Anti-PD-1 antibody preparations

Abstract

In some aspects, the present disclosure describes stable formulations comprising pembrolizumab, methods of making the stable pharmaceutical formulations, and methods of using the stable pharmaceutical formulations.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 358,033, filed July 1, 2022, entitled "ANTI-PD-1 ANTIBODY FORMULATIONS," which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (A117570000WO00-SEQ-AZW.xml; size: 8,546 bytes; created on June 27, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to stable formulations comprising pembrolizumab or a biosimilar thereof, and methods of making and using such formulations. [Background technology]

[0004] PD-1 is an inhibitory immune checkpoint present on the surface of T cells. Activation of the PD-1 signaling pathway through interaction of PD-1 with its ligands (e.g., PD-L1 or PD-L2) leads to the suppression of immune cell responses. This mechanism is observed in cancer and viral infections. For example, a mechanism used by cancer cells to evade immune cells is the secretion of ligands that target inhibitory immune checkpoints (e.g., PD-1) on the surface of T cells. Anti-PD-1 antibodies bind to PD-1 and block its interaction with its ligand, thereby preventing the inhibition of T cell responses against cancer cells. One such antibody is pembrolizumab, a recombinant monoclonal antibody specific for human PD-1. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure is directed to stable pharmaceutical formulations comprising pembrolizumab, methods of making stable pharmaceutical formulations comprising pembrolizumab, uses of the formulations disclosed herein, and methods of treating diseases comprising administering to a subject a formulation disclosed herein.

[0006] Surprisingly, some embodiments of the stable formulations disclosed herein have self-buffering capabilities and / or are stable for extended periods at 2-8°C and pH 5.3-5.8, e.g., 5.5, in the absence of one or more strong buffering agents, such as histidine, and / or one or more disaccharides. Commercially available pembrolizumab formulations are not recommended for storage at room temperature for more than 6 hours from the time of dilution, or at 2-8°C for more than 96 hours, even in the presence of one or more strong buffering agents (i.e., histidine) and disaccharides (e.g., sucrose).

[0007] In some embodiments, the disclosed formulations include an amino acid that was not previously known to be an effective stabilizing agent in monoclonal antibody formulations, let alone liquid formulations specific for PD-1 inhibitors at pH 5.2-5.8. For example, neither L-threonine nor asparagine would have been known to be an effective stabilizer in monoclonal antibody formulations, let alone PD-1-specific formulations, prior to this work. In some embodiments, the disclosure provides pharmaceutical formulations that include: (i) about 25 mg / mL to 200 mg / mL, e.g., about 25 mg / mL or about 50 mg / mL, of an anti-PD-1 antibody, such as pembrolizumab; (ii) a surfactant; and (iii) one or more stabilizers, e.g., two, three, four, or more stabilizers. In some embodiments, the disclosure provides a pharmaceutical formulation comprising: (i) an anti-PD-1 antibody, such as pembrolizumab, at about 25 mg / mL to 200 mg / mL, e.g., 25 mg / mL or 50 mg / mL; (ii) a surfactant; and (iii) one or more stabilizers, e.g., two, three, four, or more stabilizers; the formulation does not contain a buffer and has a pH of 5.2 to 5.8, e.g., 5.5.

[0008] In some embodiments, the disclosure provides a pharmaceutical formulation comprising (i) about 25 mg / mL or 50 mg / mL of pembrolizumab, (ii) a surfactant, and (iii) two stabilizers that are not disaccharide stabilizers and that do not have strong buffering capacity at pH 5.5.

[0009] In some embodiments, the anti-PD-1 antibody is pembrolizumab. It is understood that the term pembrolizumab includes biosimilars of pembrolizumab.

[0010] In some embodiments, the surfactant is a non-ionic surfactant, hi some embodiments, the surfactant is a polysorbate, such as polysorbate 80 (PS80) (comprising about 0.01% to 0.1% w / v, or 0.02% w / v, of PS80).

[0011] In some embodiments, the one or more stabilizers are amino acids. In some embodiments, the amino acid is glutamic acid, e.g., L-glutamic acid, proline, valine, L-threonine, asparagine, or any combination thereof. In some embodiments, the pembrolizumab formulation comprises two amino acids, L-glutamic acid and valine, and has a pH of about 5.2 to 5.8, e.g., about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, or about pH 5.8, including pH 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8. In some embodiments, the pembrolizumab formulation comprises two amino acids, L-glutamic acid and asparagine, and has a pH of about 5.2 to 5.8, e.g., about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, or about pH 5.8, including pH 5.2 and 5.8, e.g., 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8. In some embodiments, the pembrolizumab formulation comprises two amino acids, L-glutamic acid and L-threonine, and has a pH of about 5.2 to 5.8, e.g., about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, or about pH 5.8, including pH 5.2 to 5.8, e.g., pH 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8. In some embodiments, the amino acid stabilizer is valine. In some embodiments, the amino acid stabilizer is L-threonine.In some embodiments, the stabilizer is a high concentration of L-threonine, e.g., 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, 259 mM, 260 mM, 261 mM, 262 mM, 263 mM, 264 mM, 265 mM, 266 mM, 267 mM, 268 mM, 269 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 276 mM, 277 mM, 278 mM, 279 mM, 280 mM , 281mM, 282mM, 283mM, 284mM, 285mM, 286mM, 289mM, 290mM, 291mM, 292mM, 293mM, 294mM, 295mM, 296mM, 297mM, 298mM, 299mM, or 300mM, 100mM to 300mM, 1 In some embodiments, the stabilizer is greater than 100 mM L-threonine, including 00 mM to 271 mM, 150 mM to 300 mM, 150 mM to 271 mM, 200 mM to 300 mM, 200 mM to 271 mM, 225 mM to 275 mM, 225 mM to 271 mM, 250 mM to 275 mM, and 250 mM to 271 mM. In some embodiments, the amino acid stabilizer is proline. In some embodiments, the stabilizer is a polyol. In some embodiments, the polyol stabilizer is glycerol. In some embodiments, the polyol stabilizer is sorbitol. In some embodiments, the polyol stabilizer is mannitol. In some embodiments, the stabilizer is not a disaccharide. In other embodiments, the stabilizer is a disaccharide. In some embodiments, the disaccharide stabilizer is trehalose. In some embodiments, the disaccharide stabilizer is sucrose. In some embodiments, the formulation comprises about 10% w / v trehalose. In some embodiments, the formulation does not include sucrose, sorbitol, glycerol, and / or trehalose. In some embodiments, the formulation does not include histidine, sucrose, sorbitol, glycerol, trehalose, or any combination thereof. In some embodiments, the formulation does not include histidine, trehalose, sucrose, acetic acid, glycerol, or any combination thereof.

[0012] In some embodiments, the formulation comprises about 10 mM L-glutamic acid and about 2.5% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid and about 271 mM valine. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 65 mM valine, and about 2% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 5 mM valine, and about 2.5% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid and about 271 mM L-threonine. In some embodiments, the formulation comprises about 10 mM L-glutamic acid and about 250 mM L-threonine. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 30 mM L-threonine, and about 2% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 10 mM L-threonine, and about 2.5% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 100 mM proline, and about 1.6% w / v glycerol. In some embodiments, the formulation comprises about 100 mM proline and about 1.6% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid and about 10% w / v trehalose. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 1% w / v trehalose, and about 2% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 1% w / v sorbitol, and about 2% w / v glycerol. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 1% w / v mannitol, and about 2% w / v glycerol. In some embodiments, the formulation comprises about 2 mM acetic acid, about 10 mM L-glutamic acid, and about 2.5% w / v glycerol. In some embodiments, the formulation comprises about 10 mM lactic acid and about 2.5% w / v glycerol. In some embodiments, the formulation comprises about 10 mM lactic acid and about 271 mM L-threonine.

[0013] In some embodiments, the formulation comprises about 25 mg / mL to 50 mg / mL pembrolizumab, about 10 mM L-glutamic acid, about 271 mM valine, and about 0.02% w / v PS80 at pH 5.5.

[0014] In some embodiments, the formulation comprises about 25 mg / mL to 50 mg / mL pembrolizumab, about 10 mM L-glutamic acid, about 271 mM L-threonine, and about 0.02% w / v PS80 at pH 5.5.

[0015] In some embodiments, the formulation comprises about 25 mg / mL to 50 mg / mL pembrolizumab, about 10 mM L-glutamic acid, about 250 mM L-threonine, and about 0.02% w / v PS80 at pH 5.5.

[0016] In some embodiments, the disclosure provides a formulation comprising about 25 mg / mL or about 50 mg / mL of an anti-PD-1 antibody, wherein the formulation does not include a buffer and / or a disaccharide stabilizer.

[0017] In some embodiments, the formulation is self-buffering and / or does not contain a buffer, or the formulation is buffered at a pH that does not have strong buffering capacity.

[0018] In other embodiments, the formulation comprises one or more buffering agents, such as lactic acid or acetic acid. In some embodiments, the disclosed formulations do not comprise a histidine buffer.

[0019] In some embodiments, the present disclosure provides a method of treatment comprising administering a stable pharmaceutical formulation described herein to a subject having or at risk of developing a disease or condition. In some embodiments, the method further comprises administering a second therapeutic composition to the subject. In some embodiments, the disease or condition is selected from the group consisting of cancer, melanoma, renal cell carcinoma (RCC), non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma, endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), anaplastic thyroid cancer, and infectious diseases. In some embodiments, the subject is a mammal, hi some embodiments, the mammal is a human.

[0020] Each of the limitations of the present invention may encompass multiple embodiments of the present invention. Accordingly, it is anticipated that each of the limitations of the present invention, including any one element or combination of elements, may be included in each aspect of the present invention. The present disclosure is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways. Furthermore, the phraseology and terminology used in this application are for purposes of description and should not be regarded as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof, is meant to encompass the items listed thereafter and equivalents thereof, as well as additional items. The term "a" or "an" refers to one or more entities. [Brief explanation of the drawings]

[0021] [Figure 1]1 is a bar graph showing the aggregation rate in terms of percentage of high molecular weight species (HMW%) in exemplary formulations listed in Table 1 as measured by SE-UHPLC over a 4-week period at 5° C. On the x-axis for each sample, time points (0, 1, 2, 3, and 4 weeks) are indicated from left to right. [Figure 2] 1 is a graph showing the change in the amount of % HMW species in the formulations listed in Table 2 throughout the various steps of the scaled-down manufacturing process as measured by SE-UHPLC. [Figure 3] 1 is a graph showing the change in the amount of % HMW species in the formulations listed in Table 2 over 12 weeks at 5° C. as measured by SE-UHPLC. [Figure 4] 1 is a graph showing predicted end-to-end % HMW projections versus expected shelf life for 25 mg / mL and 50 mg / mL formulations at various time points. At post-formulation time points, the indicated 50 mg / mL samples were diluted to 25 mg / mL and monitored along with the 25 mg / mL samples through 3 years of storage. [Figure 5] 1 is a bar graph showing % HMW in 25 mg / mL and 50 mg / mL formulations as measured by SE-UHPLC over 0M, 3M, and an expected 24M time period at 5° C. [Figure 6] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) over time at 5° C. as measured by size exclusion chromatography. Glu / Thr (10 mM glutamic acid, 250 mM threonine) showed up to 0.5% less HMW aggregation compared to the RP formulation (RPF): 10 mM histidine, 7 w / v% sucrose over 104 weeks of stability at 5° C. [Figure 7] 1 is a graph showing the change in aggregation (percent high molecular weight, HMW%) from 0 to 104 weeks at 5° C. as measured by size exclusion chromatography. Glu / Thr (10 mM glutamate, 250 mM threonine; 0.1% HMW) showed less change in aggregation compared to RPF (10 mM histidine, 7% w / v sucrose; 0.2% HMW) after 104 weeks of stability at 5° C. [Figure 8] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) over time at 25° C. as measured by size exclusion chromatography. Glu / Thr (10 mM glutamate, 250 mM threonine) showed up to 0.4% less HMW aggregation compared to RPF (10 mM histidine, 7% w / v sucrose) over 24 weeks of stability at 25° C. [Figure 9] 1 is a graph showing the change in aggregation (percent high molecular weight, HMW%) from 0 to 24 weeks at 25° C. as measured by size exclusion chromatography. Glu / Thr (10 mM glutamate, 250 mM threonine; 0.1% HMW) showed comparable or less change in aggregation compared to RPF (10 mM histidine, 7% w / v sucrose; 0.2% HMW) over 24 weeks of stability at 25° C. [Figure 10] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) in various buffer / stabilizer combinations as measured by size exclusion chromatography. Glu-highThr (10 mM glutamic acid, 271 mM threonine) maintained a similar % HMW level to RPF (10 mM histidine, 7% w / v sucrose) over 4 weeks at 25° C. Glu-highThr maintained a lower % HMW than formulations with less threonine (Glu-lowThr-Glyc, Glu-medThr-Glyc) or no threonine (GluGlyc, Glyc). [Figure 11] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) in various buffer / stabilizer combinations as measured by size exclusion chromatography. Glu-highThr (10 mM glutamic acid, 271 mM threonine) maintained a similar % HMW level to RP (10 mM histidine, 7% w / v sucrose) over 8 weeks at 5° C. Glu-highThr maintained a lower % HMW than formulations with less threonine (Glu-lowThr-Glyc, Glu-medThr-Glyc) or no threonine (GluGlyc, Glyc). [Figure 12] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) over a 4 week period at 25° C. as measured by size exclusion chromatography. [Figure 13] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) over a 4 week period at 25° C. in various buffer / stabilizer combinations as measured by size exclusion chromatography. [Figure 14] 1 is a graph showing the amount of aggregation (high molecular weight percent, HMW%) over a 4 week period at 25° C. in various buffer / stabilizer combinations as measured by size exclusion chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0022] Commercially available anti-PD-1 antibody pharmaceutical formulations for pembrolizumab contain buffers, stabilizers, and surfactants for drug stability. However, it is known that some buffers are prone to self-decomposition under stress conditions, and some surfactants are decomposed by oxidation and hydrolysis, which can subsequently generate reactive oxygen species (ROS), which can cause oxidative damage to the antibody protein. Therefore, there is a need to develop a formulation that can stabilize proteins without using excipients that may affect the stability of anti-PD-1 antibody pharmaceutical formulations and that does not require the presence of a buffer.

[0023] The present disclosure provides stable pharmaceutical formulations comprising an anti-PD-1 antibody (e.g., pembrolizumab) or an antigen-binding fragment thereof, and methods of making stable pharmaceutical formulations comprising an anti-PD-1 antibody (e.g., pembrolizumab). Lyophilized forms of the stable pharmaceutical formulations disclosed herein are also provided. In other aspects, the present disclosure also provides uses of the formulations disclosed herein and methods of administering these formulations to treat diseases such as cancer and infectious diseases.

