Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1998-11-12
2001-06-26
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C424S130100, C424S177100, C435S326000, C530S388100, C530S387100, C530S390500
Reexamination Certificate
active
06252055
ABSTRACT:
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 &bgr;-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 &ggr;-globulin.’ Vox. Sang. 7, 157-174 (1962)).The first of these—IgG intravenous preparation (Gamimune®, 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° C. 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% &ggr;-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 IgG
3
monoclonal antibody having a concentration of 0.1 &mgr;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 IgG
1
, IgG
2
, IgG
3
and IgG
4
. The invention is more preferably applied to a preparation of immunoglobulins of the class IgG
4
and IgG
1
, most preferably IgG
1
.
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, a
DeCloux Amy
Glaxo Wellcome Inc.
Nixon & Vanderhye P.C.
Saunders David
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