.R. et al. (Cancer Chemother. Pharmacol. 25: 267-273, 1990) decreased the in vivo half-life of the chemotherapeutic drug, thioTEPA in human subjects. These decreases in plasma half-life correspondingly increase the need for more frequent drug administration to maintain effective plasma drug concentrations which in turn increases the cost associated with such therapy in the clinical setting.
• Liu, F. and Liu, D. demonstrated the ability of Polysorbate 80 to attenuate the clearance of parenterally administered oil-in-water emulsions.
• oil-in-water emulsions are physicochemically and biochemically different from the immune globulin proteins of the present invention.
• Immune globulin preparations with novel clinical characteristics and benefits are presented.
• Immune globulin preparations of the present invention contain an immune globulin with relatively high IgG and low aggregated protein contents and are suitable for intravenous injection or infusion.
• the immune globulin preparation contains one or more non-ionic surface active agents in a physiologically compatible buffered medium. Inclusion of one or more such non-ionic surface active agents in the preparation surprisingly prolongs the serum half-life of the immune globulin in vivo and improves the safety profile of the product.
• An immune globulin preparation with an increased serum half-life is clearly advantageous and contrary to the teachings of the prior art.
• a preparation with an extended half-life means that the active therapeutic ingredient would have a longer survival time in the bloodstream to exert its desired therapeutic effect.
• a longer serum survival time would also alow for a reduced frequency of drug administration. This results in a more convenient dosing schedule with fewer injections which thereby improves patient compliance. A consequence is to reduce indirect costs associated with parenteral administration of the drug.
• a longer serum survival time of a time may translate into the requirement of smaller maintenance doses to maintain an effective serum drug concentration thereby minimizing the direct cost of drug therapy.
• the present invention provides an immune globulin preparation comprising an immune globulin and a non-ionic surface active agent, where the non-ionic surface active agent is in a concentration sufficient to increase the serum half-life of the immune globulin.
• the immune globulin preparation may have more than one non-ionic surface active agent, so long as the non-ionic surface active agents are in a concentration sufficient to increase the serum half-life of the immune globulin.
• the immune globulin is anti-Rh 0 D immune globulin wherein the anti-Rh 0 D immune globulin has an IgG purity of greater than 95% and a monomeric protein content of greater than 94%.
• the preparation is aqueous.
• the immune globulin is anti-c immune globulin which has an IgG purity of greater than 95% and a monomeric protein content of greater than 94%.
• the preparation has a concentration of the immune globulin of about 2 weight percent to about 10 weight percent.
• the non-ionic surface active agent may be a sorbitan ester of a fatty acid selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate and sorbitan trioleate.
• the non-ionic surface active agent may also be a polyoxyethylene sorbitan ester of a fatty acid selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (4) sorbitan monolaurate; polyoxyethylene (20) sorbitan monopalmitate; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene (4) sorbitan monostearate; polyoxyethylene (20) sorbitan tristearate; polyoxyethylene (20) sorbitan monooleate; polyoxyethylene (5) sorbitan monooleate; and polyoxyethylene (20) sorbitan trioleate.
• polyoxyethylene sorbitan ester of a fatty acid selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (4) sorbitan monolaurate; polyoxyethylene (20) sorbitan monopalmitate; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene (4) sorbitan monostearate;
• the preparation will have a concentration of the non-ionic surface active agent of about 0.01 weight percent to about 0.5 weight percent.
• an aqueous immune globulin preparation wherein the immune globulin has an increased serum half-life comprising: about 3-8% human anti-
• Rh 0 D immune globulin with an IgG purity of greater than 95% and a monomeric protein content of greater than 94%; sodium chloride at about 0.25% (w/v); very low level buffer with essentially no ionic strength; Polysorbate 80 at about 0.01% to about 0.5% (w/v); and L-glycine at about 0.1M.
