Title:  Concentrated antibody preparation

United States Patent:  6,252,055

Assignee:  Glaxo Wellcome Inc. (Triangle Park, NC)

Filed:  November 12, 1998

PCT NO:  PCT/EP97/02595

102(e) Date:  November 12, 1998

PCT PUB. Date:  December 4, 1997

Foreign Application Priority Data:  May 24, 1996[GB] (9610992)

Concentrated monoclonal antibody preparations for administration to humans are described in which the antibody is present at a concentration of greater than 100 mg/ml and as high as 350 mg/ml.

Description of the Invention

The present invention relates to a concentrated antibody preparation, pharmaceutical formulations containing such a preparation, its use in human therapy and processes for its preparation.

Most commercially available immunoglobulins produced at high concentration are derived from human serum and produced by the blood products industry. The first purified human immunoglobulin G (IgG) preparation used clinically was immune serum globulin which was prepared in the 1940's (Cohn, E. J. et al `Preparation and properties of serum and plasma proteins`. J. Am. Chem. Soc. pg68, 459-475 (1946) and Oncley, J. L et al `The separation of antibodies, isoagglutinins, prothrombin, plasminogen and .beta.-lipoproteins into sub-fractions of human plasma.` J. Am. Chem. Soc. 71, 541-550 (1949)).

The next generation of purified IgG's were developed in the 1960's, and focused on preparations suitable for intravenous administration (Barandun, S.et al `Intravenous administration of human .gamma.-globulin.` Vox. Sang. 7, 157-174 (1962)).The first of these--IgG intravenous preparation (Gamimune.RTM., Cutter Biological), was formulated as a 5% (50 mg/ml) IgG solution in 0.2 M glycine, 10% maltose, pH 6.8. This solution was stable for at least 2.5 years at 5^oC. Key criteria for the acceptance of intravenous IgG (IVIG) products were that the IgG had undergone little fragmentation and that no high molecular weight aggregates were present.

Today, human therapeutic immunoglobulin products are available for either intramuscular (IMIG) or intravenous (IVIG) administration. IMIG are used principally for hepatitis A prophylaxis and sometimes for the treatment of agammaglobulinaemic patients. IVIG are used in the treatment of primary immunodeficiencies and idiopathic thrombocytopenic purpura, as well as for secondary immune deficiencies, various infections, haematological and other autoimmune diseases. In general IMIG products are marketed as 16% (w/v) (160 mg/ml) solutions and IVIG products as 5% (w/v) solutions (50 mg/ml).

Manufacturers experience with IVIG has shown that these preparations are unstable in relatively dilute solutions (<10% (w/v)), and the instability is manifested by the formation of insoluble particles by a process known as `shedding` when the material is stored at room temperature (Fernandes, P. M. and Lundband, J. L. `Preparation of a stable intravenous gamma-globulin: process design and scale up.` Vox. Sang. 39, 101-112 (1980)). Commercially available 16.5% .gamma.-globulin is usually stabilised in a buffered glycine-saline solution. The use of maltose at 5-10% as a stabiliser has been shown to be effective in protecting 5% IVIG from particulate formation (Fernandes et al supra).

In addition to shedding, concentrated (16.5%) solutions of IVIG have a tendency to aggregate during long term storage. As much as 10-30% (w/w) of the IVIG solution could be comprised of aggregates (Gronski, P.et al,`On the nature of IgG dimers. I. Dimers in human polyclonal IgG preparations: kinetic studies.` Behring Inst. Mitt. 82, 127-143 (1988)).

The majority of these aggregates are dimers produced by complexes of idiotypic and anti-idiotypic antibodies. Since monoclonal antibodies prepared from tissue culture supernatants do not contain anti-idiotype antibodies, these sort of dimers are absent. However, dimer formation in these preparations can be caused by complexation between partially denatured monomeric antibody molecules. Mechanical stress such as that encountered during tangential flow ultrafiltration used for concentrating antibody preparations can also lead to an increase in aggregation (Wang, Y.-C. J. and Hanson, M. A. `Parenteral formulations of proteins and peptides: stability and stabilisers.` J. Parenteral Sci. Technol. 42, Suppl. S3-S26 (1988)).

Concentrated (>100 mg/ml) preparations of immunoglobulins are therefore available but to date these are polyclonal antibody preparations derived from the blood processing industry, and are stabilised by the addition of various excipients such as glycine and maltose.

It is therefore surprising that monoclonal antibody preparations have been obtained at a concentration >100 mg/ml in the absence of excipients and without a concomitant increase in aggregates.

