WO2012010799A1

WO2012010799A1 - Formulation of anti-cd20 antibodies

Info

Publication number: WO2012010799A1
Application number: PCT/FR2011/051741
Authority: WO
Inventors: Laetitia Cohen-Tannoudji; Sylvain Huille
Original assignee: Lfb-Biotechnologies
Priority date: 2010-07-20
Filing date: 2011-07-19
Publication date: 2012-01-26
Also published as: AR082283A1; FR2962908A1

Abstract

The application relates to a pharmaceutical composition comprising an anti-CD20 antibody, a detergent such as polysorbate, a buffer and a nonionic osmolality agent, such as mannitol or glycine. The composition has a pH between 5.0 and 7.0.

Description

Anti-CD20 antibody formulation

The invention relates to a stable pharmaceutical formulation of anti-CD20 antibodies.

The CD20 antigen is a transmembrane protein present on pre-B lymphocytes as well as on mature B cells. The CD20 antigen is present on the surface of more than 90% of peripheral B lymphocytes and lymphoid organs. It is found on normal and tumor B lymphocytes. More particularly, the CD20 antigen is expressed on more than 90% of non-Hodgkin's lymphoma cells and is absent from hematopoietic stem cells, B-lymphocytes, plasma cells or other healthy tissues.

The role of CD20 in the proliferation and / or differentiation of B lymphocytes is poorly known, however it represents an interesting target of targeted therapy for controlling and killing B cells involved in cancers or autoimmune diseases.

IDEC Pharmaceuticals Corporation, U.S has developed a chimeric anti-CD20 monoclonal antibody designated Rituximab and marketed as Rituxan® or MabThera®. This antibody is now conventionally used in the treatment of non-Hodgkin's lymphomas and has many applications in the treatment of B cell-mediated immunological diseases, such as for example malignant tumors, such as chronic lymphocytic leukemia, autoimmune diseases, Immunes involving an antibody such as autoimmune haemolytic anemia and idiopathic thrombocytopenic purpura and inflammatory diseases such as chronic rheumatoid arthritis or multiple sclerosis.

Several anti-CD20 antibody formulations have been described (WO 2009/080541, WO 2005/044859, WO 98/56418). The application WO 2009/080541 describes in particular a formulation containing an anti-CD20 antibody, histidine, threalose and polysorbate 20. The commercial formulation of Rituxan® contains an anti-CD20 antibody, a citrate buffer, sodium chloride and polysorbate 80. According to the package leaflet which summarizes the characteristics of the product (RCP), the formulation of Rituxan® is stable for 30 months stored in the refrigerator at 2-8 ° C and protected from light. Furthermore, after dilution in 0.9% NaCl solution or 5% glucose solution, the ready-to-infuse Rituxan® solution remains physically and chemically stable for 24 hours at 2-8 ° C and for 12 hours at 15 ° C. 25 ° C (Professional Information from the Swiss Compendium of Medicines® MabThera® Roche, 2009). In particular, the formulation of Rituxan® remains sensitive to stress of temperature, agitation and oxidation which can cause an early degradation of the product. It would therefore be advantageous to have a formulation containing an anti-CD20 antibody having better stability characteristics, making its use less restrictive, in particular by increasing the storage temperature at 25 ° C instead of 2-8 ° C for the commercial formulation Rituxan®.

SUMMARY OF THE INVENTION The invention provides a liquid pharmaceutical composition, comprising an anti-CD20 antibody, a detergent, which is preferably a polysorbate, a buffer and a nonionic osmolality agent, which is preferably mannitol or glycine. The composition in liquid form has a pH of between 5.0 and 7.0.

Advantageously, the composition of the invention is free of sodium chloride. Detailed description of the invention

The pharmaceutical composition of the invention provides anti-CD20 antibody compositions having chemical and physical stability at room temperature greater than the currently marketed anti-CD20 antibody composition (Rituxan®).

In particular, the novel pharmaceutical composition in liquid form of the invention better withstands the stress conditions, in particular the temperature and oxidation stress conditions, than the reference composition (Rituxan®).

The composition is less sensitive to the degradation phenomenon of the anti-CD20 antibody and thus benefits from a less restrictive use for the users.

