Anti-human pd-1 antibody crystals and methods of using the same

Abstract

The present invention provides methods for producing crystalline anti-PD-1 monoclonal antibodies (mAbs), wherein the mAbs are pembrolizumab or pembrolizumab variants, comprising (1) mixing a solution comprising (a) a mAb, (b) polyethylene glycol (PEG), and (c) an additive selected from the group consisting of caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a biologically active gibberellin, and a pharmaceutically acceptable salt of the biologically active gibberellin to form a crystallization solution; (2) incubating the crystallization solution for a time sufficient for crystal formation; and (3) optionally harvesting the crystalline anti-PD-1 mAb from the solution. In particular embodiments, the PEG is PEG 3350, and the additive is caffeine. The present invention also relates to novel anti-human PD-1 mAb crystals produced by the methods described herein. Characterization of the resolubilized crystal suspensions using several biochemical methods indicates that the biophysical properties of the resolubilized mAb crystals are consistent with the intact antibody starting sample. The crystals and methods of the present invention are suitable for a variety of pharmaceutical applications, such as purification, storage, formulation, and drug delivery.

Description

TECHNICAL FIELD

[0001] The present invention relates to methods of producing a crystal suspension of an anti-PD-1 monoclonal antibody. The invention further relates to antibody crystals produced by the methods herein, pharmaceutical compositions comprising the crystals, and methods of using the same.

[0002] Cross Reference to Related Applications

[0003] This application claims the benefit of U.S.S.N. 62 / 753,615, filed October 31, 2018, which is incorporated by reference herein in its entirety.

[0004] Reference to Sequence Listing on File as an Electronic Submission

[0005] The Sequence Listing for this application is being submitted electronically via EFS-Web in ASCII format and is being filed herewith. The file name is "24638WOPCT-SEQLIST-17OCT2019.TXT", the creation date is October 9, 2019, and the size is 10 Kb. The Sequence Listing submitted electronically via EFS-Web is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND

[0006] Therapeutic and diagnostic antibodies have become the fastest growing sector of the biopharmaceutical industry. A key aspect of the success of antibodies as therapeutics has been the development of improved methods to express, purify, and characterize these proteins. In general, antibody therapeutics are large (typically greater than 150 kDa) and complex in nature, and thus must be administered in stoichiometric amounts rather than catalytic amounts. As a result, production and purification scales have reached levels of production previously thought impossible. Stable formulations and delivery strategies also need to be developed for such large quantities of complex molecules.

[0007] The development of stable formulations containing high concentrations of active agents, such as antibodies or antigen binding fragments, is particularly important for biological formulations intended for subcutaneous administration to patients, as the volume of solution delivered to the patient is greatly reduced. Subcutaneous administration is the preferred method of administration for many antibodies, in part because it can enable self-administration or make administration by a medical professional (e.g., a pharmacist, physician, or nurse) easier. Therapeutic antibodies have traditionally been prepared either as lyophilized or in solution. The lyophilized form can exhibit enhanced long-term stability, but requires reconstitution prior to use, making it less than ideal for self-administration. On the other hand, the development of stable liquid formulations is more challenging and typically requires refrigeration prior to use.

[0008] Immune checkpoint therapy targeting the Programmed Death-1 (PD-1) axis has made breakthrough progress in clinical responses in a variety of human cancers (Brahmer et al., N Engl J Med 2012, 366:2455-65; Garon et al. N Engl J Med 2015, 372:2018-28; Hamid et al., N Engl J Med 2013, 369:134-44; Robert et al., Lancet 2014, 384:1109-17; Robert et al., N Engl J Med 2015, 372:2521-32; Robert et al., N Engl J Med 2015, 372:320-30; Topalian et al., N Engl J Med 2012, 366:2443-54; Topalian et al., J Clin Oncol 2014, 32:1020-30; Wolchok et al., N Engl J Med 2013, 369:122-33). The interaction of the PD-1 receptor on T cells with its ligands, PD-L1 and PD-L2, on tumors and immune infiltrating cells, modulates T cell-mediated immune responses and can play a role in immune evasion of human tumors (Pardoll DM. Nat Rev Cancer 2012, 12:252-64). Binding of PD-1 to either of its ligands results in the transmission of an inhibitory stimulus to the T cell. Immune therapies targeting the PD-1 axis include monoclonal antibodies to the PD-1 receptor (KEYTRUDA TM (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ and OPDIVO TM (nivolumab), Bristol-Myers Squibb, Princeton, NJ) and those that bind the PD-L1 ligand (MPDL3280A; TECENTRIQ TM (atezolizumab), Genentech, San Francisco, CA). Both treatment approaches have demonstrated anti-tumor effects in multiple cancer types.

[0009] There is a need for improved stable formulations of anti-PD-1 antibodies for use in, for example, the treatment of cancer patients. Preferably, these antibody formulations do not require reconstitution prior to administration. In addition, these formulations enable the administration of higher antibody concentrations than are easily achieved using typical solution formulations, and preferably support high concentrations at low viscosities sufficient to facilitate subcutaneous delivery. SUMMARY

[0010] In one aspect, the present invention relates to a method of producing crystalline anti-PD-1 monoclonal antibodies (mAbs), comprising: (a) mixing: (i) an aqueous buffered solution comprising about 5 mg / mL to about 80 mg / mL of a mAb, wherein the anti-PD-1 monoclonal antibody is pembrolizumab or a pembrolizumab variant, (ii) polyethylene glycol (PEG), and (iii) an additive selected from the group consisting of caffeine, theophylline, 2’-deoxyguanosine-5’-monophosphate, a biologically active gibberellin such as gibberellin A3 and a pharmaceutically acceptable salt of gibberellin, to form a crystallization solution, wherein the crystallization solution has a pH of about 6.0 to about 8.8, and comprises about 2% to about 40% weight by volume (w / v) of PEG and about 0.1% to about 0.30% w / v of the additive; (b) incubating the crystallization solution for a period of time sufficient to form crystals; and (c) optionally harvesting the crystalline anti-PD-1 mAbs from the solution.

[0011] In some embodiments, the mAb is pembrolizumab. In further embodiments, the mAb is a pembrolizumab variant that retains the ability to bind to PD-1 and the ability to bind to the additive.

[0012] In particular embodiments, the additive is caffeine.

[0013] In some embodiments, the crystallization solution further comprises about 1% to about 10% of dextran sodium sulfate.

[0014] In one aspect, the present invention relates to an isolated anti-PD-1 crystal produced by the method of the present invention.

[0015] In another aspect, the present invention relates to an isolated crystal comprising pembrolizumab complexed with caffeine, wherein the crystal is characterized by a space group P2221 a = b = g = 90°.

[0016] In another aspect, the present invention relates to a crystalline pembrolizumab comprising pembrolizumab complexed with caffeine, characterized by a solid state NMR13C spectrum showing peaks at about 182.16, 181.54, 179.99, 109.36, 108.23, 103.58, 76.88, and 76.04 ppm. 13 C spectrum.

[0017] In another aspect, the present application relates to crystalline pembrolizumab comprising pembrolizumab complexed with caffeine, characterized by showing peaks at about 183.07, 182.16, 181.54, 180.55, 179.99, 110.70, 110.15, 109.36, 108.23, 103.58, 101.49, 99.75, 98.56, 76.88, 76.04, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26, and 11.13 ppm by solid state NMR 13 C spectra.