[0024] "Pembrolizumab" is a humanized antibody used in cancer immunotherapy and is marketed under the name KEYTRUDA®. Pembrolizumab targets the PD-1 receptor on lymphocytes. It is an IgG4 isotype antibody that blocks the protective mechanisms of cancer cells, allowing the immune system to destroy them. Pembrolizumab was first approved for pharmaceutical use in the United States in 2014 for the treatment of patients with unresectable or metastatic melanoma and disease progression after ipilimumab and, if BRAF V600-positive, a BRAF inhibitor. Additionally, pembrolizumab was approved in the US between 2014 and 2022 for the treatment of advanced non-small cell lung cancer; recurrent or metastatic squamous cell carcinoma of the head and neck; metastatic non-small cell lung cancer; classical Hodgkin lymphoma; metastatic non-squamous non-small cell lung cancer (NSCLC) (regardless of PD-L1 expression); locally advanced or metastatic urothelial carcinoma; any solid tumor with certain genetic characteristics such as mismatch repair deficiency or microsatellite instability; previously treated patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction cancer whose tumors express PD-L1; previously treated patients with recurrent or metastatic cervical cancer whose tumors express PD-L1; refractory or recurrent primary mediastinal large B-cell lymphoma (PMBCL); and EGF Treatment of patients with metastatic non-squamous NSCLC without R or ALK genomic tumor aberrations; in combination with carboplatin and either paclitaxel or nab-paclitaxel for the first-line treatment of patients with metastatic squamous non-small cell lung cancer (NSCLC); treatment of patients with hepatocellular carcinoma (HCC) previously treated with sorafenib; treatment of patients with recurrent locally advanced or metastatic Merkel cell carcinoma; adjuvant treatment of patients with melanoma, including lymph node(s) after complete resection; treatment in combination with axitinib as first-line treatment of patients with advanced renal cell carcinoma (RCC); first-line treatment of squamous cell carcinoma of the head and neck; treatment of metastatic small cell lung cancer (SCLC); treatment of recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus; treatment in combination with lenvatinib for patients with certain types of endometrial cancer; treatment of patients with BCG-refractory high-risk non-muscle-invasive bladder cancer;Treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that cannot be cured by surgery or radiation; first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer; treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL); treatment in combination with chemotherapy for patients with locally recurrent unresectable or metastatic triple-negative breast cancer whose tumors express PD-L1 (CPS ≥ 10); treatment with platinum- and fluoropyrimidine-based chemotherapy in certain patients with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) cancer; treatment in combination with trastuzumab and chemotherapy as first-line treatment of locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction adenocarcinoma; treatment in combination with trastuzumab and chemotherapy as first-line treatment of locally advanced endometrial cancer in combination with lenvatinib for patients with high-risk, early-stage triple-negative breast cancer; in combination with chemotherapy as neoadjuvant therapy followed by adjuvant therapy after surgery, followed by treatment as a single agent; in combination with lenvatinib for the first-line treatment of adult patients with advanced renal cell carcinoma (RCC); in combination with chemotherapy, with or without bevacizumab, for patients with persistent, recurrent, or metastatic cervical cancer whose tumors express PD-L1 (CPS ≥ 1); as adjuvant therapy for certain patients with renal cell carcinoma (RCC) after surgery; and in any setting, for the treatment of patients with MSI-H / dMMR advanced endometrial cancer whose disease has progressed after prior systemic therapy and who are not candidates for curative surgery or radiation therapy. Pembrolizumab can be used alone or in combination with chemotherapy. Pembrolizumab can be administered intravenously or subcutaneously.

[0025] Pembrolizumab can be produced by common methods known in the art. For example, U.S. Patent No. 9,834,605, the entire contents of which are incorporated herein by reference, describes a method that a person skilled in the art can use to prepare pembrolizumab antibodies. For example, pembrolizumab can be prepared by recombinant expression of immunoglobulin light chain and heavy chain genes in host cells.

[0026] Pembrolizumab is indicated for the treatment of melanoma, lung cancer, including non-small cell lung cancer (NSCLC), head and neck cancer, including head and neck squamous cell carcinoma (HNSCC), Hodgkin lymphoma, including classical Hodgkin lymphoma (cHL), B-cell lymphoma, including mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, renal cell carcinoma, gastric cancer, microsatellite instability-high (MSI-H) cancer, mismatch repair deficient (dMMR) cancer (including MSI-H and dMMR colorectal cancer), and other solid tumors, including cervical cancer, liver cancer, Merkel cell carcinoma (MCC), esophageal cancer, hepatocellular carcinoma (HCC), endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), and triple-negative breast cancer (TNBC). Pembrolizumab may also include a biosimilar of the active pembrolizumab present in the commercially available Keytruda. Pembrolizumab comprises a heavy chain variable region comprising SEQ ID NOs: 1-3 and a light chain variable region comprising SEQ ID NOs: 4-6. Pembrolizumab has a heavy chain CDR1 having the amino acid sequence (NYYMY) of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence (GINPSNGGTNFNEKFK) of SEQ ID NO: 2, and a heavy chain CDR3 having the amino acid sequence (RDYRFDMGFDY) of SEQ ID NO: 3, as well as a light chain CDR1 having the amino acid sequence 4 (RASKGVSTSGYSYLH), a light chain CDR2 having the amino acid sequence (LASYLES) of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence (QHSRDLPLT) of SEQ ID NO: 6. Pembrolizumab has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7. [ka] and a light chain variable region having the amino acid sequence of SEQ ID NO: 8. [ka] Includes:

[0027] As used herein, a "biosimilar," particularly a pembrolizumab biosimilar, is a biological product that is very similar to pembrolizumab, with minor differences in clinically inactive components, and there are no clinically important differences between the biological product and pembrolizumab in terms of product safety, purity, and potency. As used herein, the term "pembrolizumab" includes a pembrolizumab biosimilar.

[0028] As used herein, an "antibody" is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical and substantially full-length light (L) chains and two identical and substantially full-length heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among heavy chains of different immunoglobulin isotypes. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains (Chothia et al. J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592 (1985); Chothia et al., Nature 342:877-883 (1989)).

[0029] As used herein, "antibody fragment" includes a fragment of an antibody that binds to a target antigen. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments.

[0030] As used herein, the term "pharmaceutically effective amount" or "effective amount" means the amount of a therapeutic composition or formulation introduced into a patient that is sufficient to treat a disease or condition.

[0031] As used herein, the term "about," when modifying an amount of a substance or composition (e.g., mM or M), a percentage of a formulation component (v / v or w / v), the pH of a solution / formulation, or the value of a parameter determining a characteristic of a step in a method, refers to variations in the numerical quantity that may occur, for example, due to typical measuring, handling, and sampling procedures associated with preparing, characterizing, and / or using a substance or composition, due to errors in the instructions in these procedures, due to differences in the manufacture, source, or purity of components used to make or use the composition or perform the procedure, and the like. In certain embodiments, "about" can mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0032] As used herein, "x% (w / v)" corresponds to xg / 100ml (e.g., 5% w / v is equal to 50mg / ml).

[0033] As used herein, the term "pharmaceutical formulation" refers to a preparation in a form that allows the active ingredient to become effective. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout.

[0034] As used herein, a "stable formulation" is one in which the active ingredient therein, e.g., a protein, essentially retains its physical and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For example, in one embodiment, a stable formulation is one with no significant changes observed at refrigerated temperatures (2-8°C) for at least 12 months, such as at least 18 months or at least 24 months. In another embodiment, a stable formulation is one with no significant changes observed at refrigerated temperatures (2-8°C) for at least 18 months. In another embodiment, a stable formulation is one with no significant change observed at refrigerated temperatures (2-8°C) for at least 60 months. A stable formulation with no significant change is one in which no more than 10%, preferably no more than 5%, of the antibody monomer degrades as measured by SEC-HPLC or SEC-UHPLC. A stable formulation is colorless or clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of a stable formulation vary by no more than + / -10%. Potency is typically within 60-140%, preferably 80-120%, of the control or reference. In a stable formulation, no more than 10%, e.g., no more than 5%, of the antibody clipping is observed, e.g., as determined by HP-SEC ...

[0035] In embodiments, the stable pharmaceutical formulations described herein comprise an anti-PD-1 antibody (e.g., pembrolizumab) and one or more excipients selected such that the formulation exhibits suitable properties for use as a pharmaceutical composition. Formulations suitable for use as pharmaceutical compositions generally exhibit low amounts of high molecular weight species (referred to herein as "HMWS" or "HMW"), such as aggregates and dimers, and / or exhibit a low degree of oxidation over time. Suitable formulations may also exhibit minimal amounts of subvisible particles (e.g., <6000 per container for particles ≥10 μm in diameter, <600 per container for particles ≥25 μm in diameter) and / or non-spherical particles (e.g., particles with an aspect ratio ≥5 μm). High amounts of HMWS, oxidation, and / or particles may affect the shelf life, safety, and / or efficacy of the formulation. In some embodiments, the stable formulation can be maintained at 2-8°C, pH 5.2-5.8, e.g., pH 5.5, for at least 1 month, at least 2 months, at least 3 months, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 36 months, 48 months, or 60 months. and has a variation of HMW species of less than 5%, e.g., less than 1%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than about 0.04%, less than about 0.03%, less than 0.02%, e.g., 0.01% to 0.08%, 0.03% to 0.07%, or about 0.04% to 0.06%, e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, or 0.09%. Stable pharmaceutical formulations comprising an anti-PD-1 antibody (e.g., pembrolizumab) are described in the embodiments set forth below.

[0036] In some embodiments, the stable pharmaceutical formulations described herein are aqueous formulations. As used herein, an "aqueous" formulation contains water. Aqueous formulations can be in a liquid or frozen state, and are preferably liquid formulations.

[0037] As used herein, a "stable" formulation refers to a formulation that exhibits sufficient stability to permit administration to a patient. For example, a stable formulation may exhibit long-term stability, such as stability upon storage for three years after manufacture. Formulation stability may be assessed, for example, by the growth of high molecular weight species over time. When stability is assessed by the increase in HMWS over time, a stable formulation may exhibit less than about a 5% increase in HMWS species aggregation after storage at 2-8°C, e.g., 5°C, pH 5.2-5.8, pH 5.3-5.8, e.g., pH 5.5, for at least 4, 8, 12, 24, 36, 72, or 104 weeks, as measured by size-exclusion high-performance liquid chromatography (SE-HPLC) and / or size-exclusion ultra-high-performance liquid chromatography (SE-UHPLC). When stability is assessed by the increase in HMWS over time, a stable formulation may exhibit less than about a 5% increase in HMWS species after storage at 2-8°C, pH 5.5 for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, such as 3-36 months, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months, 36 months, 48 months, and 60 months, as measured by SE-HPLC.

[0038] The level of aggregation can be assessed using methods known to those of skill in the art, including SE-HPLC, size E (SE-HPLC), SE-UHPLC, cation exchange chromatography (CEX), and / or reduced capillary electrophoresis (rCE). In some embodiments, SE-HPLC is used to monitor monoclonal antibody aggregation. SE-HPLC is used to determine the amount of HMW species in an anti-PD-1 formulation, such as one or more of the disclosed pembrolizumab formulations, and to monitor the change in the % HMW species over a selected period of time. SE-HPLC is described herein and in the following publications: A.E. Hamielec, S.T. Balke, B.P. Leclair & S.L.Pearce; Ind. Eng. Chem., Prod. Res. Dev., 8, 54 (1969); Z. Grubistic, R. Rempp & H. Benoit; J. Polymer Sci., Part B, 5, 753 (1967); "Polymer Handbook", J. Brandrup, E.H. Immersut: John Wiley & Sons Publishers; Data Reduction in Multidetector Size Exclusion Chromatography, Y. Brun, J. Liq. Chrom&Rel. Technology, 21 (13), 1979-2015 (1998); Modern Size-Exclusion Liquid Chromatography, W.W. Yau, J.J. Kirkland, D.D.B.ly John Wiley & Sons, Inc., New This may be accomplished by techniques known to those skilled in the art, including those disclosed in The Journal of Applied Physiology or Biology, Vol. 1, No. 1, pp. 1979-2002, each of which is incorporated herein by reference in its entirety.

[0039] In some embodiments, the pharmaceutical formulations described herein comprise pembrolizumab and one or more (typically 1, 2, 3, 4, or 5) excipients described herein and are self-buffering. In some embodiments, the pharmaceutical formulations described herein comprise an anti-PD-1 antibody described herein (e.g., pembrolizumab), one or more stabilizing reagents, and one or more additional excipients.

[0040] As used herein, an "excipient" is a component of a formulation other than water and other than an active agent (e.g., an anti-PD-1 antibody) that is added to the formulation. Examples of excipients include buffers, stabilizers such as amino acids and amino acid derivatives, polyethylene glycol and polyethylene glycol derivatives, polyols, acids, amines, disaccharides or disaccharide derivatives, polysaccharides or polysaccharide derivatives, salts, and surfactants, and pH adjusters.

[0041] In some embodiments, disclosed herein are pharmaceutical formulations that are self-buffering and contain an anti-PD-1 antibody (e.g., pembrolizumab) in an environment that lacks any buffering agent or strong buffering capacity (e.g., pH 5.5 in the case of L-glutamic acid). These formulations further contain one or more (e.g., 1, 2, 3, 4, or 5) excipients described herein.

