RU2848327C1 - Formulations based on anti-cd19 antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, specifically to compositions based on CD19 antibodies and use thereof for treating lymphoma or leukaemia, which express CD19. Liquid formulation contains an anti-CD19 antibody at concentration of 20 mg/ml to 125 mg/ml, a citrate buffer at concentration of 20 mm to 50 mm, a polysorbate at concentration of 0.005 % to 0.06 % (w/v), has pH 6.0, and also contains trehalose in concentration of 180 mM to 240 mM or mannitol in concentration of 180 mM to 240 mM and sucrose in concentration of 10 mM to 50 mM. Said anti-CD19 antibody contains variable regions of the heavy and light chains with SEQ ID NO: 10 and SEQ ID NO: 11, respectively, heavy chain constant domain SEQ ID NO: 8 and constant light chain domain with SEQ ID NO: 9. Liquid formulation can be produced by reconstituting a lyophilised pharmaceutical formulation containing the above ingredients.

EFFECT: inventions provide the thermal stability of the anti-CD19 antibody and enable to obtain stable based formulations.

41 cl, 2 dwg, 5 tbl, 3 ex

Description

Field of technology to which the invention relates

The present invention relates to stable lyophilized pharmaceutical formulations based on an antibody to CD19 and provides methods for producing and methods for using such formulations.

State of the art of the invention

B cells are lymphocytes that play a major role in the humoral immune response. They are produced in the bone marrow of most mammals and constitute 5-15% of the circulating lymphoid pool. The primary function of B cells is to produce antibodies to various antigens, and they are an integral part of the adaptive immune system.

Due to their critical role in regulating the immune system, B cell dysregulation is associated with various disorders, such as lymphomas and leukemias. These disorders include non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL).

The human CD19 molecule is a structurally distinct cell surface receptor expressed on the surface of human B cells, including but not limited to pre-B cells, early-stage B cells (i.e., immature B cells), mature B cells through terminal differentiation into plasma cells, and malignant B cells. CD19 is expressed in most cases of pre-B-cell acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, acute undifferentiated lymphocytic leukemia, and some null-phenotype acute lymphoblastic leukemia (Nadler et al, J. Immunol., 131:244–250 (1983), Loken et al, Blood, 70:1316–1324 (1987), Uckun et al, Blood, 71:13–29 (1988), Anderson et al, 1984. Blood, 63:1424–1433 (1984), Scheuermann, Leuk. Lymphoma, 18:385-397(1995)). Expression of CD19 on plasma cells further suggests that it may be expressed on differentiated B-cell tumors such as multiple myeloma, plasmacytomas, and Waldenstrom's tumors (Grossbard et al., Br. J. Haematol, 102:509-15(1998); Treon et al., Semin. Oncol, 30:248-52(2003)).

Therefore, the CD19 antigen represents a target for immunotherapy in the treatment of non-Hodgkin's lymphoma (including each of the subtypes described herein), chronic lymphocytic leukemia, and/or acute lymphoblastic leukemia.

MOR208 (previously named XmAb5574) is a genetically engineered humanized Fc monoclonal antibody that binds CD19. Increased MOR208 Fc binding to FcγR, resulting from genetically engineered XmAb mutations, significantly enhances in vitro antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and antibody-dependent cell-mediated phagocytosis (apoptosis) against tumors compared to the unmodified antibody. MOR208 has not been shown to mediate complement-dependent cytotoxicity.

MOR208 has been and is currently undergoing clinical trials in CLL, ALL, and NHL. Specifically, a Phase I trial entitled "Safety and Tolerability of XmAb®5574 in Chronic Lymphocytic Leukemia" and a Phase IIa trial entitled "A Study of an Fc-Optimized Antibody to CD19 (MOR208) for the Treatment of B-Cell Acute Lymphoblastic Leukemia (B-ALL)" have been completed. Recruitment for a Phase IIa trial entitled "A Study of an Fc-Optimized Antibody to CD19 (MOR208) for the Treatment of Non-Hodgkin's Lymphoma (NHL)" has been completed. The following trials are also ongoing: a phase II/III trial entitled "A Trial to Evaluate the Efficacy and Safety of MOR208 with Bendamustine (BEN) Compared to Rituximab (RTX) with BEN in Adult Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL) (B-MIND)", a phase II trial entitled "A Study to Evaluate the Efficacy and Safety of MOR208 with Idelalisib in Patients with R/R CLL/SLL Pre-Treated with BTKi", a phase II trial entitled "A Study to Evaluate the Safety and Efficacy of Lenalidomide with MOR208 in Patients with R-R DLBCL", and a phase II trial entitled "Phase II, MOR208 in Combination with Lenalidomide for Patients with Relapsed or Refractory CLL, SLL, or PLL, or Elderly Patients with Untreated CLL, SLL, or PLL".

Therapeutic antibodies and antibody fragments are larger and more complex molecules than small molecules of traditional organic and inorganic drugs because antibodies contain multiple functional groups in addition to complex three-dimensional structures, and therefore, the formulation of such proteins poses unique challenges. For a protein to remain biologically active, the formulation must maintain the conformational integrity of at least the core amino acid sequence of the protein while protecting multiple functional groups of the protein from degradation. Antibody-based formulations can have short shelf lives, and formulated antibodies can lose biological activity as a result of chemical and physical instability during storage. The three most common protein degradation pathways are aggregation, deamidation, and oxidation (Cleland et al., Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307–377 (1993)). In particular, aggregation can potentially lead to an enhanced immune response in patients, leading to safety concerns and must be minimized or prevented.

Brief description of the invention

The aim of the present invention is to provide anti-CD19 antibody formulations, and in particular anti-CD19 antibody formulations, characterized by a suitable shelf life.

Suitable formulations for therapeutic antibodies may be an aqueous pharmaceutical composition or a lyophilisate that can be reconstituted to form a solution for administration to a patient.

Provided herein are lyophilized pharmaceutical formulations comprising an antibody. In one aspect, the formulation comprises an anti-CD19 antibody, a buffer, sucrose, and a surfactant, wherein the formulation is characterized by a pH of approximately 6.0, wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In one aspect, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of from about 20 mg/mL to about 125 mg/mL, a buffer, polysorbate at a concentration of from about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of from about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In some embodiments, said anti-CD19 antibody comprises an HCDR1 region comprising the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region with the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region with the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, said anti-CD19 antibody comprises a heavy chain variable region with the sequence and a variable region of the light chain with the sequence

In some embodiments, said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, the anti-CD19 antibody in the formulation has a concentration of from about 20 mg/mL to about 80 mg/mL. In some embodiments, the anti-CD19 antibody in the formulation has a concentration of about 40 mg/mL.