[0018] Also provided herein are compositions comprising the anti-PD-1 mAb crystals of the present application and a pharmaceutically acceptable carrier.

[0019] In one aspect, the present application provides methods of treating cancer and / or infectious diseases by administering the crystals or compositions of the present application to a patient in need thereof. In particular embodiments, the compositions are administered to the patient by intravenous infusion. In alternative embodiments, the crystals are administered to the patient by subcutaneous injection. BRIEF DESCRIPTION OF DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] Figures 1A-1C Micrographs showing crystals in a suspension of pembrolizumab crystals obtained by vapor diffusion using a Silver Bullet Bio crystallization reagent A2 precipitant solution and 12.5% w / v PEG 3350, 0.05 M HEPES buffer, pH 6.8 at 30°C. See Example 1. Micrographs at 200x magnification using SONICC TM Imaging system acquired after 30 days. Figure 1A Visible light images of crystals taken at 200x magnification are provided. Figure 1B and 1C Visible light images of crystals taken at 200x magnification are provided. TM Images produced by UV-TPEF and SHG modes of the SONICC

[0022] Figures 2A-2C Visible light micrographs taken at 200x magnification of crystals within a suspension of pembrolizumab crystals produced using droplet vapor diffusion are provided, as described in Example 2. Figure 2ACrystals formed from 0.20% caffeine, 12% PEG 3350, 50 mM HEPES, pH 6.8 are shown. Figure 2B Crystals formed from 0.2% theophylline + 0.2% ethanolamine + 10% PEG 3350 are shown. Figure 2C Crystals formed using 0.2% theophylline + 0.2% 2’-Deoxyguanosine 5-monophosphate sodium salt hydrate + 16% PEG 3350 are shown.

[0023] Figure 3 Micrographs of pembrolizumab crystals at 200x magnification are provided that were produced from a crystallization method that included incubating pembrolizumab with 9.8% PEG 3350, 45 mM HEPES, pH 7.7, 0.23% caffeine at 30 °C for 18 hours (10 mL scale).

[0024] Figures 4A-4F Images of a crystal suspension prepared as described in Example 6 with 10.18% PEG 3350, 50 mM HEPES, pH 7.2 solution are provided. The images show crystals formed from the crystallization solution after incubation at 2 °C and 50 °C, which were characterized using SONICC TM visible light mode of the imaging system Figure 4A and 4D ), UV-TPEF mode Figure 4B and 4E ), and SHG mode Figure 4C and 4F ).

[0025] Figure 5 Micrographs of pembrolizumab crystals prepared using the procedure described in Example 10 are shown. Crystals selected for complete structural characterization are shown.

[0026] Figure 6A Graphical representations of the pembrolizumab / caffeine complex in the low-salt PEG / caffeine crystal form described in Example 10 are shown. The protein backbone is shown as a ribbon; the glycosyl groups attached to the protein as well as the ordered caffeine molecules bound to the protein are depicted as sticks. In the color version, the protein backbone is shown as a ribbon colored as follows: orange for VL, magenta for CL, green for VH, cyan for CH1, yellow for CH2, and gray for CH3. Figure 6A Figure 6B Close-up view of the caffeine molecules that were found to be ordered and mediate crystal contacts is shown. The protein backbone is represented as a ribbon with side chains around the caffeine molecules, which are depicted as sticks. In the color version, the color rules are the same as Figure 6A

[0027] Figures 7A-7C ​​This demonstrates the use of batch crystallization (175 mL scale) and the conditions described in Example 11, utilizing SONICC. TM Visible light in imaging systems ( Figure 7A ), UV-TPEF Figure 7B ) and SHG ( Figure 7C Crystal images generated by the ) mode.

[0028] Figure 8A The viscosity (cP) of a 200 mg / mL pembrolizumab crystal suspension is shown compared to the shear rate (s) of BD Hypak 1 mL PFS with 27 G RW and 29 G TW x” needles. -1 See Example 12. Figure 8B The syringe injection force (N) is shown as the displacement (mm) of 200 (triangular), 175 (square), and 150 (rhomboid) mg / mL pembrolizumab crystal suspensions produced as described in Example 11.

[0029] Figure 9 The required injection force (N) for producing a 200 mg / mL crystal pembrolizumab suspension in a variety of 1 mL plastic and glass syringes over distance (mm) is provided. See Example 12. The crystal suspension was produced under the conditions described in Example 11.

[0030] Figure 10A The solid state of the pembrolizumab crystal suspension prepared as described in Example 11 is depicted. 13 C NMR CP MAS. Figure 10B Depicting Figure 10A The magnified spectral region of the spectrum.

[0031] Figure 11A and Figure 11B The image depicts a pembrolizumab-caffeine crystal suspension (solid line) and caffeine-only crystals (dashed line). 13 C( Figure 11A )and 15 N( Figure 11B CP MAS spectrum. 2- 13 C and 1,3- 15 N-isotope-labeled caffeine was used in these spectra. Detailed Implementation

[0032] The present invention provides crystalline forms of pembrolizumab antibodies and variants thereof, suspensions of these crystals, and pharmaceutical formulations of these suspensions. Highly purified pembrolizumab monoclonal antibodies were used for high throughput (HT) vapor diffusion sparse matrix screening experiments. Novel crystal suspensions were obtained using various additives at 30°C and room temperature. The present invention also provides methods for preparing the novel monoclonal antibody (mAb) crystal suspensions, such as using bulk crystallization (batch and dialysis) to prepare at high yield, where the mAb is pembrolizumab or a variant thereof.

[0033] In one aspect, the present invention relates to a method for producing crystalline anti-PD-1 mAb comprising: (a) mixing: (i) an aqueous buffered solution comprising about 5 mg / mL to about 80 mg / mL mAb, (ii) polyethylene glycol (PEG), and (iii) an additive selected from the group consisting of: caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a biologically active gibberellin, and a pharmaceutically acceptable salt of a biologically active gibberellin; to form a crystallization solution, wherein the crystallization solution has a pH of about 6.0 to about 8.8, and comprises about 5% to about 40% weight / volume (w / v) PEG and about 0.10% to about 0.30% w / v additive; (b) incubating the crystallization solution for a period of time sufficient to form crystals; and (c) optionally harvesting the crystalline anti-PD-1 mAb from the solution. The resulting crystal suspension comprises anti-PD-1 mAb crystals, such as pembrolizumab crystals, having a particle size of 0.5-200 microns after harvesting. In particular embodiments, the method further comprises a step of homogenizing the crystals formed in step (b). In still further embodiments, the crystalline anti-PD-1 mAb is harvested from the crystallization solution, or is at least partially purified from the crystallization solution, and then the harvested or purified crystals are homogenized. The resulting anti-PD-1 mAb crystals, such as pembrolizumab crystals, have a particle size of about 0.5 to about 50 microns after homogenization.

[0034] The present invention further provides various methods for preparing the crystalline pembrolizumab antibodies of the present invention, as described in more detail in Examples 1-18. Examples 1 and 2 provide vapor diffusion-based methods that can be used to perform screening to determine crystallization conditions. Such methods are also suitable for producing large crystals for X-ray diffraction studies, for example, to determine the three-dimensional structure of an anti-PD-1 antibody. In some embodiments, dextran sulfate sodium is added to the crystallization solution to allow better control of nucleation; thereby allowing growth of larger crystals.