[0042] Examples of amino acids and amino acid derivatives that can be used as excipients include L-glutamic acid (e.g., concentrations of about 5 mM to about 50 mM, about 5 mM to about 25 mM, about 10 mM to about 20 mM, about 5 mM to about 15 mM, about 7 mM to about 12 mM, about 9 mM to about 11 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM, e.g., 5 mM to 50 mM, 5 mM to 25 mM, 10 mM to 20 mM, 5 mM to about 15 mM, 7 mM to 12 mM, 9 mM to 11 mM, e.g., 5 mM, 10 mM, 15 mM, 20 mM, and / or 25 mM); M), L-threonine (e.g., concentrations of about 0.1 to about 450 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, for example, 0.1 to 450 mM, 100 mM to 300 mM, 200 mM to 280 mM, 250 mM to 275 mM, for example, 100 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM) mM), N-acetylarginine (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 90 mM to about 150 mM), citrulline (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 100 mM to about 150 mM), sarcosine (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 100 mM to about 150 mM), N-acetylproline (for example, at a concentration of about 0.1 mM to about 450 mM, about 100 mM to about 150 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, and / or about 450 mM, for example, 0.1 mM to 450 mM, 100 mM to 300 mM, 200 mM to 280 mM, and / or 250 mM to 275 mM, for example, 100 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM), N-acetylornithine (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 100 mM to about 150 mM), ornithine (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 100 mM to about 150 mM), beta-alanine (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 100 mM to about 150 mM), alanine (for example, at a concentration of about 0.1 mM to about 450 mM and / or about 80 mM to about 120 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, and / or about 450 mM, for example, 0.1 mM to 450 mM M, 100 mM to 300 mM, 200 mM to 280 mM, 250 mM to 275 mM, for example, 100 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM), asparagine (for example, concentrations of 0.1 mM to about 450 mM, about 80 mM to about 120 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, and / or about 450 mM, for example, 0.1 mM to 450 mM, 100 mM to 300 mM, 80 mM to 120 mM, 200 mM to 280 mM, 250 mM to 275 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM), isoleucine (for example, concentrations of about 0.1 mM to about 450 mM, about 80 mM to about 120 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, and / or about 450 mM, e.g., 0.1 mM to 450 mM, 100 mM to 300 mM, 80 to 120 mM, 200 mM to 280 mM, 250 mM to 275 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, or 450 mM), serine (e.g., at a concentration of about 0.1 mM to about 450 mM, about 80 mM to about 120 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, for example, 0.1 to 450 mM, 100 mM to 300 mM, 80 to 120 mM, 200 mM to 280 mM, 250 mM to 275 mM, for example, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, or 450 mM), aspartic acid (for example, concentrations of about 0.1 mM to about 450 mM, about 10 mM to about 30 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, or about 450 mM, for example, 0.1 mM to 450 mM, 10 mM to 30 mM, 100 mM 10 mM to 300 mM, 200 mM to 280 mM, 250 mM to 275 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, 450 mM), creatine (for example, concentrations about 0.1 mM to about 450 mM, about 15 mM to about 35 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, e.g., 0.1 to 450 mM, 10 mM to 30 mM, 100 mM to 300 mM, 200 mM to 280 mM, 250 mM to 275 mM, e.g., 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, 450 mM), glutamine (e.g., concentration about 0.1 mM to about 450 mM, about 40 to about 60 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, for example, 0.1 to 450 mM, 40 mM to 60 mM, 100 mM to 300 mM, 200 mM to 280 mM, 250 mM to 275 mM, for example, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM), phenylalanine (for example, concentrations of about 0.1 mM to about 450 mM, about 40 mM to about 60 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM to about 3 00 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, and / or about 450 mM, for example, 0.1 to 450 mM, 40 to 60 mM, 100 mM to 300 mM, 200 mM to 280 mM, 250 mM to 275 mM, for example, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM), tryptophan (e.g., at concentrations of about 0.1 mM to about 450 mM, about 15 mM to about 35 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, and / or about 450 mM, for example, 0.1 to 450 mM, 15 mM to 35 mM, 100 mM to 300 mM, 80 to 120 mM, 200 mM to 280 mM, 250 mM to 275 mM, e.g., 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM , 300 mM, 350 mM, 400 mM, and / or 450 mM), and arginine-HCl (e.g., concentrations of about 0.1 mM to about 450 mM, about 80 mM to about 120 mM, about 100 mM to about 300 mM, about 200 mM to about 280 mM, about 250 mM to about 275 mM, about 100 mM, about 200 mM, about 225 mM, about 250 mM, about 271 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, e.g., 0.Examples of such concentrations include 1 to 450 mM, 100 mM to 300 mM, 80 to 120 mM, 200 mM to 280 mM, and / or 250 mM to 275 mM, for example, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 200 mM, 225 mM, 250 mM, 271 mM, 300 mM, 350 mM, 400 mM, and / or 450 mM. In some embodiments, high concentrations of L-threonine, e.g., greater than 100 mM (100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, 259 mM, 260 mM, 261 mM, 262 mM, 263 mM, 264 mM, 265 mM, 266 mM, 267 mM, 268 mM, 269 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 276 mM, 277 mM, 278 mM, 279 mM, 280 mM, 281 mM, 282 mM, 283 mM, 284 mM, 285 mM, 286 mM, 287 mM, 288 mM, 289 mM, 290 mM, 291 mM, 292 mM, 293 mM, 294 mM, 295 mM, 296 mM, 297 mM, 298 mM, 299 mM, 300 mM, 301 mM, 302 mM, 303 mM, 304 mM, 305 mM, 306 mM, 307 mM, 308 mM, 309 mM, 310 mM, 311 mM, 312 mM, 313 mM, 314 mM, 315 mM, 316 mM, 317 mM, 318 mM, 319 mM, 320 mM, 321 mM, 322 mM, 323 mM, 324 mM 1mM, 262mM, 263mM, 264mM, 265mM, 266mM, 267mM, 268mM, 269mM, 270mM, 271mM, 272mM , 273mM, 274mM, 275mM, 276mM, 277mM, 278mM, 279mM, 280mM, 281mM, 282mM, 283mM, 284 100mM to 450mM, including 285mM, 286mM, 289mM, 290mM, 291mM, 292mM, 293mM, 294mM, 295mM, 296mM, 297mM, 298mM, 299mM, 300mM, 325mM, 350mM, 375mM, 400mM, 425mM, or 450mM L-threonine concentrations include: 100mM-300mM, 100mM-271mM, 150mM-300mM, 150mM-271mM, 200mM-300mM, 200mM-271mM, 225mM-275mM, 225mM-271mM, 250mM-275mM, and 250mM-271mM.

[0043] The excipient may also include suitable polyethylene glycols and polyethylene glycol derivatives, such as PEG 15 hydroxystearate (e.g., at a concentration of about 0.1% (w / v) to about 20% (w / v) or about 3% (w / v) to about 6% (w / v)), PEG 3350 (e.g., at a concentration of about 0.1% (w / v) to about 30% (w / v) or about 1% (w / v) to about 7% (w / v)), PEG 200 (e.g., at a concentration of about Examples of such an PEG-400 include, but are not limited to, PEG-400 (for example, at a concentration of about 0.1% (w / v) to about 10% (w / v) or about 0.6% (w / v) to about 4.8% (w / v)), PEG600 (for example, at a concentration of about 0.1% (w / v) to about 30% (w / v) or about 1.2% (w / v) to about 14.5% (w / v)), and PEG400 (for example, at a concentration of about 0.1% (w / v) to about 20% (w / v) or about 0.3% (w / v) to about 1.5% (w / v)).

[0044] The excipient can also include a suitable polyol, such as inositol (e.g., at a concentration of about 0.1 to about 450 mM, or about 150 to about 210 mM), glycerol (also called glycerin) (e.g., at a concentration of about 0.1% (w / v) to about 15% (w / v), or about 0.5% (w / v) to about 1% (w / v)), sucrose (e.g., at a concentration of about 0.1% (w / v) to about 15% (w / v), about 4% (w / v) to about 10% (w / v), about 6% (w / v), Examples of suitable sorbitol include, but are not limited to, sorbitol (for example, at a concentration of about 0.1% (w / v) to about 10% (w / v), about 3% (w / v) to about 5% (w / v), and / or about 4% (w / v)). Examples of suitable acids include glycolic acid (e.g., at a concentration of about 0.1 to about 300 mM or about 50 to about 70 mM), pyrrolidonecarboxylic acid (PCA) (e.g., at a concentration of about 0.1% (w / v) to about 15% (w / v) or about 0.05% (w / v) to about 2% (w / v)), medronic acid (e.g., at a concentration of about 0.1 to about 450 mM or about 100 to about 150 mM), benzenesulfonic acid (e.g., at a concentration of about 0.1 to about 300 mM or about 60 to about 90 mM), and methanesulfonic acid (MSA) (e.g., at a concentration of about 0.1 to about 150 mM, about 0.1 to about 50 mM, and / or about 10 to about 30 mM). Examples of suitable amines include monoethanolamine hydrochloride (MEA-HCl) (e.g., at a concentration of about 0.1 to about 150 mM or about 0.1 to about 40 mM), monoethanolamine (MEA) (e.g., at a concentration of about 0.1 to about 300 mM, about 0.1 to about 50 mM, and / or about 30 to about 160 mM), and triethanolamine (TEA) (e.g., at a concentration of about 0.1 to about 170 mM or about 30 to about 150 mM).

[0045] The excipient may also include a suitable polysaccharide or polysaccharide derivative, including, but not limited to, hyaluronic acid (e.g., at a concentration of about 0.05% (w / v) to about 2.5% (w / v) or about 0.1% (w / v) to about 0.05% (w / v)), sodium carboxymethylcellulose (NaCMC) (e.g., at a concentration of about 0.1% (w / v) to about 15% (w / v) or about 0.1% (w / v) to about 2% (w / v)), and dextran (e.g., at a concentration of about 0.1% (w / v) to about 15% (w / v) or about 8% (w / v) to about 12% (w / v)).

[0046] Examples of suitable surfactants include Pluronic F68 (e.g., concentrations of about 0.001% (w / v) to about 10% (w / v), about 0.005% (w / v) to about 1% (w / v), about 0.05% (w / v) to about 0.4% (w / v), about 0.05% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.2% (w / v), about 0.03% (w / v) to about 0.06% (w / v), about 0.01% (w / v), about 0.05% (w / v), about 0.06% (w / v), and and / or about 0.1% (w / v)), polysorbate 80 (for example, at a concentration of about 0.001% (w / v) to about 2% (w / v), about 0.005% (w / v) to about 0.1% (w / v), about 0.03% (w / v) to about 0.1% (w / v), about 0.04% (w / v) to about 0.1% (w / v), about 0.02 (w / v), about 0.03% (w / v), about 0.04% (w / v), for example, 0.01% (w / v), 0.02% (w / v), 0 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), 0.1% (w / v)), polysorbate 20 (for example, at a concentration of about 0.001% (w / v) to about 2% (w / v) or about 0.005% (w / v) to about 0.1% (w / v)), docusate sodium (for example, at a concentration of about 0.001% (w / v) to about 1% (w / v) or about 0.005% (w / v) to about 0.05% (w / v)), benzalkonium chloride (for example, at a concentration of about 0.001% (w / v) to about 1% (w / v) or about 0.05% (w / v) to about 0.5% (w / v)), Span 40 (sorbitan monopalmitate) (for example, at a concentration of about 0.001% (w / v) to about 1% (w / v) or about 0.05% (w / v) to about 0.5% (w / v)), and Triton X-100 (for example, at a concentration of about 0.001% (w / v) to about 1% (w / v) or about 0.005% (w / v) to about 0.1% (w / v)).

[0047] Other suitable examples of excipients include imidazole (for example, at a concentration of about 0.1% (w / v) to about 15% (w / v) or about 0.5% (w / v) to about 2% (w / v)), taurine (for example, at a concentration of about 0.1 to about 450 mM or about 100 to about 150 mM), betaine (for example, at a concentration of about 0.1 to about 450 mM or about 100 to about 150 mM), gelatin (for example, at a concentration of about 0.1% (w / v) to about 15% (w / v) or about 0.5% (w / v) to about 2% (w / v)), niacinamide (for example, at a concentration of about 0.1 to about 450 mM or about 100 to about 120 mM), polyvinylpyrrolidone (PVP), such as 10K Examples of such an agent include PVP (for example, at a concentration of about 0.001% (w / v) to about 10% (w / v) or about 0.05% (w / v) to about 2% (w / v)), guanidine hydrochloride (GnHCl) (for example, at a concentration of about 0.1 to about 150 mM or about 10 to about 30 mM), and ethanol (for example, at a concentration of about 0.05% (w / v) to about 2.5% (w / v) or about 0.25% (w / v) to about 1% (w / v)).

[0048] Optionally, the pharmaceutical formulation has a pH of about 4.8 to about 5.8, e.g., pH 5.2 to 5.8, e.g., about 4.9 to about 5.6, about 5.0 to about 5.5, about 5.1 to about 5.2, about 5.1 to about 5.4, about 5.1 to about 5.3, about 5.2 to about 5.3, about 5.2 to about 5.8, about 5.3 to about 5.5, about 5.4 to about 5.5, about 5.4 to about 5.6, about 5.5 to about 5.6, about 5.5 to about 5.7, about 5.5 to about 5.8, about 5. 5.6 to about 5.7, about 5.7 to about 5.8, 4.8 to 5.5, 5.3 to 5.6, 5.4 to 5.6, 5.4 to 5.5, 5.4 to 5.7, 5.5 to 5.6, 5.5 to 5.7, 5.5 to 5.8, such as about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, and / or about 5.8, e.g., 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8. Optionally, the pH of the stable pharmaceutical formulation is adjusted using a strong acid and / or a strong base, including, but not limited to, hydrochloric acid or sodium hydroxide.

[0049] In some embodiments, the present disclosure provides a stable pharmaceutical formulation comprising (i) an anti-PD-1 antibody, (ii) a surfactant, and (iii) a stabilizer. In some embodiments, the disclosure provides a stable pharmaceutical formulation comprising anti-PD-1, wherein the formulation does not contain a buffer or an excipient with strong buffering capacity in a particular environment, such as a particular pH. In some embodiments, the formulation does not contain histidine. In embodiments, the formulation does not contain a disaccharide, such as sucrose or trehalose, and / or a buffer, such as histidine, acetate, or citrate, or any other excipient with strong buffering capacity at pH 5.5-5.8, e.g., pH 5.5. A solution with strong or significant buffering capacity is one whose pH is ±1 unit from the pKa. In this pH range, significant amounts of both the conjugate base species and the conjugate acid species are present in the solution. Histidine has three pKas: 1.5, 6.0, and 9, acetic acid has a pKa of 4.8, and citric acid has three pKas: 3.1, 4.8, and 6.4. Therefore, histidine, acetic acid, and citric acid have significant buffering capacity at pH 5.5 and are considered strong buffers at this pH (e.g., pH 5.5 is within ±1 unit of the pKa of each molecule).

[0050] In some embodiments, the formulations described herein contain the anti-PD-1 antibody at a concentration of about 25 mg / mL, e.g., 25 mg / mL. In some embodiments, the formulations described herein contain the anti-PD-1 antibody at a concentration of about 50 mg / mL, e.g., 50 mg / mL. In some embodiments, the concentration of the anti-PD-1 antibody in the formulation is about 5 mg / mL to about 200 mg / mL, e.g., 5 mg / mL to 200 mg / mL. In some embodiments, the concentration of the anti-PD-1 antibody in the formulation is about 5 mg / mL to about 500 mg / mL, e.g., 5 mg / mL to 500 mg / mL. In some embodiments, the concentration of the anti-PD-1 antibody in the formulation is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, The concentration of the anti-PD-1 antibody in the formulation is about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 165 mg / mL, about 167 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 220 mg / mL, about 240 mg / mL, about 250 mg / mL, about 300 mg / mL, about 350 mg / mL, about 400 mg / mL, about 450 mg / mL, or about 500 mg / mL. In some embodiments, the amount of anti-PD-1 antibody in the formulation is between 200 mg / mL and 500 mg / mL, e.g., about 200 mg / mL, about 250 mg / mL, about 300 mg / mL, about 350 mg / mL, about 400 mg / mL, about 450 mg / mL, or about 500 mg / mL.In some embodiments, the amount of anti-PD-1 antibody contained in the formulation is about 5 mg / mL to about 10 mg / mL, about 5 mg / mL to about 20 mg / mL, about 5 mg / mL to about 30 mg / mL, about 5 mg / mL to about 40 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 60 mg / mL, about 5 mg / mL to about 75 mg / mL, about 10 mg / mL to about 30 mg / mL, about 10 mg / mL to about 40 mg / mL, Approximately 10 mg / mL to approximately 50 mg / mL, approximately 10 mg / mL to approximately 75 mg / mL, approximately 25 mg / mL to approximately 50 mg / mL, approximately 50 mg / mL to approximately 200 mg / mL, approximately 75 mg / mL to approximately 200 mg / mL, approximately 100mg / mL to approx. 200mg / mL, approx. 25mg / mL to approx. 175mg / mL, approx. 50mg / mL to approx. 175mg / mL, approx. 75mg / mL to approx. 175mg / mL, approx. 100mg / mL to approx. 175mg / mL mL, about 25 mg / mL to about 150 mg / mL, about 50 mg / mL to about 150 mg / mL, about 75 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, about 25 mg / mL to about 12 5mg / mL, about 50mg / mL to about 125mg / mL, about 75mg / mL to about 125mg / mL, about 25mg / mL to about 100mg / mL, about 125mg / mL to about 175mg / mL, about 125mg / mL about 200 mg / mL, about 5 mg / mL to 200 mg / mL, about 150 mg / mL to about 300 mg / mL, about 150 mL to about 400 mg / mL, about 150 mL to about 450 mg / mL, about 150 mg / mL to about 250 mg / mL, about 200 mg / mL to about 500 mg / mL, about 200 mg / mL to about 300 mg / mL, about 200 mg / mL to 400 mg / mL, or about 200 mg / mL to about 500 mg / mL.

