Human anti-PD-L1 antibody formulation

Abstract

The present disclosure relates to formulations and compositions of human anti-PD-L1 directed antibodies, such as durvalumab. [Selected figure] Figure 2

Description

[Technical field]

[0001] The present disclosure relates to formulations and compositions of human anti-PD-L1 directed antibodies, such as durvalumab. [Background technology]

[0002] Programmed death-ligand 1 (PD-L1), also known as B7H1, is a 40 kDa transmembrane protein that poses a major obstacle to anti-cancer immunity. Binding to the programmed death receptor (PD-1), PD-L1 inactivates T cells, protecting tumor cells from immune system detection and allowing unchecked proliferation of cancer cells. PD-L1 also binds to the costimulatory molecule CD80.

[0003] A wide range of tumorigenic and activated immune cell types naturally express PD-L1, including antigen-presenting cells, macrophages, monocytes, B cells, T cells, and non-hematopoietic cells. In addition, inflammatory cytokines such as interferon gamma (IFNγ) can induce the expression of PD-L1. For example, activated T cells produce IFNγ, which is the most potent inducer of PD-L1. IFNγ-induced expression of PD-L1 promotes tumor protection, a mechanism known as adaptive immune tolerance.

[0004] One strategy to suppress adaptive immune resistance and the lethality of PD-L1 is to use anti-PD-L1 antibodies. Consistent with this approach, the 149 kDa anti-PD-L1 antibody durvalumab, an affinity-optimized anti-PD-L1 monoclonal IgG1 triple mutant (TM) that blocks PD-L1 binding to PD-1, can be employed to abolish the immunosuppressive effect of PD-L1 on cytotoxic T cells. The result is a reduction in negative inhibitory signals that promote tumor growth, enhanced anti-tumor immunity, and a response that enhances tumor cell killing by the immune system.

[0005] Importantly, buffer formulations of anti-PD-L1 antibodies that maintain the stability of the liquid drug substance and the lyophilized drug product are essential to the efficacy of anti-PD-L1 antibodies. Summary of the Invention [Means for solving the problem]

[0006] In one aspect, the disclosure provides an antibody formulation comprising 40 mg / mL to 50 mg / mL of anti-PD-L1 antibody, 15 mM to 35 mM buffer, 255 mM to 275 mM disaccharide, and 0.01% (w / v) to 0.05% (w / v) surfactant, where the pH of the formulation is about pH 5.5 to about pH 7.2.

[0007] In one aspect, the disclosure provides an antibody formulation comprising 50 mg / mL human anti-PD-L1 antibody, 26 mM histidine / histidine-HCl buffer, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, where the pH of the formulation is pH 6.0.

[0008] In one aspect, the disclosure provides an antibody formulation comprising 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about pH 5.5.

[0009] In one aspect, the disclosure provides an antibody formulation comprising 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about pH 6.5.

[0010] In one aspect, the disclosure provides an antibody formulation comprising 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM trehalose dehydrate, 0.04% (w / v) polysorbate 80, where the pH of the formulation is about pH 6.0.

[0011] In one aspect, the disclosure provides an antibody formulation comprising 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM sucrose, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about pH 6.0.

[0012] In one aspect, the disclosure provides an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, and a major form of the antibody that comprises greater than 45% of the protein in the composition, as measured using capillary isoelectric focusing (cIEF) of the composition.

[0013] In one aspect, the disclosure provides a composition comprising an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, a major form of the antibody that comprises more than 45% of the protein in the composition, as measured using cIEF of the composition, and an acidic form of the antibody that comprises 45%-50% of the protein in the composition, as measured using cIEF of the composition.

[0014] In one aspect, the disclosure provides a composition comprising an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, the major form of the antibody comprising greater than or equal to 45% of the protein in the composition, as measured using cIEF of the composition, and the base form of the antibody comprising between 18% and 23% of the protein in the composition, as measured using cIEF of the composition.

[0015] In one aspect, the disclosure provides an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2; a major form of the antibody which comprises more than 45% of the protein in the composition as measured using cIEF of the composition, an acidic form of the antibody which comprises 45%-50% of the protein in the composition as measured using cIEF of the composition, and a base form of the antibody which comprises 18%-23% of the protein in the composition as measured using cIEF of the composition.

[0016] In one aspect, the disclosure provides a composition comprising an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2, wherein the glycan structures of the anti-PD-L1 antibody include G0f, G1f, G2f, and G0 glycoforms.

[0017] In one aspect, the disclosure provides a composition comprising an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2, wherein between 1.5% and 2.5% of the anti-PD-L1 antibody forms aggregates as determined by high pressure size exclusion chromatography (HP-SPEC), and 97% and 98% of the anti-PD-L1 antibody is present as a monomer as measured by HP-SEC. [Brief description of the drawings]