[0035] Examples 5, 11 and 15-17 provide crystallization methods suitable for large scale production, such as batch crystallization and body dialysis crystallization, which can be used for commercial scale production of crystalline pembrolizumab or pembrolizumab variants for therapeutic use. For example, methods for harvesting the crystals of the application using centrifugation are provided in Examples 11, 14 and 15, but filtration methods known in the art, such as hollow fiber tangential flow filtration, can also be used to harvest the crystals, for example on a commercial scale.

[0036] Although the specific embodiments disclosed employ a 1:1 and / or 1:3 mixture of antibody solution to precipitant solution, any modification of the disclosed methods that results in approximately the same concentrations of solution components in the final crystallization solution from which the crystals are produced is equivalent. For example, if a precipitant solution (a solution comprising PEG and additives, as defined herein) that is less than or greater than 50% of the final volume of the crystallization solution is used, respectively, the concentrations of components in the precipitant solution can be proportionally increased or decreased.

[0037] The crystallization methods of the application also provide a method for purifying a pembrolizumab or pembrolizumab variant antibody, even if such crystals are re-dissolved prior to use. In one embodiment, a pembrolizumab antibody is produced and at least partially purified by methods described herein and known in the art. The antibody is then crystallized, for example by batch crystallization or body dialysis. The crystalline antibody is then recovered and washed, for example as described in Example 5 (or by filtration), and re-dissolved in a buffer, for example 10 mM histidine buffer pH 5.4, or any suitable buffer for the intended use of the purified antibody. For therapeutic use, suitable pharmaceutically acceptable buffers and excipients are used.

[0038] The crystallization methods of the application also provide a method for storing a purified pembrolizumab antibody, even if such crystals are re-dissolved prior to use. In one embodiment, a pembrolizumab or pembrolizumab variant antibody is produced and at least partially purified by methods described herein and known in the art. The antibody is then crystallized, for example by batch crystallization or body dialysis. The resulting concentrated suspension of pembrolizumab crystals is stored as a stable, concentrated formulation that is suitable for shipping and re-formulation at global manufacturing sites.

[0039] The crystalline pembrolizumab antibodies of the present invention have several advantageous properties for treatment, including the ability to be formulated at high concentrations and low viscosity. Such high concentrations can enable more efficient administration to a subject, for example, by subcutaneous injection. The crystalline suspensions of the present invention can be used to prepare pharmaceutical formulations of up to 300-400 mg / mL, thereby enabling higher dosing with lower injection volumes, and thus, reduced discomfort. The crystalline suspensions of the present invention can be delivered by subcutaneous injection using a small gauge needle, for example, a 27G insulin syringe. Reduced volume, reduced viscosity, and use of a smaller needle can all reduce discomfort to the patient upon subcutaneous administration.

[0040] The crystalline pembrolizumab antibodies of the present invention also have other advantageous properties. Suspensions of the crystalline pembrolizumab antibodies show comparable stability to the starting solution formulation and can allow for a longer shelf life. Additionally, the ability to store the suspensions of the present invention crystals at room temperature can provide significant advantages in drug product handling and supply chain management.

[0041] Prior crystalline suspensions of pembrolizumab were prepared using a high salt process. See WO 2016 / 137850. The novel pembrolizumab crystals of the present invention do not require the use of high salt, which is advantageous for pharmaceutical manufacturing processes as high salt levels are not suitable for pharmaceutical formulations intended for subcutaneous administration.

[0042] I. Definitions and Abbreviations

[0043] As used throughout the specification and the appended claims, the following abbreviations apply:

[0044] CDR complementarity determining region

[0045] CHO Chinese hamster ovary

[0046] CP cross-polarization

[0047] CPS combined positive score

[0048] DFS disease-free survival

[0049] ELISA enzyme-linked immunosorbent assay

[0050] FR framework region

[0051] GRAS generally recognized as safe

[0052] HEPES hydroxyethyl-piperazineethane-sulfonic acid buffer

[0053] HT high throughput

[0054] IEX ion exchange

[0055] IHC immunohistochemistry or immunohistochemical

[0056] IPTG isopropyl β-d-1-thiogalactopyranoside

[0057] IV. Intravenous

[0058] mAb monoclonal antibody

[0059] MAS Magic Angle Spin

[0060] NCI (National Cancer Institute)

[0061] NMR (Nuclear Magnetic Resonance)

[0062] PBS (Phosphate Buffered Saline)

[0063] PD progressive disease

[0064] PD-1 programmed death 1

[0065] PD-L1 programmed cell death ligand 1

[0066] PD-L2 programmed cell death 1 ligand 2

[0067] PEG (Polyethylene Glycol)

[0068] PFS No Progression Survival

[0069] PK Pharmacokinetics

[0070] PR Partial Response

[0071] OR Overall Response

[0072] OS Overall Survival

[0073] Q2W, one dose every two weeks.

[0074] Q3W, one dose every three weeks.

[0075] QD One dose per day

[0076] RECIST Response Evaluation Criteria for Solid Tumors

[0077] RPLC (Reversed-phase liquid chromatography)

[0078] RPM (revolutions per minute)

[0079] SC subcutaneous

[0080] SD (stable disease) or standard deviation, depending on the context.

[0081] SHG Second Harmonic Generation

[0082] Second order nonlinear imaging of SONICC chiral crystals

[0083] T / C ratio of tumor volume for treatment versus control

[0084] TPS tumor proportion score

[0085] UV-TPEF ultraviolet two-photon excitation fluorescence

[0086] VH immunoglobulin heavy chain variable region

[0087] VK immunoglobulin kappa light chain variable region

[0088] w / v weight / volume

[0089] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0090] As used throughout the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0091] Reference to “or” means any one or any combination of the alternatives referred to. In some cases, “and / or” is used to emphasize one or both possibilities.

[0092] “Treatment” or “treating” means administration of a composition of the present invention to a patient to induce a positive therapeutic effect. The term does not necessarily indicate complete abolition of all disease or disorder symptoms. “Treating” a cancer or immune disorder means administering a crystal suspension or composition of the present invention to a patient having an immune disorder or cancerous condition, or diagnosed as having or being susceptible to a cancer or infection by a pathogen (e.g., virus, bacteria, fungus) to achieve at least one positive therapeutic effect, such as reduced number of cancer cells, reduced size of tumor, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth. “Treatment” can include one or more of the following: inducing / increasing an anti-tumor immune response, stimulating an immune response to a pathogen, toxin, and / or self-antigen, stimulating an immune response to a viral infection, reducing the number of one or more tumor markers, inhibiting growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, reducing the level of one or more tumor markers, improving, reducing the severity or duration of cancer, prolonging survival of a patient relative to the expected survival of a similar untreated patient.

[0093] "Immune disorder" or "immune disease" includes, for example, pathologic inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune disorder" also refers to infections, persistent infections, and proliferative disorders such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist elimination by the immune system. "Cancerous disorder" includes, for example, cancer, cancerous cells, tumors, angiogenesis, and precancerous disorders such as dysplasia.

[0094] "Inflammatory disorder" refers to a disorder or pathological condition in which pathology is caused, in whole or in part, by a change in the number, migratory capacity, or activation of cells of the immune system. Cells of the immune system include, for example, T cells, B cells, monocytes or macrophages, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils, mast cells, or any other cell that is specifically associated with immunity, such as cytokine-producing endothelial or epithelial cells.