[0051] In some embodiments, the formulations described herein contain pembrolizumab at a concentration of 25 mg / mL. In some embodiments, the formulations described herein contain pembrolizumab at a concentration of 50 mg / mL. In some embodiments, the concentration of pembrolizumab in the formulation is about 5 mg / mL to about 200 mg / mL, e.g., 5 mg / mL to 200 mg / mL. In some embodiments, the concentration of pembrolizumab in the formulation is about 25 mg / mL to about 200 mg / mL. In some embodiments, the concentration of pembrolizumab in the formulation is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 140 mg / mL , about 150 mg / mL, about 160 mg / mL, about 165 mg / mL, about 167 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, about 300 mg / mL, about 350 mg / mL, about 400 mg / mL, about 450 mg / mL, or about 500 mg / mL.In some embodiments, the amount of pembrolizumab included in the formulation is about 5 mg / mL to about 10 mg / mL, about 5 mg / mL to about 20 mg / mL, about 5 mg / mL to about 30 mg / mL, about 5 mg / mL to about 40 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 60 mg / mL, about 5 mg / mL to about 75 mg / mL, about 10 mg / mL to about 30 mg / mL, or about 10 mg / mL to about 40 mg / mL. g / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 75 mg / mL, about 50 mg / mL to about 200 mg / mL, about 75 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200mg / mL, about 25mg / mL to about 175mg / mL, about 50mg / mL to about 175mg / mL, about 75mg / mL to about 175mg / mL, about 100mg / mL to about 175mg / mL, about 25mg / mL ~ approx. 150 mg / mL, approx. 50 mg / mL ~ approx. 150 mg / mL, approx. 75 mg / mL ~ approx. 150 mg / mL, approx. 100 mg / mL ~ approx. 150 mg / mL, approx. 25 mg / mL ~ approx. 125 mg / mL , about 50 mg / mL to about 125 mg / mL, about 75 mg / mL to about 125 mg / mL, about 25 mg / mL to about 100 mg / mL, about 125 mg / mL to about 175 mg / mL, about 125 mg / mL to about 20 0 mg / mL, about 5 mg / mL to 200 mg / mL, about 150 mg / mL to about 300 mg / mL, about 150 mL to about 400 mg / mL, about 150 mL to about 450 mg / mL, about 150 mg / mL to about 250 mg / mL, about 200 mg / mL to about 500 mg / mL, about 200 mg / mL to about 300 mg / mL, about 200 mg / mL to 400 mg / mL, or about 200 mg / mL to about 500 mg / mL.

[0052] In some embodiments, the concentration of pembrolizumab in the formulation is 5 mg / mL, 10 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 140 mg / mL , 150mg / mL, 160mg / mL, 165mg / mL, 167mg / mL, 170mg / mL, 175mg / mL, 180mg / mL, 190mg / mL, 200mg / mL, 210mg / mL mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, or 500 mg / mL.In some embodiments, the amount of pembrolizumab in the formulation is 5 mg / mL to 10 mg / mL, 5 mg / mL to 20 mg / mL, 5 mg / mL to 30 mg / mL, 5 mg / mL to 40 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 60 mg / mL, 5 mg / mL to 75 mg / mL, 10 mg / mL to 30 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 75 mg / mL, 50 mg / mL to 200 mg / mL, 75 mg / mL to 200 mg / mL, 100 mg / mL to 200 mg / mL, 25 mg / mL to 175 mg / mL, 50 mg / mL to 175 mg / mL, 75 mg / mL to 175 mg / mL, 100 mg / mL to 175 mg / mL, 25 mg / mL to 30 mg / mL, 30 mg / mL to 40 mg / mL, 30 mg / mL to 50 mg / mL, 3 ...25 mg / mL~150mg / mL, 50mg / mL~150mg / mL, 75mg / mL~150mg / mL, 100mg / mL~150mg / mL, 25mg / mL~125mg / mL, 50mg / mL~125mg / mL, 75mg / mL~125mg / mL, 25mg / mL~100mg / mL, 125mg / mL~175mg / mL, 125mg / mL~200m g / mL, 5mg / mL~200mg / mL, 150mg / mL~300mg / mL, 150mL~400mg / mL, 150mL~450mg / mL, 150mg / mL~250m g / mL, 200 mg / mL to 500 mg / mL, 200 mg / mL to 300 mg / mL, 200 mg / mL to 400 mg / mL, or 200 mg / mL to 500 mg / mL.

[0053] In some embodiments, the stabilizer is an amino acid. In some embodiments, the amino acid stabilizer is valine. In some embodiments, the amino acid stabilizer is L-threonine. In some embodiments, the stabilizer is L-glutamic acid. In some embodiments, the amino acid stabilizer is proline. In some embodiments, the stabilizer is a polyol. In some embodiments, the polyol stabilizer is glycerol. In some embodiments, the polyol stabilizer is sorbitol. In some embodiments, the polyol stabilizer is mannitol. In some embodiments, the stabilizer is a disaccharide. In some embodiments, the disaccharide stabilizer is trehalose. In some embodiments, the disaccharide stabilizer is sucrose. In some embodiments, one or more stabilizers, such as two, three, four, or more, are included in the formulation. In some embodiments, two stabilizers are included in the formulation. In some embodiments, L-glutamic acid and L-threonine are included in the formulation as stabilizers that do not include trehalose and / or sucrose. In some embodiments, the one or more stabilizers are not disaccharides.

[0054] In some embodiments, the formulation includes glycerol. In some embodiments, the formulation includes trehalose. In some embodiments, the formulation includes L-glutamic acid and glycerol. In some embodiments, the L-glutamic acid included in the formulation is a stabilizer. In some embodiments, the formulation includes L-glutamic acid and valine. In some embodiments, the formulation including L-glutamic acid and valine further includes glycerol. In some embodiments, the L-glutamic acid or valine included in the formulation, or a combination of both L-glutamic acid and valine included in the formulation, is a buffer. In some embodiments, the formulation includes L-glutamic acid and L-threonine. In some embodiments, the formulation including L-glutamic acid and L-threonine further includes glycerol. In some embodiments, the formulation including L-glutamic acid and L-threonine further includes a polysorbate, such as PS20 or PS80. In some embodiments, the formulation comprising L-glutamic acid and L-threonine further comprises PS80 and does not comprise histidine, sucrose, and / or trehalose. In some embodiments, the formulation comprises L-glutamic acid and / or L-threonine, or a combination of both L-glutamic acid and L-threonine, or a combination of both L-glutamic acid and L-threonine, and the formulation has a pH of 5.2 to 5.8, e.g., pH 5.5. In some embodiments, the formulation comprises L-glutamic acid and proline. In some embodiments, the formulation comprising L-glutamic acid and proline further comprises glycerol. In some embodiments, the formulation comprises proline and glycerol. In some embodiments, the formulation comprises L-glutamic acid and trehalose. In some embodiments, the formulation comprises L-glutamic acid, trehalose, and glycerol. In some embodiments, the formulation comprises L-glutamic acid, sorbitol, and glycerol. In some embodiments, the formulation comprises L-glutamic acid, mannitol, and glycerol. In some embodiments, the formulation comprises acetic acid, L-glutamic acid, and glycerol.

[0055] In some embodiments, the formulation is at pH 5.5 and includes one or more amino acids for stabilization, but does not include amino acids with strong buffering capacity at pH 5.5. For example, the pKa of the side chain of L-glutamic acid is about 4.2, and the molecule does not have significant buffering capacity at pHs of about 5.5, including pH 5.5. Therefore, L-glutamic acid is not a strong buffering agent at this pH, nor in any of the disclosed formulations containing L-glutamic acid at pH 5.5.

[0056] In some embodiments, self-buffering formulations comprising a protein are disclosed, specifically pharmaceutically acceptable formulations comprising a pharmaceutical protein that are buffered by the protein itself and do not require additional buffering agents to maintain a desired pH, with the protein being substantially the only buffering agent (i.e., other components, if present, do not substantially act as buffering agents in the formulation).

[0057] In some embodiments, a self-buffering formulation is one in which the protein provides about 70-85% buffering capacity at pH 5.5-6.5 and about 40-60% buffering capacity at pH 5.5-4.5, assuming 50% solvent activity of His / glutamic acid side chains at lower protein levels. In some embodiments, pharmaceutical formulations with self-buffering capacity are disclosed, in which the protein provides at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5%, e.g., 70-85%, of buffering capacity over a range of ±1 pH unit from the desired pH. In some embodiments, pharmaceutical formulations are disclosed in which pembrolizumab provides at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the buffering capacity at a given pH, e.g., pH 5.5-6.5, with the remainder of the buffering capacity provided by one or more additional excipients, such as one or more amino acids, e.g., glutamic acid, e.g., 10 mM glutamic acid, including 5-25 mM L-glutamic acid. In some embodiments, the formulation also includes L-threonine, e.g., 100-300 mM L-threonine (containing 250 mM L-threonine), and a polysorbate, such as PS80 (e.g., 0.01% w / v to 0.1% w / v PS80).

[0058] In some embodiments, pharmaceutical formulations with self-buffering capacity are disclosed, wherein the buffering capacity of a protein, e.g., pembrolizumab, is at least 1.00, or 1.50, or 1.63, or 2.00, or 3.00, or 4.00, or 5.00, or 6.50, or 8.00, or 10.0, or 15.0, or 20.0, or 30.0, or 40.0, or 50.0, or 75.0, or 100, or 125, or 150, or 200, or 250, or 300, or 350, or 400, or 500, or 700, or 1,000 mEq per liter per pH unit, e.g., 1.00, 1.50, 1.63, 2.00, 3.00, 5.0, 10.0, or 20.0.

[0059] In any of the formulations disclosed herein, the concentration of the protein, e.g., pembrolizumab, may be about 5 to 500 mg / ml, or 20 to 300, or 20 to 250, or 20 to 200, or 20 to 150 mg / ml, e.g., about 20 to 400 mg / ml, about 20 to 250 mg / ml, about 20 to 150 mg / ml, e.g., 5 mg / mL, 10 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 90 mg / mL, L, 100 mg / mL, 110 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 165 mg / mL, 167 mg / mL, 170 mg / mL, 175 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, or 500 mg / mL.In some embodiments, the amount of pembrolizumab in the formulation is 5 mg / mL to 10 mg / mL, 5 mg / mL to 20 mg / mL, 5 mg / mL to 30 mg / mL, 5 mg / mL to 40 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 60 mg / mL, 5 mg / mL to 75 mg / mL, 10 mg / mL to 30 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 50 mg / mL, 10 mg / mL to 75 mg / mL, 50 mg / mL to 200 mg / mL, 75 mg / mL to 200 mg / mL, 100 mg / mL to 200 mg / mL, 25 mg / mL to 175 mg / mL, 50 mg / mL to 175 mg / mL, 75 mg / mL to 175 mg / mL, 100 mg / mL to 175 mg / mL, 25 mg / mL to 30 mg / mL, 30 mg / mL to 40 mg / mL, 30 mg / mL to 50 mg / mL, 3 ...25 mg / mL~150mg / mL, 50mg / mL~150mg / mL, 75mg / mL~150mg / mL, 100mg / mL~150mg / mL, 25mg / mL~125mg / mL, 50mg / mL~125mg / mL, 75mg / mL~125mg / mL, 25mg / mL~100mg / mL, 125mg / mL~175mg / mL, 125mg / mL~200m g / mL, 5mg / mL~200mg / mL, 150mg / mL~300mg / mL, 150mL~400mg / mL, 150mL~450mg / mL, 150mg / mL~250m g / mL, 200 mg / mL to 500 mg / mL, 200 mg / mL to 300 mg / mL, 200 mg / mL to 400 mg / mL, or 200 mg / mL to 500 mg / mL.

[0060] Exemplary methods for determining the self-buffering capacity of a protein are disclosed in U.S. Patent Application Publication No. 20130273067A1, the entire disclosure of which is incorporated herein by reference (see, e.g., the Examples section at paragraphs

[0159] to

[0208] of U.S. Patent Application Publication No. 20130273067A1).

[0061] In some embodiments, the formulation contains a buffer. As used herein, a "buffer" is a component of a formulation to which a pharmaceutical agent is added to resist changes in pH. A buffer can maintain the pH of the formulation within an acceptable range. When referring to the concentration of a buffer, the stated concentration refers to the molar concentration of the free acid form of the buffer. As described above, a solution with strong buffering capacity is one whose pH is within ±1 unit of the pKa. In this pH range, significant amounts of both conjugate base species and conjugate acid species are present in the solution. Histidine has three pKas: 1.5, 6.0, and 9; acetic acid has a pKa of 4.8; and citric acid has three pKas: 3.1, 4.8, and 6.4. Therefore, histidine, acetic acid, and citric acid have strong buffering capacity at pH 5.5 and are considered strong buffers at this pH (e.g., pH 5.5 is within ±1 unit of the pKa of each molecule). Glutamic acid has three pKa's: 2.2, 4.2, and 9.7, and does not have a strong buffering capacity at pH 5.5.

[0062] Examples of suitable buffers include acetic acid and / or acetate salts (e.g., at concentrations of about 0.1 mM to about 300 mM, about 2 mM to about 30 mM, about 5 mM to about 50 mM, about 5 mM to about 15 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 15 mM to about 25 mM, about 30 mM to about 40 mM, about 35 mM to about 45 mM, about 40 mM to about 50 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM). , lactic acid and / or lactate salts (e.g., at concentrations of about 0.1 mM to about 300 mM, about 2 mM to about 30 mM, about 10 mM to about 30 mM, about 5 mM to about 15 mM, about 7 mM to about 12 mM, about 9 mM to about 11 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM), and proline (e.g., at concentrations of about 0.1 to about 450 mM, about 50 to about 320 mM, and / or about 50 to about 300 mM). Exemplary buffers of the disclosed formulations do not contain histidine.

[0063] Other examples of possible buffers include adipic acid salts (e.g., at concentrations of about 5 mM to about 50 mM, about 10 mM to about 25 mM, about 15 mM to about 20 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM), glucuronate salts (e.g., at concentrations of about 5 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM), and HCl salts (e.g., at concentrations of about 5 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM). Examples of suitable salts include, but are not limited to, benzoates (e.g., at concentrations of about 5 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM), benzoates (e.g., at concentrations of about 5 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM), and glycolates (e.g., at concentrations of about 5 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM, about 15 mM, about 20 mM, and / or about 25 mM).

[0064] Thus, suitable buffers include, but are not necessarily limited to, acetate / acetic acid buffer (acetate buffer), lactate / lactic acid buffer (lactated buffer), and proline buffer. Other potential buffers include adipate / adipic acid buffer (adipate buffer), glucuronate / glucuronate buffer (glucuronate buffer), benzoate / benzoic acid buffer (benzoate buffer), and glycolate / glycolic acid buffer (glycolate buffer).

[0065] In some embodiments, the buffer is a lactate buffer, in some embodiments, the buffer is an acetate buffer, in some embodiments, the buffer is not a histidine buffer.

[0066] In some embodiments, the formulation comprises lactic acid and glycerol. In some embodiments, the lactic acid contained in the formulation is a buffer. In some embodiments, the formulation comprises lactic acid and L-threonine.