[0018] [Figure 1] An overview of durvalumab formulation development activities is presented. [Diagram 2] Differential scanning calorimetry (DSC) profile of durvalumab at 3 mg / mL in formulation buffer (26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0) is shown, where Tm1 was demonstrated to be 64.5°C and Tm2 was demonstrated to be 73.04°C. [Diagram 3] Capillary isoelectric focusing (cIEF) profile of durvalumab in formulation buffer (26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0). Four peaks showed a pI range of 8.3 to 8.8. The major peak had a pI of 8.6. [Figure 4] Demonstrate the liquid stability of durvalumab clone 1 or clone 2 after one month of incubation at 5°C or 40°C in formulation buffer (26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0). [Figure 5A-B] Electropherograms of durvalumab after 3 months of storage at 5° C. (FIG. 5A), 25° C. (FIG. 5B), and 40° C. (FIG. 5C) are illustrated. After 3 months of storage at 25° C. and 40° C., an additional pyroglutamic acid peak was present. [Figure 5C] Electropherograms of durvalumab after 3 months of storage at 5° C. (FIG. 5A), 25° C. (FIG. 5B), and 40° C. (FIG. 5C) are illustrated. After 3 months of storage at 25° C. and 40° C., an additional pyroglutamic acid peak was present. [Figure 6] Lyophilization microscopy is demonstrated showing the temperature of onset and complete disintegration of durvalumab in formulation buffer (26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0). [Figure 7] FIG. 1 illustrates a DSC thermogram showing the glass transition (Tg') temperature of durvalumab in formulation buffer (26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0). [Figure 8] Design space generation process model. Experimental designs are run with varied primary drying temperature, pressure, primary drying time, and maximum product temperature. Cycle midpoints (dots) and process robustness (white triangular area) at chamber pressure and shelf temperature are depicted. [Figure 9] We demonstrate performance data for durvalumab NMF lyophilization where convergence of the Pirani gauge (Δ10 μbar) occurs approximately between 103 and 115 hours of the drying process, which corresponds to a 10% safety margin. [Figure 10] We demonstrate large scale NMF freeze drying performed in an Amsco freeze dryer. Results showed convergence of the product thermocouples within the allotted primary time. [Figure 11] Demonstrates microflow imaging (MFI) results from durvalumab concussion studies. [Figure 12-1] Demonstrate ultra-performance liquid chromatography (UPLC) peak identification of 2-AB labeled oligosaccharides for durvalumab. [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 12-4] Continued from Figure 12-3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present disclosure relates to formulations and compositions of anti-PD-L1 directed antibodies, such as durvalumab.

[0020] As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0021] The term "antibody" as used herein refers to a protein capable of recognizing, and in particular binding to, an antigen. A normal or conventional mammalian antibody comprises a tetramer, which typically consists of two homologous pairs of polypeptide chains, each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain", as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 or more amino acids that is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant domain that is responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide comprises a variable domain (VH) and three constant domains (CH1, CH2, and CH3), as well as a hinge region between CH1 and CH2, where the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), where the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[0022] Within full-length light and heavy chains, the variable and constant domains are typically linked by a "J" region of about 12 or more amino acids, with the heavy chain also including an additional "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FRs) linked by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both light and heavy chains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0023] The antibody formulations disclosed herein comprise an anti-PD-L1 antibody. In certain embodiments, the formulation comprises 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, or 60 mg / mL of anti-PD-L1 antibody. In other embodiments, the formulation comprises 40 mg / mL to 50 mg / mL of anti-PD-L1 antibody. In other embodiments, the formulation comprises 50 mg / mL of anti-PD-L1 antibody. In certain embodiments, the anti-PD-L1 antibody is human.

[0024] The antibody formulations disclosed herein include one or more buffers. As used herein, "buffer" refers to an excipient for maintaining the pH of the formulation. In certain embodiments, the buffer is a histidine / histidine-HCl buffer. The buffer is present at a concentration of about 15 mM, 20 mM, 25 mM, or 30 mM. In certain embodiments, the concentration of the buffer is 26 mM.

[0025] In certain embodiments, the antibody formulation comprises a disaccharide. In certain embodiments, the disaccharide is trehalose dihydrate or sucrose. The disaccharide is present at a concentration of about 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, or 280 mM. In certain embodiments, the disaccharide concentration is 265 mM. In other embodiments, the disaccharide concentration is 275 mM.

[0026] In certain embodiments, the antibody formulation comprises a surfactant. The term "surfactant" as used herein refers to an organic substance with an amphiphilic structure; i.e., such substances are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Depending on the charge of the surface-active moiety, surfactants can be classified as anionic, cationic, and nonionic surfactants. Surfactants are frequently used as wetting, emulsifying, solubilizing, and dispersing agents for various pharmaceutical formulations and preparations of biological materials. In certain embodiments, the surfactant is polysorbate 80. The surfactant is present at a concentration of about 0.001% to about 0.5% (by volume).

[0027] In certain embodiments, disclosed herein is an antibody formulation comprising 40 mg / mL-50 mg / mL anti-PD-L1 antibody, 15 mM-35 mM buffer, 255 mM-275 mM disaccharide, 0.01% (w / v)-0.05% (w / v) surfactant, where the pH of the formulation is about pH 5.5 to about pH 7.2. In certain embodiments, the antibody formulation comprises 50 mg / mL human anti-PD-L1 antibody, 26 mM histidine / histidine-HCl buffer, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about 6.0. In another embodiment, the antibody formulation comprises 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about 5.5. In another embodiment, the antibody formulation comprises 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about 6.5. In another embodiment, the antibody formulation comprises 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM trehalose dehydrate, 0.04% (w / v) polysorbate 80, where the pH of the formulation is about 6.0.In another embodiment, the antibody formulation comprises 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM sucrose, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about 6.0. In another embodiment, the antibody formulation comprises 50 mg / mL human anti-PD-L1 antibody, 25 mM histidine / histidine-HCl buffer, 265 mM sucrose, 0.02% (w / v) polysorbate 80, where the pH of the formulation is about 6.0.

[0028] In certain embodiments, the human anti-PD-L1 antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2. In other embodiments, the human anti-PD-L1 antibody comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 3, a VH CDR2 having the amino acid sequence of SEQ ID NO: 4, a VH CDR3 having the amino acid sequence of SEQ ID NO: 5, a VL CDR1 having the amino acid sequence of SEQ ID NO: 6, a VL CDR2 having the amino acid sequence of SEQ ID NO: 7, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 8.