• the present invention provides a method of increasing the serum half-life of an immune globulin comprising administering a preparation comprising an immune globulin and a non- ionic surface active agent in a physiologically acceptable medium to an animal in need thereof.
• the present invention provides a method of reducing the elevation of neutrophil counts in a recipient comprising administering a preparation comprising an immune globulin and a non-ionic surface active agent in a physiologically acceptable medium to an animal in need thereof.
• the immune globulin preparation may be administered intravenously.
• the preparation possesses the novel characteristic of an extended serum half-life in vivo and reduced immunogenicity in comparison with equivalent immune globulin preparations not containing the non-ionic surface active agent.
• a preparation according to the present invention may be in the format of a liquid formulation or may be lyophilized to form a powder formulation.
• the liquid formulation may be administered directly, while the lyophilized powder format may be reconstituted in a physiologically compatible medium before drug administration.
• the immune globulin in a preparation of the present invention is a human immune globulin prepared by extraction from human plasma using conventional cold ethanol fractionation method followed by a method to render the preparation suitable for intravenous administration or by chromatographic procedures.
• the immune globulin or binding partner may also be a monoclonal antibody or binding partner produced by recombinant DNA or hybridoma technology.
• the immune globulin in a preparation of the present invention is an anti-D (also known as anti-Rh 0 or anti-Rh 0 D) immune globulin; an anti-C (also known as anti-rh') immune globulin; an anti-E (also known as anti-rh”) immune globulin; an anti-c (also known as anti-hr') immune globulin or anti-e (also known as anti-hr”) immune globulin.
• This immune globulin may be prepared by conventional cold ethanol fraction followed by a method to render the preparation suitable for intravenous administration, by chromatographic techniques or by recombinant DNA or hybridoma technology.
• Another aspect of the invention provides a method of extending the serum half-life or altering the immunomodulatory effect of and immune globulin comprising the addition of a sufficient amount of one or more non-ionic surface active agents to the immune globulin formulation.
• the immune globulin may a human immune globulin extracted from plasma using conventional ethanol fractionation followed by a method to render the preparation suitable for intravenous administration, by chromatographic methods, or it may be a monoclonal antibody or binding partner produced by recombinant DNA or hybridoma technology.
• the immune globulin may be an anti-D (also known as anti-Rh 0 or anti-Rh 0 D) immune globulin; an anti-C (also known as anti-rh') immune globulin; an anti-E (also known as anti-rh”) immune globulin; an anti-c (also known as anti-hr') immune globulin or anti-e (also known as anti-hr”) immune globulin.
• an anti-D also known as anti-Rh 0 or anti-Rh 0 D
• an anti-C also known as anti-rh'
• an anti-E also known as anti-rh
• an anti-c also known as anti-hr' immune globulin or anti-e (also known as anti-hr" immune globulin.
• the immune globulin formulation is administered to a mammal by parenteral injection or infusion to elevate circulating immune globulin levels.
• Figure 1 is a graph of mean anti-Rh 0 D immune globulin blood levels after an intramuscular injection of WinRho SDTM.
• Mean serum anti-Rh 0 D immune globulin results are shown for formulations with and without 0.01% (w/v) Polysorbate 80.
• the solid diamonds show the results from subjects injected with the conventional formulation of WinRho SDTM in 0.9% (w/v) sodium chloride solution.
• the shaded boxes show the results in subjects injected with new formulation of WinRho SDTM with Polysorbate 80 in 0.9% (w/v) sodium chloride solution.
• Figure 2 is a graph of mean anti-Rh 0 D immune globulin blood levels for up to 82 days after an intramuscular injection of WinRho SDTM.
• Mean serum anti-Rh 0 D immune globulin results are shown for formulations with and without 0.01% (w/v) Polysorbate 80.
• the solid diamonds show the results from subjects injected with the conventional formulation of WinRho SDTM in 0.9% (w/v) sodium chloride solution.
• the open squares show the results from subjects injected with the formulation of WinRho SDTM with Polysorbate 80 in 0.9% (w/v) sodium chloride solution.
