FIELD OF INVENTION

The present invention is related to pharmaceutical formulations of therapeutic monoclonal antibodies. Specifically, the invention is related to generation of a stable aqueous formulation for rituximab. This formulation can be subjected to lyophilization and reconstitution into an aqueous form with a suitable solvent.

BACKGROUND OF INVENTION

Unlike traditional organic or inorganic drugs, proteins are large and complex molecules. Protein drugs constitute an increasing proportion of medical therapeutics possessing multiple functional groups conferring complex secondary and tertiary structures. Hence, formulation of such complex molecules engenders special challenges. For a protein to remain biologically and therapeutically active, its formulation must preserve its intact conformation and functional integrity. Often protein preparations are unstable at very dilute, as well as at very high concentration, as the preparations tend to aggregate or degrade, resulting in formulations with shorter shelf life.

A protein formulation primarily consists of a buffer that maintains a suitable pH, along with excipients like salts, intended either for protein stability, or for isotonicity. Further, the solvent may include additional components like; lyoprotectants, or surfactants, or cryoprotectants. Further, certain lyoprotectants, at suitable concentration can also double as an isotonicity agent.

Further, other excipients include isotonicity agents such as salts like sodium chloride (NaC1), magnesium chloride (MgC12). Salts, such as, MgC12 can also act as a preservative. Chelating agents like ethylene-di-amine-tetra-acetate (EDTA), or surfactants like polysorbate may be included. Reducing and non- reducing sugars, such as the non reducing disaccharide trehalose, is often added as an excipient, and can acts as a lyoprotectant, and stabilizing agent.

Yet, despite the availability of apparently well characterized and standardized protein formulation excipients, the person skilled in art would readily appreciate that it is not possible to generalize the "optimized" composition of a protein formulation, including the protein concentration, excipient composition and concentration, solvent etc. for any protein, or class of proteins. Certain excipients may be more suitable for some proteins or class of proteins, while being unsuitable for others. Further, the choice of excipients can vary depending on the intended use of the formulation, mode of administration, storage conditions etc., as well as their potential to interact with the protein itself, other excipients or the container. Hence, protein formulations, their components, concentrations, etc, need to be optimized for each protein or class of proteins.

Several conditions and parameters need to be evaluated prior to formulating a protein, including: (a) the nature of the protein, pH, chemical and thermodynamic stability, (b) protein concentration, (c) exposed side chains residues of the protein, (d) intent and duration of use, , (e) the therapeutic use and intended mode of administration, (f) storage and shipping conditions etc.

Further protein solutions, intended for long term storage or robust handling conditions may need to be lyophilized. "Lyophilization" or "freeze-drying" is a process whereby the substance to be lyophilized, is first frozen and then the solvent removed by sublimation under vacuum leaving behind a dehydrated form of the product, this technique is frequently employed as a formulation technique. Similar to selecting protein solvents and their components, excipients for lyophilization need to be carefully chosen so that the protein of interest is unaffected by the freezing process, and its stability not compromised during storage and subsequent use. Pikal, M., Biopharm. 3(9)26-30(1990) and Arkawa et, al., Pharm. Res. 8(3):285-291 (1991). Thus, an optimal choice of buffer, excipients and technique of lyophilization can significantly enhance the stability, shelf life and cut storage and shipping costs, especially for therapeutic proteins, which otherwise demand cumbersome and expensive handling conditions.

Antibodies constitute one of the most important classes of therapeutic proteins, especially in the therapeutic areas of oncology, arthritis and other chronic diseases. Antibodies being, multimeric in nature, have a higher tendency to aggregate in solution.

Rituximab, also identified as C2B8, is a chimeric mouse/human monoclonal antibody bearing murine variable region directed against the human CD20 molecule (expressed by B cells and certain neoplastic cells), and human gamma 1 constant region. Further, it is classified as a type I anti CD20 antibody that kills its target cell by complement dependent cytotoxicity (CDC), it also exhibits considerable amount of antibody dependent cell mediated cytotoxicity (ADCC).