In some embodiments, said buffer in the composition is a citrate buffer or a phosphate buffer.

In some embodiments, said trehalose in the composition is characterized by a concentration of approximately 200 mM. In some embodiments, said trehalose in the composition is characterized by a concentration of 200 mM.

In some embodiments, said mannitol in the composition is characterized by a concentration of approximately 219 mM, and sucrose in the composition is characterized by a concentration of approximately 29 mM. In some embodiments, said mannitol in the composition is characterized by a concentration of 219 mM, and sucrose in the composition is characterized by a concentration of 29 mM.

In some embodiments, the composition has a pH of about 6.0. In some embodiments, the composition has a pH of 6.0.

In some embodiments, the polysorbate in the formulation is polysorbate 20. In some embodiments, the polysorbate (e.g., polysorbate 20) in the formulation has a concentration of from about 0.005% (w/v) to about 0.06% (w/v). In some embodiments, the polysorbate (e.g., polysorbate 20) in the formulation has a concentration of about 0.02% (w/v). In some embodiments, the polysorbate (e.g., polysorbate 20) in the formulation has a concentration of 0.02% (w/v).

In a further aspect, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, citrate at a concentration of about 25 mM, trehalose at a concentration of about 200 mM, polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0.

In a further aspect, the document provides a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate at a concentration of 25 mM, trehalose at a concentration of 200 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of approximately 6.0.

In a further aspect, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, citrate at a concentration of about 25 mM, mannitol at a concentration of about 219 mM, sucrose at a concentration of about 29 mM, polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0.

In a further aspect, the present document provides a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate at a concentration of 25 mM, mannitol at a concentration of 219 mM, sucrose at a concentration of 29 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of approximately 6.0.

In some embodiments, said anti-CD19 antibody in the formulation is not pre-lyophilized, for example, it is liquid. In some embodiments, said anti-CD19 antibody in the formulation is a monoclonal antibody. In some embodiments, said anti-CD19 antibody in the formulation is a full-length antibody. In some embodiments, said anti-CD19 antibody in the formulation is an IgG antibody. In some embodiments, said anti-CD19 antibody in the formulation is a humanized antibody or a human antibody. In some embodiments, said anti-CD19 antibody in the formulation is an antibody fragment comprising an antigen-binding region. In some embodiments, the antibody fragment is a Fab fragment or an F(ab')2 fragment.

Description of graphic materials

Figure 1: The amino acid sequences of MOR208 are shown.

Figure 2: Subvisible particles (SVP) of MOR208 were counted after 3 months at 40°C. Formulation 3 was compared with Formulation 9, and the mannitol/sucrose formulation produced more particles over time, especially in the range between 2 µm and 1000 µm.

Detailed description of the invention

The term "antibody" refers to monoclonal antibodies, including any isotype, such as IgG, IgM, IgA, IgD, and IgE. An IgG antibody consists of two identical heavy chains and two identical light chains, which are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called complementarity-determining regions (CDRs) or hypervariable regions, which are primarily responsible for binding to the antigen epitope. They are referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more conserved portions of the variable regions outside the CDRs are called framework regions. The term "antibody fragment" means a fragment of Fv, scFv, dsFv, Fab, Fab', F(ab')2 or other fragment that contains at least one heavy chain variable region or light chain variable region, each containing CDRs and framework regions.

"VH" refers to the variable region of the immunoglobulin heavy chain of an antibody or antibody fragment. "VL" refers to the variable region of the immunoglobulin light chain of an antibody or antibody fragment.

The term "CD19" refers to a protein known as CD19, which has the following synonyms: B4, B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, CV1D3, differentiation antigen CD19, MGC12802, and T-cell surface antigen Leu-12.

Amino acid sequence of human CD19:

"MOR208" is an anti-CD19 antibody. The amino acid sequence of the variable domains is shown in Figure 1. The amino acid sequence of the Fc regions of the heavy and light chains of MOR208 is shown in Figure 1. "MOR208" and "XmAb 5574" are used synonymously to describe the antibody shown in Figure 1. The MOR208 antibody is described in U.S. Patent Application Ser. No. 12/377,251, which is incorporated by reference in its entirety.

Additional antibodies specific for CD19 are described in U.S. Patent No. 7,109,304 (Immunomedics), which is incorporated herein by reference in its entirety; U.S. Patent Application Ser. No. 11/917,750 (Medarex), which is incorporated herein by reference in its entirety; U.S. Patent Application Ser. No. 11/852,106 (Medimmune), which is incorporated herein by reference in its entirety; U.S. Patent Application Ser. No. 11/648,505 (Merck Patent GmbH), which is incorporated herein by reference in its entirety; U.S. Patent No. 7,968,687 (Seattle Genetics), which is incorporated herein by reference in its entirety; and U.S. Patent Application Ser. No. 12/710,442 (Glenmark Pharmaceuticals), which is incorporated herein by reference in its entirety.

In addition, additional antibodies specific for CD19 are described in WO2005012493 (US 7109304), WO2010053716 (US 12/266999) (Immunomedics); WO2007002223 (US US 8097703) (Medarex); WO2008022152 (12/377251) and WO2008150494 (Xencor), WO2008031056 (US 11/852106) (Medimmune); WO2007076950 (US 11/648505) (Merck Patent GmbH); WO 2009/052431 (US 12/253895) (Seattle Genetics) and WO 2010095031 (12/710442) (Glenmark Pharmaceuticals), WO 2012010562 and WO 2012010561 (International Drug Development), WO 2011147834 (Roche Glycart) and WO 2012/156455 (Sanofi), all of which are incorporated by reference in their entireties.

The term "pharmaceutical formulation" refers to a drug intended for administration to subjects. Such subjects may include humans.

A "stable" formulation is one that can be administered to patients after storage. In aspects, the formulation substantially retains its physical and chemical properties, as well as its biological activity, during storage. Various analytical methods for measuring protein stability are available in the art and are discussed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).

