MXPA98009645A - Concentrated preparation of anticuer - Google Patents

Abstract

The present invention relates to a concentrated preparation of antibodies, pharmaceutical formulations containing such preparation, its use in the therapy of humans and processes for its preparation.

Description

Concentrated Antibody Preparation FIELD OF THE INVENTION The present invention relates to a concentrated preparation of antibodies, pharmaceutical formulations containing such a preparation, their use in the therapy of humans and processes for their preparation.

BACKGROUND OF THE INVENTION The most commercially available immunoglobulins, produced in high concentration, are derived from human serum and are produced by the blood products industry. The first immunoglobulin preparation of human, purified G (IgG) used clinically was serum globulin, immune, which was prepared in the 1940's (Cohn, EJ et al. Preparation and properties of serum and plasma proteins. Chem. Soc. Pages 68, 459-475 (1946) and Oncley, JL and collaborators The separation of antibodies, isoagglutinins, prothrombin, plasminogen and ß-lipoproteins into sub-fractions of human plasma 'J. Am. Chem. Soc. 71, 541-550 (1949)). The next generation of purified IgG 's is REF .: 28868 - -developed in the 1960's, and focused on the appropriate preparations for intravenous administration (Barandun, S. and collaborators xIntravenous administration of human? -globulin 'Vox. Sang. 7, 157-174 (1962 )). The first of these - intravenous IgG preparation (Gamimune®, Cutter Biological), was formulated as a solution of 5% IgG (50 mg / ml) in 0.2 M glycine, 10% maltose, pH 6.8. This solution was stable for at least 2.5 years at 5 ° C. The key criteria for the acceptance of intravenous IgG products (IVIG) were that the IgG had undergone little fragmentation and high molecular weight aggregates were not present. Today, the therapeutic immunoglobulin products, from human, are available for either intramuscular (IMIG) or intravenous administration (IVIG). IMIGs are used primarily for the prophylaxis of hepatitis A and sometimes for the treatment of agammaglobulinemic patients. IVIG is used in the treatment of primary deficiencies and idiopathic thrombocytopenic purpura, as well as secondary immunodeficiencies, various infections, hematological diseases and other autoimmune diseases. In general, IMIG products are marketed as 16% (w / v) solutions (160 mg / ml) and IVIG products as 5% (w / v) solutions (50 mg / ml).

The experience of manufacturers with the 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 through a process known as? Sedimentation or settlement 'when the material is stored at room temperature (Fernandes, PM and Lundband, JL xPreparation of a stable intravenous gamma-globulin: process design and scale up' Vox.Sange 39, 101-112 (1980)). The commercially available 16.5%? -globulin is usually stabilized in a buffered glycine-saline solution. The use of 5-10% maltose as a stabilizer has been shown to be effective in the protection of IVIG at 5% particle formation (Fernandes et al. Supra). In addition to sedimentation or settlement, the concentrated solutions (16.5%) of IVIG have a tendency to aggregate for a long time of 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).) Most of these aggregates are dimers produced by idiotypic and -antiidiotypic antibody complexes, since monoclonal antibodies prepared from tissue culture supernatants do not contain anti-idiotype antibodies, these classes of dimers are absent, however, the formation of dimers in these preparations can be caused by the formation of complexes between monomeric, partially denatured antibody molecules.

Mechanical strength such as that found during tangential flow ultrafiltration used for the concentration of 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 stabilizers. 'J.