The term "pharmaceutical composition" refers to preparations allowing the biological activity of the active ingredients and containing no additional toxic components for the subjects to which the composition is administered. The term "anti-CD20 antibody" includes all forms of antibodies specifically binding to the CD20 antigen. This includes but is not limited to human antibodies, humanized antibodies, genetically engineered antibodies such as monoclonal antibodies, chimeric antibodies, recombinant antibodies, transgenic antibodies as well as fragments of such antibodies as long as the specific binding properties of anti-CD20 antibodies are present. According to a particular embodiment, the anti-CD20 antibody used is the EMAB603 antibody as described in the patent application WO2006 / 064121. This antibody is produced by the clone R603 deposited on November 29, 2005 under the registration number CNCM 1-3529 to the National Collection of Cultures of Microorganisms (CNCM, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15).

The term "antibody fragments" includes a portion of an entire antibody, generally at least the antigen-binding portion or variable region thereof. Examples of antibody fragments include diabodies, single chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used in the above definition refers to an antibody obtained from a substantially homogeneous antibody population, ie, each of the antibodies in the population are identical, they may have natural mutations present in amount minor. Monoclonal antibodies are highly specific because they are directed against a single antigenic site. In addition, unlike polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant. The term "monoclonal" used herein indicates the character of the antibody to be obtained from a substantially homogeneous antibody population and does not define the method of producing the antibody. Thus, the defined antibody can be produced by any method described in the prior art.

The term "chimeric antibody" refers to a monoclonal antibody comprising a variable region, ie, a binding region, a source or species, and at least a portion of a constant region derived from a source or a a different species, generally prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. "Transgenic antibodies" are monoclonal antibodies produced from transgenic animals. Applications WO1995017085 and WO2007048077 describe in particular transgenic antibodies and methods for preparing transgenic antibodies.

"Humanized antibodies" are chimeric antibodies containing a minimal sequence derived from non-human immunoglobulin. The humanized antibodies are, in large part, human immunoglobulins in which hypervariable region residues are replaced by hypervariable region residues of immunoglobulins of nonhuman species such as mice, rats, rabbits or nonhuman primates with specificity. , a desired affinity and capacity. The "hypervariable regions" mentioned above refer to the amino acid residues of an antibody that are responsible for the binding of the antibody-antigen.

The term "human antibody" includes antibodies having variable regions and constant regions derived from immunoglobulin sequences of human germ lines.

The term "recombinant human antibody" includes all human antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from host cells such as NSO, CHO or YB2 / 0 cells or from an animal ( for example a transgenic mouse for human immunoglobulin genes or antibodies expressed by means of a recombinant expression vector transfected into a host cell. Such recombinant antibodies have variable regions and constant regions derived from human germline immunoglobulin sequences in a rearranged form. Human recombinant antibodies may be subject to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions, although derived or related to VH and VL sequences of human germ lines, may not exist in the repertoire of human antibody germline in vivo. The term "specific binding" previously used refers to an antibody specifically binding the CD20 antigen. Preferably, the binding affinity has a KD value of 10 ^{~ 9} mol / L or less (preferably 10 ^{~ 10} mol / L), preferentially with a KD value of 10 ^{~ 10} mol / L or less (preferably 10 ^{~ 12} mol / L). Binding affinity is determined with a standard binding assay, such as surface plasmon resonance (Biacore®). The term "CD20" or "CD20 antigen" refers to all variants, isoforms and homologues of human CD20 species naturally expressed by cells or expressed by cells transfected with the CD20 gene. Binding an antibody of the invention to the CD20 antigen results in the death of CD20 expressing cells (such as a tumor cell) by inactivating CD20.

The term "stable composition" here means that the formation of aggregates (insoluble or soluble) is minimized, and / or that the chemical degradation is reduced, the pH is maintained and the conformation of the antibody is not substantially modified during the production or storage of the compositions of the invention so that the biological activity and stability of the antibody are maintained. Various analytical techniques measuring the stability of a protein are available in the prior art and are grouped in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, Pubs (1991) and Jones, A Adv. Drug Delivery Rev. 10: 29-90 (1993) for example. Stability can be measured at a given temperature for a given time.