The Derwent Abstract of JP01268646A (AN89-359879) reports that the application describes an injection preparation of an IgG₃ monoclonal antibody having a concentration of 0.1 .mu.g to 100 mg/ml. Subject matter disclosed in these publications is outside the scope of the instant invention.

The present invention therefore provides a monoclonal antibody preparation for administration to a human characterised in that the antibody in said preparation is at a concentration of 100 mg/ml or greater, preferably greater than 100 mg/ml. Above a concentration of 350 mg/ml the preparation can be very viscous and recovery rates become unacceptably low. The ideal concentration is between 100 and 300 mg/ml.

Preparations according to the invention are substantially free from aggregate. Acceptable levels of aggregated contaminants would be less that 5% ideally less than 2%. Levels as low as 0.2% are achievable, although approximately 1% is more usual. The preparation is also preferably free from excipients traditionally used to stabilise polyclonal formulations, for example glycine and/or maltose.

The present invention therefore provides a monoclonal antibody preparation for administration to a human characterised in that the antibody in said preparation is at a concentration of 100 mg/ml or greater, preferably greater than 100 mg/ml and the preparation is substantially free from aggregate.

Recombinant antibodies by their very nature are produced in a synthetic and unnatural cell culture environment. Expression systems which are used to generate sufficient quantities of the protein for commercialisation are routinely based on myeloma or chinese hamster ovary (CHO) host cells.

In order to culture such cells, complex synthetic media which are devoid of contaminating animal protein have been devised resulting in glycosylation patterns of the protein which would not be expected to arise in nature. It is therefore all the more surprising that a complex glycoprotein produced under such synthetic conditions can be prepared at concentrations several times greater than would occur in normal human serum with all its buffering capabilities.

The present invention therefore provides a monoclonal antibody preparation for administration to a human characterised in that the antibody in said preparation is a recombinant antibody and is at a concentration of 100 mg/ml or greater, preferably greater than 100 mg/ml. The preparation is preferably substantially free from aggregate.

During the production of purified antibodies whether for therapeutic or diagnostic use, it is important that the antibody is sufficiently stable on storage and various chemical entities may have an adverse effect on the stability of the antibody. For example, trace amounts of copper (Cu++) are now known to have a destabilising effect on immunoglobulin molecules on storage (WO93/08837), and that this effect can be eliminated by formulating the immunoglobulin molecule with a suitable chelator of copper ions, for example EDTA or citrate ion.

The present invention is applicable to a preparation of immunoglobulins of all classes, i.e. IgM, IgG, IgA, IgE and IgD, and it also extends to a preparation of Fab fragments and bispecific antibodies. The invention is preferably applied to a preparation of immunoglobulins of the class IgG, which includes the sub-classes IgG₁, IgG₂, IgG₃ and IgG₄. The invention is more preferably applied to a preparation of immunoglobulins of the class IgG₄ and IgG₁, most preferably IgG₁.

The invention finds particular application in the preparation of recombinant antibodies, most particularly chimaeric antibodies or humanised (CDR-grafted) antibodies. Particular examples of these include chimaeric or humanised antibodies against CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD23, CD25, CD33, CD54, and CDw52 antigen. Further examples include chimaeric or humanised antibodies against various tumour cell markers e.g 40 kd (J.Cell Biol. 125 (2) 437-446 (1994)) or the antigens of infectious agents such as hepatitis B or human cytomegalovirus. Particularly preferred examples include chimaeric or humanised antibodies against CDw52, CD4 and CD23 antigen.

Immunoglobulins intended for therapeutic use will generally be administered to the patient in the form of a pharmaceutical formulation. Such formulations preferably include, in addition to the immunoglobulin, a physiologically acceptable carrier or diluent, possibly in admixture with one or more other agents such as other immunoglobulins or drugs, such as an antibiotic. Suitable carriers include, but are not limited to, physiologic saline, phosphate buffered saline, glucose and buffered saline, citrate buffered saline, citric acid/sodium citrate buffer, maleate buffer, for example malic acid/sodium hydroxide buffer, succinate buffer, for example succinic acid/sodium hydroxide buffer, acetate buffer, for example sodium acetate/acetic acid buffer or phosphate buffer, for example potassium dihydrogen orthophosphate/disodium hydrogen orthophosphate buffer. Optionally the formulation contains Polysorbate for stabilisation of the antibody. Alternatively the immunoglobulin may be lyophilised (freeze dried) and reconstituted for use when needed by the addition of water and/or an aqueous buffered solution as described above.

The preferred pH of the pharmaceutical formulations according to the invention will depend upon the particular route of administration. However, in order to maximise the solubility of the antibody in the concentrated solution, the pH of the solution should be different from the pH of the isoelectric point of the antibody.