Parenteral Sci. Technol. 42, Suppl. S3-S26 (1988)). Therefore, the concentrated preparations (> 100 mg / ml) of immunoglobulins are available but to date these are preparations of polyclonal antibodies, derived from the blood processing industry, and are stabilized by the addition of various excipients such as glycine and maltose. Therefore, it is surprising that the monoclonal antibody preparations have been obtained in a concentration > 100 mg / ml in the absence of excipients and without a concomitant increase in the aggregates. The Derwent summary of Japanese Patent Application No. 01268646A (AN89-359879) reports that the application describes an injection preparation of an IgG3 monoclonal antibody having a concentration of 0.1 μg to 100 mg / ml. The content described in these publications is outside the scope of the present invention. Therefore, the present invention provides a preparation of monoclonal antibodies for administration to a human, characterized in that the antibody in the 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 the recovery rates become unacceptably low. The ideal concentration is between 100 and 300 mg / ml. The preparations according to the invention are substantially free of aggregate. The levels of added contaminants would be less than 5% ideally less than 2%. Levels as low as 0.2% are achieved, although approximately 1% is more usual. The preparation is also, preferably, free of excipients traditionally used to stabilize the polyclonal formulations, for example glycine and / or maltose. Therefore, the present invention provides a preparation of monoclonal antibodies for administration to a human, characterized in that the antibody in the preparation is at a concentration of 100 mg / ml or greater, preferably greater than 100 mg /. ml and the preparation is substantially free of aggregates. The recombinant antibodies, by their very nature, are produced in a non-natural and synthetic cell culture environment. The expression systems which are used to generate sufficient amounts of the protein for commercialization are routinely based on a myeloma or Chinese Hamster Ovary Host Cell (CHO). In order to cultivate such cells, it has been planned that complex synthetic media that are free of contaminating animal protein will result in protein glycosylation patterns that would not be expected to originate anywhere. Therefore, what is most surprising is that a complex glycoprotein produced under such synthetic conditions can be prepared in concentrations several times greater than would occur in a normal human serum with all its buffering capabilities. Therefore, the present invention provides a preparation of monoclonal antibodies for administration to a human, characterized in that the -antibody in the preparation is a recombinant antibody and is in a concentration of 100 mg / ml or greater, preferably greater than 100 mg / ml. The preparation is preferably substantially free of aggregate. During the production of purified antibodies either for therapeutic or diagnostic use, it is important that the antibody be sufficiently stable in storage and that various chemical entities may have an adverse effect on the stability of the antibody. For example, it is now known that very small amounts of copper (Cu ++) have a stabilizing effect on the immunoglobulin molecules in storage (W093 / 08837), and that this effect can be eliminated by formulating the immunoglobulin molecule with a protein-forming agent. Copper ion chelates, suitable, for example EDTA or citrate ion. The present invention is applicable to a preparation of immunoglobulins of all kinds, ie IgM, IgG, IgA, IgE and IgD, and also extends to a preparation of Fab fragments and bispecific antibodies. The invention, preferably, is applied to a preparation of immunoglobulins of the IgG class, which includes the subclasses IgGi, IgG2, IgG3 and IgG. The invention is more preferably applied to a preparation of immunoglobulins of the class IgG4 and IgGi, more preferably IgGi. The invention finds particular application in the preparation of recombinant antibodies, more particularly chimeric antibodies or humanized antibodies (CDR graft). Particular examples of these include chimeric or humanized antibodies against antigen CD2, CD3, CD4, CD5, CD7, CD8, CDlla, CDllb, CD18,CD19, CD23, CD25, CD33, CD54 and CDw52. Additional examples include chimeric or humanized antibodies against various tumor cell markers eg 40kd (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 chimeric or humanized antibodies against CDw52, CD4 and CD23 antigen. The proposed immunoglobulins for therapeutic use in general will 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 antibiotics. Suitable carriers include, but are not limited to, physiological saline, phosphate-buffered saline, glucose and buffered saline, citrate-buffered saline, citrate / sodium citrate buffer, maleate buffer, e.g. malic acid / sodium hydroxide, 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 