[0095] Active therapeutic effects of cancer can be measured in a variety of ways (see W. A. Weber, J. Nucl. Med. 50: 1 S-10S (2009)). For example, with respect to tumor growth inhibition, according to NCI criteria, T / C < 42% is the minimum level of antitumor activity. T / C < 10% is considered a high level of antitumor activity, where T / C (%) = median tumor volume of treated / median tumor volume of control x 100. In some embodiments, the treatment achieved by administration of the formulations of the application is any one of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as "time to tumor progression," refers to the length of time during and after treatment that the cancer does not grow, and includes the amount of time a patient is in complete response or partial response, as well as the amount of time a patient is in stable disease. DFS refers to the length of time a patient remains free of disease during and after treatment. OS refers to the prolongation of life expectancy compared to the original or untreated individual or patient. While embodiments of the formulations, methods of treatment, and uses of the application can not effectively achieve an active therapeutic effect in every patient, they should achieve an active therapeutic effect in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi2test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0096] The term "patient" (or "subject" or "individual" as referred to herein) refers to a mammal (e.g., a rat, a mouse, a dog, a cat, a rabbit) that can be treated with the formulations of the application, most preferably a human. The term "patient" can also include non-human animals, including livestock animals and domestic animals, including but not limited to, bovines, equines, ovines, porcines, caprines, lagomorphs, felines, canines, and other mammals in need of treatment. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient. A "patient in need of treatment" is one who has been diagnosed with, is suspected of having, or is predisposed to a disease or disorder in which the crystal suspensions or compositions of the application are intended to treat, or a patient in need of prevention of a disorder.

[0097] The term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies. A "parental antibody" refers to an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for the intended use, e.g., humanization of an antibody for use as a human therapeutic antibody.

[0098] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable regions of each light / heavy chain pair interact to form the antibody binding site. Thus, generally, a complete antibody has two binding sites. The carboxy-terminal portion of the heavy chain can be defined as a constant region primarily responsible for effector functions. Usually, the light chains of a human antibody are classified as kappa and lambda light chains. Furthermore, the heavy chains of a human antibody are classified as mu, delta, gamma, alpha, or epsilon, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0099] Generally, the variable domains of each of the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FRs). The CDRs are aligned to form an antigen binding site in conjunction with the framework regions. Generally, from N- to C-terminus, the variable domains of the light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Generally, the assignment of amino acids to each domain is according to the Kabat et al. (1991) numbering system. Sequences of Proteins of Immunological InterestKabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0100] An antibody that "specifically binds" to a particular target protein is one that exhibits preferential binding to that target as compared to other proteins, although such specificity does not require absolute binding specificity. An antibody is considered "specific" for its intended target if its binding establishes the presence of the target protein in a sample, e.g., without producing undesirable results such as false positives. Antibodies or binding fragments thereof useful in the present application bind to the target protein, i.e., human PD-1, with an affinity that is at least two-fold higher, preferably at least ten-fold higher, more preferably at least 20-fold higher, and most preferably at least 100-fold higher than the affinity for non-target proteins. As used herein, an antibody is said to specifically bind to a polypeptide comprising a given amino acid sequence (e.g., the amino acid sequence of a mature human PD-1 molecule) if it binds to a polypeptide comprising that sequence but does not bind to a protein lacking that sequence.

[0101] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic composition or formulation sufficient to effect a treatment of a disease or condition thereby introduced into a patient. One skilled in the art recognizes that this level can vary depending on the characteristics of the patient (e.g., age, weight, etc.). When used in connection with a crystal suspension or composition of the present application, the term "effective amount" refers to an amount of the suspension or composition sufficient to treat the pathological condition (e.g., a cancerous condition or an inflammatory disorder) for which it is intended. An "effective amount" of a crystal or composition of the present application refers to an amount sufficient to elicit the response that is sought in a cell, tissue, system, animal, or human. In one embodiment, an effective amount is a "therapeutically effective amount" for alleviating the symptoms of the disease or condition being treated. When the active compound (i.e., active ingredient) is administered as a salt, the amount of the active ingredient refers to the free acid or free base form of the compound.

[0102] The term "about" when used in reference to the amount of a material or composition (e.g., mM or M), the percentage of a formulation component (v / v or w / v), the pH of a solution / formulation, or the value of a parameter in a method of characterization, etc., refers to the variation of a numerical quantity that can occur, e.g., through typical measurement, manipulation, and sampling procedures involved in the preparation, characterization, and / or use of the material or composition; through incidental error in these procedures; through differences in the manufacture, source, or purity of the ingredients used to make or use the composition; etc. In certain embodiments, "about" can refer to a variation of ±0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 of the appropriate unit. In certain embodiments, "about" can refer to a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%. In certain embodiments, for the purposes of solid state NMR, the term "about" refers to ±0.1 ppm.

[0103] The terms "cancer," "cancerous," or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More particular examples of such cancers include squamous cell cancer, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer

[0104] "Concentration," when used in reference to a crystalline antibody suspension of the present application, refers to the amount of antibody (e.g., pembrolizumab) present in a given macroscopic unit volume of solution. Although suspensions have inherent heterogeneity compared to traditional solutions, the term concentration is used in its customary sense. The concentration of antibody in a crystalline suspension is equivalent to the concentration of an equivalent sample in which the antibody is not in crystalline form.

[0105] "Anti-PD-1 mAb crystal" or "crystalline anti-PD-1 mAb," as used herein, refers to a crystal comprising antibodies arranged in a three-dimensionally periodically repeating lattice structure. In contrast, a solid, amorphous form of a monoclonal antibody, such as produced by lyophilizing a mAb dissolved in a solution, does not exhibit the optical properties typical of the crystalline antibody form, such as refractive index and birefringence.

[0106] "Antibody solution" refers to a solution of an anti-human PD-1 antibody (e.g., pembrolizumab) used to generate the crystalline antibody of the application. "Precipitant solution" refers to a second solution that is mixed with the antibody solution, typically in a 1 : 1 volume ratio (i.e., equal volumes of the two solutions are mixed), to generate a "crystallization solution" from which the antibody crystals grow. For convenience, concentrations of the antibody and precipitant solutions for a 1 : 1 mixture are provided herein, but one skilled in the art will recognize that the volume ratio used to make the mixture can vary, and thus the concentrations of the solutions that make up the mixture can also vary. Such modifications are within the scope of the application if they result in the same crystallization conditions (i.e., the same crystallization solution) as the mixtures described herein.

[0107] With respect to the dialysis-based crystallization method, "dialysis solution" refers to the solution against which the solution of pembrolizumab ("antibody solution") is dialyzed to drive the formation of the crystalline antibody of the application. "Retentate" refers to the antibody solution after dialysis, which can include the harvested antibody crystals. The antibody solution / retentate is on one side of the dialysis membrane, and the dialysis solution is on the opposite side.

[0108] The term "homogenization" refers to the use of mechanical means to reduce the size of the crystal particles; thereby producing smaller particles that are more uniform and evenly distributed. Homogenization can be performed by any known means, for example, by using a homogenizer, or by forcing the crystalline particles through smaller pores (venturi effect), such as a syringe, to break the particles into smaller sizes.

[0109] The terms "micron" and "millionth of a meter" are used interchangeably herein, and both refer to 1 / 1,000,000 of a meter.

