WO2005072772A1

WO2005072772A1 - Pharmaceutical compositions

Info

Publication number: WO2005072772A1
Application number: PCT/FI2005/000065
Authority: WO
Inventors: Jaakko Parkkinen
Original assignee: Suomen Punainen Risti Veripalvelu
Priority date: 2004-01-30
Filing date: 2005-01-31
Publication date: 2005-08-11

Abstract

Liquid composition of antibodies, such as immunoglobulin G, immunoglobulin M and immunoglobulin A, formulated for parenteral administration, wherein the antibodies are mixed in an aqueous medium with a stabilizing agent and optionally with other auxiliary agents known per se. The composition contains trehalose as a stabilizing agent at a concentration, which effectively prevents polymerization of the antibodies.

Description

PHARMACEUTICAL COMPOSITIONS

Background of the Invention

Field of the Invention

The present invention relates to pharmaceutical compositions. In particular, the present invention concerns parenterally-administrable formulations of antibodies. The invention also concerns a method of producing such formulations and the uses thereof.

Description of Related Art

Immunoglobulins, also called antibodies, are the main effector molecules of the humoral immune response. They have a basic four-peptide structure of two identical heavy and two identical light chains, which are joined by interchain disulfide bonds. The most common immunoglobulin class is IgG, which accounts for about 75 % of the total immunoglobulins in plasma of healthy individuals. IgG is further divided into subclasses with different heavy chain isotypes: IgGl, IgG2, IgG3 and IgG4. Other immunoglobulin classes comprise IgM, IgA, IgD and IgE.

The biological activities of antibodies are related to inactivation and clearance of infectious agents and their products, e.g. bacteria, viruses and toxins. Antibodies exert two major effector functions: activation of complement and opsonisation, i.e. the induction of phagocytosis. Immunoglobulins have a "Y" shaped structure with two antigen binding Fab parts and an Fc part, which mediates the effector functions induced as a result of binding of the Fab part to the antigen. A monoclonal antibody has a single type of antigen-binding Fab part, whereas polyclonal antibodies consist of a number of different immunoglobulin molecules with variable Fab parts.

The antimicrobial activities of antibodies are exploited therapeutically in prevention and treatment of various infectious diseases. Additionally, immunoglobulins exert different immunomodulatory activities, which form the basis for their therapeutic use in a number of clinical indications. Furthermore, monoclonal antibodies with a defined antigen-binding specificity have been developed for various targets ranging from infectious agents to body's own components and their therapeutic use covers a wide range of clinical disorders.

Antibodies can be extracted from blood of human blood donors and they can be produced by hybridoma technology and recombinant DNA technology. In view of their broad scope of biological activity, antibodies are valuable therapeutic agents.

For pharmaceutical uses, antibodies are primarily formulated for parenteral, in particular intravenous, administration. Immunoglobulin purified from normal human plasma is typically given intravenously ("Imrnune globulin intravenous human" or "Human normal immunoglobulin for intravenous administration"). Such "intravenous immunoglobulin" (IVIG) is formulated into liquid compositions having high purity with respect to the immunoglobulin component and low or practically no content of polymers and aggregates.

Generally, a liquid formulation of antibodies, such as of IVIG, should be formulated by keeping the following goals in mind: 1. The osmolarity should be close to physiological to make intravenous infusion of even large volumes possible; 2. Formation of polymeric IgG should be prevented during storage; and 3. The substance added for stabilization and adjustment of osmolarity should be compatible with IgG: there must be no chemical reactions with the protein

Mostly, compositions of antibodies formulated for intravenous administration, as well as other parenteral compositions of therapeutically useful antibodies, are provided in the form of liquid formulations, in which the active component is mixed with suitable excipients and adjuvants, including stabilizers, osmolarity regulating agents and pharmaceutical carriers. Stabilizers commonly used include sugars, such as maltose, glucose and sucrose.