• the present inventors have found that the addition of a non-ionic surface active agent to a preparation of an immune globulin favourably alters the pharmacokinetics of the immune globulin as a therapeutic agent.
• the inclusion of said non-ionic surface active agent in the formulation prolongs the survival time or serum half- life of said immune globulin.
• the present invention provides an immune globulin preparation comprising an immune globulin and a non-ionic surface active agent, where the non-ionic surface active agent is in a concentration sufficient to increase the serum half-life of the immune globulin.
• the immune globulin preparation may have more than one non-ionic surface active agent, so long as the non-ionic surface active agents are in a concentration sufficient to increase the serum half- life of the immune globulin.
• surface active agent means an agent that reduces surface tension when dissolved in a solution, such as in water or an aqueous solution.
• surfactant is synonomous.
• sufficient to increase the serum half-life of the immune globulin means that the serum half-life of the immune globulin with at least one surface active agent is increased as compared to the serum half-life of the immune globulin when administered without a surface active agent.
• the immune globulin of the present invention can be any immune globulin including IgG (all subclasses), IgA, IgD, IgE, and IgM and includes fragments of the immune globulins such as Fab' and F(ab') 2 fragments.
• the immune globulin is preferably non-aggregated (over about 94% monomeric immune globulin) and has a purity of greater than about 95%. More preferably, the immune globulin preparation is in a form suitable for intravenous injection or infusion.
• Rh immune globulin that can be used in the present invention is Rh immune globulin or Rh antibodies.
• Rh antibodies include anti-D (also known as anti-Rh 0 or anti-Rh 0 D); anti-C (also known as anti-rh'); anti-E (also known as anti-rh”); anti-c (also known as anti-hr') and anti-e (also known as anti-hr”).
• the Rh antibodies of the present invention may be preparations from plasma enriched for Rh antibodies, polyclonal antibodies, monoclonal antibodies, antibody fragments (e.g. Fab, and F(ab') 2 ), and those produced by recombinant DNA technology.
• immune globulin preparations suitable for intravenous injection or infusion such as varicella zoster immune globulin (Varitect ® by Biotest Pharma) or cytomegalovirus immune globulin (Cytogam ® by Connaught), can also benefit from the present invention.
• varicella zoster immune globulin Varitect ® by Biotest Pharma
• cytomegalovirus immune globulin Cytogam ® by Connaught
• the inventors have also found that the addition of a non- ionic surface active agent to an immune globulin reduced the elevation of patient neutrophil counts observed with a conventional immune globulin without the non-ionic surface active agent.
• Preparations with a high Rh antibody content suitable for intravenous injection or infusion may be isolated as an immune globulin fraction from plasma, preferably human plasma, using conventional techniques. For example, they may be isolated using: (a) the Cohn cold ethanol fractionation method or modifications thereto (see Huchet, J. et al., Rev. Fr. Transfus. 13:231, 1970; Chown, B. et al., Can. Med. Assoc. ⁇ . 100:1021, 1969; Jouvenceaux, A. et al., Rev. Fr. Transfus. 12 (suppl.): 341, 1969; Barandun, S. et al., Vox Sang.
• anti-Rh 0 D immune globulin preparations may also be used in the methods.
• anti-Rh 0 D preparations such as WinRho ® or WinRho SD ® (Cangene Corporation) may be used in the present invention.
• an anti-Rh 0 D immune globulin fraction is prepared by contacting an aqueous plasma fraction containing IgG with one or more chromatographic separation columns to produce a purified IgG-rich fraction.
• the aqueous plasma fraction used in the process may be normal non-immunized plasma from an animal source, preferably a human source, or hyperimmune plasma such as plasma from Rh alloimmunized donors.
• Rh 0 D antigen is used to immunize an animal through intramuscular, subcutaneous, intraperitoneal, or intraocular injection, with or without an adjuvant such as Freund's complete or incomplete adjuvant.