[0110] "PD-L1" or "PD-L2" expression refers to any detectable level of expression of the specified PD-L protein on the surface of a cell or of the specified PD-L mRNA within a cell or tissue. PD-L protein expression can be detected in an immunohistochemical (IHC) analysis of a tumor tissue section with a diagnostic PD-L antibody or by flow cytometry. Alternatively, PD-L protein expression by tumor cells can be detected by PET imaging using a binding agent (e.g., an antibody fragment, an affibody, etc.) that specifically binds to the desired PD-L target (e.g., PD-L1 or PD-L2). Techniques for detecting and measuring PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0111] Several methods have been described to quantify PD-L1 protein expression in IHC analysis of tumor tissue sections. See, e.g., R. H., et al., Proc. Natl. Acad. Sci USA 101(49): 17174-17179 (2004); Thompson, R. H. et al., Cancer Res. 66:3381-3385 (2006); Gadiot, J., et al., Cancer 117:2192-2201 (2011); Taube, J. M. et al., Sci Transl Med 4:127ra37 (2012); and Toplian, S. L. et al., New Eng. J Med. 366(26):2443-2454 (2012).

[0112] One method employs a simple binary endpoint of PD-L1 expression positive or negative, with positive results defined by the percentage of tumor cells showing histological evidence of cell surface membrane staining. Tumor tissue sections are counted as PD-L1 expression positive if at least 1%, preferably 5%, of the total tumor cells express PD-L1.

[0113] In another method, PD-L1 expression in tumor tissue sections is quantified both in tumor cells and in infiltrating immune cells that predominantly comprise lymphocytes. The percentage of tumor cells and infiltrating immune cells that exhibit membrane staining are quantified as <5%, 5-9%, and then in increments of 10% up to 100% respectively. In some embodiments, PD-L1 expression in tumor cells is counted as negative if the score is less than 5% score, and positive if the score is >5%. Expression of PD-L1 in immune infiltrates is reported as a semi-quantitative measure, which is referred to as the adjusted inflammation score (AIS), which is determined by multiplying the percentage of membrane-stained cells by the intensity of the infiltrate, which is graded as none (0), mild (score 1, rare lymphocytes), moderate (score 2, tumor locally infiltrated by lymphoid cell aggregates), or severe (score 3, diffuse infiltrate). Tumor tissue sections are counted as positive for PD-L1 expression in immune infiltrates if the AIS is >5.

[0114] Tissue sections from tumors that have been stained by IHC with a diagnostic PD-L1 antibody can also be scored for PD-L1 protein expression by assessing PD-L1 expression in tumor cells and infiltrating immune cells in the tissue section using a scoring process. See WO 2014 / 165422. One PD-L1 scoring process involves examining each tumor nest in the tissue section that is stained and assigning one or both of a modified H-score (MHS) and a modified proportion score (MPS) to the tissue section. To assign a MHS, four separate percentages are estimated for all viable tumor cells and stained mononuclear inflammatory cells in all examined tumor nests: (a) cells with no staining (intensity = 0), (b) cells with weak staining (intensity = 1+), (c) cells with moderate staining (intensity = 2+), and (d) cells with strong staining (intensity = 3+). Cells must have at least partial membrane staining to be included in the weak, moderate, or strong staining percentages. The estimated percentages, which sum to 100%, are then input into the formula 1x(percent of weakly stained cells) + 2x(percent of moderately stained cells) + 3x(percent of strongly stained cells) and the result is assigned as the MHS to the tissue section. The MPS is assigned by estimating the percentage of cells with at least partial membrane staining of any intensity in all viable tumor cells and stained mononuclear inflammatory cells in all examined tumor nests, and the resulting percentage is assigned as the MPS to the tissue section. In some embodiments, a tumor is designated as PD-L1 expression positive if the MHS or MPS is positive.

[0115] "CPS" or "combined positive score" refers to an algorithm used to determine a PD-L1 expression score from a tumor sample of a patient. CPS can be used to select patients for treatment with a particular treatment regimen, including a treatment method that involves administration of an anti-PD-1 antibody, where expression of PD-L1 is associated with a higher response rate in a particular patient population relative to the same patient population that does not express PD-L1. CPS is determined by determining the number of viable PD-L1 positive tumor cells, the number of viable PD-L1 negative tumor cells, and the number of viable PD-L1 positive mononuclear inflammatory cells (MICs) in tumor tissue of a patient having a tumor and calculating the CPS using the following formula:

[0116]

[0117] TPS or "tumor proportion score" refers to the percentage of tumor cells expressing PD-L1 on the cell membrane. TPS generally includes the percentage of tumor cells expressing PD-L1 at any intensity (weak, moderate, or strong), which can be determined by immunohistochemical analysis using diagnostic anti-human PD-L1 mAbs (e.g., antibody 20C3 and antibody 22C3) as described above. Cells are considered to express PD-L1 if there is membrane staining, including cells with partial membrane staining.

[0118] The level of PD-L mRNA expression can be compared to the mRNA expression level of one or more reference genes (e.g., ubiquitin C) commonly used in quantitative RT-PCR.

[0119] In some embodiments, the PD-L1 expression level (protein and / or mRNA) of malignant cells and / or infiltrating immune cells within a tumor is determined to be "overexpressed" or "elevated" based on comparison to the PD-L1 expression level (protein and / or mRNA) of an appropriate control. For example, the control PD-L1 protein or mRNA expression level can be the level quantified in non-malignant cells of the same type or in a section from a matched normal tissue. In some preferred embodiments, the PD-L1 expression is determined to be elevated in a tumor sample if the PD-L1 protein (and / or PD-L1 mRNA) is at least 10%, 20%, 30%, 40%, or 50% higher in the tumor sample than in the control sample.

[0120] "Pembrolizumab" is an IgG4 monoclonal antibody, the structure of which is described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) (Merck Sharp & Dohme Corp., Whitehouse Station, NJ). Each light chain of pembrolizumab comprises light chain complementarity determining regions (CDRs) comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, and heavy chain CDRs comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6. The variable light chain (VL) and variable heavy chain (VH) of pembrolizumab are set forth in SEQ ID NOs: 7 and 8, respectively. L ) and heavy chain (V H) respectively comprising the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, and the full-length light and heavy chains respectively comprising or consisting of the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10. Pembrolizumab is approved by the U.S. FDA for the treatment of patients with unresectable or metastatic melanoma, as an adjuvant treatment for melanoma patients with evidence of nodal involvement following complete surgical resection, and for the treatment of certain patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC), classical Hodgkin’s lymphoma (cHL), urothelial carcinoma, gastric cancer, cervical cancer, primary mediastinal large B-cell lymphoma, microsatellite instability-high (MSI-H) cancer, esophageal cancer, hepatocellular cancer, Merkel cell carcinoma, renal cell cancer, endometrial cancer, small cell lung cancer, and non-small cell lung cancer, as described in Prescribing Information for KEYTRUDA® TM (Merck & Co., Inc., Whitehouse Station, NJ USA; initial U.S. approval 2014, September 2019 update).