buffer / disodium acid orthophosphate. Optionally, the formulation contains Polysorbate for the stabilization of the antibody. Alternatively, the immunoglobulin can be lyophilized (frozen and then dehydrated in vacuum) and can be reconstructed for use when necessary 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 on the particular route of administration. However, in order to maximize the solubility of the antibody in the concentrated solution, the pH of the solution must be different from the pH of the isoelectric point of the antibody. Thus, according to a further aspect, the invention provides a preparation of monoclonal antibodies for administration to a human, characterized in that the antibody in the 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 parenterally routinely, including injection or intravenous, intramuscular, and intraperitoneal delivery. However, the preparation is especially useful in the generation of subcutaneous formulations which should be low in volume eg about 1 ml in volume per dose. To ensure that the therapeutic dosage can be achieved in such a formulation, a concentrated preparation will invariably be invariably necessary. Preferred concentrations for the subcutaneous 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 subcutaneous preparation has the advantage that it can be self-administered, thus avoiding the need for hospitalization for intravenous administration. Preferably, the subcutaneous formulations according to the invention are isotonic and will be buffered to a particular pH. The preferred pH range for a subcutaneous formulation will generally range from pH 4 to pH 9. The preferred pH and therefore the buffer will depend on the isoelectric point of the related antibody as discussed above. Thus, in the case of subcutaneous 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, for example pH 5.5 and in the case of antibodies anti-CD23 in the range of pH 4 to pH 6.5. In this way, the preferred buffers for use in subcutaneous formulations containing anti-CD4 antibodies are maleate, succinate, acetate or, more preferably, phosphate buffers. The shock absorbers are preferably used in a concentration of 50 mM to 100 mM. Subcutaneous 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 preparation of monoclonal antibodies for subcutaneous administration to a human, characterized in that the antibody in the 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 considered for use in human therapy. Various human disorders such as cancer or infectious diseases can be treated, for example those mentioned above, and immune dysfunction such as T cell-mediated disorders including severe vasculitis, rheumatoid arthritis, systemic lupus, also autoimmune disorders such as multiple sclerosis, graft against host disease, psoriasis, juvenile onset diabetes, Sjogrens disease, thyroid disease, myasthenia gravis, transplant rejection, inflammatory bowel disease and asthma. Therefore, the invention provides the use of a monoclonal antibody preparation, concentrated as described herein in the preparation of a medicament for the treatment of any of the disorders mentioned above. A method for treating a human being having any disorder of this kind is also provided, which comprises administering to the individual a therapeutically effective amount of a preparation according to the invention. The doses of such antibody preparations will vary with the conditions being treated and the recipient or recipient of the treatment, but will be in the range of 50 to about 2000 mg for a preferentially adult patient 100-1000 mg administered - either daily or weekly during a period between 1 and 30 days and will be repeated as necessary. The doses can be administered as single or multiple doses. An antibody preparation can be concentrated by various means such as transverse flow (tangential) or agitated ultrafiltration, the preferred route is by tangential flow ultrafiltration. Low rates of recovery and formation of the precipitate can be a problem when the antibody is concentrated. The present invention solves this particular problem by a concentration method involving reducing the shear stresses of the transverse flow ultrafiltration at high circulation speeds (500 ml / min). Reduction of recirculation for example at 250 ml / min leads to the successful concentration of the antibody at > 150 mg / ml and the high recovery of the material. Therefore, the invention 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%. The concentrated antibody preparations, prepared according to the above process, may contain additional ingredients such as buffers, salts, polysorbate and / or EDTA. These additional agents can 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 to concentrated antibody preparations containing phosphate buffer or maleate using this method. The invention also provides "a concentrated preparation of antibodies obtainable by such methods." The following are non-limiting examples of the invention.