• samples of serum are collected and tested for reactivity to the antigen in standard assays (described below).
• polyclonal antisera which will give a signal on one of the assays that is at least three times greater than background. Once the titre of the animal has reached a plateau in terms of antigen reactivity, larger quantities of the antisera may be obtained readily either by periodic bleeding or by exsanguinating the animal.
• Human anti-Rh 0 D immune globulin may also be produced in human volunteers.
• an anti-Rh 0 D immune globulin preparation may be obtained from a subject who is naturally immunized (e.g. from an Rh incompatible pregnancy) or artificially immunized using Rh-positive blood cells or Rh 0 D antigen.
• Anti-Rh 0 D immune globulin-containing plasma collected from animal or human is modified to the ionic strength and pH of the initial buffer used with a chromatographic separation column.
• an aqueous animal plasma fraction is contacted with one or more, preferably one to two, anionic exchangers to produce a purified IgG-rich fraction.
• an aqueous animal or human plasma fraction is applied to an anion exchange column which may contain an agarose cross-linked anionic exchange resin such as DEAE-Sepharose CL6B, TMAE Fractogel or DEAE Sephadex A-50.
• An IgG-rich fraction is obtained from the column by eluting with an equilibrating buffer.
• the IgG-rich fraction may be concentrated, for example, by ultrafiltration.
• the purified IgG protein may optionally be treated with a solvent and detergent to inactivate lipid envelope viruses. Suitable solvents and detergents which may be used include Triton X-100 and tri(n-butyl) phosphate (Horowitz, B., Curr. Stud. Hematol. Blood Transfus. 56: 83-96, 1989).
• the solvents and detergents may be removed using conventional methods such as reverse phase chromatography.
• Monoclonal immune globulins may also be readily produced using recombinant and hybridoma techniques (see Canadian Patent number 1,303,534; Canadian Patent number 1,303,533; European Patent Application 87302620.7 published as EP 239,400; European Patent Application 93102609.0 published as EP 557,897; Fletcher, A. and Thompson, A., Transfus. Med. Rev. 9: 314-326, 1995; Alting-Mees, M. et al., Strat. Mol. Biol. 3: 1-9, 1990; Huse, W.D. et al., Science 246: 1275-1281, 1989; Sastry, L. et al., Proc. Natl. Acad. Sci.
• binding partners or domains may be constructed using recombinant DNA techniques to incorporate the variable regions of a gene encoding a specific antibody (see PCT Patent Application PCT/GB93/00605 published as WO 93/19172; PCT Patent Application PCT/GB93/02492 published as WO 94/13804; PCT Patent Application PCT/EP90/01964 published as WO 91/07492; Bird et al., Science 242: 423- 426, 1988). It will be apparent to one skilled in the art that fractionation and recombinant approaches may be applied to diverse types of immune globulins. For example, specific monoclonal immune globulins against different antigens may be generated by techniques based on the same principle of recombinant DNA technology.
• Non-ionic surface active agents of the present invention can be any agent that can prolong the serum half-life of an immune globulin.
• the surface active agent is of the Tween ® or Span ® type surface active agents.
• Span ® type agents are partial esters of common fatty acids and sugar alcohol anhydrides derived from sorbitol.
• the common fatty acids derived from sorbitol are preferably lauric acid, palmitic acid, stearic acid or oleic acid derived from sorbitol.
• Span 20 is sorbitan monolaurate
• Span 40 is sorbitan monopalmitate
• Span 60 is sorbitan monostearate
• Span 65 is sorbitan tristearate
• Span 80 is sorbitan monooleate
• Span 85 is sorbitan trioleate.
• Tween ® type agents are derivatives of Span ® products with polyoxyethylene chains attached to non-esterified hydroxyl groups.
• the common fatty acids derived from sorbitol are preferably lauric acid, palmitic acid, stearic acid or oleic acid derived from sorbitol.