[0121] As used herein, a "pembrolizumab variant" refers to a derivative of the pembrolizumab antibody that (1) substantially maintains its biological activity of binding to and inhibiting the activity of the antigen (i.e., human PD-1) (e.g., blocking the binding of PD-1 to PD-L1 and / or PD-L2) and (2) maintains the ability of the antibody to bind to an additive used in the crystallization solution in the methods of the present application, wherein the additive is caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a biologically active gibberellin such as gibberellin A3, or a pharmaceutically acceptable salt thereof. In embodiments of the present application, the pembrolizumab variant comprises the same sequence as the light chain and heavy chain sequences in pembrolizumab (SEQ ID NOs: 9 and 10, respectively), except for up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions located outside of the light chain CDRs and outside of the heavy chain CDRs, e.g., in the framework regions or constant regions. In further embodiments, the pembrolizumab variant has up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions located outside of the pembrolizumab light chain and heavy chain CDRs and further outside of the pembrolizumab residues that bind to caffeine, i.e., outside of TYR 436 and ASN 434 of the pembrolizumab heavy chain (positions 434 and 436 of SEQ ID NO: 10). In other words, the pembrolizumab and the pembrolizumab variant comprise the same CDR sequences but differ from each other by conservative amino acid substitutions at no more than ten other positions in their full-length light chain and heavy chain sequences, respectively. The pembrolizumab variant is substantially identical to pembrolizumab with respect to the following properties: binding affinity to PD-1, ability to block the binding of each of PD-L1 and PD-L2 to PD-1, and ability to bind to an additive selected from the group consisting of: caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a biologically active gibberellin (such as gibberellin A3), and a pharmaceutically acceptable salt of the biologically active gibberellin.

[0122] A "precipitant" is a compound that decreases the solubility of a polypeptide (e.g., an antibody) in a concentrated solution. In a batch crystallization method, the precipitant can be included in a "precipitant solution," while in a dialysis method, the precipitant can be included in a "dialysis solution." The precipitant induces crystallization by forming an energetically unfavorable precipitant-depleted layer around the polypeptide molecule. To minimize the relative amount of this depleted layer, the polypeptides associate and eventually form crystals. This process is explained in Weber (1991) Advances in Protein Chemistry 41 : 1. Various precipitants are known in the art. In the methods of the present application, the precipitant is polyethylene glycol (e.g., PEG 3350).

[0123] In addition to the precipitant, one or more additives that promote crystallization can be added to the polypeptide-precipitant solution or crystallization solution, selected from the group consisting of: caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a biologically active gibberellin, and a pharmaceutically acceptable salt of a biologically active gibberellin. Two of the additives (caffeine and theophylline) that were found to be useful in the methods of the present application share structural similarities as shown below:

[0124]

[0125] It is also demonstrated herein that gibberellin A3 (or, GA3 or gibberellic acid) is a useful reagent in the crystallization methods of the methods of the present application. Gibberellins (also known as GAs) are a class of hormones found in plants that share a common diterpene acid structure and regulate various developmental processes. "Biologically active gibberellins" are involved in different aspects of plant germination and share the following structural features: 1) a hydroxyl group on C-3 beta, 2) a carboxyl group on C-6, and 3) a lactone between C-4 and C-10 (see below). Based on the similar structure and function of "biologically active gibberellins" (which include gibberellin Al (GA1), gibberellin A3 (GA3), gibberellin A4 (GA4), and gibberellin A7 (GA7)) or a pharmaceutically acceptable salt thereof, any biologically active gibberellin or a pharmaceutically acceptable salt thereof is expected to be useful in the methods of the present application.

[0126]

[0127]

[0128] In addition to the precipitant, one or more additional excipients can be added to the polypeptide-precipitant solution or crystallization solution. Excipients include buffers (e.g., Tris or HEPES) to adjust the pH of the solution (and thus the surface charge on the peptide), salts (e.g., sodium chloride, lithium chloride, and sodium citrate) to decrease the solubility of the polypeptide.

[0129] A "tissue section" refers to an individual portion or small piece of a tissue sample, e.g., a thin slice of tissue cut from a sample of normal tissue or a tumor.

[0130] "Tris" (2-amino-2-hydroxymethyl-propane-l,3-diol) as used herein is synonymous with TRIS, Tris base, Trizma, Trisamine, THAM, Aminotrisol, Trolamine, Tris Base, and TROM.

[0131] "Tumor," when applied to a subject diagnosed with or suspected of having cancer, refers to a malignant or potentially malignant tumor or tissue mass of any size, and includes primary tumors and secondary neoplasm. A solid tumor refers to an abnormal growth or mass of tissue that typically does not contain cysts or fluid areas. Different types of solid tumors are named for the type of cell that forms them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) usually do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0132] "Tumor burden," also referred to as "tumor load," refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumors within the entire body, including lymph nodes and bone marrow. Tumor burden can be determined by a variety of methods known in the art, for example, by measuring the size of a tumor upon removal from a subject, for example, using calipers, or while in the body, using imaging techniques (e.g., ultrasound, bone scan, computed tomography (CT) or magnetic resonance imaging (MRI) scan).

[0133] The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, for example, by measuring the size of a tumor upon removal from a subject, for example, using calipers, or while in the body, using imaging techniques (e.g., bone scan, ultrasound, CT or MRI scan).

[0134] "Humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized forms of a rodent antibody generally will comprise the same CDR sequences of the parental rodent antibody, although certain amino acid substitutions can be included to improve affinity, stability, or for other reasons.

[0135] Antibodies that can be used in the compositions of the application also include antibodies with modified (or blocked) Fc regions to provide altered effector function. See, e.g., U.S. Patent No. 5,624,821; WO 2003 / 086310; WO 2005 / 120571; WO 2006 / 0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modifications can be used to enhance or suppress various responses of the immune system, which can have beneficial effects in diagnosis and therapy. Changes to the Fc region include amino acid alterations (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fc. Changes to the Fc can also alter the half-life of the antibody in a therapeutic antibody, and a longer half-life will result in a lower frequency of dosing, with increased convenience and reduced use of materials. See Presta (2005) J. Allergy Clin. Immunol. 116:731, 734-35.

[0136] "hypervariable region" refers to amino acid residues of an antibody that are responsible for antigen binding and are variable among different antibodies. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) in the heavy chain variable domain as determined by the Rabat numbering system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and / or those residues from a "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). The term "framework" or "FR" residues as used herein refer to those variable domain residues other than the hypervariable region residues as defined herein as CDR residues. CDR and FR residues are determined in accordance with the standard sequence definition set forth by Rabat. Rabat et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md.

[0137] "Conservatively modified variants" or "conservative substitutions" refer to those amino acid substitutions that are known to those of skill in the art and that generally can be made in a polypeptide without altering the biological activity of the resulting molecule, even in essential regions of the polypeptide. These exemplary substitutions are preferably made according to those listed in Table 1 below:

[0138] Table 1. Exemplary conservative amino acid substitutions

[0139]

[0140]

[0141] In addition, one of skill will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter biological activity. See, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.).

[0142] The phrase "consisting essentially of or variations thereof such as "consists essentially of or "consists essentially," as used throughout the specification and claims, indicates that the specified dosage regimen, method or composition can include additional elements not specified, but that such additional elements do not materially alter the basic or novel properties of the specified dosage regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of a specified amino acid sequence can also include one or more amino acids that do not materially affect the properties of the binding compound, including substitution of one or more amino acid residues.

[0143] "Comprise" or variations such as "comprises" or "comprising," as used throughout the specification and claims, are used in the inclusive, open sense, that is, specified features are present, unless otherwise indicated by the context, but additional features can not be present or added.