The term "physical stability" of the anti-CD20 antibody refers to the reduction or absence of formation of insoluble or soluble aggregates of the dimeric, oligomeric or polymeric forms of the anti-CD20 antibody, reduction or absence of any structural denaturation of the molecule, as well as the reduction or absence of any precipitation of the molecule. Signs of aggregation, denaturation and / or precipitation are visually identifiable by the color and / or clarity of the solution, or measured by the absorbance at 400 nm, by membrane filtration 0.22 μιη, by measurement of dynamic light scattering by steric exclusion chromatography, SDS PAGE or microcalorimetry.

The term "chemical stability" refers to the reduction or absence of any chemical modification of the anti-CD20 antibody that may alter its biological activity during storage, in a liquid or solid formulation, under accelerated conditions. For example, the phenomena of hydrolysis, deamidation, and / or oxidation are avoided or delayed. The oxidation of sulfur-containing amino acids is limited. Chemical stability can be assessed by detecting and quantifying the chemically altered forms of the antibody. Chemical alterations can lead to changes in the size and charge of the antibody that can be evaluated, for example, by size exclusion chromatography, ion exchange, SDS PAGE or electrical isofocusing (IEF). The pharmaceutical composition of the invention comprises an anti-CD20 antibody, a polysorbate, a buffer and mannitol and / or glycine and has a pH of between 5.0 and 7.0.

Preferably the pH of the composition is maintained between 5.5 and 7.0, preferably between 6.0 and 7.0, more preferably between 6.2 and 6.7, more preferably at about 6.5.

In a preferred embodiment, the anti-CD20 antibody content in the composition of the invention may be as follows: between 5 and 20 g / l, preferably between 8 and 12 g / l, preferably between 9 and 11 g / L and preferably about 10 g / L.

The term "detergent" includes, in particular, Pluronic F68 and polysorbates, especially polyoxyethylene sorbitan monolaurate (synonymous with polysorbate 20, sold under the trademark Tween 20 ™) and polyoxyethylene sorbitan monooleate (synonymous with polysorbate 80, sold under the trademark Tween 80 ™). These detergents may be used at concentrations ranging from 70 to 1000 ppm, preferably at a concentration of 100 ppm to 800 ppm, preferably from 300 to 800 ppm, preferably from 500 to 800 ppm and even more preferably at a concentration of about 700 ppm. Preferably, polysorbate 80 is used.

The term "buffer" includes a pharmaceutically acceptable buffer. A pharmaceutically acceptable buffer includes, but is not limited to, histidine buffers or preferably a salt of an alkali or alkaline earth metal, or a transition metal. The salts are preferably in citrate, succinate, acetate or phosphate form. For example, trisodium citrate or sodium phosphate may be used. Preferably, the buffer used is a citrate buffer. The buffer may also, preferably, be a sodium phosphate buffer. Histidine may be added preferentially as an excipient in a lower proportion than the main buffer which is preferably trisodium citrate or sodium phosphate. The buffer concentrations may be between 1 and 100 mM, preferably between 5 mM and 50 mM and still preferably at about 25 mM. Regardless of the buffer used, the pH is adjusted to a value between 5.0 and 7.0 and preferably to about 6.5 by adjusting with a base or an acid known in the prior art or by using buffer component mixtures. adequate or both. The term "non-ionic osmolality agent" includes especially sugars such as polyols and sucrose, and nonionic amino acids at pH 5-7, such as proline, glycine, alanine, cysteine isoleucine, leucine, serine, valine, phenylalanine, methionine, asparagine, tyrosine and threonine. Preferably, the nonionic osmolality agent is mannitol or glycine.

Mannitol is preferably used in an amount of about 25 to 500 mM, preferably between 200 and 400 mM, preferably between 270 and 330 and more preferably about 300 mM.

Glycine is preferably used in an amount of about 25 to 500 mM, preferably between 200 and 400 mM, preferably between 200 and 330 and more preferably about 240 or 300 mM.

In a preferred embodiment, the pharmaceutical composition of the invention is free of sodium chloride.