[0029] In certain embodiments, the human anti-PD-L1 antibody comprises a light chain variable domain comprising an amino acid sequence that is less than 100% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain variable domain comprising an amino acid sequence that is less than 100% identical to the amino acid sequence of SEQ ID NO:2. In other embodiments, the human anti-PD-L1 antibody comprises a light chain variable domain comprising an amino acid sequence that has 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the amino acid sequence of SEQ ID NO:1, and a heavy chain variable domain comprising an amino acid sequence that has 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0030] The terms "MEDI4736" and "durvalumab" as used herein refer to an antibody that selectively binds human anti-PD-L1 and interferes with the binding of PD-L1 to the PD-1 and CD80 receptors, as disclosed in U.S. Patent Nos. 8,779,108 and 9,493,565, each of which is incorporated by reference in its entirety. The fragment crystallizable (Fc) domain of durvalumab contains a triple mutation in the constant region of the IgG1 heavy chain that reduces binding to complement components C1q and Fcγ receptors, which are involved in mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Durvalumab can relieve PD-L1-mediated suppression of human T cell activation in vitro and inhibits tumor growth in xenograft models via a T cell-dependent mechanism.

[0031] As used herein, the phrases "pharmaceutical formulation," "formulation," and "antibody formulation" are used interchangeably and refer to a composition comprising an anti-PD-L1 antibody and one or more suitable buffers and / or excipients. Suitably, the pharmaceutical formulations described herein will be "pharmaceutical acceptable" and thus meet any necessary approval requirements required by federal or state government regulatory authorities or set forth in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and particularly humans.

[0032] The antibody formulations disclosed herein may be formulated as liquid, frozen, lyophilized, or reconstituted formulations.

[0033] Lyophilization may occur by drying in an oven, vacuum centrifuge, or other means known to those of skill in the art. Lyophilized durvalumab retains the activity of the anti-PD-L1 antibody when reconstituted.

[0034] The terms "stability" and "stable", as used herein in the context of a formulation comprising an anti-PD-L1 antibody, refer to the resistance of the antibody in the formulation to aggregation, degradation, or fragmentation under given manufacturing, preparation, shipping, and storage conditions. A "stable" formulation retains biological activity under given manufacturing, preparation, shipping, and storage conditions. Antibody stability can be assessed by the extent of aggregation, degradation, or fragmentation as measured by high pressure size exclusion chromatography (HP-SEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS binding techniques, in comparison to a reference formulation. The overall stability of formulations comprising human PD-L1 antibodies can be assessed by a variety of immunological assays, including, for example, ELISA and radioimmunoassays using isolated antigen molecules.

[0035] In certain embodiments, less than about 1% of the anti-PD-L1 antibody forms aggregates upon storage at 40°C for about one month, as determined by HP-SPEC. In other embodiments, at least 97% of the human anti-PD-L1 antibody is present as a monomer after storage at about 40°C for about one month, as determined by HP-SPEC. In other embodiments, at least 99% of the human anti-PD-L1 antibody is present as a monomer after storage at about 40°C for about one month, as determined by HP-SPEC. In other embodiments, at least 98% of the human anti-PD-L1 antibody is present as a monomer after storage at about 5°C for about one month, as determined by HP-SPEC. In certain embodiments, the antibody formulation maintains stability after at least three freeze / thaw cycles.

[0036] In certain embodiments, a composition disclosed herein comprises an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2; wherein the predominant form of the antibody comprises 45% or more of the protein in the composition as measured using capillary isoelectric focusing (cIEF) of the composition. In other embodiments, a composition disclosed herein comprises an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2; wherein the predominant form of the antibody comprises 45% or more of the protein in the composition as measured using cIEF of the composition; and wherein the acidic form of the antibody comprises 45%-50% of the protein in the composition as measured using cIEF of the composition. In another embodiment, a composition disclosed herein comprises an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; where the major form of the antibody comprises greater than 45% of the protein in the composition, as determined using cIEF of the composition; and where the basic form of the antibody comprises between 18% and 23% of the protein in the composition, as determined using cIEF of the composition. In another embodiment, a composition disclosed herein comprises an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; where the major form of the antibody comprises greater than 45% of the protein in the composition, as measured using cIEF of the composition; where the acidic form of the antibody comprises 45%-50% of the protein in the composition, as measured using cIEF of the composition; and where the base form of the antibody comprises 18%-23% of the protein in the composition, as measured using cIEF of the composition.

[0037] In certain embodiments, the compositions disclosed herein comprise an anti-PD-L1 antibody comprising a light chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2; wherein the glycan structure of the anti-PD-L1 antibody comprises G0f, G1f, G2f, and G0 glycoforms. In other embodiments, the glycan structure of the anti-PD-L1 antibody has a content of greater than about 90% for the G0f, G1f, G2f, and G0 forms. In some embodiments, the anti-PD-L1 antibody composition comprises a G0f content of about 65-75%, a G1f content of 13-23%, a G2f content of 0-3%, and a G0 content of 0-4%. In another embodiment, the anti-PD-L1 antibody composition comprises a G0f content of about 71.9%, a G1f content of 18.4%, a G2f content of 1.5%, and a G0 content of 1.9%.

[0038] In certain embodiments, the compositions disclosed herein comprise an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; where 1.5%-2.5% of the anti-PD-L1 antibody forms aggregates as determined by High Pressure Size Exclusion Chromatography (HP-SPEC), and where 97%-98% of the anti-PD-L1 antibody is present as a monomer as measured by HP-SEC.