There are some problems associated with the known substances. In particular, it has been found that reducing sugars may interact with immunoglobulin, leading to the formation of Schiff bases and giving rise to additional chemical reactions, including Maillard reactions. Such reactions shorten the shelf life of the immunoglobulin compositions. Another group of stabilizers include amino acids, such as glycine. Surfactants such as polysorbate may also be used. Thus, US Patent Specification No. 5,945,098 teaches intravenous immunoglobulin preparations, which are essentially protein-free apart from the immunoglobulin and which comprise aqueous solutions of immunoglobulin, about 0.1 M to 0.3 M glycine, about 0.0005 % to 0.01 % polysorbate and less than about 0.2 gram % PEG.

The high levels of amino acids needed for adjustment of physiological osmolarity may, however, cause side effects in intravenous immunoglobulin. Furthermore, amino acids are not compatible with reducing sugars, but may lead to the formation of Schiff bases.

Thus, as apparent from the above overview, there is a need for improved parenteral formulations of antibodies, including intravenous immunoglobulin and other therapeutically useful immunoglobulins, which exhibit increased shelf-life.

Summary of the Invention

It is an object of the present invention to provide new liquid compositions of antibodies formulated for parenteral administration.

It is another object of the invention to provide a new method of producing novel antibody compositions.

It is still a third object of the invention to provide a new use of trehalose.

A fourth object of the invention comprises a method of providing mammals with 1) passive immunization against infectious diseases, 2) immunomodulation to alleviate and cure various clinical disorders and 3) targeted therapy with monoclonal antibodies.

These and other objects, together with the advantages thereof over known compositions and methods, are achieved by the present invention, as hereinafter described and claimed.

The invention is based on the idea of using a specific non-reducing disaccharide, viz. trehalose as a stabilizing agent at a concentration, which effectively prevents polymerization and aggregation of the antibodies in the liquid parenteral compositions. Being a non-reducing sugar, trehalose does not react with amino acids or proteins as part of Maillard browning.

In connection with the present invention it has been found that no formation of polymers takes place during storage of antibodies, such as IgG, in liquid formulations even at elevated temperatures in the presence of trehalose, whereas detectable amounts of polymers are formed in the presence of previously described stabilizers. Trehalose is capable of stabilizing proteins during freeze-drying and spray-drying. The present invention is based on the new finding that trehalose is capable of preventing polymer formation of proteins during storage in solution in a liquid formulation. When antibodies can be stored in a stable liquid formulation, their clinical use is facilitated and made more reliable, because reconstitution of a dried powder becomes superfluous.

Thus, in the method according to the present invention for preparing therapeutically useful compositions, antibodies are admixed in an aqueous medium with trehalose and, optionally, other auxiliary agents known per se, at a concentration of about 0.001 M to 0.5 M to provide a parenterally-administrable liquid composition. Such compositions can, e.g., be administered intravenously.

The present invention also provides for the use of trehalose in a liquid formulation of antibodies at a concentration, which effectively prevents polymerization of the antibodies.

In view of the above features, the present invention also provides a new method of providing passive immunization of mammals against infectious diseases, which method comprises parenterally administering to the mammal a liquid immunoglobulin composition, which contains trehalose as a stabilizer in a concentration sufficient to effectively prevent polymerization of the immunoglobulin.

More specifically, the liquid compositions according to the present invention are mainly characterized by what is stated in the characterizing part of claim 1. The method according to the invention for preparing the liquid compositions is characterized by what is stated in the characterizing part of claim 11 and the use comprises the features of claim 13.

The therapeutic methods according to the invention are characterized by what is stated in the characterizing parts of claims 16 to 18.

Surprisingly, a very beneficial stabilizing effect on the parenteral compositions can be obtained by the present invention. Not only does trehalose effectively prevent polymerization and other undesired reactions of the antibodies, it also acts as an osmolarity regulating agent and partially or totally eliminates the need for additional osmolarity regulating agents in the compositions.

Further features and advantages of the present invention will become apparent from the following detailed description and the appended working example and drawings.