Preferably the detergent (preferably polysorbate 80), the buffer (preferably citrate or phosphate) and the nonionic osmolality agent (preferably mannitol or glycine) are the only excipients of the formulation (which comprises also the antibody, and water).

In an exemplary embodiment, the composition may comprise:

an anti-CD20 antibody;

polysorbate 80;

- trisodium citrate;

mannitol.

In another exemplary embodiment, the composition may comprise:

an anti-CD20 antibody;

polysorbate 80;

trisodium citrate;

glycine.

In another example, the composition may comprise:

an anti-CD20 antibody; polysorbate 80;

sodium phosphate;

mannitol or glycine. More particularly, the composition may comprise:

8 to 12 g / L of anti-CD20 antibody;

100 to 1000 ppm of polysorbate 80;

1 to 100 mM trisodium citrate or 1 to 100 mM sodium phosphate;

25 to 500 mM mannitol or 25 to 500 mM glycine;

at a pH of 5.0 to 7.0.

Preferably, the pharmaceutical composition comprises:

10 g / L of anti-CD20 antibody;

700 ppm of polysorbate 80;

25 mM trisodium citrate or 25 mM sodium phosphate;

300 mM mannitol or 240 or 300 mM glycine;

at a pH of 6.5.

The concentrations are determined with respect to the compositions in liquid form, before drying, or after reconstitution in the form of an injectable preparation. The composition for pharmaceutical use does not comprise sodium chloride, an ingredient that is nevertheless used in the usual manner as an osmolality agent in pharmaceutical formulations comprising an anti-CD20 antibody.

The replacement of sodium chloride with a nonionic osmolality agent, preferably mannitol or glycine, offers several advantages. On the one hand, the marked increase in turbidity observed after heating in a formulation containing sodium chloride is avoided when it is replaced by mannitol or glycine, more advantageously with mannitol. The presence of mannitol or glycine in the anti-CD20 antibody solution prevents the aggregation of proteins. Mannitol and glycine improving the stability of the anti-CD20 antibody solution with respect to thermal phenomena. Moreover, glycine plays a protective role vis-à-vis the oxidation of the anti-CD20 antibody solution. Degradation is more marked with a solution containing sodium chloride. Thus, the anti-CD20 antibody solution formulated with glycine as the osmolality agent undergoes less degradation. Glycine improves the stability of the anti-CD20 antibody solution with respect to oxidation phenomena.

The composition of the invention can be obtained using any conventional technique. In particular, the composition of the invention can be obtained by implementing a process comprising mixing the anti-CD20 antibody with a buffer solution, adjusting the pH if necessary, filtration to obtain a liquid form,

The composition according to the invention may advantageously be subjected to a method for eliminating or inactivating the infectious agents, for example by acid pH or nanofiltration treatment.

The following figures and examples illustrate the invention without limiting its scope.

Legend of figures

Figure 1 is a photograph showing the appearance of solutions Fl, F2 and F3 after heating to 57 ° C. The influence of the osmolality agent on the turbidity of the anti-CD20 antibody solutions is observed.

Figure 2 shows thermograms showing the denaturation and enthalpy transition temperature values of anti-CD20 antibody solutions in citrate / NaCl, citrate / mannitol or citrate / glycine environment.

Figure 3 shows the monitoring of the optical density (OD) turbidity at 400nm, after stability of the formulations at 25 ° C.

Figure 4 shows the follow-up of the percentage of kappa / IgG chains, after stability of the formulations at 40 ° C.

Figure 5 represents the monitoring of the evolution of the fragment level, after stability of the formulations at 40 ° C. EXAMPLES

Example 1: Preparation of solutions and analyzes at T0

1.1 Preparation of solutions

Anti-CD20 LFB-R603 antibody (described in WO2006064121) at an approximate concentration of 10 g / L was formulated with 25 mM sodium citrate buffer pH 6.5, with 700 ppm Tween 80 and with an agent. ionic osmolality, namely NaCl (formulation F1) at 9 g / L or a neutral osmolality agent, namely mannitol at 300 mM (formulation F2) or glycine at 300 mM (formulation F3). These three formulations were then filtered on Milex Durapore 0.22 μιη. Then the osmolality and pH values were checked and grouped in Table 1.