[0039] Without limiting the disclosure, a number of embodiments of the present disclosure are described below for illustrative purposes. The following examples illustrate specific embodiments of the present invention and various uses thereof. They are provided for illustrative purposes only and are not to be considered as limiting the present invention. For example, the present invention encompasses the following embodiments: [Embodiment 1] (a) 40 mg / mL to 50 mg / mL of an anti-PD-L1 antibody; (b) 15 mM to 35 mM buffer; (c) 255 mM to 275 mM disaccharides; and (d) 0.01% (w / v) to 0.05% (w / v) of a surfactant; An antibody formulation comprising: The antibody formulation herein has a pH of from pH 5.5 to pH 7.2. [embodiment 2] The anti-PD-L1 antibody is: (a) a light chain variable domain comprising the amino acid sequence of SEQ ID NO:1, and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:2; or (b) a VH CDR1 having the amino acid sequence of SEQ ID NO:3; VH CDR2 having the amino acid sequence of SEQ ID NO:4; VH CDR3 having the amino acid sequence of SEQ ID NO:5; VL CDR1 having the amino acid sequence of SEQ ID NO:6; VL CDR2 having the amino acid sequence of SEQ ID NO:7; and VL CDR3 having the amino acid sequence of SEQ ID NO:8, 2. The antibody formulation of embodiment 1, comprising: [Embodiment 3] The antibody formulation of embodiment 1, wherein the buffer is a histidine / histidine-HCl buffer. [Embodiment 4] The antibody formulation of embodiment 1, wherein the disaccharide is trehalose dihydrate. [Embodiment 5] The antibody formulation of embodiment 1, wherein the disaccharide is sucrose. [Embodiment 6] The antibody formulation of embodiment 1, wherein the surfactant is polysorbate 80. [Embodiment 7] The antibody formulation of any one of embodiments 1 to 6, wherein the formulation is a liquid formulation, a frozen formulation, a lyophilized formulation, or a reconstituted formulation. [Embodiment 8] The antibody formulation of embodiment 1, wherein less than about 1% of the anti-PD-L1 antibody forms aggregates upon storage at 40°C for about one month, as measured by high pressure size exclusion chromatography (HP-SPEC). [Embodiment 9] The antibody formulation of embodiment 1, wherein at least 97% of the anti-PD-L1 antibody is present as a monomer after storage at about 40°C for about one month, as measured by high pressure size exclusion chromatography (HP-SEC). [Embodiment 10] The antibody formulation of embodiment 1, wherein at least 99% of the anti-PD-L1 antibody is present as a monomer after storage at about 40°C for about one month, as measured by high pressure size exclusion chromatography (HP-SEC). [Embodiment 11] The antibody formulation of embodiment 1, wherein at least 98% of the anti-PD-L1 antibody is present as a monomer after storage at about 5°C for about one month, as measured by high pressure size exclusion chromatography (HP-SEC). [Embodiment 12] The antibody formulation of embodiment 1, wherein the antibody formulation remains stable after at least three freeze / thaw cycles. [Embodiment 13] (a) 50 mg / mL of a human anti-PD-L1 antibody; (b) 26 mM histidine / histidine-HCl buffer; (c) 275 mM trehalose dihydrate; and (d) 0.02% (w / v) polysorbate 80; An antibody formulation comprising: wherein the pH of the formulation is pH 6.0. [Embodiment 14] (a) 50 mg / mL of a human anti-PD-L1 antibody; (b) 25 mM histidine / histidine-HCl buffer; (c) 265 mM trehalose dihydrate; and (d) 0.02% (w / v) polysorbate 80; An antibody formulation comprising: wherein the pH of the formulation is pH 5.5. [Embodiment 15] (a) 50 mg / mL of a human anti-PD-L1 antibody; (b) 25 mM histidine / histidine-HCl buffer; (c) 265 mM trehalose dihydrate; and (d) 0.02% (w / v) polysorbate 80; An antibody formulation comprising: An antibody formulation wherein the pH of the formulation is pH 6.5. [Embodiment 16] (a) 50 mg / mL of a human anti-PD-L1 antibody; (b) 25 mM histidine / histidine-HCl buffer; (c) 265 mM trehalose dihydrate; and (d) 0.04% (w / v) polysorbate 80; An antibody formulation comprising: wherein the pH of the formulation is pH 6.0. [Embodiment 17] (a) 50 mg / mL of a human anti-PD-L1 antibody; (b) 25 mM histidine / histidine-HCl buffer; (c) 265 mM sucrose; and (d) 0.02% (w / v) polysorbate 80; An antibody formulation comprising: wherein the pH of the formulation is pH 6.0. [Embodiment 18] The antibody formulation of any of Embodiments 13 to 17, wherein the human anti-PD-L1 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 1, and a heavy chain having the amino acid sequence of SEQ ID NO: 2. [embodiment 19] The human anti-PD-L1 antibody comprises: VH CDR1 having the amino acid sequence of SEQ ID NO:3; and VH CDR2 having the amino acid sequence of SEQ ID NO:4; and A VH CDR3 having the amino acid sequence of SEQ ID NO:5; and VL CDR1 having the amino acid sequence of SEQ ID NO:6; and VL CDR2 having the amino acid sequence of SEQ ID NO:7; and VL CDR3 having the amino acid sequence of SEQ ID NO:8, 18. The antibody formulation of any one of embodiments 13 to 17, comprising: [Embodiment 20] The antibody formulation of any one of