Brief Description of the Drawings

Figure 1 shows in graphical form the stabilizing effect of various sugars and glycine against polymer formation as a function of storage time in liquid formulations of antibodies (ρH 4.0);

Figure 2 gives a similar graphical representation of formation of chemical reactions products of various stabilizers as a function of storage time in liquid formulations of antibodies (pH 4.0); and Figure 3 gives a similar graphical representation of the stabilizing effect of trehalose and glycine against polymer formation as a function of storage time in liquid formulations of antibodies (pH 5.3).

Detailed Description of the Invention

The present invention provides novel liquid compositions of antibodies formulated for parenteral administration, wherein the antibodies are mixed in an aqueous medium with a stabilizing agent and optionally with other auxiliary agents known per se. The stabilizing agent is, according to the present invention, at least partially comprised of trehalose, which is added in a concentration sufficient to provide for effectively preventing polymerization of the antibodies during storage. The shelf life of immunoglobulin compositions typically is 1-3 years in refrigerator, i.e. at temperatures +2 - +8 °C. By contrast, the liquid compositions according to the present invention can be stored at room temperature (at about 25 °C) for extended periods of time (at least 6 months and up to 3 years), which makes the storage and handling easier and less risky particularly in outpatient use. Furthermore, the novel liquid composition makes the immunoglobulin stable also at higher temperatures than room temperature, to which the immunoglobulin may be exposed during transportation and travelling of patients. The novel composition makes it possible to manufacture intravenous immunoglobulin, in which no polymerization of antibodies takes place even during storage for 2-6 months at 37 °C. By the expression "no polymerization of immunoglobulin takes place", it is meant that the level of polymerized immunoglobulin species is 0.1 mole-% or less and no increase in amount of polymerized species (during storage) can be found by size exclusion liquid chromatography.

To achieve that goal, the concentration of trehalose is typically 0.001 M to 0.5 M, in particular 0.01 - 0.4 M, in the present composition.

It should be pointed out that different manufacturing processes yield final products containing varying amounts of aggregated IgG. Thus, the novel process described in our copending patent application titled "Process for the Manufacture of Virus-Safe Immunoglobulin" results in a product, which is essentially free of polymeric proteins, whereas the product of known processes, such as that disclosed in US Patent No. 5,886,154 may contain up to 0.5 % polymeric proteins. The use of trehalose as a stabilizer prevents the increase of protein polymers during storage.

Within the scope of the present invention, the terms "antibody" or "antibodies" are used for designating any monoclonal and polyclonal antibodies of the IgG, IgA and IgM classes of animal or human origin. More specific explanations of the term "antibodies of human origin" can be found in US Patent No. 5,807,734, the contents, of which is herewith incorporated by reference.

However, the antibodies may also be chimeric antibodies, such as humanized mouse antibodies. Further, modified antibodies, such as divalent and monovalent antibody fragments, single chain antibodies and single domain antibodies, are also included in the meaning of the terms "antibody" of "antibodies". Further, the antibodies or modified antibodies may also be conjugated with other molecules, such as pharmaceutically actives effector units, including radionucleides, drugs and toxins, thereby forming therapeutically useful conjugates. Conjugated antibodies for parenteral administration may also be used for diagnostic purposes, such radionucleide-labelled antibodies for imaging.

The term "immunoglobulin" designates monoclonal and polyclonal immunoglobulins selected from the group of IgG, IgM and IgA.

In the following description, the invention is described in more detail using human polyclonal IgG and F(ab') fragments of human polyclonal IgG as an example. However, it should be noted that the invention is applicable to other polyclonal and monoclonal antibodies suitably modified if necessary to take into account the different sources and therapeutic use of the immunoglobulin.