Table 1: Characteristics of test solutions

1.2 Measurements of the exact concentration of anti-CD20 antibodies

The determination of the total anti-CD20 antibody concentration was performed for each formulation by measuring the absorbance at 280 nm. The molar extinction coefficient for the anti-CD20 antibody is 1.61 Lg ^ .com ^"1.

The total protein concentrations of the three solutions were measured and correspond to the expected values (F1: 9.50 g / L, F2: 9.35 g / L and F3: 9.42 g / L). he

1.3 Measurements of turbidity

The turbidity of the solutions was evaluated by measuring the OD at 400 nm, it is an indirect measure of the diffused intensity which makes it possible to follow the phenomena of macroscopic aggregation. The turbidity values obtained are low for the three formulations (Fl: 0.015, F2: 0.016 and F3: 0.015), which means a lack of aggregate.

1.4 DLS measurements (Dynamic Light Scattering)

The dynamic light scattering measurement makes it possible to measure the hydrodynamic rays of the proteins and aggregates present in solution. This measurement makes it possible to follow aggregation phenomena at early stages of formation, since the sizes of the detected objects range from nanometer or micron. The percentage of the main population, composed mainly of monomers, can be reported. These are non-quantitative readings, as the intensity of diffusion does not depend solely on the concentration of objects but also on their size. The results showed that solutions Fl, F2 and F3 are all free of even sub-micron aggregates at T0.

1.5 Conclusion

The set of analyzes at T0 confirms that the three solutions are equivalent with respect to the pH, the osmolality, the concentration and the initial state of aggregation. Example 2: Temperature Stress

2.1 Stress conditions

Thermal stress is used to accelerate the chemical and physical degradation phenomena and to compare the stability of the different formulations.

Thermal stress was achieved by heating in a water bath at 57 ° C. For each formulation, the stress was carried out on 1 mL of solution in a hermetically sealed hemolysis tube to limit evaporation for 1 hour (or 45 minutes). minutes) and 3 hours. 2.2 Visual Observations

Any macroscopic change in the solution, such as the appearance of microbubbles, the change in color, the appearance of filaments or particles, can be identified by visual observation. Regarding the tracking of aggregation phenomena, only objects larger than 50 μιη are detected visually.

After one hour of heating, a clear increase in turbidity was observed for solution F1. Solutions F2 and F3 are very slightly turbid. After 3 hours of heating, the trend is confirmed for the solution Fl which remains turbid. Solution F2 appears slightly more degraded than solution F3 (see Figure 1). 2.3 Measurement of turbidity

The values presented in Table 2 confirm the visual observations. The turbidity value makes it possible to establish a classification of the osmolality agents according to their impact on the stability of the solution. The formulation F2 is more stable than the formulation F3, itself much more stable than the formulation F1. Table 2: OD values at 400 nm - Measurement of the turbidity

2.4 Determination of the percentage of aggregated proteins

Materials and methods

The determination of the amount of aggregated proteins was carried out after membrane filtration 0.22 μιη of the heated solution. This step removes aggregates larger than 220 nm. A total protein assay performed in the filtrate was then performed and compared to T0. When heating at 57 ° C, an evaporation phenomenon occurs, this must be taken into account in the interpretation of the concentration obtained after heating and filtration. This phenomenon has the effect of underestimating the percentage of proteins approved. The value obtained takes into account the aggregated proteins that were retained on the membrane during filtration, but also the evaporation of the solution during stress and the amount of non-aggregated proteins on the membrane.

Results The protein concentration values obtained after 1 hour or 3h of heating are sometimes higher than at T0, which reveals a high evaporation during stress. If we assume that evaporation is the same for all solutions, it is possible to compare them with each other at a time T and not with respect to T0.

After 1 hour and 3 hours of heating, the presence of mannitol or glycine (F2 and F3 formulations) prevents the aggregation of proteins. The solution containing NaCl (Fl formulation) is, for its part, the most aggregated with nearly 33% of aggregated proteins (Table 3).