embodiments 13 to 17, wherein the formulation is a liquid formulation, a frozen formulation, a lyophilized formulation, or a reconstituted formulation. [Embodiment 21] The antibody formulation of any of Embodiments 13-17, wherein less than about 1% of the human anti-PD-L1 antibodies form aggregates when stored at 40°C for about 1 month, as determined by high pressure size exclusion chromatography (HP-SPEC). [Embodiment 22] The antibody formulation of any of Embodiments 13-17, wherein at least 97% of the human anti-PD-L1 antibody is present as a monomer after storage at about 40°C for about one month, as measured by high pressure size exclusion chromatography (HP-SEC). [Embodiment 23] The antibody formulation of any of Embodiments 13-17, wherein at least 99% of the human anti-PD-L1 antibody is present as a monomer after storage at about 40°C for about 1 month, as measured by high pressure size exclusion chromatography (HP-SEC). [Embodiment 24] The antibody formulation of any of Embodiments 13-17, wherein at least 98% of the human anti-PD-L1 antibody is present as a monomer after storage at about 5°C for about one month, as measured by high pressure size exclusion chromatography (HPSEC). [Embodiment 25] The antibody formulation of any one of embodiments 13 to 17, wherein the antibody formulation maintains stability after at least three freeze / thaw cycles. [Embodiment 26] A composition comprising: (a) an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; and (b) a major form of the antibody that comprises 45% or more of the protein in the composition as determined using capillary isoelectric focusing (cIEF) of the composition; A composition comprising: [Embodiment 27] A composition comprising: (a) an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; (b) a major form of the antibody that comprises 45% or more of the protein in the composition as measured using cIEF of the composition; and (c) an acidic form of the antibody comprising 45% to 50% of the protein in the composition as measured using cIEF of the composition; A composition comprising: [Embodiment 28] A composition comprising: (a) an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; (b) a major form of the antibody that comprises 45% or more of the protein in the composition as measured using cIEF of the composition; and (c) the basic form of the antibody comprises between 18% and 23% of the protein in the composition as measured using cIEF of the composition; A composition comprising: [Embodiment 29] A composition comprising: (a) an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; (b) a major form of said antibody that comprises greater than 45% of the protein in said composition as measured using cIEF of said composition; (c) an acidic form of the antibody that comprises 45% to 50% of the protein in the composition as measured using cIEF of the composition; and (d) the basic form of the antibody comprises between 18% and 23% of the protein in the composition as measured using cIEF of the composition; A composition comprising: [Embodiment 30] A composition comprising: (a) an anti-PD-L1 antibody comprising a light chain that has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain that has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; and (b) the glycan structure of the anti-PD-L1 antibody comprises G0f, G1f, G2f, and G0 glycoforms. [Embodiment 31] The composition of embodiment 30, wherein the glycan structure of the anti-PD-L1 antibody has a content of the G0f, G1f, G2f, and G0 glycoforms that is greater than 90%. [Embodiment 32] The composition of embodiment 30, wherein the anti-PD-L1 antibody comprises a G0f content of 71.9%, a G1f content of 18.4%, a G2f content of 1.5%, and a G0 content of 1.9%. [Embodiment 33] A composition comprising: (a) an anti-PD-L1 antibody comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and a heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2; (b) between 1.5% and 2.5% of the anti-PD-L1 antibody forms aggregates, as determined by high pressure size exclusion chromatography (HP-SPEC); and (c) a composition, wherein 97% to 98% of the anti-PD-L1 antibody is present as a monomer, as measured by HP-SEC. [embodiment 34] The PD-L1 antibody comprises: VH CDR1 having the amino acid sequence of SEQ ID NO:3; and VH CDR2 having the amino acid sequence of SEQ ID NO:4; and A VH CDR3 having the amino acid sequence of SEQ ID NO:5; and VL CDR1 having the amino acid sequence of SEQ ID NO:6; and VL CDR2 having the amino acid sequence of SEQ ID NO:7; and VL CDR3 having the amino acid sequence of SEQ ID NO:8, 34. The composition of any one of embodiments 26 to 33, comprising: [Embodiment 35] (a) 50 mg / mL of a human anti-PD-L1 antibody; (b) 26 mM histidine / histidine-HCl buffer; (c) 275 mM trehalose dihydrate; and (d) 0.02% (w / v) polysorbate 80; An antibody formulation comprising: an antibody formulation, wherein the pH of the formulation is pH 6.0, and wherein the human anti-PD-L1 antibody comprises a light chain having the amino acid sequence of SEQ ID NO:1, and a heavy chain having the amino acid sequence of SEQ ID NO:2. EXAMPLES