Rituximab is indicated for treatment of B-cell non-Hodgkin's lymphoma, rheumatoid arthritis and chronic lymphocytic leukemia and other autoimmune diseases. Given the importance of rituximab, there is a need for a stable formulation of the drug which is suitable for therapeutic use. There is also a need for a stable pharmaceutical formulation which can be easily administered, and contains a high antibody concentration.

So far anti-CD20 antibodies are largely available only as an aqueous formulation at a concentration of 10mg/ml in sodium citrate buffer pH 6.0, containing sodium chloride and polysorbate 80 (http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5312lbl. pelf). US6991790 discloses an aqueous formulation of anti-CD20 consisting of acetate buffer at a pH of 4.8-5.5 along with polyols, surfactant and lacking tonicifying amount of sodium chloride. WO2009009407 discloses an aqueous formulation wherein the formulation comprises sodium acetate, sodium chloride, argininefree base, EDTA, and polysorbate. US20100015157 claims a stable pharmaceutical formulation of a group of antibodies in histidine acetate buffer which is neither lyophilized and nor subjected to prior lyophilization.WO2009080541 claims a lyophilized formulation of a type II anti CD20 antibody (e.g. Tositumomab). However, there are no prior-art disclosing any formulation of rituximab that can be used as such and that can also be lyophilized. The present invention provides a stable therapeutically effective aqueous formulation of rituximab, that can be used as such, and that can also be lyophilized and reconstituted with a suitable solvent prior to administration.

SUMMARY OF INVENTION

The present invention provides (a) a stable aqueous formulation of rituximab comprising a therapeutically effective amount of the antibody, lyoprotectant, and surfactant in a suitable buffer, and that can further be subjected to lyophilization, and (b) the lyophilized formulation that can be reconstituted with an appropriate solvent prior to use, without affecting physiochemical properties of the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Cation exchange chromatography profile of rituximab before and after lyophilization. The inset shows indistinguishable superimposed profiles of the non-lyophilized formulation, and the formulation obtained by reconstitution of the lyophilized composition in both tubular and molded vials.

Figure 2: Comparison of the size exclusion profiles of the formulation obtained by reconstitution of the lyophilized composition in tubular and molded vials in comparison to non lyophilized formulation, exhibiting similar peaks.

Figure 3: Hydrophobic interaction chromatography profile of the formulation obtained by reconstitution of the lyophilized composition rituximab in molded or tubular glass vials shown, in comparison to non-lyophilized formulation. Significantly similar species distribution is observed in all the samples.

Figure 4: 4-12% gradient SDS-PAGE followed by silver staining, of rituximab formulation obtained by reconstitution of the lyophilized composition in molded or tubular vials against non-lyophilized rituximab of demonstrating uniformity of protein species in all samples.

Marker

Lane 1. Non-lyophilzied rituximab

Lane 2. Rituximab formulation obtained by reconstitution after lyophilization in tubular glass vials

Lane 3. Rituximab formulation obtained by reconstitution after lyophilization in molded glass vials

Figure 5: Thermal unfolding of rituximab; (A) non-lyophilized formulation, and (B) formulation obtained by reconstitution after lyophilization.

Figure 6: Second derivative FT-IR spectra of rituximab lyophilized in tubular or molded glass vial, followed by reconstitution, in comparison to non-lyophilized rituximab.

Figure 7: Far UV-CD spectra of rituximab lyophilized in tubular or molded glass vial followed by reconstitution, in comparison to non-lyophilized rituximab.

DETAILED DESCRIPTION OF THE INVENTION

CD20 is a pan-B cell marker that plays a significant role in the activation and differentiation of B cells across various mammalian species. This marker is expressed at very high levels on neoplastic B cells. Thus, CD20 serves as a suitable target for treatment of B-cell lymphomas. Anti CD20 antibody that specifically binds to the CD20 surface antigen is used in the treatment of patients with indications of B cell lymphoma (US 5736137, US5677180, BLA 103705, BLA 103737) and rheumatoid arthritis (EP1176981, EMEA/H/C/000165). Anti CD20 therapy has also been reported for treatment of a cohort of autoimmune conditions: such as systemic lupus erythematosus (SLE) (Sfikakis P. P., et. al., Curr Opin Rheumatol 2005;17(5):550-557), multiple sclerosis (Hauser S. L., et. al., N Engl J Med. 2008 Feb 14;358(7):676-88), idiopathic thrombocytopenic purpura (Braendstrup P., et. al., Am J Hematol 2005;78:275-80).