[0067] In some embodiments, the formulations described herein comprise an amino acid at a concentration of about 2 mM to about 300 mM. In some embodiments, the concentration of amino acid in the formulations described herein is about 2 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 510 mM, about 520 mM, about 530 mM, about 540 mM, about 550 mM, about 560 mM, about 570 mM, about 580 mM, about 590 mM, about 600 mM, about 610 mM, about 620 mM, about 50 mM, about 160 mM, about 170 mM, about 175 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 271 mM, about 272 mM, about 273 mM, about 274 mM, about 275 mM, about 280 mM, about 290 mM, or about 300 mM. In some embodiments, the concentration of amino acids in the formulations described herein is about 2 to about 10 mM, about 5 to about 10 mM, about 8 to about 12 mM, about 8 to about 15 mM, about 10 to about 20 mM, about 15 to about 30 mM, about 20 to about 30 mM, about 25 to about 40 mM, about 40 to about 50 mM, about 40 to about 75 mM, about 50 to about 100 mM, about 75 to about 100 mM, about 100 to about 110 mM, about 100 to about 115 mM, about 110 to about 120 mM, about 115 to about 130 mM, about 130 to about 140 mM, about 140 to about 150 mM, about 150 to about 160 mM, about 160 to about 170 mM, about 170 to about 180 mM, about 180 to about 200 mM, about 180 to about 210 mM, about 180 to about 220 mM, about 180 to about 230 mM, about 180 to about 240 mM, about 180 to about 250 mM, about 190 to about 260 mM, about 260 to about 270 mM, about 270 to about 280 mM, about 280 to about 300 mM, about 290 to about 310 mM, about 310 to about 320 mM, about 320 to about 330 mM, about 330 to about 340 mM, about 340 to about 350 mM, about 350 to about 360 mM, about 360 to about 370 mM, about 370 to about 380 mM, about 380 to about 400 mM, about The concentration is about 130 mM, about 120 to about 130 mM, about 125 to about 140 mM, about 140 to about 150 mM, about 140 to about 175 mM, about 150 to about 200 mM, about 175 to about 200 mM, about 200 to about 215 mM, about 210 to about 220 mM, about 215 to about 230 mM, about 220 to about 230 mM, about 225 to about 240 mM, about 240 to about 250 mM, about 240 to about 275 mM, about 250 to about 300 mM, or about 270 to about 300 mM. In some embodiments, the amino acid is valine. In some embodiments, the amino acid is L-glutamic acid. In some embodiments, the amino acid is L-threonine.

[0068] In some embodiments, the formulations described herein include an amino acid at a concentration of 2 mM to 450 mM, e.g., 2 mM to 300 mM. In some embodiments, the concentration of amino acid in the formulations described herein is 2 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, 550 mM, 560 mM, 570 mM, 580 mM, 590 mM, 600 mM, 610 mM, 620 mM, 630 mM, 640 mM, 650 mM, 660 mM, 670 mM, 680 mM, 690 mM, 700 mM, 71 70 mM, 175 mM, 180 mM, 190 mM, 200 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 240 mM, 250 mM, 255 mM, 260 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 280 mM, 290 mM, 300 mM, 350 mM, 400 mM, or 450 mM. In some embodiments, the concentration of amino acids in the formulations described herein is about 2 to about 10 mM, about 5 to about 10 mM, about 8 to about 12 mM, about 8 to about 15 mM, about 10 to about 20 mM, about 15 to about 30 mM, about 20 to about 30 mM, about 25 to about 40 mM, about 40 to about 50 mM, about 40 to about 75 mM, about 50 to about 100 mM, about 75 to about 100 mM, about 100 to about 110 mM, 100 to 115 mM, 110 to 120 mM, 115 to 130 mM, or 140 to 150 mM. M, 120-130mM, 125-140mM, 140-150mM, 140-175mM, 150-200mM, 175-200mM, 200-215mM, 210-220mM, 215-230mM, 220-230mM, 225-240mM, 240-250mM, 240-275mM, 250-300mM, 250mM-280mM, 250mM-271mM, 270-300mM, 250mM-400mM.

[0069] In some embodiments, the amino acid is valine. In some embodiments, the formulations described herein include valine at a concentration of 2 mM to 450 mM, e.g., 2 mM to 300 mM. In some embodiments, the concentration of valine in the formulations described herein is 2 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, 550 mM, 560 mM, 570 mM, 580 mM, 590 mM, 600 mM, 610 mM, 620 mM, 630 mM, 640 mM, 650 mM, 660 mM, 670 mM, 680 mM, 6 70 mM, 175 mM, 180 mM, 190 mM, 200 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 240 mM, 250 mM, 255 mM, 260 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 280 mM, 290 mM, 300 mM, 350 mM, 400 mM, or 450 mM. In some embodiments, the concentration of valine in the formulations described herein is about 2 to about 10 mM, about 5 to about 10 mM, about 8 to about 12 mM, about 8 to about 15 mM, about 10 to about 20 mM, about 15 to about 30 mM, about 20 to about 30 mM, about 25 to about 40 mM, about 40 to about 50 mM, about 40 to about 75 mM, about 50 to about 100 mM, about 75 to about 100 mM, about 100 to about 110 mM, 100 to 115 mM, 110 to 120 mM, 115 to 130 mM, or 140 to 150 mM. M, 120-130mM, 125-140mM, 140-150mM, 140-175mM, 150-200mM, 175-200mM, 200-215mM, 210-220mM, 215-230mM, 220-230mM, 225-240mM, 240-250mM, 240-275mM, 250-300mM, 250mM-280mM, 250mM-271mM, 270-300mM, 250mM-400mM.

[0070] In some embodiments, the amino acid is L-glutamic acid. In some embodiments, the formulations described herein include L-glutamic acid at a concentration of 2 mM to 450 mM, e.g., 2 mM to 300 mM. In some embodiments, the concentration of L-glutamic acid in the formulations described herein is 2 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, or the like. , 170mM, 175mM, 180mM, 190mM, 200mM, 210mM, 215mM, 220mM, 225mM, 230mM, 240mM, 250mM, 255mM, 260mM, 270mM, 271mM, 272mM, 273mM, 274mM, 275mM, 280mM, 290mM, 300mM, 350mM, 400mM, or 450mM. In some embodiments, the concentration of L-glutamic acid in the formulations described herein is about 2 to about 10 mM, about 5 to about 10 mM, about 8 to about 12 mM, about 8 to about 15 mM, about 10 to about 20 mM, about 15 to about 30 mM, about 20 to about 30 mM, about 25 to about 40 mM, about 40 to about 50 mM, about 40 to about 75 mM, about 50 to about 100 mM, about 75 to about 100 mM, about 100 to about 110 mM, 100 to 115 mM, 110 to 120 mM, 115 to 130 mM, or 135 to 140 mM. 0mM, 120-130mM, 125-140mM, 140-150mM, 140-175mM, 150-200mM, 175-200mM, 200-215mM, 210-220mM, 215-230mM, 220-230mM, 225-240mM, 240-250mM, 240-275mM, 250-300mM, 250mM-280mM, 250mM-271mM, 270-300mM, 250mM-400mM.

[0071] In some embodiments, the amino acid is L-threonine. In some embodiments, the formulations described herein include L-threonine at a concentration of 2 mM to 450 mM, e.g., 2 mM to 300 mM. In some embodiments, the concentration of L-threonine in the formulations described herein is 2 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, 550 mM, 560 mM, 570 mM, 580 mM, 590 mM, 600 mM, 610 mM, 620 mM, 630 mM, 640 mM, 650 mM, 660 mM, 670 mM , 170mM, 175mM, 180mM, 190mM, 200mM, 210mM, 215mM, 220mM, 225mM, 230mM, 240mM, 250mM, 255mM, 260mM, 270mM, 271mM, 272mM, 273mM, 274mM, 275mM, 280mM, 290mM, 300mM, 350mM, 400mM, or 450mM. In some embodiments, the concentration of L-threonine in the formulations described herein is about 2 to about 10 mM, about 5 to about 10 mM, about 8 to about 12 mM, about 8 to about 15 mM, about 10 to about 20 mM, about 15 to about 30 mM, about 20 to about 30 mM, about 25 to about 40 mM, about 40 to about 50 mM, about 40 to about 75 mM, about 50 to about 100 mM, about 75 to about 100 mM, about 100 to about 110 mM, 100 to 115 mM, 110 to 120 mM, 115 to 130 mM, or 130 to 150 mM. 0mM, 120-130mM, 125-140mM, 140-150mM, 140-175mM, 150-200mM, 175-200mM, 200-215mM, 210-220mM, 215-230mM, 220-230mM, 225-240mM, 240-250mM, 240-275mM, 250-300mM, 250mM-280mM, 250mM-271mM, 270-300mM, 250mM-400mM.In some embodiments, a high concentration of L-threonine is used, for example, greater than 100 mM L-threonine, for example, 100 mM to 300 mM, 100 mM to 271 mM, 150 mM to 300 mM, 150 mM to 271 mM, 200 mM to 300 mM, 200 mM to 271 mM, 225 mM to 275 mM, 225 mM to 271 mM, 250 mM to 275 mM, 250 mM to 271 mM, e.g., 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, M, 259mM, 260mM, 261mM, 262mM, 263mM, 264mM, 265mM, 266mM, 267mM, 268mM, 26 9mM, 270mM, 271mM, 272mM, 273mM, 274mM, 275mM, 276mM, 277mM, 278mM, 279mM, 2 80 mM, 281 mM, 282 mM, 283 mM, 284 mM, 285 mM, 286 mM, 289 mM, 290 mM, 291 mM, 292 mM, 293 mM, 294 mM, 295 mM, 296 mM, 297 mM, 298 mM, 299 mM, or 300 mM L-threonine is used.

[0072] In some embodiments, the concentration of the amino acid in the formulations described herein is 5 mM. In some embodiments, the concentration of the amino acid in the formulations described herein is 10 mM, e.g., 10 mM L-glutamic acid. In some embodiments, the concentration of the amino acid in the formulations described herein is 30 mM. In some embodiments, the concentration of the amino acid in the formulations described herein is 65 mM. In some embodiments, the concentration of the amino acid in the formulations described herein is 100 mM. In some embodiments, the concentration of the amino acid in the formulations described herein is 250 mM, e.g., 250 mM L-threonine. In some embodiments, the concentration of the amino acid in the formulations described herein is 271 mM, e.g., 271 mM L-threonine. In some embodiments, the concentration of the amino acid is 10 mM L-glutamic acid and 250 mM L-threonine, wherein the formulation also includes 0.02% w / v of a polysorbate, such as PS80. In some embodiments, the concentrations of amino acids are 10 mM L-glutamic acid and 271 mM L-threonine, where the formulation also includes 0.02% w / v of a polysorbate, such as PS80.In some embodiments, the amino acid concentration is 10-30 mM L-glutamic acid, e.g., 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM glutamic acid, and 200-300 mM L-threonine, e.g., 200 mM, 201 mM, 202 mM, 203 mM, 204 mM, 205 mM, 206 mM, 207 mM, 208 mM, 209 mM, 210 mM, 211 mM, 212 mM, 213 mM, 214 mM, 215 mM, 216 mM, 217 mM, 218 mM, 219 mM, 220 mM, 221 mM, 222 mM, 223 mM, 224 mM, 225 mM, 226 mM, 227 mM, 228 mM, 229 mM, 230 mM, 231 mM, 232 mM, 233 mM, 234 mM, 235 mM, 236 mM, 237 mM, 238 mM, 239 mM, 240 mM, 241 mM, 242 mM, 243 mM, 244 mM, 245 mM, 246 mM, 247 mM, 248 mM, 249 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, 259 mM, 260 mM, 261 mM, 262 mM, 263 mM, 264 mM, 265 mM, 266 mM, 267 mM, 268 mM, 269 mM, 270 mM, 271 4mM, 215mM, 216mM, 217mM, 218mM, 219mM, 220mM, 221mM, 222mM, 223mM, 224mM, 225mM, 226mM, 227mM, 228mM, 229m M, 230mM, 231mM, 232mM, 233mM, 234mM, 235mM, 236mM, 237mM, 238mM, 239mM, 240mM, 241mM, 242mM, 243mM, 244mM, 2 45mM, 246mM, 247mM, 248mM, 249mM, 250mM, 251mM, 252mM, 253mM, 254mM, 255mM, 256mM, 257mM, 258mM, 259mM, 260 mM, 261mM, 262mM, 263mM, 264mM, 265mM, 266mM, 267mM, 268mM, 269mM, 270mM, 271mM, 272mM, 273mM, 274mM, 275mM, 276mM, 277mM, 278mM, 279mM, 280mM, 281mM, 282mM, 283mM, 284mM, 285mM, 286mM, 289mM, 290mM, 291mM, 292mM, 293mM, 294mM, 295mM, 296mM, 297mM, 298mM, 299mM, or 300mM L-threonine, wherein the formulation also contains 0.02% w / v of a polysorbate such as PS80.

[0073] In some embodiments, one or more (e.g., two, three, or four) amino acids are included in the formulations described herein. In some embodiments, the amino acids in the formulations described herein are selected from the group consisting of L-glutamic acid, valine, L-threonine, proline, and any combination thereof. In some embodiments, the formulations include L-threonine and L-glutamic acid.

[0074] In some embodiments, the concentration of the disaccharide in the formulations described herein is about 0.5% to about 50% w / v, hi some embodiments, the concentration of the disaccharide in the formulations described herein is about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, about 40%, or about 50% w / v. In some embodiments, the concentration of the disaccharide in the formulations described herein is about 0.5% w / v to about 1% w / v, about 0.5% w / v to about 5% w / v, about 1% w / v to about 10% w / v, about 5% w / v to about 8% w / v, about 5% w / v to about 10% w / v, about 10% w / v to about 12% w / v, about 10% w / v to about 15% w / v, about 10% w / v to about 20% w / v, about 15% w / v to about 25% w / v, about 20% w / v to about 30% w / v, about 25% w / v to about 40% w / v, about 20% w / v to about 50% w / v, or about 40% w / v to about 50% w / v. In some embodiments, the concentration of the disaccharide in the formulations described herein is about 1% w / v. In some embodiments, the concentration of the disaccharide in the formulations described herein is about 7% w / v. In some embodiments, the concentration of the disaccharide in the formulations described herein is about 10% w / v. In some embodiments, the disaccharide in the formulations described herein is trehalose or sucrose.

[0075] In some embodiments, the concentration of polyol in the formulations described herein is about 0.5% to about 20% w / v. In some embodiments, the concentration of polyol in the formulations described herein is about 0.5%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% w / v. In some embodiments, the concentration of polyol in the formulations described herein is about 0.5% w / v to about 1% w / v, about 0.5% w / v to about 2% w / v, about 1% w / v to about 2% w / v, about 1% w / v to about 2% w / v, about 1% w / v to about 2.5% w / v, about 2% w / v to about 3% w / v, about 2% w / v to about 5% w / v, about 5% w / v to about 10% w / v, about 8% w / v to about 12% w / v, about 10% w / v to about 15% w / v, about 12% w / v to about 15% w / v, about 12% w / v to about 20% w / v, about 15% w / v to about 20% w / v, or about 17% w / v to about 20% w / v. In some embodiments, the concentration of polyol in the formulations described herein is about 1% w / v. In some embodiments, the concentration of polyol in a formulation described herein is about 1.6% w / v. In some embodiments, the concentration of polyol in a formulation described herein is about 2% w / v. In some embodiments, the concentration of polyol in a formulation described herein is about 2.5% w / v. In some embodiments, one or more (e.g., 2, 3, or 4) polyols are included in a formulation described herein. In some embodiments, the polyol in a formulation described herein is selected from the group including glycerol, sorbitol, mannitol, and any combination thereof.