Example 1 Campath Concentration ÍH The Minitan ultrafiltration equipment (Minitan XX42 ASY MT Ultrafiltration System, Millipore) was joined with 2 NMWCO polysulfone 30K filter plates (Minitan PTTK 30K NMWCO, Millipore), and the pipe and plates were sanitized for 30 minutes with 0.1M NaOH according to the instructions of the manufacturers (System of Minitan Ultrafiltration: Assembly, Operation, Maintenance Instructions, Millipore Corporation, P15076).

The sterilizing substance was removed by flooding with 1-2 liters of phosphate buffered saline (PBS), pH 7. 2. Campath-ΔH (a humanized antibody against the antigen CDw52: Reichmann et al. Nature, 332, 323-327 (1988)) (2200 ml to 16. 4 mg / ml in 50 mM sodium citrate, pH 6.0), was circulated through the side of the retained product of the membranes at a flow rate of 600 ml / min at a back pressure of 2-2.5 bar. The back pressure was maintained at this value throughout the remainder of the experiment, and the impregnated flow rate was measured at various time intervals. The antibody samples were removed from the product retained from the container at various time points and tested for antibody concentration, turbidity,% aggregate and viscosity. Because the filtration rate was too slow in this experiment, it was necessary to carry out the concentration for 3 days. The system was flooded with PBS, and the concentrate stored overnight at 4 ° C. After day 2, thiomersal was added at 0.01% (w / v) to the concentrate before storage overnight to prevent microbial contamination. At the end of the concentration, the system was flooded with 500 ml of PBS, then a 500 ml flood of additional PBS was circulated around the side of the retained product of the membranes for 30 minutes. The concentration of antibodies in these floods was determined by measuring the absorbance at 280 nm. The total time taken for the concentrated Campath-IH from 16.4 mg / ml to 257 mg / ml using only 2 plates in the Minitan was 17.25 hours. The Table 1 (a) shows the change in the concentration of Campath-IH during this time ..

Time (h) 0 5 6.5 9.5 11.5 12.5 14.5 16 17 17.25 Conc. 16 34 41 79 106 136 190 230 301 257 (mg / ml) Table 1 (a) The concentration increased exponentially to a peak of 300 mg / ml after 17 hours. The final concentration was slightly lower than this peak value; the discrepancy probably due to the difficulty in obtaining a representative sample of a very viscous liquid. Table 1 (b) shows that the concentration of Campath-IH was accompanied by a reciprocal decrease in the flow rate of the impregnated.

Conc. 16 34 41 79 106 136 189 301 257 (mg / ml) Flow 4 3 2.5 1.5 1.25 0.9 0.5 (ml / min) Viscosity 1 1 1 1 1 1 0.96 4.85 8.1 (cPs) Table 1 (b) This table also shows that there was a dramatic increase in the viscosity of the remaining concentrate above a concentration of 189 mg / ml. Table l (c) shows that the recovery was high up to a concentration of 190 mg / ml, but began to decline markedly above this concentration as the increase in viscosity led to the material sticking to the glassware and the pipeline and that it was lost during the flood before storing overnight. The final recovery of 257 mg / ml of material Conc. 16 41 106 190 257 (mg / ml) Rec. (%) 100 97 97 85 63 Table 1 (c) (excluding material removed during sampling and loss in washings) was 63.4%. An additional 14.6% was recovered in the first wash with PBS of the system and 0.5% in the second, the wash was circulated with PBS. Therefore, in total 78.5% of the initial material was recovered at the end of the experiment (excluding the material removed during sampling and the loss in the washes), leaving a loss of 21.5% mainly due to the adherence of the material to the glassware and plastics The turbidity of the Campath-IH solution was calculated during the concentration. The absorbance of the 1.0 ml aliquots, properly diluted, from the antibody samples at 650 nm was used as a measure of turbidity. Table 1 (d) shows that there was no increase. Conc. 16 41 79 106 136 190 301 257 (mg / ml) Rec. (%) 0.96 1.16 1.1 0.91 1 1.01 1.11 1.01 Added 0.002 0.015 0.023 0.032 0.042 0.032 0.035 (%) Table 1 (d) The samples for the determination of aggregates were diluted to a protein concentration of 1 mg / ml using PBS and aliquots of 50 μl or 100 μl injected in a size-exclusion-CLAP column TSK-GEL G3000SWXL. The column was developed with 0.05% NaN3 and 0.1 M Na2SO4 in 0.1 M phosphate buffer, pH 6.7 at a flow rate of 1.0 ml / min. The amount of the aggregate was determined by integrating the absorbance peaks at 280 nm and it was found that they remain around 1% completely.