Based on the binding and biological properties of anti CD20 antibodies have been classified into two types; type I and type II, and can be distinguished according to Cregg, M.S., et al, Blood 103(2004) 2738-2743; and Cragg, M.S., et al, Blood 101 (2003) 1045-1051. Rituximab is an example of type I anti-CD20 antibody that has a binding affinity of about 8.0 nM, and that is a potent inducer of complement dependent cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC), whereas type II anti-CD20 antibodies are thought to induce caspase independent cell death to their targets.

Rituximab is indicated in the treatment of patients with relapsed or refractory, low-grade or follicular, CD20-positive, B-cell, non-Hodgkin's lymphoma. Rituximab in combination with methotrexate is indicated in the treatment of moderate to severely active rheumatoid arthritis (EP 1613350). Rituximab has been recently approved for treatment of patients suffering from chronic lymphocytic leukemia (CLL) (US 20100080769, EP 1616572, http://www.accessdata.fda.gov/druqsatfda docs/labe)/2010/103705s5312)b).p df, http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Procedural_steps_taken_and_scientific_information_after_authorisation/hu man/000165/WC500025820.pdf).

The present invention provides a stable and therapeutically active aqueous formulation of rituximab. This aqueous formulation can be subjected to lyophilization. In one embodiment, invention comprises a therapeutically effective amount of rituximab in a buffer at a pH of about 6.0.

In another embodiment, the invention relates to a formulation of rituximab in a buffer at a pH of about 6.0, comprising a lyoprotectant.

In yet another embodiment, the invention relates to a formulation comprising a therapeutically effective formulation of rituximab in a suitable buffer at a pH of about 6.0, comprising a lyoprotectant and a suitable surfactant.

In another embodiment, the stable therapeutically effective formulation of rituximab in a suitable buffer at a pH of about 6.0, comprises a lyoprotectant, suitable surfactant, and isotonicity agent.

In a preferred embodiment, the buffer is a histidine buffer at a pH of about 6.0, and the formulation further comprises a surfactant, and a non-reducing sugar as lyoprotectant.

In yet another preferred embodiment, the invention relates to a formulation comprising a therapeutically effective amount of rituximab in histidine buffer at a pH of about 6.0, trehalose, and polysorbate. In a preferred embodiment, the concentration of the antibody in the formulation is more than 10mg/ml to about 25mg/ml.

The rituximab formulation prepared in accordance to the invention can lyophilized for extended storage, and reconstituted in a suitable solvent just prior to administration.

The solvent of reconstitution may be sterile water for injection (WFI). The lyophilized rituximab may be reconstituted using water for injection to a final concentration of more than 10mg/ml_, or 15mg/ml, or 20mg/ml_, or 40mg/ml_ or 50mg/mL.
Lyophilization of the formulation prepared in accordance to the invention, does not affect the physiochemical properties of rituximab, as assessed by reconstituting the lyophilized composition in water for injection and subjecting the reconstituted formulation to various analytical techniques such as size exclusion chromatography (SEC), far UV-CD spectroscopy, ion exchange chromatography, hydrophobic interaction chromatography (HIC), SDS-PAGE, and Fourier transformed Infrared spectroscopy (FT-IR). The results of the analysis are shown figures 1 - 7, where the respective physico-chemical parameters of the non-lyophilized formulation is compared with that of the formulation obtained by reconstitution of the lyophilized composition (herein and henceforth referred to as simply "lyophilized formulation".)

The formulation prepared in accordance to the present invention, can be used for the treatment of patients suffering from CD20 or B cell related disorders. B cell disorders comprising of, cancerous conditions like, chronic lymphocytic leukemia (CLL), B cell Non-Hodgkin's lymphoma or autoimmune conditions comprising rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis, or Sjojren's syndrome, or idiopathic thrombocytopenic purpura. The formulation prepared in accordance to the invention , may also be used in synergistic combination with other cancer or tumor treating agents such as cisplatin (Cis P), 5 Fluro-Uracil (5FU), Melphalan, mitomycin C, cytarabine, or other anti-proliferative drugs like protein kinase inhibitors, or other pro-apoptotic agents, or cell-cycle blockers.