[0144] "Isolated antibody" and "isolated antibody fragment" refer to a purified state and in context refer to the named molecule being substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates or other materials (such as cell debris and growth media). Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffers or salts, unless the amount present is so significant as to seriously interfere with experimental or therapeutic uses of the binding compound described herein.

[0145] "Monoclonal antibody" or "mAb" or "Mab", as used herein, refers to a population of antibody molecules that are substantially uniform in their amino acid sequences, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that can be present in minor amounts. In contrast, a conventional (polyclonal) antibody preparation typically includes a multitude of different antibodies that have different amino acid sequences in their variable domains, particularly their CDRs, which typically confer specificity for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0146] The term "buffering agent" encompasses those agents that maintain the pH of a solution of a formulation of the application within acceptable ranges, or for a lyophilized formulation of the application, provide an acceptable solution pH prior to lyophilization.

[0147] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the active ingredient to be effective and to be free of additional components that would be deleterious to the recipient of the drug.

[0148] "Pharmaceutically acceptable" means that which is reasonably safe for use in contact with the subject to provide an effective amount of the active ingredient used, and is "generally regarded as safe" e.g., an excipient (vehicle, additive), and composition that is generally regarded as safe, e.g., when used in a human being, physiologically tolerable, and generally not producing an allergic or similar untoward reaction (such as gastric upset etc.) In another embodiment, the term refers to molecular entities and compositions approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0149] "Room temperature" or "RT" as used herein refers to a temperature in the range of about 18°C to about 25°C (about 64 to about 77 degrees Fahrenheit).

[0150] A "stable" formulation is one in which the protein therein essentially maintains its physical stability 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, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured over a selected period of time at a selected temperature. For example, in one embodiment, a stable formulation is one in which no significant change is observed for at least 12 months at refrigerated temperatures (2-8°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 18 months at refrigerated temperatures (2-8°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 3 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 6 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 12 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 18 months at room temperature (23-27°C).

[0151] "Substantially pure" as used herein suitably means that at least about 60% by weight, typically at least about 70% by weight, preferably at least about 80% by weight, more preferably at least about 90% by weight (e.g., about 90% to about 99% by weight), even more preferably at least about 95% by weight (e.g., about 95% to about 99% by weight, or about 98% to 100% by weight), and most preferably at least about 99% by weight (e.g., 100% by weight) of a product comprising a crystalline anti-PD-1 antibody (e.g., a crystalline pembrolizumab or a variant thereof) or a salt thereof (e.g., a product isolated from a reaction mixture providing the crystalline anti-PD-1 antibody or salt) consists of the crystalline anti-PD-1 antibody or salt. The level of purity of the crystalline anti-PD-1 antibody and salt can be determined using standard analytical methods, such as thin layer chromatography, gel electrophoresis, high performance liquid chromatography, and / or mass spectrometry. If more than one analytical method is employed and the methods provide experimentally significant differences in the level of purity determined, the method providing the highest level of purity prevails. A crystalline anti-PD-1 antibody or salt that is 100% pure is one that is free of detectable impurities as determined by standard analytical methods.

[0152] II. Anti-PD-1 antibodies for use in the methods of the invention

[0153] In the methods of producing a crystal of an anti-PD-1 mAb and the methods of use / treatment methods of the present application, the anti-human PD-1 antibody is pembrolizumab or a pembrolizumab variant. The amino acid sequence of pembrolizumab is provided in Table 2.

[0154] Table 2. Pembrolizumab antibody sequences

[0155]

[0156]

[0157] The crystal anti-PD-1 mAb of the present application comprises three light chain CDRs (CDRL1, CDRL2, and CDRL3) and three heavy chain CDRs (CDRH1, CDRH2, and CDRH3). In one embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the three heavy chain CDRs are SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0158] In certain embodiments, the present application provides a crystal anti-PD-1 mAb comprising a light chain variable region (V L ) and a heavy chain variable region (V H ), wherein the light chain variable region comprises SEQ ID NO: 7 or a variant of SEQ ID NO: 7, and the heavy chain variable region comprises SEQ ID NO: 8 or a variant of SEQ ID NO: 8. In some embodiments, the variant light chain or heavy chain variable region sequence is identical to the reference sequence except for having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. In particular embodiments, the amino acid substitutions are conservative amino acid substitutions. The substitutions in the pembrolizumab variant are in the framework region (i.e., outside of the CDRs) or in the constant region, and are outside of any residues that inhibit binding of the pembrolizumab variant to the additive used in the methods herein, thereby inhibiting crystallization.

[0159] In one embodiment of the present application, the crystal anti-human PD-1 antibody comprises a light chain variable region (V L ) and a heavy chain variable region (V H ), the light chain variable region comprises or consists of SEQ ID NO: 7, and the heavy chain variable region comprises or consists of SEQ ID NO: 8.

[0160] In another embodiment, the crystal anti-PD-1 mAb of the present application comprises a V L region or V H region having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence homology to the above-described V Ldomain and / or V H domain, and exhibits specific binding to PD-1. In another embodiment, the crystalline anti-PD-1 mAb comprises a V L domain and V H domain, and exhibits specific binding to PD-1.

[0161] In any of the above embodiments, the anti-PD-1 crystal of the present application can comprise a full-length anti-PD-1 antibody (e.g., pembrolizumab), or can be a short truncated antigen-binding fragment comprising (1) the light chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and the heavy chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, (2) specifically binds human PD-1, and (3) specifically binds the additive used in the method of the present application. In certain embodiments, the anti-PD-1 antibody is a full-length anti-PD-1 antibody selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. IgG of any isotype can be used, including IgGl, IgG2, IgG3, and IgG4. Different constant domains can be attached to the V L domain and V H region. For example, if the particular intended use of the antibody (or fragment) of the present application requires altered effector function, a heavy chain constant domain other than IgGl can be used. Although IgGl antibodies provide longer half-lives and effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, these activities can not be required for all uses of the antibody. In these cases, for example, an IgG4 constant domain can be used.

[0162] In embodiments of the present application, the crystalline anti-PD-1 mAb is an anti-PD-1 antibody comprising a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 9 and a heavy chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 10. In some embodiments of the present application, the crystalline anti-PD-1 mAb of the present application is a crystalline pembrolizumab or a pembrolizumab biosimilar.

[0163] In further embodiments, the crystalline anti-PD-1 mAb is a pembrolizumab variant having up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions outside of the CDRs of the light and heavy chains of pembrolizumab, and further outside of the residues of pembrolizumab that bind to caffeine (i.e., conservative amino acid substitutions outside of TYR 436 and ASN 434 of the heavy chain of pembrolizumab (434 and 436 of SEQ ID NO: 10).

[0164] Generally, the amino acid sequence variants of the crystalline pembrolizumab variants of the application have at least 90% amino acid sequence identity to the amino acid sequence (e.g., heavy chain, light chain, V H or V L sequences) of the reference antibody, more preferably at least 95%, 98%, or 99%. Identity or homology with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the anti-PD-1 residues, after aligning the sequences, introducing gaps if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal, C-terminal, or internal extensions, deletions, or insertions in the antibody sequences are construed as affecting sequence identity or homology.