Stability can be assessed qualitatively and/or quantitatively in a variety of ways, including assessment of aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); assessment of charge heterogeneity using ion exchange chromatography (IEC), size exclusion chromatography (HP-SEC), SDS-PAGE analysis to compare reduced and intact antibody; assessment of the biological activity or antigen-binding function of the antibody, etc. Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), truncation/hydrolysis/fragmentation (e.g., hinge fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

As used herein, the term "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen. This may additionally include binding of the antibody to the antigen and may result in a measurable biological response that can be measured in vitro or in vivo.

As used herein, the term "buffer" refers to a buffered solution that resists changes in pH due to the combined action of its acidic and basic components. The buffer of the present invention preferably has a pH in the range of about 4.5 to about 7.0, preferably from about 5.6 to about 7.0. In one embodiment, the buffer has a pH of about 6.0, or the pH is 6.0. For example, citrate buffer and phosphate buffer are examples of buffers that will control the pH in this range.

As used herein, the term "surfactant" refers to a surface-active agent. Preferably, the surfactant is a non-ionic surfactant. Examples of surfactants herein include polysorbate (e.g., polysorbate 20 and polysorbate 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl, myristyl, linoleyl, or stearyl sulfobetaine; lauryl, myristyl, linoleyl, or stearyl sarcosine; linoleyl, myristyl, or cetyl betaine; lauroamidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmidopropyl, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl, palmidopropyl, or isostearamidopropyl dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol, and ethylene-propylene glycol copolymers (e.g., Pluronics, PF68, etc.), etc. In one embodiment, the surfactant herein is polysorbate 20.

"Fc region" refers to the constant region of an antibody, which in humans may belong to the IgG1, 2, 3, 4, or other subclasses. Sequences of human Fc regions are available from IMGT, Human IGH C-REGIONs, http://www.imgt.org/IMGTrepertoire/Proteins/protein/human/IGH/IGHC/Hu_IGHCallgenes.html (accessed May 16, 2011).

"Administered" or "administration" includes, without limitation, delivery by an injectable form such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route of delivery, or a mucosal route of delivery such as, for example, as a nasal spray or aerosol for inhalation, or as an swallowed solution, capsule or tablet.

A "therapeutically effective amount" of a compound or combination refers to an amount sufficient to at least partially suppress the clinical manifestations of a given disease or disorder and its complications. The amount effective for a particular therapeutic purpose will depend on the severity of the disease or injury, as well as the weight and general condition of the subject. Clearly, the appropriate dosage can be determined using routine experimentation, constructing a table of values, and testing various points in the table; all of these techniques are common skills of a trained physician or clinical scientist.

"CDRs" in this document are defined by either Chothia et al. or Kabat et al. See Chothia C, Lesk AM. (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol., 196(4):901–17, which is incorporated herein by reference in its entirety. See Kabat EA, Wu TT, Perry HM, Gottesman KS, and Foeller C. (1991). Sequences of Proteins of Immunological Interest. 5th ed., NIH Publication no. 91-3242, US Dept. of Health and Human Services, Washington, DC, which is incorporated herein by reference in its entirety.

"Cross-competes" means the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to CD19 in a standard competitive binding assay. The ability or extent to which an antibody or other binding agent can interfere with the binding of another antibody or binding molecule to CD19, and hence its ability to cross-compete in accordance with the present invention, can be determined using standard competitive binding assays. One suitable assay uses Biacore technology (e.g., using a BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the degree of interaction using surface plasmon resonance technology. Another assay uses an ELISA-based approach to measure cross-competition. A high-throughput process for "epitope binning" of antibodies based on their cross-competition is described in International Patent Application No. WO 2003/48731.

The term "epitope" includes any protein determinant capable of specifically binding to an antibody or otherwise interacting with the molecule. Epitope determinants typically consist of chemically active surface moieties of molecules, such as the side chains of amino acids, carbohydrates, or sugars, and may have specific properties of three-dimensional structures, as well as certain properties due to the presence of an electrical charge. An epitope can be "linear" or "conformational." The term "linear epitope" refers to an epitope in which all interaction points between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein (continuous). The term "conformational epitope" refers to an epitope in which discontinuous amino acids are combined together in a three-dimensional conformation. In a conformational epitope, the interaction points are arranged along amino acid residues on the protein so that they are separated from one another.

"Binds the same epitope as" means that the antibody or other binding agent binds to CD19 and contains the same epitope as the exemplary antibody. The epitopes of the exemplary antibody and other anti-CD19 antibodies can be determined using standard epitope mapping techniques. Epitope mapping techniques well known in the art include the epitope mapping protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined by, for example, simultaneously synthesizing a large number of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still bound to the supports. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al, (1984) Proc. Natl. Acad. Sci. USA 8:3998–4002; Geysen et al, (1985) Proc. Natl. Acad. Sci. USA 82:78–182; Geysen et al, (1986) Mol. Immunol. 23:709–715. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, such as hydrogen/deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols above. Antigenic regions of proteins can also be identified using standard antigenicity and hydrophobicity plots, such as those calculated using, for example, the Omiga version 1.0 software available from the Oxford Molecular Group. This computer program uses the Hopp/Woods method, Hopp et al. (1981) Proc. Natl. Acad. Sci USA 78:3824–3828, to determine antigenicity profiles and the Kyte-Doolittle method, Kyte et al. (1982) J. MoI. Biol. 157: 105–132, to plot hydrophobicity plots.

Implementation options

In another embodiment, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of from about 20 mg/mL to about 125 mg/mL, a buffer, polysorbate at a concentration of from about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of from about 180 mM to about 240 mM, or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In another embodiment, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of from about 20 mg/mL to about 125 mg/mL, a citrate buffer or a phosphate buffer, polysorbate at a concentration of from about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of from about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In some embodiments, the concentration of said buffer in the formulation is from 10 to 75 mM. In some embodiments, the concentration of said buffer in the formulation is from 20 to 50 mM. In some embodiments, the concentration of said buffer in the formulation is from 20 to 40 mM. In some embodiments, the concentration of said buffer in the formulation is from 20 to 30 mM. In some embodiments, the concentration of said buffer in the formulation is approximately 25 mM. In some embodiments, the concentration of said buffer in the formulation is 25 mM. In some embodiments, said buffer in the formulation is a citrate buffer or a phosphate buffer.