• Tween 20 is polyoxyethylene (20) sorbitan monolaurate
• Tween 21 is polyoxyethylene (4) sorbitan monolaurate
• Tween 40 is polyoxyethylene (20) sorbitan monopalmitate
• Tween 60 is polyoxyethylene (20) sorbitan monostearate
• Tween 61 is polyoxyethylene (4) sorbitan monostearate
• Tween 65 is polyoxyethylene (20) sorbitan tristearate
• Tween 80 is polyoxyethylene (20) sorbitan monooleate
• Tween 81 is polyoxyethylene (5) sorbitan monooleate
• Tween 85 is polyoxyethylene (20) sorbitan trioleate.
• Non-ionic surface agents such as sorbitan esters or polyoxyethylene sorbitan esters of fatty acids may be prepared by methods well known in the art. Such surface active agents may also be obtained commercially from J.T. Baker Inc. (Phillipsburg, New Jersey, USA), ICI Atkemix (Brantford, Ontario, Canada), Van Waters and Rogers Ltd. (Richmond, British Columbia, Canada), or Nikkol Co. (Japan). Immune Globulin Preparation
• Formulation of anti-Rh 0 D immune globulin of the present invention involves the addition of an amount of a non-ionic surface active agent sufficient to extend the serum half-life or to alter the immunomodulatory effect of the immune globulin to the IgG-rich concentrate obtained as described above.
• the immune globulin preferably is at least about 95% pure, more preferably about 99.5% pure, contains at least 94% monomeric IgG, and has not been subjected to chemical or enzymatic modification.
• a preferred non-ionic surface active agent is Polysorbate 80 which is added to a final concentration of about 0.01% to about 0.5%. Sodium chloride may be added to the formulation to a final concentration of up to about 0.9%.
• An additional stabilizing agent such as L-glycine or L-histidine may be added to a final concentration of about 0.025M to 0.05M.
• a preferred preparation contains the following: a pharmacologically effective amount of human anti-Rh 0 D immune globulin (about 3-8%); sodium chloride at about 0.25% (w/v); no or very low level buffer with essentially no ionic strength; Polysorbate 80 at a concentration of about 0.01%-0.02% (w/v); and L-glycine at a concentration of about 0.1M.
• the anti-Rh 0 D immune globulin formulation is aseptically filtered again through a 0.22 micron filter and put into vials or ampoules.
• filling operations are conducted under aseptic conditions and the fill volume per vial is calculated so that each vial contains a pharmacologically effective amount of anti-Rh 0 D immune globulin. This specific amount or volume can vary depending upon the intended route of administration and therapeutic use.
• the target filling volume is also calculated with sufficient excess to allow for variation in the potency assay and /or possible loss of potency during storage.
• the final aqueous formulation may be lyophilized using a
• Virtus 251 SRC-5 Sublimator (or equivalent).
• Lyophilization also termed vacuum freeze-drying or sublimation
• the lyophilization process is often described as being divided into three stages: freezing; primary drying (also termed ice sublimation); and secondary drying (also termed water desorption).
• the starting aqueous solution containing the protein is frozen and the ice is subsequently sublimed thereby leaving a dry porous mass of protein which is stable and can be reconstituted rapidly in water.
• the technical parameters of the lyophilization process including temperature (eutectic and collapse), vacuum pressure and atmospheric gas composition, are fully automated by the Virtus 251 SRC-5 Sublimator.
• the powder comprising the immune globulin and the non-ionic surface active agent is reconstituted in a physiologically compatible diluent such as sterile water for injection or saline before parenteral administration.
• a physiologically compatible diluent such as sterile water for injection or saline before parenteral administration.
• 120 ug (600 IU) or 300 ug (1,500 IU) of a commercial anti- Rh 0 D immune globulin product, WinRho SDTM is reconstituted in 2.5 mL diluent.
• Therapeutic dosage of immune globulin preparation Dosages of anti-Rh 0 D immune globulin in formulations of the present invention depend on individual needs, on the protein content /concentration of the immune globulin preparation, on the desired effect in a particular therapeutic indication, and on the chosen route of drug administration.