Polyclonal immunoglobulin can be obtained by processing of plasma obtained from blood donors. Most methods are based on the cold ethanol precipitation process ("Cohn process"), but methods based on other precipitating agents, such as caprylic acid and PEG, and chromatographic methods have been developed. Polymers and aggregates can be removed from immunoglobulin by pepsin treatment, PEG precipitation and chromatography. Suitable methods of producing intravenous immunoglobulin are described in, e.g., US Patent No. 5,945,098, US Patent No 6,281,336, and in our copending patent application titled "Process for the Manufacture of Virus-Safe Immunoglobulin", the contents of which are herewith incorporated by reference. Preferably, the protein product is subjected to virus inactivation during the production process. Virus inactivation can be achieved by different methods, such as treatment with SD substances, caprylic acid or heat. The process may contain effective virus removal steps such as precipitations, chromatography and virus filtration.

Monoclonal antibodies can be produced by hybridoma and recombinant technology. Similar purification methods can be applied as for polyclonal immunoglobulin preparations, including caprylic acid and PEG precipitation and chromatography. Suitable methods of producing monoclonal antibodies are described in, e.g., US Patent Nos. 5,807,734 and 6,600,022.

Generally, the immunoglobulin is recovered in the form of a liquid composition having a concentration of 1 to 250 g 1, although the suitable concentration of polyclonal immunoglobulin for its conventional therapeutic uses is in the range of some 10 to 250 g/1, e.g. 50 to 200 g/1. The present invention makes it possible to produce such highly concentrated polyclonal Ig (e.g. IgG, IgA and IgM) compositions, which allows for facile administration of large amounts of immunoglobulins not only intraveneously but also subcutaneously and intramuscularly. One particular advantage of subcutaneous administration is that it for home-treatment of patients with high doses of immunoglobulins.

It is also possible to recover and purify the immunoglobulin and to store it after a separate freeze-drying step. In that case, the protein needs to be resuspended into an aqueous solution, which contains trehalose or to which trehalose is added once the protein has been dispersed in the solution.

The parenteral formulations according to the present invention are readily produced from the obtained immunoglobulin-containing liquid compositions by adding trehalose as such or a mixture of trehalose and other conventional stabilizers to the IgG solution and pH is adjusted if necessary. The solution is then sterile filtered and filled aseptically to final containers, such as vials.

The stabilizing sugar used, trehalose, is a unique, naturally occurring disaccharide containing two glucose molecules bound in an , -1, 1 linkage. This structure results in a chemically stable, non-reducing sugar with many important functional characteristics. Trehalose is found in nature in hundreds of plants and animals. It is an important source of energy, and has been shown to be a primary factor in stabilization of organisms during times of freezing and desication. Trehalose is consumed as part of a normal diet. Thus, the use thereof in the present invention is pharmacologically safe.

According to an embodiment of the invention, the osmolarity of the composition is in excess of about 150 mOsmol/kg, preferably > 240 mOsmol/kg. This osmolarity range can be obtained by adding trehalose in a concentration sufficient to render the composition the desired osmolarity. According to a preferred embodiment, trehalose is added in a concentration sufficient as such to render the composition a physiological osmolarity (i.e. an osmolarity of about 200 mOsmol/kg or more). The pH of the composition is 3.8-7.4. It is further possible to complement the compositions according to the present invention with conventional excipients and pharmaceutical carriers, such as a second stabilizer and/or a further osmolarity regulating component. The conventional stabilizer can be selected from suitable sugars and amino acids, exemplified by maltose, glucose, sucrose and glycine; and it can be a polyol, such as a sugar alcohol. Other conventional pharmaceutical excipients and adjuvants known in the art can be added to the composition. Examples of such carriers include, but are not limited to, albumin, liposomes, buffers, such as phosphate, acetate, succinate and maleate, electrolytes, glycerol, hydroxymethylcellulose, propylene glycol, polyethylene glycol, polyoxyethylenesorbitan, other surface active agents, vegetable oils, conventional anti-bacterial or anti-fungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Antioxidative agents may also be added, such metal chelators and oxygen radical scavengers. A pharmaceutically-acceptable carrier within the scope of the present invention meets industry standards for sterility, stability, and non-pyrogenicity.