[0040] Example 1 Materials and Methods 1. Protein Concentration Determination Durvalumab protein concentration was determined by measuring absorbance at 280 nm on an Aligent UV-V spectrophotometer. Dilutions were made in formulation buffer. Protein concentration was calculated by using a theoretical extinction coefficient of 1.55 (mg / mL)-1 cm-1. For some of the determinations, an empirical coefficient of 1.52 (mg / mL)-1 cm-1 was used in the calculation.

[0041] 2. High-pressure size-exclusion chromatography (HP-SEC) analysis Samples for HP-SEC analysis were initially eluted isocratically with 0.1 M disodium phosphate pH 6.8 containing 0.1 M sodium sulfate at a flow rate of 1.0 mL / min. Eluted protein was detected by absorbance at 280 nm. Results were reported as the percent area of ​​the product monomer peak compared to all other peaks. A buffer-related peak observed at approximately 12 minutes was excluded from the reported results. Any peaks eluting earlier than the monomer peak were recorded as percent aggregation. Peaks eluting after the monomer peak were recorded as percent fragments.

[0042] 3. Visual Analysis Visual inspection of the samples was performed using a light box with both a light and dark background to examine the glass vials for color, clarity, and the presence of particulates and fibers. Visible particles and clarity were evaluated.

[0043] 4. Visible particle analysis using high accuracy (HIAC) liquid particle counter Samples were diluted to 5 mg / mL with filtered formulation buffer and then allowed to sit for 30 minutes before analysis. Samples were prepared in Falcon tubes that were rinsed three times with ultrapure water. Samples with protein concentrations <5 mg / mL were analyzed as is. Six readings were taken for each sample using a Pacific Standard HIAC Royco 3000A and an 8-channel particle counter 8000A. The average of the final three readings was recorded as the final sub-visible particle count. The cumulative particle counts >10-25 μm were recorded.

[0044] 5. Osmolarity and pH Osmolality was measured using a Gonotec Osmomat 030-D osmometer. Solution pH was measured using a PHM220 Lab pH meter.

[0045] 6. Karl Fischer Analysis The presence of residual moisture in the lyophilized formulations was measured using Karl Fischer titration (Mettler Toledo). The lyophilized material was reconstituted using dry methanol. The residual moisture was determined based on the amount of water in the dry methanol and the weight of the total solids.

[0046] 7. Capillary Isoelectric Focusing (cIEF) Samples were adjusted to 0.25 mg / mL with HPLC grade water. Samples were digested with Carboxypeptidase B (CBP) for 10 minutes at 37° C. and then diluted with 1% methylcellulose solution, Pharmalyte pH 3-10, pI marker 9.46, and pI marker 5.85. Samples were loaded onto an iCE280 Analyzer and focused at 1500V for 1 minute, followed by 3000V for 7 minutes. The resulting electropherograms were analyzed using EZChrom software and compared to reference standards.

[0047] 8. Reducing and Non-reducing Gel Electrophoresis An Agilent 2100 BioAnalyzer with Protein 230 LabChip technology (Agilent) was used to analyze durvalumab by reducing and non-reducing gel electrophoresis. The flow paths of the LabChip allowed separation, staining, destaining, and detection. Samples and standards were adjusted to 4 mg / mL in PBS and mixed 1:1 with SDS, which denatured the sample buffer, in the presence of 60 mM N-ethylmaleimide (non-reduced) or 60 mM dithiothreitol (reduced). Samples were then heated, centrifuged, diluted with water, and loaded into wells on the LabChip. In the first dimension, proteins were separated with a resolution comparable to a 4-20% gradient gel. Proteins were separated in the second dimension by molecular weight. Fluorescent dyes present in the sample buffer were excited at 633 nm.

[0048] 9. Differential Scanning Calorimetry Differential scanning calorimetry (DSC) was used to determine the melting temperature (Tm1).

[0049] 10.Viscosity The viscosity of the samples and buffer solutions was measured using an Anton Paar AMVn Viscometer. Measurements were performed at target concentrations.

[0050] 11. Amino acid sequence analysis for hot spots The amino acid sequence of durvalumab was analyzed using BLAZE software to identify hotspot amino acid residues or potential modification sites. Hotspot reactivity was assigned a high, medium, or low risk score consistent with empirical sequence liability criteria using other monoclonal antibodies.

[0051] 12. Identification of N-linked oligosaccharides in durvalumab N-linked oligosaccharides in durvalumab, detected as significant peaks in ultra-performance liquid chromatography (UPLC), were identified using a Waters UPLC system with an FLR detector. Durvalumab reference standard (10.2 mg / mL; 100 μg), formulated in 26 mM histidine / histidine-HCl, 275 mM trehalose dihydrate, 0.02% (w / v) polysorbate 80, pH 6.0, was reconstituted to 0.5 mg / mL in 50 mM Tris buffer, pH 7.8. Samples were digested with 2 μL PNGase F (Promega), labeled with the fluorescent tag 2-aminobenzamide (2-AB; Sigma Aldrich), washed with a HILIC SPE cartridge, and eluted into water for UPLC profiling. The 2-AB labeled oligosaccharides were further digested with various exo-glycosides including fucosidase, sialidase A, mannosidase, β-galactosidase, and β-N-acetylhexosaminidase for peak identification.

[0052] Samples were injected onto an Acquity UPLC® BEH Glycan column (1.7 μM, 2.1×150 mm) on a Waters UPLC system using 50 mM ammonium formate, pH 4.4, as mobile phase A and 100% acetonitrile as mobile phase B. Data was collected and profiles of glycosidase digested samples were carefully compared to undigested samples to identify glycans and their oligosaccharide linkages.

[0053] Example 2 Development research The durvalumab formulation was determined after feasibility studies including hot spot analysis, melting temperature, isoelectric point (pI), and stability determination (Figure 1).

[0054] 1. Hotspot Analysis The stability of durvalumab during storage is essential for the efficacy of monoclonal antibodies. The risk of antibody storage is loss of activity due to the primary degradation pathway via aggregation. In addition, alterations of amino acid residues during long-term storage may affect the stability and activity of the antibody.

[0055] Loss of activity of durvalumab was assessed by homogeneous time-resolved fluorometry (HTRF). No loss of activity was observed after 1 month of incubation at 40°C in both high and low pH buffers. Only trace levels of Asp isomerization at D54(G) was detected by peptide mapping after 1 month in pH 6.0 buffer at 40°C.