Table 3: Concentration of Anti-CD20 Antibody After Aggregate Filtration - Influence of the Osmolality Agent

Mannitol and glycine are found to be osmolality agents that promote stability of the anti-CD20 antibody solution under heat stress conditions.

Example 3 Oxidation Stress

3.1 Stress conditions

Oxidation stress makes it possible to measure the stability of a protein solution with respect to the degradation process represented by the oxidation. Thus, a strong oxidant, hydrogen peroxide, was added to the anti-CD20 antibody solutions at a concentration of 30 mM. The mixtures were incubated for 24 hours at 37 ° C. 3.2 SDS-PAGE analysis

Materials and methods

The SDS-PAGE electrophoresis technique separates proteins according to their molecular weight. It makes it possible to know if the stress carried out fragments the protein or leads to covalent aggregates. The analysis was carried out under reducing and non-reducing conditions with staining in Coomassie blue. Oxidized solutions were compared to T0 solutions. The amount of protein per well was set at 2 μg. The size marker used is Mark 12.

Results The analysis of the electrophoresis gels shows that:

At T0, the various solutions tested have an electrophoretic profile under non-reducing conditions, comprising a majority band at 150 kDa and two band groups at about 130 kDa and 110 kDa.

After 24 hours of oxidation, the appearance of bands at about 105, 75 and 45 kDa under non-reducing conditions and an intensification of the bands at 70 and 25 kDa approximately. The proportion of these bands is greater for oxidized anti-CD20 antibody solutions containing NaCl (F1) and mannitol (F3). Under reducing conditions, the solution containing glycine (F2) has an electrophoretic profile comparable to that observed at T0, whereas the solutions containing NaCl or mannitol (Fl and F3) show a significant increase in the proportion of the 125 kDa band (7%).

The SDS-PAGE analysis results show that glycine plays a protective role vis-à-vis oxidation with less marked degradation than for solutions containing NaCl or mannitol (Fl and F3).

3.3 HP-SEC Materials and Methods

Steric exclusion chromatography in the liquid phase makes it possible to separate the objects according to their hydrodynamic radius. The detection is carried out by measurement of OD at 280 nm. The integration of the UV chromatogram makes it possible to determine the percentages of monomers, dimers, polymers and fragments. Results

Table 4 presents the results. The proportions of each entity obtained for the samples at TO are comparable regardless of the osmolality agent considered. Under oxidation stress, degradation of the anti-CD20 antibody is observed in the presence of NaCl and mannitol (Fl and F3), corresponding to the appearance of new entities (peak 4 and peak 6) and an increase fragments. The F2 solution appears less degraded after oxidation, where only a slight increase in the level of fragments is observed.

Table 4: Results of HP-SEC for oxidized TO samples - Results of integration of UV chromato grams - Influence of the osmolality agent

The SDS-PAGE and HP-SEC results indicate that under oxidation stress, the anti-CD20 antibody solution formulated with glycine as the osmolality agent undergoes less degradation after 24 hours. at 37 ° C.

Example 4: Microcalorimetry

Materials and methods The microcalorimetry tests were carried out on the Microcal VP-DSC apparatus. The analysis consists in measuring the heat flux released during a controlled temperature sweep in the sample containing the protein compared to its formulation buffer. This heat flux reflects the changes of states of the protein and thus informs on its denaturation. Differences in thermogram profiles tell us about the relative thermal stability of the protein in different formulation environments. Results

The anti-CD20 antibody thermograms of the F2 and F3 solutions, shown in FIG. 2, show the same transitions indicating that the denaturation of the protein proceeds according to the same decoupled mechanism in 4 transitions. The total enthalpy necessary to completely denature the protein is equivalent for these two formulations (952 Kcal / mol for F2 vs 950 Kcal / mol for F3). The profile of the anti-CD20 antibody thermogram in citrate / NaCl formulation is significantly different with the appearance of an additional transition, a decrease in the denaturation start temperature and a decrease in the total enthalpy of denaturation (866 Kcal / mol). These elements mean that the anti-CD20 antibody in citrate / NaCl medium is less thermally stable in this formulation.