The formulation prepared in accordance to the invention, may be used in conjugation to radiotherapy from an external or internal radioactive source, at the discretion of the physician.

Likewise the formulation prepared in accordance to the invention, may also be used in conjugation of radiotherapy from an external or internal radioactive source, along with other chemotherapeutic, anti-inflammatory, or anti-proliferative therapy at the discretion of the physician

The formulation prepared in accordance to the invention, may be used in combination with other biopharmaceuticals at their respective therapeutic dosage and, dose regimen, such as cytokines like IFNa, IL18, and other therapeutic antibodies such as anti IL6, anti IL18, anti TNFa.

The formulation prepared in accordance to the invention, may also be introduced for therapeutic purposes into a suitable patient by any parental route such as intra-venous (i.v), intra-peritoneal (i.p), subcutaneous (s.c) or any other technique known to the pharmaceutical art.

Definitions: The term "CD20" and 'CD20 antigen" are used interchangeably herein, and include any variants, isoforms and species homologues of human CD20 which are naturally expressed by cells or are expressed on cells transfected with the CD20 gene. Synonyms of CD20, as recognized in the art, include B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5. The term "anti-CD20 antibody" according to the invention is an antibody that binds specifically to CD20 antigen.

Example 1

Antibody Preparation

Rituximab, produced in Chinese Hamster Ovary cells. The cells expressing rituximab were expanded from the master cell bank with at least three stages of spinners and one stage of seed reactor, prior to inoculation in a production reactor.

For efficient cell growth and viability, the cells where cultured in PF-CHO media at the spinner stage. Initial inoculum of 0.2 million cells/mL was used at the spinner flask stage. The spinner bottles were incubated at 37°C and the pH was maintained at 7.0. When an integral viable cells count of ^ 8 million cells/mL was attained, feed addition (HyQ 15.4 feed) coupled with incubation temperature shift from 37°C to 31°C was done. The culture was harvested after a period of 12 days, and the supernatant containing the crude cell extract was collected.

This supernatant was clarified, and the cell culture broth containing the desired antibody was purified by Protein A affinity chromatography, followed by two Ion exchange (Anion and Cation) chromatography steps. The purified antibody was concentrated by Tangential Flow Filtration (TFF) using 30kDa MWCO membrane at a protein concentration of > 12.5mg/mL containing 5mM histidine, trehalose dehydrate and polysorbate 20 to generate the formulated drug substance (FDS).

Example 2

Aqueous antibody formulation

The antibody was formulated to contain from 12.5mg/mL, up to 25mg/ml rituximab, 60mM trehalose, 0.01% polysorbate 20, and 5mM histidine, pH 6.0 (sterile filtration by 0.22um filter). Aqueous formulated solution was filled into depyrogenated, sterile 15ml and 50ml tube glass vials (20mm neck finish) to a final strengths of 150mg per vial respectively, with partial stoppered. The aqueous formulation was lyophilized in MiniFast 10, BOC MBE-6060 freeze dryer. It may be possible to formulate the antibody up to higher strength such as up to 200mg, 250 mg or 300mg or 400mg or 450mg per vial and subjected to lyophilization.

Example 3

Lyophilization of the antibody

The lyophilization cycle was performed in two types of vials, 1) molded finish glass and 2) tubular glass vials, wherein the sample was loaded at a shelf temperature of +10°C. Subsequently the vials were cooled to -50°C, where the shelf temperature was ramped over 2.33h (~24°C/h) and held for 5h before proceeding with primary drying. After the chamber was evacuated to the pressure set point of 150u bar, lyophilization shelf was warmed to the temperature set point of -10°C over a period of 1h. Primary drying was done by ice sublimation for either 20h or 40h for 15ml or 50ml vials respectively, with constant temperature and pressure set points (Table 1), wherein the chamber pressure was regulated with nitrogen gas. Following primary drying, secondary drying was performed to remove the residual moisture at a set point of +15°C where the shelf temperature was raised from -10°C to +15°C over a period of 1h and held for 15h (Table 1).

The chamber pressure was maintained constant at 150u bar throughout primary and secondary drying. Vials where then capped under partial vacuum (750u bar) under nitrogen environment.