[0056] 2. Determination of Melting Temperature, Isoelectric Point (pI), and Stability The melting point of the antibody was determined using differential scanning calorimetry (DSC) of durvalumab at 3 mg / mL in formulation buffer. The pI and stability of durvalumab were determined using capillary isoelectric focusing and high pressure size exclusion chromatography (HPSEC), respectively.

[0057] The DSC profile of durvalumab showed a Tm1 of 64.5 °C and a Tm2 of 73.04 °C (Figure 2). Four peaks ranging from 8.3 to 8.8 were detected in the capillary isoelectric focusing (cIEF) profile of durvalumab. The major peak had an isoelectric point of 8.6 (Figure 3). Liquid stability studies showed that clone 1 and clone 2 were stable in the default formulation after one month of incubation at either 5 °C or 40 °C (Figure 4). The results of the developability study are summarized in Table 1.

[0058] [Table 1]

[0059] Example 3 Lyophilized preparations Five formulations were screened to evaluate the stability of the liquid drug substance and the lyophilized drug product. The formulations screened are shown in Table 2.

[0060] An initial level of P80 (i.e., 0.02% w / v) was used. During subsequent formulation screening for lyophilized formulations, 0.04% (w / v) P80 (formulation 4) was also included if particles were observed upon reconstitution of formulations containing 0.02% (w / v) P80. Based on the data from this study, 0.02% (w / v) P80 was found to be the optimal surfactant level for durvalumab.

[0061] [Table 2]

[0062] The formulations were lyophilized for formulation screening studies. The formulations underwent freezing, annealing, primary drying, and secondary drying in a Virtis Genesis 25EL Freeze-Dryer. The parameters of the lyophilization cycle are provided in Table 3.

[0063] [Table 3]

[0064] 1. Stability After one month at 40° C., the rate of monomer loss for all lyophilized formulations was <0.3% per month (Table 4A). At 5° C., the rate of monomer loss per month for all formulations was <0.1% per month. All formulations were comparable in terms of aggregation after four weeks at 40° C. (Table 4B). Aggregation was not present for any of the formulations after four weeks at 40° C. (Table 4C).

[0065] Freeze-thaw studies were performed on all five formulations. The formulations underwent 0, 1, and 3 freeze-thaw cycles, consisting of freezing at -70°C and thawing at ambient temperature. Samples were visually inspected and analyzed by HP-SEC at the end of the cycles. HP-SEC results showed no significant change in purity by HP-SEC in any of the samples. All samples were substantially free of visible particles after three cycles.

[0066] [Table 4]

[0067] [Table 5]

[0068] [Table 6]

[0069] 2. Stability after reconstitution All five lyophilized formulations were tested for stability after storage and reconstitution at 5° C. and 25° C. for up to 24 hours. None of the five formulations showed a change in the number of visible particles or a significant decrease in monomer purity as assessed by SEC-HPLC. The results at TO are shown in Table 5A, at 5° C. for 24 hours in Table 5B, and at 25° C. for 24 hours in Table 5C.

[0070] [Table 7]

[0071] [Table 8]

[0072] [Table 9]

[0073] Freeze-thaw studies on durvalumab in a formulation of 3.26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0 The stability of durvalumab was investigated by assessing the degradation of the antibody structure on repeated freeze / thaw cycles. Consistent with this, the stability of durvalumab was tested using a freeze-thaw study.

[0074] A freeze-thaw study of durvalumab unformulated drug substance (UDS) was conducted on the formulation (26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0). Durvalumab was subjected to 0, 1, and 3 uncontrolled freeze-thaw cycles in 8 mL Nalgene bottles filled at 5.6 mL or in 30 mL Celsius Pak bags filled at 20 mL. Nalgene bottles were frozen at -80°C and Celsius Pak bags at -40°C, and both were thawed at ambient temperature. Samples were then analyzed for purity by high pressure size exclusion chromatography (HP-SEC) analysis, protein concentration as determined by A280, and appearance assessed after each freeze-thaw cycle.

[0075] Durvalumab was found to be stable after three freeze-thaw cycles, as shown below in Tables 6A (Nalgene bottle) and 6B (Celsius Pak).

[0076] [Table 10]

[0077] [Table 11]

[0078] These results indicate that durvalumab was stable after repeated freeze / thaw cycles in both Nalgene bottles and Celsius Paks.

[0079] Example 4: Formation and detection of pyroglutamic acid Durvalumab has an N-terminal glutamic acid on the heavy and light chains. Over time, the glutamic acid cyclizes to pyroglutamic acid. This conversion was detected in a cIEF assay after 3 months of storage at 25°C and 40°C as a peak at pI 8.8. A peak similarly occurred after 24 months at 2-8°C (Figure 4). Cyclization occurred in both lyophilized and liquid forms. A mutant durvalumab was created indicating that cyclization did not affect the potency of the antibody.

[0080] Example 5: Final lyophilized formulation parameters and stability The lyophilized formulation of 26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% (w / v) polysorbate 80, pH 6.0 contained 50 mg / mL durvalumab. The formulation parameters of 26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% polysorbate 80, pH 6.0 are shown in Table 7 and the stability of the lyophilized drug product in the final formulation is provided in Table 8. The solubility profile of the lyophilized drug substance in the final formulation in polypropylene tubes is shown in Table 9.

[0081] [Table 12]

[0082] [Table 13]

[0083] [Table 14]

[0084] Example 6 Freeze-drying cycle development Development of the lyophilization cycle resulted in the parameters shown in Table 10.

[0085] [Table 15]

[0086] Freeze-drying microscopy was used to assess the freeze-drying cycle temperatures for the onset of collapse (-33°C) and complete collapse (-27.3°C; Figure 6). The glass transition temperature (Tg') was determined to be -27.14°C by differential scanning calorimetry (DSC; Figure 7).