In some embodiments, the concentration of said citrate buffer in the composition is from 10 to 75 mM. In some embodiments, the concentration of said citrate buffer in the composition is from 20 to 50 mM. In some embodiments, the concentration of said citrate buffer in the composition is from 20 to 30 mM. In some embodiments, the concentration of said citrate buffer in the composition is approximately 25 mM. In some embodiments, the concentration of said citrate buffer in the composition is 25 mM.

In some embodiments, the concentration of said phosphate buffer in the composition is from 10 to 75 mM. In some embodiments, the concentration of said phosphate buffer in the composition is from 20 to 50 mM. In some embodiments, the concentration of said phosphate buffer in the composition is from 20 to 30 mM. In some embodiments, the concentration of said phosphate buffer in the composition is approximately 25 mM. In some embodiments, the concentration of said phosphate buffer in the composition is 25 mM.

In another embodiment, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of from about 20 mg/mL to about 125 mg/mL, a citrate buffer or a phosphate buffer at a concentration of from 20 mM to 50 mM, polysorbate at a concentration of from about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of from about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In another embodiment, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 20 mg/mL to about 125 mg/mL, a citrate buffer or a phosphate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of about 180 mM to about 240 mM, or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In another embodiment, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 20 mg/mL to about 125 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In another embodiment, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 20 mg/mL to about 125 mg/mL, a phosphate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM.

In a further embodiment, said anti-CD19 antibody is a full-length antibody. In a further embodiment, said anti-CD19 antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

In another embodiment, said anti-CD19 antibody is a humanized antibody or a human antibody. In a further embodiment, said anti-CD19 antibody is an antibody fragment comprising an antigen-binding region. In a further embodiment, said antibody fragment is a Fab fragment or an F(ab')2 fragment.

In another embodiment, said stable lyophilized pharmaceutical formulation is stable at 2-8°C for at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months.

In another embodiment, said stable lyophilized pharmaceutical formulation is stable at about 40°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more weeks. In some embodiments, said stable lyophilized pharmaceutical formulation is stable at about 40°C for at least about 1, 2, 3, 4, 5 or more months. In some embodiments, said stable lyophilized pharmaceutical formulation is stable at about 25°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.

In some embodiments, said stable lyophilized pharmaceutical formulation is stable at about 5°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more months. In some embodiments, said stable lyophilized pharmaceutical formulation is stable at about 5 +/- 3°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more months. In some embodiments, said stable lyophilized pharmaceutical formulation is stable at about -20°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more months. In some embodiments, said stable lyophilized pharmaceutical formulation is stable at 5°C or -20°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more months.

In a further embodiment, the anti-CD19 antibody in the formulation retains at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of its biological activity after storage. In some embodiment, the biological activity is measured by binding of the antibody to CD19. In some embodiment, the biological activity is measured by binding of the antibody to CD19 in a FACS CD19 binding assay. In some embodiment, the biological activity is measured by ADCC activity of said anti-CD19 antibody. In some embodiment, the stable lyophilized pharmaceutical formulation is sterile.

In a further embodiment, said stable lyophilized pharmaceutical formulation is suitable for administration to a subject. In a further embodiment, said stable lyophilized pharmaceutical formulation is suitable for intravenous (IV) administration or subcutaneous administration.

In another aspect, the present document provides an article of manufacture comprising a container containing a stable lyophilized pharmaceutical formulation as disclosed herein. In one embodiment, said container is a glass vial or a metal alloy container. In a further embodiment, the metal alloy is 316L stainless steel or Hastelloy.

In another aspect, the present document provides a method for treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a composition disclosed herein, wherein the disease or disorder is cancer. In another aspect, the present document provides the use of a stable lyophilized pharmaceutical formulation as disclosed herein for treating a disease or disorder in a subject, comprising administering to the subject an effective amount of said composition, wherein the disease or disorder is cancer. In a further aspect, the present document provides the use of a stable lyophilized pharmaceutical formulation as disclosed herein for the manufacture of a medicament for treating a disease or disorder in a subject, comprising administering to the subject an effective amount of said composition, wherein the disease or disorder is cancer. In one embodiment, the disease or disorder is non-Hodgkin's lymphoma (including each of the subtypes described herein), chronic lymphocytic leukemia, and/or acute lymphoblastic leukemia. In embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosal lymphoid tissue lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and mantle cell lymphoma.

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 20 mg/mL to about 125 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM,

wherein said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region with the sequence RMSNLNS (SEQ ID NO: 5) and an LCDR3 region with the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 20 mg/mL to about 125 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM, wherein said antibody to CD19 comprises a heavy chain variable region with the sequence and a variable region of the light chain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 20 mg/mL to about 125 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 6.0, wherein the formulation further comprises a) trehalose at a concentration of about 180 mM to about 240 mM,

or

b) mannitol at a concentration of from about 180 mM to about 240 mM and sucrose at a concentration of from about 10 mM to about 50 mM, wherein said CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, a polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises

a) trehalose at a concentration of approximately 200 mM, or

b) mannitol at a concentration of about 219 mM and sucrose at a concentration of about 29 mM, wherein said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region with the sequence RMSNLNS (SEQ ID NO: 5) and an LCDR3 region with the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, a polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises

a) trehalose at a concentration of approximately 200 mM,

or

b) mannitol at a concentration of approximately 219 mM and sucrose at a concentration of approximately 29 mM, wherein said CD19 antibody comprises a heavy chain variable region with the sequence and a variable region of the light chain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, a polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises

a) trehalose at a concentration of approximately 200 mM,

or

b) mannitol at a concentration of approximately 219 mM and sucrose at a concentration of approximately 29 mM,

wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, a polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises

a) trehalose at a concentration of approximately 200 mM,

or

b) mannitol at a concentration of approximately 219 mM and sucrose at a concentration of approximately 29 mM,

wherein said antibody to CD19 comprises a heavy chain variable region characterized by at least 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence and a light chain variable region having at least 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, a polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises

a) trehalose at a concentration of approximately 200 mM,

or

b) mannitol at a concentration of approximately 219 mM and sucrose at a concentration of approximately 29 mM,