Generally, the molar ratio between the trehalose and any further stabilizer (e.g. sugar, polyol, inorganic salt and/or amino acid) is greater than 0.05 (in other words, trehalose stands for at least 5 mole-% of the total amount of stabilizer component of the composition). As apparent to a person skilled in the art, some advantages in terms of simplicity are obtained by using trehalose as sole or at least the main stabilizing component. For intravenous immunoglobulin preparations, which typically are administered in large volumes to patients, it is possible to limit the use of the present novel stabilizer at about less than 50 mole-%, and to use a balance of known stabilizers, e.g. sugars or amino acids, necessary for achieving the aimed osmolarity. In case of monoclonal antibodies, minor amounts (e.g. 0.01 - 0.5 mg/ml) of stabilizers of the polysorbate type can be employed, as compared to about 50 to 500 mmol trehalose/1 (corresponding to about n. 17 - 170 mg/ml).

The absolute concentration of the immunoglobulin varies depending on the actual immunoglobulin species and the intended use. Generally, the concentration is in the range of 1 to 250 g/1. For traditional pharmaceutical use, e.g. for passive immunization of mammals against infectious diseases, the concentration is typically about 50 to 160 g/L Thus, a suitable pharmaceutical composition comprises 50 to 160 g/1 immunoglobulin G and 0.1 to 0.4 M trehalose in a aqueous medium having an osmolarity > 240 mOsmol/kg. For monoclonal antibodies the concentration is typically less than 10 g/1.

Within the scope of the present invention, the pharmaceutical compositions are administered to patients parenterally for passive immunization against infectious diseases, for immunomodulation in the treatment of different clinical disorders and for other antibody mediated activities, such as targeted use of monoclonal antibodies. The administered dosage of antibodies is in the range of about 0.001 mg to 10 g/kg body weight, in particular about 0.1 to 1.0 g/kg body weight for intravenous immunoglobulin, and about 0.001 mg to 10 mg/kg body weight for monospecific polyclonal antibodies, such as anti-D immunoglobulin, and monoclonal antibodies.

The parenteral administration routes include: intravenous, intramuscular, subcutaneous, rectal, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal. In particular, the present liquid compositions are formulated for intravenous, subcutaneous or intramuscular administration.

The following non-limiting example illustrates the invention:

In the examples, the following analytical methods were used: IgG was determined by immunoturbidimetry with a kit from ThermoClinical Labsystems. The proportion of polymers was determined by size-exclusion liquid chromatography according to Ph. Eur. 3rd Ed. 1997:0338. The formation of chemical reaction products was monitored by absorbance at 340 nm.

Example 1

Polyclonal IgG was purified from Cohn fraction II of human plasma by DEAE-Sephadex chromatography, pepsin treatment, SD treatment, CM Sepharose chromatography and ultrafiltration. The pure antibody solution contained about 12% F(ab')₂ fragments and 5% IgG dimers and no detectable IgG polymers. It was formulated to solutions containing 100 g/1 antibodies at pH 4.0 and one of the following stabilizers: 0.3 M trehalose, 0.3 M maltose, 0.3 M sucrose, 0.3 M glucose or 0.2 M glycine. The solutions were sterile filtered and filled aseptically into glass vials. The vials were stored at 25 °C and 37 °C, and analyzed for polymers and chemical reaction products after 1, 2, 3 and 6 months. There was a clear increase in polymers after storage for one month at 37 °C in the presence of other stabilizers than trehalose, whereas no clear increase took place in the presence of trehalose during 6 months (Figure 1). Similarly, the formation of chemical reaction products was clearly faster in the presence of other stabilizers than trehalose (Figure 2). .