Example 2 Concentration of Anti-CD4 Antibody - Method A The Minitan ultrafiltration equipment was joined and sterilized as in Example 1, except that 8 filter plates 30K NMWCO of polysulfone were used instead of 2, and the complete equipment was placed in a sterile bell. The anti-CD4 antibody (2142 ml at 13.9 mg / ml in 50 mM sodium citrate, pH 6.0) was circulated through the side of the retained product of the membranes at a flow rate of 190 ml / min at a backpressure of 2-2.5 bars. The back pressure was maintained at this value throughout the remainder of the experiment, and the flow rate of the impregnated was measured at various time intervals. The antibody samples were removed from the product retained from the container at various time points and tested for antibody concentration, CD4 binding, turbidity,% aggregate and viscosity. At the end of the experiment, the retained product was pumped out of the Minitan equipment and the side of the retained product from the membranes was flooded with 500 ml of 50 mM sodium citrate, 0.05 mM EDTA, pH 6.0 and 50 ml of fractions of the flood. They collected. Finally, the system was flooded by circulating 500 ml of 50 mM sodium citrate again, 0.05 M EDTA, pH 6.0 around the side of the product retained from the membrane for 30 minutes. The antibody concentration of the flood fractions was determined by measuring their absorbance at 280 nm. The results are shown in Tables 2 (a) - (d). The increase in the number of plates used for the concentration led to a decrease in the time taken to achieve a concentration of 250 μg / ml - 250 mg / ml at 6 h compared to 17.25 h for the concentration of Campath-IH (see Table 2 (a)). Table 2 (a) also shows that the viscosity of the Anti-CD4 antibody did not increase measurably until a concentration of 113 mg / ml was achieved. Above this concentration, the viscosity increased dramatically.

Time (h) 0 2 3.5 5 6 Conc. 13.9 47.2 83 112.8 252 mg / ml Viscosity 1 1 1 1 9.7 cps Table 2 (a) At concentrations above 83 mg / ml there was a noticeable opalescence in the concentrated material, and this caused a precipitate to form as the concentration increased above this value. This led to the decrease in impregnation flow rates shown in Table 2 (b) and also the rise in turbidity shown in Table 2 (c). The aggregate level remained very low at all concentrations, being less than 0.2% completely (see Table 2 (c)). Table 2 (c) shows that the recoveries were high until the viscosity increased and the precipitate occurred, where they fell dramatically to a final recovery in the product retained after the removal of the equipment of 50%.

Conc. 13.9 47.2 83 112.8 252 mg / ml Rec (%) 100.0 100.0 100.0 103.0 51.4 Flow 13.5 3.2 2.0 2.5 (ml / min) Table 2 (b) Conc. 13.9 47.2 83 112.8 252 mg / ml Turb. 0.011 0.012 0.030 0.185 A650nm Agr (%) 0.140 0.150 0.150 0.160 0.170 Table 2 (c) This poor recovery was due to the high viscosity of the concentrated Anti-CD4 antibody which makes it sticky to the tubing and membranes of the ultrafiltration system. All the loss of Anti-CD4 antibodies in this form could be recovered subsequently by flooding the system with the buffer. Table 2 (d) shows the recovery of the anti-CD4 antibody in washing fractions of 50 ml, successive during the flooding of the Minitan equipment at the end of the experiment. The first fraction contains 11.7 g of anti-CD4 antibody at a concentration of 235 mg / ml, so that it could be combined with 12.6 g of concentrate initially recovered from the equipment at 252 mg / ml without significantly diluting the total concentration. The remaining washing fractions contained a total of 5.1 g of anti-CD4 antibody, but this was at a concentration of less than 57 mg / ml, so that it could not be combined with the concentrated material. The total recovery in the concentrate and the first washing fraction was 90%. It was observed that after storage of the anti-CD4 antibody, concentrated, final, overnight at 4 ° C led to some redissolution of the precipitate. ml 50 100 150 200 250 300 350 400 450 500 mg 11727 2828 866 379 245 202 175 151 132 125 Table 2 (d) Therefore, an experiment was exposed to determine the concentration at which the Anti-CD4 antibodies, precipitates were completely redissolved. A 10 ml aliquot of 250 mg / ml Anti-CD4 antibody was progressively diluted by the addition of 50 mM sodium citrate, 0.05 mM EDTA, pH 6.0. The absorbance of the 1.0 ml aliquots, properly diluted, of antibody samples at 650 nm was used as a measure of turbidity. The results are shown in Table 3.