wherein said antibody to CD19 comprises a heavy chain constant domain characterized by at least 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence NO: 8), and a light chain constant domain characterized by at least 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v) and a pH of about 6.0, wherein the formulation further comprises trehalose at a concentration of about 200 mM, wherein said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region with the sequence RMSNLNS (SEQ ID NO: 5) and the LCDR3 region with the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v) and at a pH of about 6.0, wherein the formulation further comprises mannitol at a concentration of about 219 mM and sucrose at a concentration of about 29 mM, wherein said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), a LCDR2 with the sequence RMSNLNS (SEQ ID NO: 5) and the LCDR3 region with the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises trehalose at a concentration of about 200 mM, wherein said anti-CD19 antibody comprises a heavy chain variable region with the sequence and a variable region of the light chain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v) and a pH of about 6.0, wherein the formulation further comprises mannitol at a concentration of about 219 mM and sucrose at a concentration of about 29 mM, wherein said anti-CD19 antibody comprises a heavy chain variable region having the sequence and a variable region of the light chain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises trehalose at a concentration of about 200 mM, wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a citrate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v) and a pH of about 6.0, wherein the formulation further comprises mannitol at a concentration of about 219 mM and sucrose at a concentration of about 29 mM, wherein the anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a phosphate buffer at a concentration of 20 to 40 mM, polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises trehalose at a concentration of about 200 mM, wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a phosphate buffer at a concentration of 20 to 40 mM, polysorbate at a concentration of about 0.02% (w/v) and a pH of about 6.0, wherein the formulation further comprises mannitol at a concentration of about 219 mM and sucrose at a concentration of about 29 mM, wherein the anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a phosphate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v), and a pH of about 6.0, wherein the formulation further comprises trehalose at a concentration of about 200 mM, wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of about 40 mg/mL, a phosphate buffer at a concentration of about 25 mM, polysorbate at a concentration of about 0.02% (w/v) and a pH of about 6.0, wherein the formulation further comprises mannitol at a concentration of about 219 mM and sucrose at a concentration of about 29 mM, wherein the anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, a citrate buffer at a concentration of 25 mM, a polysorbate at a concentration of 0.02% (w/v) and a pH of 6.0, wherein the formulation further comprises trehalose at a concentration of 200 mM, wherein said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region with the sequence RMSNLNS (SEQ ID NO: 5), and LCDR3 region with sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v) and a pH of 6.0, wherein the formulation further comprises mannitol at a concentration of 219 mM and sucrose at a concentration of 29 mM, wherein said anti-CD19 antibody comprises an HCDR1 region with the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region with the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region with the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region with the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region with the sequence RMSNLNS (SEQ ID NO: 5) and the LCDR3 region with the sequence MQHLEYPIT (SEQ ID NO: 6).

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of 6.0, wherein the formulation further comprises trehalose at a concentration of 200 mM, wherein said anti-CD19 antibody comprises a heavy chain variable region having the sequence and a variable region of the light chain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of 6.0, wherein the formulation further comprises mannitol at a concentration of 219 mM, and sucrose at a concentration of 29 mM, wherein said anti-CD19 antibody comprises a heavy chain variable region having the sequence

and a variable region of the light chain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of 6.0, wherein the formulation further comprises trehalose at a concentration of 200 mM, wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, a phosphate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of 6.0, wherein the formulation further comprises trehalose at a concentration of 200 mM, wherein said anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, citrate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of 6.0, wherein the formulation further comprises mannitol at a concentration of 219 mM, and sucrose at a concentration of 29 mM, wherein the anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

In some embodiments, provided herein is a stable lyophilized pharmaceutical formulation, wherein the formulation comprises an anti-CD19 antibody at a concentration of 40 mg/mL, a phosphate buffer at a concentration of 25 mM, polysorbate at a concentration of 0.02% (w/v), and a pH of 6.0, wherein the formulation further comprises mannitol at a concentration of 219 mM, and sucrose at a concentration of 29 mM, wherein the anti-CD19 antibody comprises a heavy chain constant domain with the sequence and a light chain constant domain with the sequence

Demonstration examples

Example 1: Determination of biophysical characteristics of MOR208

The biophysical properties of MOR208 were analyzed for characterization, component screening prior to formulation, and high concentration application.

To assess the structural stability of MOR208, thermal melting experiments were performed using differential scanning fluorimetry (DSF) and CD spectroscopy. MOR208 samples were diluted to a concentration of 1.1 mg/mL. The hydrophobic dye sypro-orange was added to detect protein unfolding, and the corresponding melting curves were obtained. The samples exhibited a relatively low melting point of 47°C, determined by a stepwise increase in fluorescence intensity.

MOR208 is a humanized monoclonal antibody that binds to CD19 on B cells and their progenitor cells. The Fc region was genetically engineered (S239D/I332E) to enhance effector functions that support B cell depletion. According to the current working hypothesis, this Fc region format results in flexibility of the CH2 domain, leading to enhanced ADCC activity but may be accompanied by a decrease in the melting temperature of the Fc domain.

Furthermore, a pH screening study was conducted using DSC, RALS, and ITF to identify the most stable pH range for the protein. The study covered a pH range of 3.5 to 8.0. Measurements were performed in a mixed buffer system covering the required pH range. Based on the results obtained, the most stable pH range for MOR208 was identified between pH 6.0 and 7.0.

Consequently, two buffer systems have been identified that provide sufficient buffering capacity in the pH range between pH 6.0 and 7.0 and are pharmaceutically acceptable for parenteral use: citrate (pH 5.5; 6.0); histidine (pH 6.0; 6.5; 7.0).

Additional DSC, RALS, and ITF measurements were conducted to test the thermal stability of MOR208 in 25 mM citrate and 25 mM histidine buffers. Only citrate at pH 5.5 demonstrated significantly lower transition temperatures, while all other tested samples demonstrated comparable thermal stability. This conclusion was also confirmed by RALS and ITF testing.

In addition, the effects of ionic strength, sugars, polyols, and polysorbate 20 on the thermal stability of MOR208 were tested. A 25 mM citrate buffer at pH 6.0 was selected as the primary buffer system to generate samples containing NaCl, sugar, or polysorbate 20. Measurements demonstrated that the addition of NaCl increased the thermal stability of MOR208, depending on the first transition temperature of 52°C. This observation was confirmed by ITF measurements. RALS testing results did not reveal any effect of NaCl on the thermal stability of MOR208.

The effects of sugars (trehalose and sucrose) and mannitol on the thermal stability of MOR208 were tested using concentrations of 200 mM trehalose, 210 mM sucrose, and a 180 mM mannitol/45 mM sucrose mixture. These concentrations were chosen to shift the composition into the isoosmotic range. Results from DSC, RALS, and ITF testing indicated that trehalose and sucrose had comparable effects on the thermal stability of MOR208, with the mannitol/sucrose mixture slightly reducing the first transition temperature by approximately 0.8°C.