[0076] In some embodiments, the formulation does not contain a buffer. In other embodiments, the formulation contains a buffer. In some embodiments, the concentration of the buffer in the formulations described herein is from about 1 mM to about 300 mM. In some embodiments, the concentration of the buffer in the formulations described herein is from about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, or about 130 mM. mM, about 130mM, about 140mM, about 150mM, about 160mM, about 170mM, about 175mM, about 180mM, about 190mM, about 200mM, about 210mM, about 215mM, about 220mM, about 225mM, about 230mM, about 240mM, about 250mM, about 260mM, about 270mM, about 275mM, about 280mM, about 290mM, or about 300mM. In some embodiments, the concentration of the buffer in the formulations described herein is about 1 to about 5 mM, about 1 to about 10 mM, about 2 to about 10 mM, about 5 to about 10 mM, about 8 to about 12 mM, about 8 to about 15 mM, about 10 to about 20 mM, about 15 to about 30 mM, about 20 to about 30 mM, about 25 to about 40 mM, about 40 to about 50 mM, about 40 to about 75 mM, about 50 to about 100 mM, about 75 to about 100 mM, about 100 to about 110 mM, about 100 to about 115 mM, about 110 to about 120 mM, or about 130 to about 140 mM. mM, about 115 to about 130 mM, about 120 to about 130 mM, about 125 to about 140 mM, about 140 to about 150 mM, about 140 to about 175 mM, about 150 to about 200 mM, about 175 to about 200 mM, about 200 to about 215 mM, about 210 to about 220 mM, about 215 to about 230 mM, about 220 to about 230 mM, about 225 to about 240 mM, about 240 to about 250 mM, about 240 to about 275 mM, about 250 to about 300 mM, or about 270 to about 300 mM.

[0077] In some embodiments, the concentration of the buffer in the formulations described herein is 2 mM to 20 mM. In some embodiments, the concentration of the buffer in the formulations described herein is 5 mM. In some embodiments, the concentration of the buffer in the formulations described herein is 10 mM. In some embodiments, the concentration of the buffer in the formulations described herein is 20 mM.

[0078] In some embodiments, the formulation is unbuffered or at a pH where the excipient does not have strong buffering capacity, such as L-glutamic acid in the formulation at pH 5.5. In some embodiments, the formulation contains two or more excipients that act as stabilizers rather than buffers. In some embodiments, the formulation is self-buffering. In some embodiments, the formulation contains at least one amino acid that has self-buffering capacity and does not have strong buffering capacity at pH 5.5.

[0079] In some embodiments, the formulation comprises about 2.5% w / v glycerol and about 0.2% w / v PS80, e.g., 2.5% w / v glycerol and 0.2% w / v PS80. In some embodiments, the formulation comprises about 10% w / v trehalose and about 0.2% w / v PS80, e.g., 10% w / v trehalose and 0.02% or 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 2.5% w / v glycerol, and about 0.2% w / v PS80, e.g., 10 mM L-glutamic acid, 2.5% w / v glycerol, and 0.02% or 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 271 mM valine, and about 0.2% w / v PS80. In some embodiments, the formulation comprises 10 mM L-glutamic acid, about 271 mM valine, and 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 65 mM valine, about 2% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises 10 mM L-glutamic acid, 65 mM valine, 2% w / v glycerol, and 0.2% w / v PS80. In some embodiments, the formulation comprises 10 mM L-glutamic acid, 65 mM valine, 2% w / v glycerol, and 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 5 mM valine, about 2.5% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 271 mM L-threonine, and about 0.2% w / v PS80. In some embodiments, the formulation comprises 10 mM L-glutamic acid, 271 mM L-threonine, and 0.2% w / v PS80. In some embodiments, the formulation comprises 10 mM L-glutamic acid, 271 mM L-threonine, and 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 30 mM L-threonine, about 2% w / v glycerol, and about 0.2% w / v PS80.In some embodiments, the formulation comprises 10 mM L-glutamic acid, 30 mM L-threonine, 2% w / v glycerol, and 0.2% w / v PS80 or 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 10 mM L-threonine, about 2.5% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises 10 mM L-glutamic acid, 10 mM L-threonine, 2.5% w / v glycerol, and 0.2% w / v PS80 or 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 100 mM proline, about 1.6% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 100 mM proline, about 1.6% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 10% w / v trehalose, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 1% w / v trehalose, about 2% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 1% w / v sorbitol, about 2% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 10 mM L-glutamic acid, about 1% w / v mannitol, about 2% w / v glycerol, and about 0.2% w / v PS80. In some embodiments, the formulation comprises about 2 mM acetic acid, about 10 mM L-glutamic acid, about 2.5% w / v glycerol, and about 0.2% w / v PS80 or about 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM lactic acid, about 2.5% w / v glycerol, and about 0.2% w / v PS80 or about 0.02% w / v PS80. In some embodiments, the formulation comprises about 10 mM lactic acid, about 271 mM L-threonine, and about 0.2% w / v PS80 or about 0.02% w / v PS80.

[0080] In some embodiments, one or more of the disclosed formulations comprises an antioxidant. Exemplary antioxidants include amino acids such as methionine, L-cysteine, L-carnitine, or mixtures thereof; vitamins such as vitamin A, vitamin C, vitamin E, or mixtures thereof; coenzymes such as coenzyme Q10; and / or glutathione, methylsulfonyl sulfate, or mixtures thereof. In some embodiments, one or more of the disclosed formulations does not comprise an antioxidant.

[0081] In some embodiments, one or more of the disclosed formulations (e.g., a formulation comprising threonine and / or glutamic acid, e.g., 25 mg / mL pembrolizumab, 10 mM L-glutamic acid, 250 or 271 mM L-threonine, and 0.02% (w / v) PS80 at pH 5.5, or a formulation comprising 50 mg / mL pembrolizumab, 10 mM L-glutamic acid, 250 or 271 mM L-threonine, and 0.02% (w / v) PS80 at pH 5.5) possesses a long shelf life (e.g., at least 2 years, e.g., 2 years, 3 years, 4 years, 5 years, or more) in a liquid state at pH 5.5 at 2-8°C in the absence of a disaccharide stabilizer and / or buffer. The disclosed formulations are stable and can be stored at 2-8°C for an extended period of time, e.g., at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, at least 60 months, e.g., about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, about 48 months, about 60 months, e.g., 6-36 months, 6-12 months, 6-9 months, 9-12 months, 12-18 months, 12- The product can be stored for 24 months, 12 to 18 months, 18 to 24 months, 12 to 36 months, 24 to 36 months, for example, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 36 months, or longer.

[0082] In some embodiments, one or more disclosed formulations exhibit a change in the total amount of HMW species in the liquid state at 5° C. of 0.1% or less at 104 weeks (2 years) or less compared to HMW species at week 0.

[0083] In some embodiments, one or more disclosed formulations exhibit a change in total amount of HMW species in the liquid state at 5° C. of 0.08% or less at week 72 compared to HMW species at week 0.

[0084] In some embodiments, one or more disclosed formulations exhibit a change in total amount of HMW species in the liquid state at 5° C. of 0.07% or less at week 36 compared to HMW species at week 0.

[0085] In some embodiments, one or more disclosed formulations exhibit a change in total amount of HMW species in the liquid state at 5° C. of 0.05% or less at 24 weeks compared to HMW species at 0 weeks.

[0086] In some embodiments, one or more disclosed formulations exhibit a change in total amount of HMW species in the liquid state at 5° C. of 0.03% or less at week 12 compared to HMW species at week 0.

[0087] In some embodiments, one or more disclosed formulations exhibit a change in total amount of HMW species in the liquid state at 5° C. of 0.03% or less at week 8 compared to HMW species at week 0.

[0088] In some embodiments, one or more disclosed formulations exhibit a change in total amount of HMW species in the liquid state at 5° C. of 0.02% or less at week 4 compared to HMW species at week 0.

[0089] In some embodiments, one or more of the disclosed formulations have a total amount of HMW species in the liquid state of 0.2% or less at 5° C. for a given period, such as 4 weeks, 8 weeks, 12 weeks, 24 weeks, 36 weeks, 72 weeks, or 104 weeks.

[0090] In some embodiments, one or more of the disclosed formulations have a total amount of HMW species in the liquid state of 0.1% or less at 25° C. for a given period of time, such as 2 weeks or 4 weeks.

[0091] In some embodiments, one or more of the disclosed formulations have a total amount of HMW species in the liquid state of 0.4% or less at 25° C. for a given period, such as 4 weeks, 8 weeks, 12 weeks, or 24 weeks.

[0092] In some embodiments, the present disclosure provides a method of treatment comprising administering a pharmaceutical formulation described herein (or a lyophilized formulation thereof once reconstituted, e.g., with sterile water for injection) to a subject having or at risk of developing a disease or condition. In some embodiments, the method further comprises administering a second therapeutic composition to the subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the present disclosure provides a composition comprising any of the pharmaceutical formulations described herein (or a lyophilized formulation thereof) for the treatment of a disease or disorder. In some embodiments, the disclosure provides use of a pharmaceutical formulation described herein (or a lyophilized formulation thereof) for the preparation of a medicament for the treatment of a disease or condition.

[0093] Exemplary diseases or conditions include, but are not limited to, cancer, melanoma, renal cancer, non-small cell lung cancer, bladder cancer, head and neck cancer, anaplastic thyroid cancer, and infectious diseases, including, but not limited to, malaria, acquired immunodeficiency syndrome (AIDS), cytomegalovirus infection, and influenza. Exemplary diseases or conditions also include, but are not limited to, melanoma, lung cancer, including non-small cell lung cancer (NSCLC), head and neck cancer, including head and neck squamous cell carcinoma (HNSCC), Hodgkin lymphoma, including classical Hodgkin lymphoma (cHL), B-cell lymphoma, including mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, renal cell carcinoma, gastric cancer, microsatellite instability-high (MSI-H), mismatch repair deficient (dMMR) cancer (including MSI-H and dMMR colorectal cancer), as well as solid tumors, cervical cancer, liver cancer, Merkel cell carcinoma (MCC), esophageal cancer, hepatocellular carcinoma (HCC), endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), and the like.

[0094] In some embodiments, the disclosed pharmaceutical formulations further comprise an endoglycosidase hydrolase enzyme. In some embodiments, the endoglycosidase hydrolase enzyme is one disclosed in U.S. Patent Application Publication Nos. 20220233693A1, 20220089738A1, or WO 2020197230A1, each of which is incorporated by reference in its entirety, such as a recombinant human hyaluronidase disclosed in U.S. Patent Application Publication Nos. 20220233693A1, 20220089738A1, or WO 2020197230A1. In some aspects, the pharmaceutical composition comprises at least about 5 U to at least about 100,000 U of the endoglycosidase hydrolase enzyme. In some embodiments, the pharmaceutical composition comprises at least about 5 U, at least about 10 U, at least about 20 U, at least about 30 U, at least about 40 U, at least about 50 U, at least about 75 U, at least about 100 U, at least about 200 U, at least about 300 U, at least about 400 U, at least about 500 U, at least about 750 U, at least about 1000 U, at least about 2000 U, at least about 3000 U, at least about 4000 U, at least about 5000 U, The pharmaceutical composition comprises at least about 6000 U, at least about 7000 U, at least about 8000 U, at least about 9000 U, at least about 10,000 U, at least about 20,000 U, at least about 30,000 U, at least about 40,000 U, at least about 50,000 U, at least about 60,000 U, at least about 70,000 U, at least about 80,000 U, at least about 90,000 U, or at least about 100,000 U of endoglycosidase hydrolase enzyme. In some embodiments, the pharmaceutical composition comprises about 20,000 U of endoglycosidase hydrolase enzyme. In some embodiments, the pharmaceutical composition comprises at least about 500 U / mL to at least about 5000 U / mL of endoglycosidase hydrolase enzyme.In some aspects, the pharmaceutical composition comprises at least about 1500 U / mL, at least about 1600 U / mL, at least about 1700 U / mL, at least about 1800 U / mL, at least about 1900 U / mL, at least about 2000 U / mL, at least about 2100 U / mL, at least about 2200 U / mL, at least about 2300 U / mL, at least about 2400 μM, at least about 2500 μM, at least about 3000 μM, at least about 3500 μM, at least about 4000 μM, at least about 4500 U / mL, or at least about 5000 U / mL of the endoglycosidase hydrolase enzyme. In some embodiments, the pharmaceutical composition comprises about 2000 U / mL of the endoglycosidase hydrolase enzyme.

[0095] In some embodiments, the pharmaceutical formulations described herein (or lyophilized formulations thereof) can be administered subcutaneously, intravenously, parenterally, intradermally, intramuscularly, and / or intraperitoneally using standard techniques. For example, the stable pharmaceutical formulations described herein can be prepared for subcutaneous administration using a pre-filled syringe. Specifically, any of the formulations described herein may be administered subcutaneously, intravenously, parenterally, intradermally, intramuscularly, and / or intraperitoneally for indefinite periods of time, at therapeutically effective doses, and in the formulations described herein, for the treatment of the above-mentioned diseases and conditions, once every week, or 6-8 days, or 7-10 days, or once every other week, or once every 2 weeks, or 12-16 days, or 7-14 days, or 13-15 days, or once every 3 weeks or 19-23 days, or once every month, or 26-30 days, or once every 29-31 days, or once every 5 weeks or 33-34 days, or once every 6 weeks or 40-44 days, or once every 7 weeks or 47-51 days, or once every 2 months or 54-58 days.

[0096] The administration and dosing regimen of the stable pharmaceutical formulations (or lyophilized formulations thereof) described herein can be adjusted to provide an effective amount for optimal therapeutic response. For example, a single bolus may be administered, two or more divided doses may be administered over time, or the dose may be proportionally increased or decreased depending on the exigencies of the therapeutic situation. For example, a unit dose may be administered over two consecutive days every two weeks. A unit dose refers to a physically discrete amount of an anti-PD-1 antibody (e.g., pembrolizumab) suitable as a single dose for a patient to be treated, each unit containing a predetermined amount of active biopharmaceutical calculated to produce a desired therapeutic effect.

[0097] A dosing regimen for a stable pharmaceutical formulation (or a reconstituted lyophilized formulation thereof) described herein may include administering a given dose on day 1, followed by administering the same dose every other week. A dosing regimen for a stable pharmaceutical formulation (or a lyophilized formulation thereof) described herein may include administering a given initial dose on day 1, or split over two consecutive days, followed by administering the same dose or a reduced dose, e.g., half the initial dose, two weeks later (day 15). The dosing regimen may further include administering the same dose or a further reduced dose, e.g., one-quarter the initial dose, two weeks later (day 29), which is continued once every two weeks as a maintenance dose.

[0098] For example, a dosing regimen for treating at least melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, renal cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer, MSI-H or dMMR colorectal cancer (CRC), gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), liver cancer, Merkel cell carcinoma (MCC), endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), and / or triple-negative breast cancer (TNBC) can be 200 mg once every three weeks or 400 mg once every six weeks. Additionally, for pediatrics, the dosing regimen for at least melanoma, cHL or PMBCL, MSI-H or dMMR cancer, MCC, and / or TMB-H cancer can be 2 mg / kg (maximum 200 mg) every three weeks. The dosing regimen for treating renal cell carcinoma (RCC) can be 200 mg every three weeks or 40 mg every six weeks, either as a single agent in the adjuvant setting or in the advanced setting, with 5 mg of axitinib orally twice daily or 20 mg of lenvatinib orally once daily. The dosing regimen for endometrial cancer can be 200 mg every three weeks or 40 mg every six weeks, with 20 mg of lenvatinib orally once daily.

[0099] In some embodiments, the present disclosure provides methods for preparing the pharmaceutical formulations described herein, comprising combining an aqueous solution comprising one or more excipients with a therapeutically effective amount of an anti-PD-1 antibody (e.g., pembrolizumab) using techniques standard in the art. The present invention further provides methods for preparing the stable lyophilized pharmaceutical formulations described herein, comprising lyophilizing an aqueous pharmaceutical formulation comprising one or more excipients and a therapeutically effective amount of an anti-PD-1 antibody (e.g., pembrolizumab) using techniques standard in the art.