Conc. 237.7 149.5 110.4 88.6 76.3 60.3 mg / ml Turb. 1.17 0.096 0.082 0.074 0.03 0.027 (A650nm) Table 3 - - The precipitate was redissolved, but the turbidity and opalescence did not completely disappear until an anti-CD4 antibody concentration of approximately 80 mg / ml was achieved. It was above this concentration that the opalescence was first observed during the concentration, so that the precipitate appears to be reversible and concentration dependent.

Example 3 Anti-CD4 Antibody Concentration - Method B Anti-CD4 Antibody Buffer Adjustment The Anti-CD4 antibody (1460 ml, 24 g) was prepared in 50 mM sodium citrate, 0.05 mM EDTA, pH 6.0. This buffer was completed with sodium citrate ~ 100 mM, EDTA 0.05 mM, pH 6.0 by adding solid citric acid to the antibody preparation and adjusting the pH to 6.0 with NaOH. The resulting preparation was sterile, filtered through a 0.22 μm filter and stored as 2 aliquots of ~ 12 g.

Concentration of the Anti-CD4 antibody in Filtron Ultrasette The Filtron Mini-Ultrasette and the Watson-Marlo pump were placed in a cold environment. The Mini-Ultrasette (Cross flow ultrafilter, shutter, 30 K Filtron) was flooded with water, then sterilized during minutes with 0.1 M NaOH according to the instructions of the manufacturers. (Mini Ultrasette Tangential Flow Device Operating Instructions. & Mini Ultrasette Care and Use Manual., Filtron Technology Corporation). The sterilizing substance was removed by flooding with sterile water followed by 1-2 liters of sterile PBS, pH 7.2 until the pH of the effluent was 7.2. The anti-CD4 was circulated through the side of the retained product of the membranes at a flow rate of 250 ml / min completely. After concentrating the Anti-CD4 antibody at ~ 150 mg / ml, the retained product was pumped out of the Mini-Ultrasette and the side of the retained product from the membranes was flooded with 3 x 20 ml of 50 mM sodium citrate, 0.05 EDTA mM, pH 6.0 and each 20 ml fraction of the flood was collected. The concentration of antibodies to the flood fractions was determined by measuring their absorbance at 280 nm as described in Example 1. The results suggested that the reduction of the flow rate of the retained product can provide a method for the concentration of antigen. CD4 at ~150 mg / ml by transverse flow ultrafiltration and avoiding any precipitation. This was tested using the Filtron Mini-Ultrasette and a flow rate of the retained product of 250 ml / min. A suitable isotonic buffer for this work was 100 mM sodium citrate, 0.05 M EDTA, pH 6.0. Therefore the remaining 1460 ml of anti-CD4 in 50 mM sodium citrate, 0.05 mM EDTA, pH 6.0 was reformulated upon addition of 16.8 g of citric acid and the pH of the final solution was adjusted with NaOH. This material was sterile filtered and divided into 2 equal aliquots which were then separately concentrated in the Filtron ultrafiltration device using a recirculation rate of 250 ml / min. The results are shown in table 4.