The effect of polysorbate 20 on the thermal stability of MOR208 was tested using concentrations of 0.02%, 0.04%, 0.08%, and 0.12%. DSC and RALS measurements showed no significant effect of polysorbate 20 at the tested concentrations on the thermal stability of MOR208.

The effect of _MgCl2 on the thermal stability of MOR208 was tested using concentrations of 1, 5, and 10 mM. DSC and RALS measurements showed no significant effect of _MgCl2 at the tested concentrations on the thermal stability of MOR208.

Case Study 2: Liquid Formulation Stability Study for MOR208

Based on the biophysical characterization of MOR208, the following 6 different liquid formulations were tested in a stability study.

The following formulations were selected for stability studies:

composition F1: 10 mM citrate, pH 6.0 + 0.01% PS 20 + 150 mM NaCl;

composition F2: 25 mM citrate, pH 6.0 + 0.02% PS 20 + 125 mM NaCl;

composition F3: 25 mM citrate, pH 6.0 + 0.02% PS 20 + 200 mM trehalose;

composition F4: 25 mM citrate, pH 6.0 + 0.02% PS 20 + 210 mM sucrose;

composition F5: 25 mM histidine, pH 6.0 + 0.02% PS 20 + 140 mM NaCl;

composition F6: 25 mM histidine, pH 6.0 + 0.02% PS 20 + 230 mM trehalose;

composition F7: 25 mM succinate, pH 6.0 + 0.02% PS 20 + 215 mM trehalose;

composition F8: 25 mM histidine, pH 6.5 + 0.02% PS 20 + 240 mM trehalose.

The protein concentration was 20 mg/ml for all formulations. Ten ml of each formulation was dispensed into a 10 ml test tube, sealed with a coated rubber stopper, and stored upside down at a controlled temperature of 25°C for 12 weeks. Analytical testing was performed before storage (t0), and after 4, 8, and 12 weeks.

Formulations 1, 3, 5, and 7 produced fewer particles visible to the naked eye over 12 weeks compared to the other formulations. Particles were measured using MFI. Formulation 5 demonstrated the lowest SVP count in the >10 µm and >25 µm ranges.

The aggregate and monomer levels in the formulations were tested using HP SEC. Formulations 2, 3, 7, and 8 demonstrated higher aggregate increase over 12 weeks compared to the other formulations.

The osmolality of composition 4 increased after 4 weeks of storage, which most likely indicates the breakdown of sucrose contained in the composition.

The pH shift also indicates the breakdown of sucrose in compound 4.

Additional HIC testing revealed that formulations 6, 7, and 8 demonstrated a greater reduction in peak 3 area % compared to the other formulations. In particular, formulation 8 demonstrated a significant reduction from 66.1% (baseline) to 52.2% after 12 weeks.

By determining the physicochemical characteristics of MOR208, the most stable pH range was identified between pH 6.0 and 7.0.

Citrate, histidine, and succinate buffers were selected as pharmaceutically acceptable buffer systems within the specified pH range. All buffer systems were used at a concentration of 25 mM, providing sufficient buffering capacity for a protein concentration of 20 mg/mL. The addition of NaCl at a minimum concentration of 125 mM had a slight positive effect on the thermal stability of MOR208 (based on DSC data). The addition of sucrose, trehalose, mannitol/sucrose, and polysorbate 20 did not alter the thermal stability of MOR208.

Based on the analytical results, compositions F1, F3 and F5 were identified as the most stable compositions.

Composition F1: 10 mM citrate, pH 6.0 + 0.01% PS 20 + 150 mM NaCl;

composition F3: 25 mM citrate, pH 6.0 + 0.02% PS 20 + 200 mM trehalose;

composition F5: 25 mM histidine, pH 6.0 + 0.02% PS 20 + 140 mM NaCl.

Samples of these three formulations were further stored at 25°C for up to 8 months and then tested again. Formulations 1 and 5 demonstrated the highest stability, but all liquid formulations formed particles invisible to the naked eye after 8 months, exceeding the pharmacopoeial requirements (NMT 6000 particles ≥10 μm in each tube and NMT 600 particles ≥25 μm in each tube). Consequently, the required shelf life of at least 24 months at 2-8°C was not achieved by one of the tested liquid formulations with a protein concentration of 20 mg/mL.

This fact led to the decision to begin developing a lyophilized dosage form.

Demonstration example 3. Study of the possibility of using lyophilization

Formulation 3 and the new Formulation 9 were included in the study for the possibility of using lyophilization with a concentration of MOR208 of 40 mg/mL.

Composition 3: 25 mM citrate;

200 mM trehalose dihydrate;

0.02% polysorbate 20;

pH 6.0.

Composition 9: 25 mM citrate;

219 mM mannitol;

29 mM sucrose;

0.02% polysorbate 20;

pH 6.0.

Appearance of the lyophilized drug

The appearance of the lyophilized tablets for both formulations was acceptable. The formulation containing trehalose was completely amorphous and exhibited a higher degree of shrinkage, which is only an external observation and is generally not related to product quality or stability. The formulation containing mannitol was partially crystalline and produced a tablet with a high degree of pharmaceutical elegance without shrinkage.

The quality of lyophilized MOR208 was tested and compared with the quality of the product before lyophilization. Table 1 below summarizes the results for formulations 3 and 9.

An additional lyophilization study was conducted, focusing solely on non-visible particles (SVPs) before and after lyophilization. The results are listed in Table 2 and clearly indicate that the lyophilization process did not result in an increase in SVPs.

Based on the test results, no negative impact of the lyophilization process on product quality was observed. Both formulations provided an acceptable tablet appearance. Reconstitution time was less than 60 seconds for the formulation containing 40 mg protein/ml. Reconstitution was performed by adding 5 ml of water for injection. The moisture content of the mannitol/sucrose formulation was higher than that of the trehalose-containing formulation, due to the higher density of the lyophilized tablet. Osmolality and protein content (UV scanning) decreased after lyophilization, as the product was diluted during the reconstitution process. The level of aggregation did not increase during lyophilization, and a small number of particles invisible to the naked eye were counted after reconstitution. As a result, both formulations were suitable for lyophilization of MOR208, and the lyophilization feasibility study was successfully completed. Following this study, an accelerated degradation stability study was conducted with both formulations at 40°C for a period of 3 months.