[0087] A design space model of the freeze-drying process was generated using Design Expert 7 using a design of experiments (DoE) approach. A robust conservative cycle of <7 days is achieved using the DoE approach. Selected operating conditions representing the midpoint (approximately Tg') and the four "corners" of the experimental space are listed in Table 11.

[0088] [Table 16]

[0089] All experimental cycles had the same ramp rate, freezing temperature, and secondary drying conditions as described in the final cycle. The final durvalumab lyophilization cycle conditions are shown in Table 12.

[0090] [Table 17]

[0091] The primary criteria in developing a lyophilization cycle were: (1) to maintain a product temperature above the collapse temperature (approximately equal to Tg'); (2) to ensure that the primary drying time did not exceed 115 hours to keep the cycle time below 6 days; and (3) to provide sufficient robustness for scale-up (shelf temperature of 2-3°C and chamber pressure of 40 mTorr).

[0092] The experimental runs in Table 11 yielded a design space for the freeze-drying process. The design space generated from five experimental runs is illustrated in Figure 8. Briefly, cycle midpoints and process robustness at chamber and shelf temperatures were determined. Data from the NMF Edwards freeze dryer showed convergence of the Pirani gauges (Δ10 ubar) at approximately 103-115 hours of primary drying. This equates to an approximate 10% safety margin (Figure 9). This is consistent with previous large scale runs, where the Amsco freeze dryer showed convergence of the product thermocouples within the allotted primary time (Figure 10).

[0093] Example 7 Liquid formulation development 1. Shaking studies to optimize surfactant levels The suitability of the liquid formulation of polysorbate 80 was evaluated through a shaking study in vials. Samples containing 0, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, and 0.05% (w / v) polysorbate 80 were placed in tubes with a worst case air to liquid volume ratio of 6.5 mL. The tubes were vigorously agitated at 600 rpm for 4 hours at ambient temperature. Samples were then examined by visual analysis, A280, BioA, HP-SEC, and microflow imaging (MFI). Control unpermeated tubes placed at 2-8°C were also analyzed.

[0094] No observable changes were noticed in the purity percentage as analyzed by BioA and HP-SEC. No changes in A280 protein concentration were caused by shaking. MFI analysis showed that all of the samples had low levels of >10 μm and >25 μm sub-visible particles (FIG. 11). Sub-visible particle levels were generally low in all tubes, and 0.02% polysorbate 80 was selected as the optimal surfactant level for the liquid formulation.

[0095] 2. Controlled Freeze-Thaw Studies A formulation of 26 mM histidine / histidine-HCl, 275 mM trehalose dehydrate, 0.02% polysorbate 80, pH 6.0 was subjected to five controlled freeze-thaw cycles, thawed at 5° C., and frozen at −40° C. At the end of the fifth freeze / thaw cycle, purity, appearance, sub-visible particles, and protein concentration were determined. No significant changes in product quality after the fifth freeze-thaw cycle were observed (Table 13).

[0096] [Table 18]

[0097] 3. Overview of formulation stability The stability profile of the lyophilized drug product in the final formulation is provided in Table 14. The results demonstrate that lyophilized durvalumab in the final formulation remains stable after long-term storage.

[0098] [Table 19]

[0099] Example 8 Identification of N-linked oligosaccharides The Fc region of durvalumab contains N-linked oligosaccharide chains attached to a single site on the heavy chain at Asn-301. Structural characterization of the oligosaccharides on durvalumab is essential for understanding the structural microheterogeneity of the product and is also important for quality control in the event of process variations.

[0100] Oligosaccharides cleaved from durvalumab that were present in the 2-AB labeled N-linked oligosaccharide profile were characterized by ultra-performance liquid chromatography (UPLC) and contained minor glycoforms. Digestion of the 2-AB labeled oligosaccharides with exo-glycosidases was performed to verify non-reducing terminal monosaccharide residues as described in Table 15. Oligosaccharide profiling was completed as shown in Table 16. LC / MS analysis was used to verify the molecular weights.

[0101] [Table 20]

[0102] [Table 21]

[0103] The N-linked oligosaccharides in durvalumab detected as significant peaks in UPLC were identified and the results are shown in Figure 12. The predominant glycoforms of durvalumab are fucosylated, biatennary, complex-type oligosaccharides without terminal galactose residues (G0f), or mono-galactosylated (G1f) and di-galactosylated (G2f) forms. The minor complex glycoforms were afucosylated G0 and G1, truncated G0f and G0 forms without N-acetylglucosamine (GlcNAc), sialylated G1f and G2f forms (G1f+NAc, G2f+NAc, or G2f+2NAc), and bisected structures G0fb and G1fb. Depending on the maturation of Chinese hamster ovary (CHO) cells when the IgG was harvested, high mannose glycoforms (Man4, Man5, Man6, Man7, and Man8) were also present.

[0104] It should be understood that the foregoing disclosure highlights certain specific embodiments of the present invention, and that all equivalent variations or alternatives are within the spirit and scope of the present invention as defined by the appended claims.

[0105] [Table 22]

Claims

[Claim 1] (a) durvalumab at 50 mg / mL; (b) 25 mM histidine / histidine-HCl buffer; (c) 265 mM sucrose; and (d) 0.02% (w / v) polysorbate 80; A stable liquid antibody formulation comprising: wherein the pH of the formulation is pH 6.0, and wherein the liquid formulation is stable over at least three freeze / thaw cycles.

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