Example 5: Stability of the compositions

To study the stability of an anti-CD20 antibody formulation according to the invention at a concentration of 10 mg / ml, batches were placed, protected from light, in temperature-controlled enclosures at +5 ° C ± 3 ° C and + 25 ° C ± 2 ° C for a duration of 24 months (Table 5). Table 5: Formulations put in stability

The following parameters are evaluated: visual appearance, purity, molecular size distribution (monomers, polymers, dimers and fragments), isoform profile, pH, study of the possible absorption of Polysorbate 80 on the plug, the study of the possible migration of aluminum from the primary packaging, the "free" kappa chains and the total protein concentration. The functional activity of the antibody is evaluated over time. Functional activity is determined by known classical methods those skilled in the art, for example by measuring antibody-mediated cytotoxicity (ADCC), binding to the CD16 receptor.

The results show that batches of anti-CD20 antibody at 10 mg / ml are stable for 24 months at + 5 ° C ± 3 ° C. Lots formulated in glycine (lots 2 and 3) are, moreover, stable for 6 months at + 25 ° C ± 2 ° C.

Example 6: Comparative Analyzes

Stability comparisons were made between the following formulations (containing 10 mg / ml of anti-CD20 antibody R603):

Table 6: Compared Formulations, Stability

The batches were placed, protected from the light, in enclosures thermostated at 5 ° C, 25 ° C or 40 ° C.

The same parameters as those shown in Example 5 were evaluated.

Several parameters showed a marked improvement with the addition of the nonionic osmolality agent (glycine). This improvement is particularly significant in terms of aggregation, in particular revealed by a measurement of turbidity (Figure 3), percentage of "free" Kappa chains (Figure 4), and fragmentation rate (Figure 5). Overall, these data show that the nonionic osmolality agent (glycine) has a stabilizing effect compared to the Rituxan® type formulation allowing for storage at 25 ° C longer and allows the reduction in surfactant Tween 80.

Claims

A pharmaceutical liquid composition comprising:

at. an anti-CD20 antibody;

b. a detergent;

vs. a tampon ;

d. a nonionic osmolality agent;

and having a pH of 5.0 to 7.0.

2. Composition according to claim 1, characterized in that it does not comprise sodium chloride.

3. Composition according to one of claims 1 and 2, wherein the detergent is polysorbate, preferably polysorbate 80.

4. Composition according to one of claims 1 to 3, wherein the nonionic osmolality agent is mannitol or glycine.

The composition of any one of claims 1 to 4, wherein the buffer is a citrate or phosphate buffer.

6. Composition according to claim 5, characterized in that it comprises between 1 and 100 mM of trisodium citrate or sodium phosphate.

7. Composition according to any one of claims 1 to 6, characterized in that it comprises between 8 and 12 g / L of anti-CD20 antibody.

8. Composition according to any one of claims 1 to 7, characterized in that it comprises between 100 and 1000 ppm of polysorbate as a detergent.

9. Composition according to any one of claims 1 to 8, characterized in that it comprises between 25 and 500 mM of mannitol or glycine as nonionic osmolality agent.

The composition of any one of claims 1 to 9, comprising:

at. 10 g / L of anti-CD20 antibody;

b. 700 ppm of polysorbate 80;

vs. 25 mM trisodium citrate;

d. 300 mM mannitol;

and having a pH of 5.0 to 7.0.

11. Composition according to any one of claims 1 to 9, comprising:

at. 10 g / L of anti-CD20 antibody;

b. 700 ppm of polysorbate 80;

vs. 25 mM trisodium citrate;

d. 240 or 300 mM glycine;

and having a pH of 5.0 to 7.0.

The composition of any one of claims 1 to 11, wherein the pH is about 6.5.

13. Composition according to any one of claims 1 to 12, wherein the anti-CD20 antibody is a monoclonal antibody produced by the clone R603 filed November 29, 2005 under the registration number CNCM 1-3529 to the National Collection of Microorganism Cultures (CNCM, Institut Pasteur, 25 rue du

Dr. Roux, 75724 Paris Cedex 15).

14. A solid pharmaceutical composition obtainable by desiccating a liquid composition according to one of claims 1 to 13.