Table 4: Concentration of Anti-CD4 at greater than 100 mg / ml in a Transverse Flow Ultrafiltration Cell at a Recirculation Rate of 250 ml / min Parameter Before Concentration Concentration Concentration i 2 Concentration - 169 156 Maximum Achieved (mg / ml) Concentration 14.4 106.4 100.5 Final Product (mg / ml) Recovery - 90 95 after concentration (%) Time taken - 11 for concentration (h) Aggregate (%) * 4.14 3.95 3.97 Turbidity 0.003 0.01 0.037 (A650nm) Osmolality 281 288 306 (mOs / kg) CD4 link 20 98.8 68.3 (mg / ml) * Analysis of the aggregate using the Size Exclusion CLAP Samples for the determination of the aggregate were diluted to a protein concentration of 1 mg / ml using PBS and aliquots of 50 μl or 100 μl injected into a size exclusion CLAP column TSK-GEL G3000SWXL. The column was developed with 0.05% NaN3 and 0.1 M Na2SO4 in 0.1 M phosphate buffer, pH 6.7 at a flow rate of 1.0 ml / min. The quantity of the aggregate was determined by integrating the absorbance peaks at 280 nm. Both concentrations achieved a maximum concentration of > 150 mg / ml in the ultrafiltration apparatus without deleterious effects on the solubility of the antibody. The concentrations took 9-11 hours. The final concentrations of ~ 100 mg / ml were a result of the dilution with the washings required to maximize the recovery of the ultrafiltration apparatus. Total recoveries were 90-95% and no visible precipitate or increase in aggregate levels was observed. The slight elevation in turbidity after concentration as measured by the absorbance at 650 nm caused a slight opacity of the final concentrate, but this was removed in the formulation with Polysorbate 80 and sterile filtration and was not considered significant. The CD4 big activity for concentration 1 was almost 100 mg / ml as expected, but a much lower value was obtained for concentration 2. The final osmolality of the combined material of concentrations 1 and 2 was approximately 297 mOs / kg and the combination was a clean, clear solution that could easily pass through a sterile 0.2 μm filter.

Concentration of Anti-CD4 antibodies in Agitated Cells An aliquot of 330 ml of Anti-CD4 antibody (as above) was concentrated at 5 ° C in a stirred ultrafiltration cell, Amicon (equipped with Amicon YM30 membrane) at a final concentration of 170 mg / ml when applying a pressure of 1.5 bar using nitrogen gas. The anti-CD4 antibody in the ultrafiltration cell was sampled in intervals and the concentration was determined by measuring the absorbance at 280 nm and turbidity by measuring the absorbance at 650 nm. At the end of the experiment, the concentrated material was removed from the ultrafiltration cell and the sterile filtrate through a 0.22 μm filter. To overcome the high shear forces generated in the cross-flow ultrafiltration apparatus, Filtron, the concentration was carried out in a stirred ultrafiltration cell using 50 mM sodium citrate, 0.05 M EDTA, pH 6.0 as the buffer. Table 5 shows the results of this experiment.

- - Table 5: Concentration by Ultrafiltration of the Anti-CD4 in an Amicon Agitated Cell Time (Volume of Concentration Flow velocity h) Product approximate of that of the retentate (ml) retained product Ultrafiltration (mg / ml) (ml / h) 0 330 16.7 - 6 150 37 30 9 100 55 17 11 75 73 12 14 46 120 - 10 38 40 134 0.25 54 27 171 * 0.81 * The actual concentration determined by measuring the absorbance at 280 nm. In total, the concentration took 2.5 days, and the flow rates declined rapidly as the viscosity of the concentrated antibody increased. A final concentration of 171 mg / ml was achieved successfully without evidence of precipitation. This material was removed from the ultrafiltration cell and the membrane was washed with sufficient 50 mM sodium citrate, 0.05 mM EDTA, pH 6.0 to give a final concentration of 100 mg / ml when combined with the concentrate. This material passed easily through a sterile 0.2 μm filter. The actual measured concentration of this combined material was 94.3 mg / ml in a volume of 46 ml. This corresponded to a recovery through the ultrafiltration step of 79%. Therefore, this experiment provided evidence that 50 mM sodium citrate, 0.05 mM EDTA, pH 6.0 was a suitable buffer for anti-CD4 concentration at at least 171 mg / ml, and that it was probably high cutting forces which were causing precipitation in the original cross-flow ultrafiltration experiments, in both the Minitan and the Filtron Mini-Ultrasette observed above.