Thus, according to a further aspect the invention provides a monoclonal antibody preparation for administration to a human characterised in that the antibody in said preparation is at a concentration of 100 mg/ml or greater and the pH of the preparation is different from the pH of the isoelectric point of the antibody.

Routes of administration are routinely parenteral, including intravenous, intramuscular, and intraperitoneal injection or delivery. However, the preparation is especially useful in the generation of sub-cutaneous formulations which must be low in volume for example approximately 1 ml in volume per dose. To ensure that therapeutic dosage can be achieved in such a formulation, a concentrated preparation will invariably be necessary. Preferred concentrations for sub-cutaneous preparations are for example in the range of 100 mg/ml to 200 mg/ml, for example 150 mg/ml to 200 mg/ml. A sub-cutaneous preparation has the advantage that it can be self-administered thus avoiding the need for hospitalisation for intravenous administration.

Preferably, sub-cutaneous formulations according to the invention are isotonic and will be buffered to a particular pH. The preferred pH range for a sub-cutaneous formulation will in general range from pH 4 to pH 9. The preferred pH and hence buffer will depend on the isoelectric point of the antibody concerned as discussed above. Thus, in the case of sub-cutaneous preparations containing anti-CD4 antibodies the pH will preferably be in the range of pH 4 to pH 5.5, for example pH 5.0 to pH 5.5 e.g. pH 5.5, and in the case of anti-CD23 antibodies in the range of pH 4 to pH 6.5. Thus, preferred buffers for use in sub-cutaneous formulations containing anti-CD4 antibodies are maleate, succinate, acetate or, more preferably phosphate buffers. Buffers are preferably used at a concentration of 50 mM to 100 mM.

Sub-cutaneous formulations according to the invention may also optionally contain sodium chloride to adjust the tonicity of the solution.

Thus, according to a further aspect of the invention provides a monoclonal antibody preparation for sub-cutaneous administration to a human characterised in that the antibody in said preparation is at a concentration of 100 mg/ml or greater and the pH of the preparation is different from the pH of the isoelectric point of the antibody.

In a further aspect of the invention the monoclonal preparation is envisaged for use in human therapy. Various human disorders can be treated such as cancer or infectious diseases for example those mentioned above, and immune disfunction such as T-cell-mediated disorders including severe vasculitis, rheumatoid arthritis, systemic lupis, also autoimmune disorders such as multiple sclerosis, graft vs host disease, psoriarsis, juvenile onset diabetes, Sjogrens' disease, thyroid disease, myasthenia gravis, transplant rejection, inflammatory bowel disease and asthma.

The invention therefore provides the use of a concentrated monoclonal antibody preparation as described herein in the manufacture of medicament for the treatment of any of the aforementioned disorders. Also provided is a method of treating a human being having any such disorder comprising administering to said individual a therapeutically effective amount of a preparation according to the invention.

The dosages of such antibody preparations will vary with the conditions being treated and the recipient of the treatment, but will be in the range 50 to about 2000 mg for an adult patient preferably 100-1000 mg administered daily or weekly for a period between 1 and 30 days and repeated as necessary. The doses may be administered as single or multiple doses.

An antibody preparation may be concentrated by various means such as cross flow (tangential) or stirred ultrafiltration, the preferred route is by tangential flow ultrafiltration. Low recovery rates and precipitate formation can be a problem when concentrating antibody. The present invention solves this particular problem by a method of concentration which involves reducing shear stresses of cross flow ultrafiltration at high circulation rates (500 ml/min). Reducing the recirculation for example to 250 ml/min leads to successful concentration of antibody to >150 mg/ml and to the high recovery of material.

The invention therefore provides a process for the preparation of a concentrated antibody preparation as described herein. The recovery of the antibody in the concentrated preparation is preferably greater than 70% but is routinely greater than 90%.

Concentrated antibody preparations prepared according to the above process may contain additional ingredients such as buffers, salts, Polysorbate and/or EDTA. These additional agents may not be required in the final pharmaceutical formulation in which case they can be removed or exchanged using diafiltration according to conventional methods known in the art. For example, concentrated antibody preparations containing citrate buffer and EDTA can be converted into concentrated antibody preparations containing phosphate or maleate buffer using this method.

Claim 1 of 5 Claims

What is claimed is:

1. A method of producing a concentrated antibody preparation comprising the steps of:

(a) providing an antibody preparation;

(b) filtering said antibody preparation through a retentate side of a filtering membrane using ultrafiltration to produce a filtrate; and

(c) circulating said filtrate back to said retentate side of the filtering membrane at a circulation rate of less than 500 ml/min thereby reducing sheer stress on said filtrate.

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