• Daily dosages of an anti-Rh 0 D immune globulin preparation (3% to 8% wt-solution) for humans by intramuscular or intravenous injection generally vary between about 50 IU (10 ug) to 2,000 IU (400 ug) per kg body weight.
• the preferred dosage is about 100 IU (20 ug) to 2,000 IU (400 ug) per kg body weight.
• the preferred dosage is about 50 IU (10 ug) to 1,000 IU (200 ug) per kg body weight, preferably 250 IU ( 50 ug) per kg body weight.
• the recommended dosage of Biotest Pharma's intravenous varicella zoster immune globulin preparation, Varitect ® is about 50 IU per kg body weight for shingles therapy and are lower (about 12 to 25 IU per kg body weight) for chickenpox prophylaxis.
• the recommended dosages of general intravenous immune globulin products such as Gamimune ® , Iveegam ® or Sandoglobulin ® are significantly higher at about 100 mg to 800 mg per kg body weight.
• the Pharmacokinetics of anti-Rh 0 D immune globulin formulations with and without Polysorbate 80 were assessed in a single- centre, randomized, parallel arm study. Twenty-four human subjects (normal, healthy male and female volunteers of age 18 to 55 years) were randomized into two groups to receive 600 ⁇ g (3,000 IU) of a commercial brand of anti-Rh 0 D immune globulin, WinRho SD ® . Twelve subjects received the conventional WinRho SD ® formulation without Polysorbate 80, and the other 12 subjects received the WinRho SD ® formulation with 0.01% (w/v) Polysorbate 80.
• Each formulation was given intramuscularly as two 1.25 mL injections with the test articles being lyophilized human anti-Rh 0 D immune globulin in 0.9% (w/v) saline for intravenous injection with or without Polysorbate 80.
• the mean time to peak anti-Rh 0 D immune globulin levels after intramuscular injection of 600 ⁇ g of WinRho SDTM was about 3 days and peak serum anti-Rh 0 D immune globulin levels that were achieved were about 70 ng/mL. There was no statistically significant difference between the two formulations and the mean time to peak or peak levels of drug. Similarly, the AUC 2 s day was about 1250 days»ng/mL and there was no difference between the two formulations in these values.
• the mean half-life was different for the two formulations of WinRho SDTM.
• Body temperature was elevated by 0.3 ⁇ 0.4 °C at 1 hour, 0.3 ⁇ 0.3 °C at 3 hours and 0.5 ⁇ 0.5 °C at 8 hours after WinRho SDTM injection compared to increases in this group of 0.2 ⁇ 0.4 °C at 24 hours, 0.1 ⁇ 0.6 °C at 7 days and 0.1+0.4 °C at 28 days after drug administration.
• the study subjects who received WinRho SDTM with polysorbate 80 had body temperature increases of 0.0+0.5 °C, 0.1+0.5 °C, 0.2+0.5 °C, 0.0 ⁇ 0.4 °C, 0.1 ⁇ 0.7 °C and 0.1 ⁇ 0.4 °C at the assessments after drug administration.
• the new WinRho SDTM formulation with Polysorbate 80 leads to higher passive anti-Rh 0 D immune globulin levels for long times after drug administration in prophylaxis of Rh Immunization of Rh negative patients.
• inclusion of Polysorbate 80 in the anti- Rh 0 D immune globulin preparation significantly minimized drug- induced elevations of neutrophil counts in the recipients and altered the immunomodulatory effect of the immune globulin.

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• 1998
  • 1998-04-07 WO PCT/CA1998/000325 patent/WO1998044948A2/en not_active Application Discontinuation
  • 1998-04-07 AU AU69149/98A patent/AU6914998A/en not_active Abandoned
  • 1998-04-07 EP EP98914750A patent/EP0973549A2/de not_active Withdrawn

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