[0100] The above detailed description is not intended to define every aspect of the present disclosure, and other features and advantages of the present disclosure will become apparent to those skilled in the art. It should be understood that the present disclosure is intended to be related as an integrated document, and that all combinations of features described herein are contemplated, even if the combinations of features do not appear together in the same sentence, paragraph, or section of the disclosure. Additionally, the present disclosure includes, as an additional aspect, any embodiment of the present invention narrower in scope than the variations specifically mentioned above. With respect to aspects of the present disclosure described or claimed with "a" or "an," these terms should be understood to mean "one or more" unless the context clearly dictates a more limited meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. When aspects of the present disclosure are described as "comprising" a feature, it is also contemplated that the embodiment "consists of" or "consisting essentially of" that feature. Additional features and variations of the present disclosure will become apparent to those skilled in the art from the present application as a whole, and all such features are contemplated as aspects of the present disclosure.

[0101] The present disclosure will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting. [Example]

[0102] The following examples illustrate exemplary formulations of the present disclosure.

[0103] Example 1: Evaluation of Aggregation Rates in Exemplary Pembrolizumab Formulations Novel pembrolizumab formulations were made using a series of excipients, including different buffers and stabilizers. Exemplary formulations containing 25 mg / mL of pembrolizumab biosimilar and varying amounts of various excipients are listed in Table 1A. Using high-throughput screening and long-term stability studies, the stability of the exemplary formulations was tested and compared to that of the reference product (RP, Keytruda®) formulation (25 mg / mL pembrolizumab, 10 mM histidine, 7% sucrose, 0.02% polysorbate 80 (w / v)).

[0104] [Table 1]

[0105] Prepared samples of the exemplary formulations were sterile filtered and aseptically filled into 6R vials. Samples were incubated at temperatures between 2-8°C or 25°C for up to 4 weeks. At each time point, the level of aggregation in each sample was assessed using size-exclusion ultra-high performance liquid chromatography (SE-UHPLC), cation exchange chromatography (CEX), and reduced capillary electrophoresis (rCE).

[0106] SE-UHPLC can be performed to monitor monoclonal antibody aggregation, such as anti-PD1 antibody aggregation, including the pembrolizumab formulations disclosed herein. Size exclusion chromatography (SEC) is a common technique for protein analysis. SEC columns can be used to evaluate protein aggregation profiles. For example, SEC can be used to determine the amount of HMW species in one or more anti-PD-1 formulations, such as the disclosed pembrolizumab formulations, and monitor changes in the % amount of HMW species over a selected period of time. SEC can be performed by techniques known to those skilled in the art. General teachings on how SEC can be performed to determine aggregation can be found in A.E. Hamielec, S.T. Balke, B.P. Leclair & S.L.Pearce; Ind. Eng. Chem., Prod. Res. Dev., 8, 54 (1969); Z. Grubistic, R. Rempp & H. Benoit; J. Polymer Sci., Part B, 5, 753 (1967); "Polymer Handbook", J. Brandrup, E.H. Immersut: John Wiley & Sons Publishers; Data Reduction in Multidetector Size Exclusion Chromatography, Y. Brun, J. Liq. Chrom&Rel. Technology, 21(13), 1979-2015 (1998); Modern Size-Exclusion Liquid Chromatography, W.W. Yau, J.J. Kirkland, D.D.Bly John Wiley & Sons, Inc., New York, 1979 (each of which is incorporated herein by reference in its entirety).

[0107] Figure 1 shows comparable aggregation rates (<0.05% HMW difference) for the various formulations and RP formulations tested at 5°C at each time point up to 4 weeks. Pembrolizumab biosimilar (25 mg / mL) formulations containing 0.02% (w / v) PS80 that have aggregation rates over time similar to those of the RP formulations include Tre (10% w / v trehalose, pH 5.5), Pro-Glyc (100 mM proline, 1.6% w / v glycerol, pH 5.5), Glu-high Val (10 mM L-glutamic acid, 271 mM valine, pH 5.5), Glu-high Treh (10 mM L-glutamic acid, 10% w / v trehalose, pH 5.5), and Glu-high Thr (10 mM L-glutamic acid, 271 mM L-threonine, pH 5.5) formulations.

[0108] Additional formulations were evaluated, as provided in Table 1B below. All test formulations were solution formulations containing 25 mg / mL pembrolizumab at pH 5.5.

[0109] [Table 2]

[0110] Example 2: Stability of exemplary pembrolizumab formulations To further evaluate the stability of the formulations within a representative manufacturing setting, five exemplary formulations containing 25 mg / mL pembrolizumab were subjected to a manufacturing process model. The formulations tested are listed in Table 2 and include a reference product formulation for the marketed pembrolizumab formulation (Keytruda®).

[0111] [Table 3]

[0112] Figure 2 shows that the manufacturing process had little effect on aggregation levels in the tested formulations. Further stability analysis evaluating the change in HMW% at 5°C at 0, 2, 4, 8, and 12 weeks showed that all five tested formulations had comparable aggregation rates at 5°C at the corresponding time points (Figure 3). These results confirm that the buffer-free Glu-Glyc (10 mM L-glutamic acid, 2.5% w / v glycerol), Glu-high Thr (10 mM L-glutamic acid, 271 mM L-threonine), Pro-Glyc (100 mM proline, 1.6% w / v glycerol), and Tre (10% w / v trehalose) formulations have stability similar to that of RPF. Additional studies showed that at all time points tested (2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks), the % change in HMW species was dramatically lower for the 25 mg / mL pembrolizumab, 0.02% (w / v) PS80, Glu-high Thr (10 mM L-glutamic acid, 250 mM L-threonine) formulation (the precision of the method for detecting %HMW was 0.1%), as shown in Table 3 below.

[0113] [Table 4]

[0114] Example 3: Stability of Pembrolizumab Formulations with Manufacturing Processing Stress and Over Time To further evaluate the stability of the formulations, formulations containing 25 mg / mL or 50 mg / mL of the pembrolizumab biosimilar were subjected to a worst-case manufacturing process model. One formulation contained 25 mg / mL of the pembrolizumab biosimilar, 10 mM L-glutamic acid, 250 mM L-threonine, and 0.02% (w / v) PS80 (pH 5.5), and the second formulation contained 50 mg / mL of the pembrolizumab biosimilar, 10 mM L-glutamic acid, 250 mM L-threonine, and 0.02% (w / v) PS80 (pH 5.5). The RPF is 25 mg / mL pembrolizumab, 10 mM L-histidine, 7% (w / v) sucrose, and 0.02% (w / v) PS80 (pH 5.5).

[0115] Figure 4 shows that expected worst-case laboratory-scale processing stresses did not affect the aggregation rate of monoclonal antibody monomers in the tested formulations. The extended arm study did not affect the quality of the pembrolizumab biosimilar product. The HMW of these formulations is predicted to remain within the quality target product profile at the end of their shelf life. Stability data at recommended storage conditions (RSC) show no impact on product quality after worst-case processing stresses.

[0116] Stability analysis evaluating changes in HMW% showed that the worst-case lab-scale post-processing formulation was stable at 2-8°C at the time tested. Experimental values demonstrate that the disclosed formulations can maintain stability throughout storage and processing. Typically, disaccharide stabilizers such as sucrose or trehalose, and / or strong buffers are required for stable liquid monoclonal antibody formulations. Surprisingly, disclosed herein are formulations (e.g., formulations comprising threonine, e.g., high concentrations of threonine, and / or glutamic acid, e.g., 25 mg / mL pembrolizumab, 10 mM L-glutamic acid, 250 or 271 mM L-threonine, and 0.02% (w / v) PS80 (pH 5.5), or 50 mg / mL pembrolizumab, 10 mM L-glutamic acid, 250 or 271 mM L-threonine, and 0.02% (w / v) PS80 (pH 5.5)) that have a long shelf life (e.g., at least 2 years, e.g., 2 years, 3 years, 4 years, 5 years, or more) when in a liquid state at pH 5.5 and at 2-8°C in the absence of a disaccharide stabilizer and / or a strong buffer. The disclosed formulations are stable and can be stored at 2-8°C for extended periods of time, e.g., at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, at least 60 months, e.g., about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, about 48 months, about 60 months, e.g., 6-36 months, 6-12 months, 6-9 months, 9-12 months, 12-18 months, 12-24 months, 12-18 months, 18-24 months, 12-36 months, 24-36 months, e.g., 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 30 months, 36 months, or more.

[0117] Table 4 shows the amount of aggregation (HMW%) in various formulations at 0, 4, 8, 12, 24, 36, 72, or 104 weeks at 5°C. Data from Table 4 is shown in Figure 6. Table 5 shows the change in aggregation (HMW%) in various formulations at 0, 4, 8, 12, 24, 36, 72, or 104 weeks at 5°C. Data from Table 5 is shown in Figure 7. Table 6 shows the amount of aggregation (HMW%) in various formulations at 0, 4, 8, 12, or 24 weeks at 25°C. Data from Table 6 is shown in Figure 8. Table 7 shows the change in aggregation (HMW%) at 0, 4, 8, 12, or 24 weeks at 25°C. Data from Table 7 is shown in Figure 9. Table 8 shows the amount of aggregation (HMW%) in various formulations at 0, 2, or 4 weeks at 25°C. Data from Table 8 is shown in Figure 10. Table 9 shows the amount of aggregation (HMW%) in various formulations at 0, 1, 2, 3, 4, or 8 weeks at 5° C. The data from Table 9 is shown in FIG. 11. Table 10 shows the amount of aggregation (HMW%) in various formulations at 0, 2, or 4 weeks at 25° C. The data from Table 10 is shown in FIG. 12. Table 11 shows the amount of aggregation (HMW%) in various formulations at 0, 2, or 4 weeks at 25° C. The data from Table 11 is shown in FIG. 13. Table 12 shows the amount of aggregation (HMW%) in various formulations at 0, 2, or 4 weeks at 25° C. The data from Table 12 is shown in FIG. 14.

[0118] [Table 5]

[0119] [Table 6]

[0120] [Table 7]

[0121] [Table 8]

[0122] [Table 9]

[0123] [Table 10]

[0124] [Table 11]

[0125] [Table 12]

[0126] [Table 13]

[0127] Example 4: Further evaluation of 25 mg / mL and 50 mg / mL pembrolizumab formulations A 50 mg / mL biosimilar pembrolizumab formulation containing 250 mM L-threonine, 10 mM L-glutamic acid, and 0.02% (w / v) PS80 was compared with a 25 mg / mL biosimilar pembrolizumab formulation containing the same excipients. After 12 weeks, the HMW of the 50 mg / mL pembrolizumab biosimilar formulation was not significantly higher than that of the 25 mg / mL HMW species, and a similar trend was observed compared to the scale-down data. Figure 5 shows the HMW% at 5°C for 25 mg / mL and 50 mg / mL at 0, 3, and 24 months (projected). A formulation containing 25 mg / mL pembrolizumab at pH 5.5 and 0.02% w / v polysorbate 80 was tested for up to 104 weeks (2 years). Figure 6 shows the results of the amount of aggregation in a 104-week stability study at 5°C (earlier time points than described in Example 2). The amount of aggregation, expressed as percent high molecular weight, or HMW%, over time at 5°C was measured by size exclusion chromatography. Glu / Thr (10 mM glutamic acid, 250 mM threonine) showed up to 0.5% less HMW aggregation compared to RPF (10 mM histidine, 7% w / v sucrose, 0.2% HMW) after 104 weeks of stability at 5°C. Figure 7 shows the results of the change in aggregation in a 104-week stability study at 5°C. The change in aggregation, expressed as percent high molecular weight, or HMW%, over time at 5°C was measured by size exclusion chromatography. Glu / Thr (10 mM glutamic acid, 250 mM threonine, 0.1% HMW) showed less change in aggregation compared to RPF (0.2% HMW) after 104 weeks of stability at 5°C. Figure 8 shows the results of the amount of aggregation in a 24-week stability study at 25°C. The amount of aggregation, expressed as percent high molecular weight, or HMW%, over time at 25°C was measured by size exclusion chromatography. Glu / Thr (10 mM glutamic acid, 250 mM threonine) showed up to 0.4% less HMW aggregation compared to RPF (0.2% HMW) after 24 weeks of stability at 25°C. Figure 9 shows the results of the change in aggregation in a 24-week stability study at 25°C.The change in aggregation, expressed as percent high molecular weight, or HMW%, over time at 25° C. was measured by size exclusion chromatography. Glu / Thr (10 mM glutamic acid, 250 mM threonine; 0.1% HMW) showed less change in aggregation compared to RPF (0.2% HMW) after 24 weeks of stability at 25° C.

[0128] Results with varying amounts of excipients were also observed. A formulation containing 25 mg / mL pembrolizumab at pH 5.5 and 0.02 w / v% polysorbate 80 was tested. Figure 10 shows the results of the amount of aggregation in a 4-week stability study at 25°C. The amount of aggregation, expressed as high molecular weight percent, or HMW%, in various buffer / stabilizer combinations was measured by size exclusion chromatography. Glu-highThr (10 mM glutamic acid, 271 mM threonine) maintained a similar HMW level % to RPF (10 mM histidine, 7 w / v% sucrose) over 4 weeks at 25°C. Glu-highThr maintained a lower HMW% than formulations with less threonine (Glu-lowThr-Glyc, Glu-medThr-Glyc) or no threonine (GluGlyc, Glyc). Figure 11 shows the results of the amount of aggregation in an 8-week stability study at 5°C. The amount of aggregation, expressed as high molecular weight percent, or HMW%, in various buffer / stabilizer combinations was measured by size exclusion chromatography. Glu-highThr (10 mM glutamic acid, 271 mM threonine) maintained a similar HMW level % to RPF over 8 weeks at 5°C. Glu-highThr maintained a lower HMW% than formulations with less threonine (Glu-lowThr-Glyc, Glu-medThr-Glyc) or no threonine (GluGlyc, Glyc). Figure 12 shows the amount of aggregation, expressed as high molecular weight percent, or HMW%, measured by size exclusion chromatography over 4 weeks at 25°C. Figure 13 shows the amount of aggregation, expressed as high molecular weight percent, or HMW%, in various buffer / stabilizer combinations over 4 weeks at 25°C, measured by size exclusion chromatography. FIG. 14 shows the amount of aggregation, expressed as high molecular weight percent, or HMW%, in various buffer / stabilizer combinations over a 4-week period at 25° C., as measured by size exclusion chromatography.

[0129] Embodiment The following embodiments are within the scope of the present disclosure. Furthermore, the present disclosure encompasses all variations, combinations, and permutations of these embodiments in which one or more limitations, elements, clauses, and descriptive terms from one or more enumerated embodiments are introduced into another enumerated embodiment in this section. For example, any enumerated embodiment that depends on another embodiment can be modified to include one or more limitations found in any other enumerated embodiment in this section that depends on the same base embodiment. Where elements are presented as a list, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element can be removed from the group. In general, when the present disclosure or aspects of the disclosure are recited as including particular elements and / or features, it is understood that the particular embodiment or aspect of the invention consists of or consists essentially of such elements and / or features. It should also be noted that the terms "comprise" and "contain" are intended to be open and allow for the inclusion of additional elements or steps. Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can assume any particular value or subrange within the ranges set forth in different embodiments of the invention to one-tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise.

[0130] A1. A pharmaceutical formulation comprising: (i) about 25 mg / mL to about 200 mg / mL of pembrolizumab or a biosimilar thereof; (ii) a surfactant; and (iii) one or more stabilizers, wherein the formulation is a liquid having a pH of about 5.2 to 5.8 and is stable in the liquid state at 2 to 8°C for at least 18 months; and further, the formulation does not contain histidine and / or a disaccharide.