Example 2

Polyclonal IgG was purified from Cohn fraction π+III of human plasma by caprylic acid precipitation, PEG precipitation, anion exchange chromatography and ultrafiltration as described in our copending patent application titled "Process for the Manufacture of Virus- Safe Immunoglobulin". The pure IgG was formulated to solutions containing 100 g/1 of IgG at pH 5.3 and 0.2 M trehalose or 0.2 M glycine as a stabilizer. The solutions were sterile filtered and filled aseptically into glass vials. The vials were stored at 25 °C and 37 °C, and analyzed for polymers and chemical reaction products. There was a clear increase in polymers after storage for one month at 37 °C in the presence of glycine, whereas no increase took place in the presence of trehalose during 2 months (Figure 3).

Similarly, the formation of chemical reaction products was faster in the presence of glycine than trehalose.

Claims

1. Liquid composition of antibodies formulated for parenteral administration, wherein the antibodies are mixed in an aqueous medium with a stabilizing agent and optionally with other auxiliary agents known per se, c h a r a c t e r i z e d in that it contains trehalose as a stabilizing agent at a concentration, which effectively prevents polymerization of the antibodies.

2. The liquid composition according to claim 1, wherein the concentration of trehalose is 0.001 M to 0.5 M.

3. The liquid composition according to claim 1 or 2, wherein the antibodies comprise monoclonal or polyclonal antibodies of animal or human origin, chimeric antibodies, modified antibodies, such as divalent and monovalent antibody fragments and single chain antibodies and single domain antibodies, and conjugates of antibodies.

4. The liquid composition according to claim 3, wherein the antibodies comprise immunoglobulin selected from the group of immunoglobulin G, immunoglobulin M and immunoglobulin A.

5. The liquid composition according to claim 3 or 4, wherein the concentration of the antibodies is 1 to 250 g/1.

6. The liquid composition according to any of the previous claims, wherein the osmolarity of the composition is > 240 mOsmol/kg.

7. The liquid composition according to any of the previous claims, wherein the concentration of trehalose is sufficient to render the composition a physiological osmolarity.

8. The liquid composition according to any of claims 1 to 6, comprising at least a second osmolarity regulating component selected from sugars, polyols, inorganic salts and amino acids.

9. The liquid composition according to any of the previous claims, comprising 50 to 200 g/1 immunoglobulin G and 0.1 to 0.4 M trehalose in a aqueous medium having an osmolarity of about 200 mOsmol/kg, in particular > 240 mOsmol/kg.

10. The liquid composition according to any of the previous claims, wherein it is formulated for intravenous, subcutaneous or intramuscular administration.

11. Method of formulating a liquid composition of antibodies for parenteral administration, wherein the antibodies are mixed in an aqueous medium with a stabilizing agent and optionally with auxiliary agents known per se, c h a r a c t e r i z e d using trehalose as a stabilizing agent at a concentration, which effectively prevents polymerization of the antibodies.

12. The method according to claim 11, wherein trehalose is used at a concentration of 0.001 M to 0.5 M.

13. Use of trehalose in a liquid composition of antibodies selected from the group of immunoglobulin G, immunoglobulin M and immunoglobulin A in a concentration, which effectively prevents polymerization of the immunoglobulin.

14. The use according to claim 13, wherein the liquid composition is formulated for intravenous administration.

15. The use according to claim 13, wherein the liquid composition is formulated for subcutaneous or intramuscular administration.

16. Method of providing passive immunization of mammals against infectious diseases, comprising parenterally administering to the mammal a liquid immunoglobulin composition, which contains trehalose as a stabilizer in a concentration sufficient to effectively prevent polymerization of the immunoglobulin.

17. Method of providing immunomodulation to alleviate and cure clinical disorders in mammals, comprising parenterally administering to the mammal a liquid immunoglobulin composition, which contains trehalose as a stabilizer in a concentration sufficient to effectively prevent polymerization of the immunoglobulin.

18. Method of providing targeted monoclonal antibodies to mammals, comprising parenterally administering to the mammal a liquid antibody composition, which contains trehalose as a stabilizer in a concentration sufficient to effectively prevent polymerization of the immunoglobulin.