Table 1

#20hr hold time Tor 15ml vials/*40hr hold time required tor 50ml vial

Example 4

Reconstitution and analysis of lyophilized antibody

The type I anti-CD20 antibody formulation lyophilized by the above mentioned process in moulded finish glass vials and tubular glass vials were reconstituted into an aqueous form, using water for injection (WFI) to a final concentration of up to 22.59 mg/mL (Table 2). Reconstitution of the lyophilate up to higher concentration of rituximab such as 40mg/ml, 60mg/ml, 100mg/ml, 150mg/ml or more than 200mg/ml may be possible. Various parameters like cake appearance and reconstitution time was analyzed for this formulation. While post reconstitution parameters including, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, Fourier transformed infrared spectroscopy, UV CD spectra, and differential scanning calorimetry was performed in comparison to the non-lyophilized aqueous counterpart of rituximab. Reconstitution parameters seem to be indistinguishable between the molded or tubular glass vials (Table 2).
Table 2

Ion exchange chromatography of lyophilized rituximab showed no difference from that of the non-lyophilized product (Figure 1), exhibiting similar charge distribution pre and post lyophilization. The superimposed CEX profile (Figure 1) substantiates the conclusion. Further the vial type used for lyophilization did not seem to alter the quality of the final product.

Size exclusion high performance liquid chromatography (SE-HPLC) of lyophilized and non-lyophilized rituximab formulation exhibited no significant differences in terms of species distribution and lyophilization did not cause any aggregation or degradation in rituximab (Figure 2).

Hydrophobic interaction chromatography (Figure 3) and SDS-PAGE (Figure 4) also demonstrated significant amount of similarity between lyophilized and the non-lyophilized rituximab. Thermodynamic stability was of the lyophilized product was ascertained in comparison with the non-lyophilized product by differential scanning calorimetry (DSC) (Figure 5). The reconstituted rituximab was diluted to 5mg/ml in formulation buffer, and the temperature was raised from 20°C to 90°C with an increment of 1°C/min. Resulting thermogram was analyzed using MN2 state curve fitting algorithm to estimate the transition temperature (Tm). Lyophilized and the non-lyophilized rituximab showed similar Tm, indicating similar thermodynamic stability between lyophilized and non-lyophilized rituximab (Figure 5).

Conformational stability of the lyophilized rituximab Vs non-lyophilized rituximab was also investigated. Fourier Transformed Infrared spectroscopy (FTIR) (Figure6) and UV CD spectroscopy (Figure 7) were employed. ATR-FT-IR was performed using 21mg/mL of liquid reconstituted rituximab in comparison with non-lyophilized sample. Second derivative spectrum was obtained by performing second derivative operation on the raw data with 29 points using IR solution software. No smoothing function and baseline correction was applied on the raw data as well as on derivative spectra. Second derivative of amide I FT-IR spectra indicated change in the 0-sheet content of rituximab before and after lyophilization, while rest of the secondary structural elements remained constant (Figure 6). This was probed further by Far-UV CD spectroscopy. Lyophilized and non-lyophilized rituximab demonstrated very similar CD spectra (Figure 7).

We claim:

1. An aqueous rituximab formulation comprising antibody, a buffer, a lyoprotectant, wherein the lyoprotectant is trehalose and the antibody concentration is more than 10 mg/ml.

2. The formulation of claim 1 wherein the buffer is an amino acid buffer at a pH of about 6.0.

3 The formulation of claim 1 additionally comprises a surfactant.

4. A lyophilized rituximab formulation comprising therapeutically effective amount of the antibody, at least one lyoprotectant, a buffer, wherein the lyoprotectant is trehalose.

5 An aqueous formulation obtained by reconstituting the formulation of claim 4, wherein the formulation is reconstituted to a final concentration of more than 10 mg/ml of rituximab.

6 The formulation of claim 4 additionally comprises a surfactant.

7 The formulation of claim 5, wherein the formulation is reconstituted in less than about 2 minutes.

8. A formulation wherein the thermodynamic stability of rituximab as determined by differential scanning calorimetry is similar between the aqueous formulation of claim 1 and the aqueous formulation of claim 5.