[0131] A2. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers is at least one amino acid stabilizer.

[0132] A3. The pharmaceutical formulation of either embodiment A1 or embodiment A2, wherein the one or more stabilizers are at least two amino acid stabilizers.

[0133] A4. The pharmaceutical formulation of embodiment A3, wherein the at least two amino acid stabilizers are selected from the group consisting of L-glutamic acid, valine, L-threonine, and asparagine.

[0134] A5. The pharmaceutical formulation of any one of embodiments A1-A3, wherein the one or more stabilizers are two amino acid stabilizers, a first amino acid stabilizer is L-glutamic acid, and a second amino acid stabilizer is valine, asparagine, or L-threonine.

[0135] A6. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers is a polyol stabilizer.

[0136] A7. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid and about 271 mM valine, eg, 10 mM L-glutamic acid and 271 mM valine.

[0137] A8. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid, about 65 mM valine, and about 2 w / v% glycerol, e.g., 10 mM L-glutamic acid, 65 mM valine, and 2 w / v% glycerol.

[0138] A9. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid, about 5 mM valine, and about 2.5 w / v% glycerol, for example, 10 mM L-glutamic acid, 5 mM valine, and 2.5 w / v% glycerol.

[0139] A10. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid and about 271 mM L-threonine, such as 10 mM L-glutamic acid and 271 mM L-threonine.

[0140] A11. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid and about 250 mM L-threonine, such as 10 mM L-glutamic acid and 250 mM L-threonine.

[0141] A12. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid, about 10 mM L-threonine, and about 2.5 w / v% glycerol, e.g., 10 mM L-glutamic acid, 10 mM L-threonine, and 2.5 w / v% glycerol.

[0142] A13. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 10 mM L-glutamic acid, about 100 mM proline, and about 1.6 w / v% glycerol, such as 10 mM L-glutamic acid, 100 mM proline, and 1.6 w / v% glycerol.

[0143] A14. The pharmaceutical formulation of embodiment A1, wherein the one or more stabilizers comprise about 100 mM proline and about 1.6 w / v % glycerol, for example 100 mM proline and 1.6 w / v % glycerol.

[0144] A15. The pharmaceutical formulation of any one of embodiments A1-A14, wherein the formulation does not contain a buffer.

[0145] A16. The pharmaceutical formulation of any one of embodiments A1 to A15, wherein the surfactant is a polysorbate, such as polysorbate 80 (PS80).

[0146] A17. The pharmaceutical formulation of any one of embodiments A1 to A16, wherein the surfactant is about 0.02 w / v% Polysorbate 80 (PS80).

[0147] A18. The pharmaceutical formulation of any one of embodiments A1 to A17, wherein the pH is about 5.5, for example 5.5.

[0148] A19. A method of treatment comprising administering to a subject having or at risk of developing a disease or condition a pharmaceutical formulation according to any one of embodiments A1-A19 above.

[0149] A20. The method of embodiment A19, further comprising administering to said subject a second therapeutic composition.

[0150] A21. The method of any one of embodiments A19 or A20, wherein the disease or condition is selected from the group consisting of cancer, e.g., melanoma, renal cell carcinoma (RCC), non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma, endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), anaplastic thyroid cancer, and / or infectious disease.

[0151] A22. The method of any one of embodiments A19-A21, wherein the subject is a mammal.

[0152] A23. The method of embodiment A22, wherein the mammal is a human.

[0153] A24. A pharmaceutical formulation of any one of embodiments A1 to A23, or the use thereof in any one of embodiments A1 to A23, wherein the formulation has a total amount of high molecular weight (HMW) species in the liquid state of 0.1% or less, as measured by size exclusion chromatography, after storage at 5°C for up to 24 weeks.

[0154] A25. A pharmaceutical formulation of any one of embodiments A1 to A23, or the use thereof in any one of embodiments A1 to A23, wherein the formulation has a total amount of HMW species in the liquid state of 0.2% or less, as measured by size exclusion chromatography, after storage at 5°C for up to 104 weeks.

[0155] A26. A pharmaceutical formulation according to any one of embodiments A1 to A23, or the use thereof in any one of embodiments A1 to A23, wherein the formulation exhibits a change in the total amount of HMW species in the liquid state at 5°C of less than 0.1% at week 104 compared to HMW species at week 0.

[0156] A27. A pharmaceutical formulation according to any one of embodiments A1 to A23, or the use thereof in any one of embodiments A1 to A23, wherein the formulation has a change in the total amount of HMW species in the liquid state at 5°C at week 36 compared to HMW species at week 0 of less than 0.07%.

[0157] A28. A pharmaceutical formulation according to any one of embodiments A1 to A27, or the use thereof in any one of embodiments A1 to A27, wherein the formulation further comprises an endoglycosidase hydrolase enzyme.

[0158] A29. The pharmaceutical preparation according to A28, wherein the endoglycosidase hydrolase enzyme is recombinant human hyaluronidase.

[0159] B1. A pharmaceutical formulation comprising: (i) about 25 mg / mL or 50 mg / mL of pembrolizumab or a biosimilar thereof; (ii) about 10 mM L-glutamic acid; (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine; and (iv) about 0.02 w / v% polysorbate 80, wherein the formulation is in a liquid state with a pH of about 5.2 to about 5.8 and is stable in the liquid state at 2 to 8°C, wherein the stable formulation does not contain a strong buffer and / or a disaccharide stabilizer, and wherein the stable formulation shows a change in the total amount of HMW species in the liquid state at 2 to 8°C by 6 months compared to the HMW species at week 0 of less than about 0.06%.

[0160] B2. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 25 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02% w / v polysorbate 80, and further, the formulation is stable at 2-8° C. for at least 18 months and has a total amount of high molecular weight (HMW) species of 0.2% or less.

[0161] B3. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 25 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02 w / v% polysorbate 80, and further, the formulation is stable at 2-8° C. for at least 2 years and has a total amount of high molecular weight (HMW) species of 0.2% or less.

[0162] B4. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 25 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02 w / v% polysorbate 80, and further, the formulation is stable at 2-8° C. for at least 3 years.

[0163] B5. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 50 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02% w / v polysorbate 80, and further, the formulation is stable at 2-8° C. for at least 18 months and has a total amount of high molecular weight (HMW) species of 0.2% or less.

[0164] B6. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 50 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02% w / v polysorbate 80, and further, the formulation is stable at 2-8°C for at least 2 years and has a total amount of high molecular weight (HMW) species of 0.2% or less.

[0165] B7. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 50 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02 w / v% polysorbate 80, and further, the formulation is stable at 2-8° C. for at least 3 years, e.g., 3 years, 4 years, or 5 years.

[0166] B8. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 25 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02 w / v% polysorbate 80, and further wherein the formulation is stable at 2-8° C. for up to 5 years, e.g., 5 years.

[0167] B9. The pharmaceutical formulation of embodiment B1, wherein the formulation consists essentially of (i) 50 mg / mL of pembrolizumab biosimilar, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, about 271 mM valine, about 65 mM valine, or about 100 mM asparagine, and (iv) about 0.02 w / v% polysorbate 80, and further wherein the formulation is stable at 2-8° C. for up to 5 years, e.g., 5 years.

[0168] B10. The pharmaceutical formulation of any one of embodiments B1-B9, wherein the formulation does not include histidine, sucrose, trehalose, and / or glycerol.

[0169] B11. The pharmaceutical formulation of any one of embodiments B1-B9, wherein the formulation does not contain histidine or sucrose.

[0170] B12. The pharmaceutical formulation of any one of embodiments B1-B9, wherein the formulation does not include histidine, sucrose, or trehalose.

[0171] B13. The pharmaceutical formulation of any one of embodiments B1-B12, wherein the formulation does not further comprise an antioxidant.

[0172] B14. The pharmaceutical formulation of any one of embodiments B1-B13, wherein the pH is about 5.5, eg, pH 5.5.

[0173] B15. A method of treatment comprising administering to a subject having or at risk of developing a disease or condition a stable pharmaceutical formulation according to any one of embodiments B1-B14 above.

[0174] B16. The method of embodiment B15, further comprising administering to said subject a second therapeutic composition.

[0175] B17. The method of any of embodiments B15 or B16, wherein the disease or condition is selected from the group consisting of cancer, e.g., melanoma, renal cell carcinoma (RCC), non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma, endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), anaplastic thyroid cancer, and / or an infectious disease.

[0176] B18. The method of any one of embodiments B15-B17, wherein said subject is a mammal.

[0177] B19. The method of embodiment B18, wherein the mammal is a human.

[0178] B20. The pharmaceutical formulation of any one of embodiments B1-B14, wherein the formulation further comprises an endoglycosidase hydrolase enzyme.

[0179] B21. The pharmaceutical formulation of B20, wherein said endoglycosidase hydrolase enzyme is recombinant human hyaluronidase.

[0180] C1. A pharmaceutical formulation comprising pembrolizumab, 5 mM to 25 mM L-glutamic acid, 100 mM to 300 mM L-threonine, and 0.01 w / v% to 0.1 w / v% polysorbate, wherein the formulation is liquid and has a pH range of 5.2 to 5.8; the formulation does not contain histidine, sucrose, or trehalose; and the formulation has a change in the total amount of liquid-state high molecular weight (HMW) species at 5°C at week 36 compared to HMW species at week 0 of less than 0.07%; and / or the formulation has 0.2% or less liquid-state high molecular weight (HMW) species after 104 weeks of storage at 2°C to 8°C, e.g., at 5°C.

[0181] C2. The pharmaceutical formulation of embodiment C1, wherein the formulation consists essentially of (i) 50 mg / mL pembrolizumab, (ii) about 10 mM L-glutamic acid, (iii) about 250 mM L-threonine, and (iv) 0.02% w / v polysorbate 80, wherein the formulation has a change in the total amount of liquid-state high molecular weight (HMW) species at 5°C of less than 0.07% at week 36 compared to HMW species at week 0; and / or the formulation has 0.2% or less liquid-state high molecular weight (HMW) species after 104 weeks of storage at 5°C.

[0182] C3. The pharmaceutical formulation of embodiment C1 or C2, wherein the formulation does not comprise histidine, sucrose, trehalose, and / or glycerol.

[0183] C4. The pharmaceutical formulation of embodiment C1 or C2, wherein the formulation does not contain histidine or sucrose.

[0184] C5. The pharmaceutical formulation of embodiment C1 or C2, wherein the formulation does not comprise histidine, sucrose, or trehalose.

[0185] C6. The pharmaceutical formulation of any one of embodiments C1-C5, wherein the formulation does not further comprise an antioxidant.

[0186] C7. The pharmaceutical formulation of any one of embodiments C1-C6, wherein the pH is about 5.5.

[0187] C8. A method of treatment comprising administering to a subject having or at risk of developing a disease or condition a stable pharmaceutical formulation of any one of embodiments C1-C7 above.

[0188] C9. The method of embodiment C8, further comprising administering to the subject a second therapeutic composition.

[0189] C10. The method of any of embodiments C8 or C9, wherein the disease or condition is selected from the group consisting of cancer, e.g., melanoma, renal cell carcinoma (RCC), non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma, endometrial cancer, tumor mutation burden-high (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), anaplastic thyroid cancer, and / or an infectious disease.

[0190] C11. The method of any one of embodiments C8-C10, wherein the subject is a mammal.

[0191] C12. The method of embodiment C11, wherein the mammal is a human.

[0192] C13. The pharmaceutical formulation of any one of embodiments C1-C12, wherein the formulation further comprises an endoglycosidase hydrolase enzyme.

[0193] C14. The pharmaceutical preparation of C13, wherein the endoglycosidase hydrolase enzyme is recombinant human hyaluronidase.

[0194] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0195] equivalent Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of the present disclosure, that certain changes and modifications can be made thereto without departing from the spirit or scope of the disclosed embodiments. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. Pembrolizumab, 5 mM to 25 mM L-glutamic acid, L-threonine in concentrations of 100 mM to 300 mM, 0.01% to 0.1% w / v polysorbate A pharmaceutical formulation comprising the above, wherein the formulation is a liquid, has a pH in the range of 5.2 to 5.8, does not contain histidine, sucrose, or trehalose, and, after being stored at 2°C to 8°C for 104 weeks, contains 0.2% or less of high molecular weight (HMW) species in a liquid state when measured by size exclusion chromatography.

2. Pembrolizumab, 10 mM L-glutamic acid, 65 mM or 271 mM valine, 0.01% to 0.1% w / v polysorbate A pharmaceutical formulation comprising the above, wherein the formulation is a liquid, has a pH in the range of 5.2 to 5.8, and after being stored at 2°C to 8°C for a maximum of 6 months, the change in the total amount of high molecular weight (HMW) species in the liquid state is 0.6% or less when measured by size exclusion chromatography compared to the HMW species at week 0.

3. The pharmaceutical formulation according to claim 1, wherein the concentration of pembrolizumab is 25 to 200 mg / mL.

4. The pharmaceutical formulation according to claim 1, wherein the concentration of pembrolizumab is 25 mg / mL.

5. The pharmaceutical preparation according to claim 1, wherein the concentration of L-glutamic acid is 10 mM.

6. The pharmaceutical preparation according to claim 1, wherein the concentration of threonine is 250 mM or 271 mM.

7. The pharmaceutical formulation according to claim 1, wherein the polysorbate is polysorbate 80.

8. The pharmaceutical preparation according to claim 1, wherein the amount of polysorbate is 0.02% w / v or 0.07% w / v polysorbate 80.

9. The pharmaceutical preparation according to claim 1, wherein the pH is 5.

5.

10. The pharmaceutical preparation according to claim 1, which does not contain histidine, sucrose, trehalose, or a combination thereof.

11. The pharmaceutical formulation according to claim 1, further comprising glycerol.

12. The pharmaceutical preparation according to claim 1, which does not contain sucrose, sorbitol, glycerol, trehalose, or any combination thereof.

13. The pharmaceutical preparation according to claim 1, which does not contain an antioxidant.

14. The pharmaceutical preparation according to claim 1, suitable for intravenous administration.

15. The pharmaceutical formulation according to claim 1, wherein, after being stored at 2°C to 8°C for 104 weeks, the change in the total amount of HMW species in liquid state is 0.1% or less when measured by size exclusion chromatography compared to the HMW species at week 0.

16. The pharmaceutical formulation according to claim 1, wherein, after being stored at approximately 25°C for 24 weeks, it contains 0.4% or less of HMW species in a liquid state when measured by size exclusion chromatography.

17. A pharmaceutical formulation according to any one of claims 1 to 16, for use in a method of treating cancer in a subject, wherein the method comprises administering an effective amount of the pharmaceutical formulation according to any one of claims 1 to 16 to the subject.

18. The pharmaceutical formulation according to claim 17, wherein the cancer is selected from the group consisting of melanoma, renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), bladder cancer, head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high microsatellite instability or mismatch repair deficiency cancer, high microsatellite instability or mismatch repair deficiency colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma, endometrial cancer, tumor gene mutational burden high score (TMB-H) cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), and undifferentiated thyroid cancer.

19. The pharmaceutical formulation according to claim 17, wherein the effective amount comprises a regimen of administering 200 mg of pembrolizumab every three weeks over the course of treatment, or a regimen of administering 400 mg of pembrolizumab every six weeks over the course of treatment.

20. The pharmaceutical formulation according to claim 17, wherein the method further comprises administering to the subject a second therapeutic agent selected from the group consisting of a chemotherapeutic agent, sorafenib, axitinib, lenvatinib, ipilimumab, and trastuzumab.

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