Demonstration example 4. Stability study with accelerated drug degradation

After the lyophilization feasibility study was successfully completed, the first stability study was conducted at 40°C (75% relative humidity) for 3 months to compare both formulations. Stability testing included color and visible particulate matter testing. Additionally, the products were tested using HP-SEC (aggregation), HIC, IEC, binding assay (CD 16 Biacore), MFI (invisible particles), and SDS-Page.

HP-SEC testing demonstrated a higher rate of aggregate increase for F9 after 3 months of storage (Table 3). Aggregate levels for F3 increased from 0.8% to 1.7%, while aggregate levels for F9 increased from 0.8% to 2.6%.

Invisible to the naked eye particles were tested at t0 and after 4, 8, and 12 weeks using the MFI method. When comparing both formulations, it was evident that the mannitol/sucrose formulation formed more particles over time, particularly in the range between 2 µm and 1000 µm (Figure 2).

Based on the analytical results, formulation 3 containing trehalose exhibited higher stability at 40°C compared to formulation 9. Differences in stability were observed in SVP (MFI) and aggregate level (HPSEC).

Conclusion

A lyophilization feasibility study demonstrated that MOR208 can be lyophilized without affecting product quality using both formulations and a protein concentration of 40 mg/mL. Stability conditions were 40°C (75% relative humidity) for a period of 3 months. During this stability study, the trehalose-containing formulation was identified as having superior stability compared to the mannitol/sucrose formulation.

Example 5. Determining the shelf life

For long-term stability studies, MOR208 in Formulation 3 was stored in real time at 5°C±3°C and tested under accelerated storage at 25°C±2°C.

The stability studies conducted included a methodology suitable for stability assessment and modern methods for monitoring medicinal products in terms of concentration, potency, purity, pharmaceutical and microbiological parameters during storage.

The following parametric methods were used, which are considered to be the main tests suitable for stability assessment:

- Determination of purity using HP-SEC

The stability properties of HP-SEC were demonstrated by analyzing the corresponding sample under stress conditions. Furthermore, the ability to detect aggregates was verified by analytical ultracentrifugation.

- Determination of homogeneity and purity using IEC and CGE under reducing/non-reducing conditions

CGE was used to detect fragments under non-reducing conditions; chemical modifications that lead to the formation of compounds with different charges due to deamidation were determined using IEC. Activity assays

Product specific activity assays, CD19 binding assay (FACS), CD16 binding assay (SPR) and ADCC activity assay demonstrated sensitivity to the respective sample under stress conditions.

The results of real-time storage at 5°C±3°C and accelerated storage at 25°C±3°C are summarized in Table 4 and Table 5, respectively.

Discussion of results

Content and activity

The functional activity of MOR208 was monitored using three different activity assays: a CD19 binding assay (FACS), a CD16 binding assay (SPR), and an ADCC-based activity assay. The combination of these three assays covers antigen binding, corresponding effector binding, and the primary mechanism of action (ADCC).

Both binding assays do not demonstrate clear or significant trends over time. The ADCC-based activity assay shows a slight increase over time (both in real time and under accelerated degradation conditions), but since the assay also exhibits higher variability, the trend is not yet considered significant. Therefore, all content and activity assays are within the limits of the description and do not indicate any critical changes in product quality over 36 months.

Purity

Over 36 months of storage at the intended storage temperature of 5 ± 3°C, none of the purity assays (i.e., HP-SEC, IEC, and CGE under reducing and non-reducing conditions) indicate critical changes in product purity. Under accelerated degradation conditions at 25°C ± 2°C, HP-SEC shows only one change in values at the last test point (a decrease in monomer values/an increase in fragment values for one increment), but this is not reflected in the long-term data and is therefore considered insignificant. IEC does not show any trends under long-term testing conditions, but does show a clear trend toward a decrease in the main peak group/an increase in the main peak group under accelerated degradation conditions.

Thus, all purity analyses are within the description and do not indicate critical changes in the quality of the product over 36 months.

Pharmaceutical tests

During 36 months of storage at the intended storage temperature of 5±3°C, none of the pharmaceutical tests indicated any critical changes over time. Therefore, all pharmaceutical tests are within the scope of the description and do not indicate any critical changes in product quality over 36 months.

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SEQUENCE LIST

<110> MorphoSys AG

<120>Compositions based on antibodies to CD19

<130> MS247

<160> 11

<170> BiSSAP 1.0

<210> 1

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 1

Ser Tyr Val Met His

1 5

<210> 2

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 2

Asn Pro Tyr Asn Asp Gly

1 5

<210> 3

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 3

Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr

1 5 10

<210> 4

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 4

Arg Ser Ser Lys Ser Leu Gln Asn Val Asn Gly Asn Thr Tyr Leu Tyr

1 5 10 15

<210> 5

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 5

Arg Met Ser Asn Leu Asn Ser

1 5

<210> 6

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 6

Met Gln His Leu Glu Tyr Pro Ile Thr

1 5

<210> 7

<211> 556

<212> PROTEIN

<213> Homo sapiens

<220>

<223> CD19

<400> 7

Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met

1 5 10 15

Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp

20 25 30

Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln

35 40 45

Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu

50 55 60

Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile

65 70 75 80

Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu

85 90 95

Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr

100 105 110

Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp

115 120 125

Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro

130 135 140

Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala

145 150 155 160

Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro

165 170 175

Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro

180 185 190

Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser

195 200 205

Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser

210 215 220

Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp

225 230 235 240

Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala

245 250 255

Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu

260 265 270

Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly

275 280 285

Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu

290 295 300

Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg

305 310 315 320

Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val

325 330 335

Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu

340 345 350

Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala

355 360 365

Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp

370 375 380

Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly

385 390 395 400

Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu

405 410 415

Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu

420 425 430

Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly

435 440 445

Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu

450 455 460

Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser

465 470 475 480

Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly

485 490 495

Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln

500 505 510

Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala

515 520 525

Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp

530 535 540

Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg

545 550 555

<210> 8

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175

Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val 180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205

Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Thr Lys Gly 210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270

Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330

<210> 9

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 13

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 10

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Ser Ser Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic construction

<400> 11

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Lys Ser Leu Gln Asn Val

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Gln Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Asn Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105 110