Example 4 Subcutaneous formulations for anti-CD4 and anti-CD23 antibodies a) Antibody Anti-CD4 or Anti-CD23 0. 15 g Potassium diacid orthophosphate, KH2P? 4 0. 0656 g (anhydrous) Disodium acid orthophosphate, Na2HP04. 12H20 0. 0673 g NaCl 0. 6263 g - - Polysorbate 80 (weight% of the total formulation) 0.01 Water at lOOg b) Antibody Anti-CD4 or Anti-CD23 0.15 g Acetate of Na 3.674 g Glacial acetic acid, 10% solution 0.315 g NaCl 0.630g Polysorbate 80 (% of the weight of the total formulation) 0.01 Water at 100 g c) Antibody Anti-CD4 or Anti-CD23 0.15 g Maleic acid 0.227 g NaOH 0.5M 6.09 g NaCl 0.777 g Polysorbate 80 (% of the weight of the total formulation) 0.01 Water at lOOg d) Antibody Anti-CD4 or Anti-CD23 0.15 g Succinic acid 0.203 g NaOH 0.5M 6.54 g NaCl 0.779 g - - Polysorbate 80 (% of the weight of the total formulation) 0.01 Water at lOOg NB Each of formulations a) b) c) or d) may optionally contain 0.05 mM EDTA.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (16)

- - CLAIMS

1. A preparation of monoclonal antibodies for administration to a human, characterized in that the antibody in the preparation is in a concentration of 100 mg / ml or greater.

2. A preparation of monoclonal antibodies for administration to a human characterized in that it comprises the antibody in a concentration of 100 mg / ml or greater wherein the preparation is substantially free of aggregates.

3. An antibody preparation according to claim 1 or 2, characterized in that the antibody is present in a concentration greater than 100 mg / ml.

4. An antibody preparation according to any of the preceding claims, characterized in that the antibody is present in a concentration between 100 and 350 mg / ml.

5. An antibody preparation according to any of the preceding claims, characterized in that the antibody is of the IgG isotype.

6. An antibody preparation according to any of the preceding claims, characterized in that the antibody is a recombinant antibody.

7. An antibody preparation according to claim 6, characterized in that the antibody is an altered antibody.

8. An antibody preparation according to claim 7, characterized in that the antibody is a chimeric antibody or a CDR graft.

9. An antibody preparation according to any of the preceding claims, characterized in that it binds a cancer antigen to a T cell.

10. An antibody preparation according to any of the preceding claims, characterized in that the pH of the preparation is different from the pH of the isoelectric point of the antibody.

11. A pharmaceutical formulation, characterized in that it comprises a monoclonal antibody in a concentration of 100 mg / ml or greater and a pharmaceutically acceptable excipient.

12. A pharmaceutical formulation according to claim 11, characterized in that it is adapted for subcutaneous administration.

13. A preparation of monoclonal antibodies according to any of claims 1 to 10, characterized in that it is for use in the therapy of humans.

14. Use of a monoclonal antibody preparation wherein the antibody in the preparation is at a concentration of 100 mg / ml or greater in the preparation of a medicament for the treatment of disorders mediated by the T cell.

15. A process for the preparation of a monoclonal antibody preparation, characterized in that the antibody in the preparation is in a concentration of 100 mg / ml or greater by tangential flow ultrafiltration.

16. A preparation of monoclonal antibodies, characterized in that it comprises the antibody in a concentration of 100 mg / ml or greater that can be obtained by tangential flow ultrafiltration.