<---

Claims (54)

1. A liquid formulation for the treatment of lymphoma or leukemia that expresses CD19, comprising an antibody to CD19 at a concentration of 20 to 125 mg/ml, a citrate buffer at a concentration of 20 to 50 mM, polysorbate at a concentration of 0.005 to 0.06% (w/v), and with a pH of 6.0, wherein the formulation further comprises a) trehalose at a concentration of 180 to 240 mM, or b) mannitol at a concentration of 180 to 240 mM and sucrose at a concentration of 10 to 50 mM, and wherein said antibody to CD19 comprises a heavy chain variable region with the sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 10) and a light chain variable region with the sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 11), a heavy chain constant domain with the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) and light chain constant domain with sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9). 2. The liquid composition according to claim 1, wherein the concentration of said antibody to CD19 in the liquid composition is 40 mg/ml. 3. The liquid composition according to claim 1 or 2, wherein the concentration of said citrate buffer in the liquid composition is 25 mM. 4. A liquid composition according to any one of claims 1-3, wherein the concentration of said trehalose in the liquid composition is 200 mM. 5. A liquid composition according to any one of claims 1-3, wherein the concentration of said mannitol in the liquid composition is 219 mM, and the concentration of said sucrose in the liquid composition is 29 mM. 6. The liquid composition according to any one of claims 1-5, wherein said polysorbate in the liquid composition is polysorbate 20. 7. A liquid composition according to any one of paragraphs 1-6, wherein the concentration of said polysorbate in the liquid composition is 0.02%. 8. The liquid formulation of claim 1, comprising said CD19 antibody in an amount of 40 mg/ml, citrate buffer at a concentration of 25 mM, trehalose at a concentration of 200 mM, and polysorbate at a concentration of 0.02% (w/v), wherein said liquid formulation has a pH of 6.0. 9. The liquid formulation of claim 1, comprising said CD19 antibody in an amount of 40 mg/ml, citrate buffer at a concentration of 25 mM, mannitol at a concentration of 219 mM and sucrose at a concentration of 29 mM, and polysorbate at a concentration of 0.02% (w/v), wherein said liquid formulation has a pH of 6.0. 10. Use of a liquid formulation according to any one of claims 1-9 in a method of treating lymphoma or leukemia that expresses CD19 in a subject, wherein the treatment comprises administering to the subject an effective amount of the liquid formulation. 11. The use of claim 10 in a method for treating non-Hodgkin's lymphoma in a subject. 12. The use according to claim 11, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma. 13. The use according to claim 11, wherein the non-Hodgkin's lymphoma is follicular lymphoma. 14. The use according to claim 11, wherein the non-Hodgkin's lymphoma is a small cell lymphocytic lymphoma. 15. The use according to claim 11, wherein the non-Hodgkin's lymphoma is a lymphoma of the lymphoid tissue of the mucous membranes. 16. The use according to claim 11, wherein the non-Hodgkin's lymphoma is marginal zone lymphoma. 17. The use according to claim 11, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma. 18. The use according to claim 11, wherein the non-Hodgkin's lymphoma is mantle cell lymphoma. 19. The use of claim 10 in a method for treating chronic lymphocytic leukemia in a subject. 20. The use of claim 10 in a method for treating acute lymphoblastic leukemia in a subject. 21. A method of treating lymphoma or leukemia that expresses CD19 in an individual in need thereof, comprising administering an effective amount of a liquid formulation according to any one of claims 1-9. 22. The method of claim 21, wherein the individual has non-Hodgkin's lymphoma. 23. The method of claim 22, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma. 24. The method of claim 22, wherein the non-Hodgkin lymphoma is follicular lymphoma. 25. The method of claim 22, wherein the non-Hodgkin's lymphoma is a small cell lymphocytic lymphoma. 26. The method according to claim 22, wherein the non-Hodgkin's lymphoma is a mucosal lymphoid tissue lymphoma. 27. The method of claim 22, wherein the non-Hodgkin's lymphoma is marginal zone lymphoma. 28. The method of claim 22, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma. 29. The method of claim 22, wherein the non-Hodgkin's lymphoma is mantle cell lymphoma. 30. The method of claim 21, wherein the individual has chronic lymphocytic leukemia. 31. The method of claim 21, wherein the individual has acute lymphoblastic leukemia. 32. Use of a stable lyophilized pharmaceutical composition containing: - an antibody to CD19 containing a heavy chain variable region with the sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSS (SEQ ID NO: 10) and a light chain variable region with the sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 11), a heavy chain constant domain with the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) and light chain constant domain with sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9); - citrate buffer; - polysorbate; and - (a) trehalose, or (b) mannitol and sucrose, for obtaining a liquid composition for the treatment of lymphoma or leukemia that express CD19, according to claim 1. 33. The use according to claim 32, wherein the concentration of said antibody to CD19 in the resulting liquid composition is 40 mg/ml. 34. The use according to claim 32 or 33, wherein the concentration of said citrate buffer in the resulting liquid composition is 25 mM. 35. The use according to any one of paragraphs 32-34, wherein the concentration of said trehalose in the resulting liquid composition is 200 mM. 36. The use according to any one of claims 32-34, wherein the concentration of said mannitol in the resulting liquid composition is 219 mM, and the concentration of said sucrose in the resulting liquid composition is 29 mM. 37. The use according to any one of paragraphs 32-36, wherein said polysorbate in the resulting liquid composition is polysorbate 20. 38. The use according to any one of paragraphs 32-37, wherein the concentration of said polysorbate in the resulting liquid composition is 0.02%. 39. The use according to any one of claims 32-38, wherein the stable lyophilized pharmaceutical formulation is stable at 2-8°C for at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months. 40. The use according to claim 32, wherein in the resulting liquid composition the amount of said antibody to CD19 is 40 mg/ml, the concentration of said citrate buffer is 25 mM, the concentration of said trehalose is 200 mM, the concentration of polysorbate 20 is 0.02% (w/v), and said liquid composition is characterized by a pH of 6.0. 41. The use according to claim 32, wherein in the resulting liquid composition the amount of said antibody to CD19 is 40 mg/ml, the concentration of said citrate buffer is 25 mM, the concentration of said mannitol is 219 mM, and the concentration of said sucrose is 29 mM, the concentration of polysorbate 20 is 0.02% (w/v), and said liquid composition is characterized by a pH of 6.0.