[0165] Sequence identity refers to the extent to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Sequence identity can be determined using the BLAST algorithm, with the parameters of the algorithm selected to give the maximum match between the respective sequences over the entire length of the respective reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J.M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., "A model of evolutionary change in proteins." Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., "Matrices for detecting distant relationships." Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. "M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al.(1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36: 290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22: 2022-2039; and Altschul, S.F. "Evaluating the statistical significance of multiple distinct local alignments." Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

[0166] III. Methods of producing crystalline antibody suspensions

[0167] In one aspect, the present application relates to a method of producing crystalline anti-PD-1 monoclonal antibodies (mAbs), comprising: (a) mixing: (i) an aqueous buffered solution comprising about 5 mg / mL to about 80 mg / mL of a mAb, wherein the anti-PD-1 mAb is pembrolizumab or a pembrolizumab variant, (ii) polyethylene glycol (PEG), and (iii) an additive selected from the group consisting of: caffeine, theophylline, 2’-deoxyguanosine-5’-monophosphate, a biologically active gibberellin, and a pharmaceutically acceptable salt of a gibberellin; to form a crystallization solution, wherein the crystallization solution has a pH of about 6.0 to about 8.8, and comprises about 2% to about 40% weight / volume (w / v) of PEG and about 0.1% to about 0.30% w / v of the additive; (b) incubating the crystallization solution for a period of time sufficient to form crystals; and (c) optionally harvesting the crystalline anti-PD-1 mAb from the solution.

[0168] In particular embodiments of the present application, the method comprises the step of harvesting the crystalline anti-PD-1 mAb from the solution. Methods of harvesting crystals are known to those skilled in the art, and include centrifugation, decanting, lyophilization, and filtration, such as hollow fiber tangential flow filtration.

[0169] In some embodiments, the method further comprises a step of homogenizing the anti-PD-1 mAb crystals after harvesting the anti-PD-1 mAb crystals from the crystallization solution. The homogenization step provides anti-PD-1 mAb crystals having a smaller particle size (e.g., 0.5 to 50 microns). These smaller particle crystals can be used, for example, in high concentration drug formulations.

[0170] In some embodiments, the method further comprises a step of homogenizing the anti-PD-1 mAb crystals without first harvesting the crystals from the crystallization solution. In this method, the crystallization solution can be homogenized after incubation for a time sufficient to form crystals, e.g., forced through a syringe without first harvesting. The smaller size anti-PD-1 mAb crystals can be optionally harvested after homogenization.

[0171] In particular embodiments of the application, the PEG and the additive are mixed together to form a precipitant solution prior to mixing with the aqueous buffered solution comprising the mAb. The precipitant solution and the aqueous buffered solution comprising the mAb are then mixed together to form the crystallization solution.

[0172] In alternative embodiments of the application, the PEG is mixed into the aqueous buffered solution comprising the mAb to form a PEG-mAb solution. The additive, as a solid or a solution, is then added to the PEG-mAb solution to form the crystallization solution.

[0173] In other embodiments, the aqueous buffered solution comprising the mAb is mixed with the additive to form an aqueous buffered solution comprising the mAb and the additive. This solution is then mixed with the PEG, whether as a solid or a solution.

[0174] In any of the above embodiments, the additive is caffeine, theophylline, 2'-deoxyguanosine-5'-monophosphate, a biologically active gibberellin, or a pharmaceutically acceptable salt of a gibberellin.

[0175] In one embodiment, the additive is caffeine.

[0176] In another embodiment, the additive is theophylline.

[0177] In yet another embodiment, the additive is 2'-deoxyguanosine-5'-monophosphate.

[0178] In further embodiments, the additive is a biologically active gibberellin or a pharmaceutically acceptable salt thereof. In particular embodiments, the biologically active gibberellin is gibberellin Al, a pharmaceutically acceptable salt of gibberellin Al, gibberellin A3, a pharmaceutically acceptable salt of gibberellin A3, gibberellin A4, a pharmaceutically acceptable salt of gibberellin A4, gibberellin A7, or a pharmaceutically acceptable salt of gibberellin A7.

[0179] In particular embodiments, the additive is gibberellin A3 or a pharmaceutically acceptable salt thereof. In some embodiments, the additive is gibberellin A3. In other embodiments, the additive is a sodium salt of gibberellin A3. In other embodiments, the additive is a potassium salt of gibberellin A3. In other embodiments, the additive is an ammonium salt of gibberellin A3.

[0180] The amount of additive in the final crystallization solution is about 0.10% to about 0.30% w / v. In other embodiments, the amount of additive is about 0.15% to about 0.30% w / v, about 0.175% to about 0.30% w / v, about 0.20% to about 0.30% w / v, about 0.225% to about 0.30% w / v, about 0.25% to about 0.30% w / v, about 0.10% to about 0.25% w / v, about 0.10% to about 0.275% w / v, about 0.10% to about 0.25% w / v, about 0.10% to about 0.225% w / v, or about 0.10% to about 0.20% w / v. In further embodiments, the amount of additive is about 0.10% w / v, about 0.125% w / v, about 0.15% w / v, about 0.175% w / v, about 0.20% w / v, about 0.225% w / v, about 0.25% w / v, about 0.275% w / v, or about 0.30% w / v.

[0181] In one embodiment, the additive is caffeine, which is present in the final crystallization solution in an amount of about 0.15% w / v to about 0.30% w / v.

[0182] In another embodiment, the additive is theophylline, which is present in the final crystallization solution in an amount of about 0.25% w / v to about 0.30% w / v.

[0183] In any of the above embodiments, the crystallization solution can further comprise about 1% to about 10% w / v dextran sulfate sodium, which reduces the nucleation rate and allows for growth of larger crystals. In certain instances, it can be desirable to form larger crystals, for example, for characterization studies such as x-ray crystallography. In further embodiments, the crystallization solution comprises about 1%, about 1.5% w / v, about 2% w / v, about 2.5% w / v, about 3% w / v, about 3.5% w / v, about 4% w / v, about 4.5% w / v, about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, or about 10% w / v dextran sulfate sodium. In alternative embodiments, the crystallization solution comprises about 1% to about 9% w / v, about 1% to about 8% w / v, about 1% to about 7% w / v, about 1% to about 6% w / v, about 1% to about 5% w / v, about 1% to about 4% w / v, about 1% to about 3% w / v, about 1% to about 2% w / v, about 2% to about 10% w / v, about 2% to about 9% w / v, about 2% to about 8% w / v, about 2% to about 7% w / v, about 2% to about 6% w / v, about 2% to about 5% w / v, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8% w / v, about 3% to about 7% w / v, about 3% to about 6% w / v, about 3% to about 5% w / v, about 3% to about 4% w / v, about 4% to about 10% w / v, about 4% to about 9% w / v, about 4% to about 8% w / v, about 4% to about 7% w / v, about 4% to about 6% w / v, about 4% to about 5% w / v, about 5% to about 10% w / v, about 5% to about 9% w / v, about 5% to about 8% w / v, about 5% to about 7% w / v, about 5% to about 6% w / v, about 6% to about 10% w / v, about 6% to about 8% w / v, about 6% to about 7% w / v, about 7% to about 10% w / v, about 7% to about 9% w / v, about 7% to about 8% w / v, about 8% to about 10% w / v, about 8% to about 8% w / v, or about 9% to about 10% w / v dextran sulfate sodium.

[0184] In any of the above embodiments of the present application, the crystallization solution comprises about 2% to about 40% w / v of PEG. The PEG has an average molecular weight of about 2500 to about 35,000. In particular embodiments, the PEG is PEG 3350. In alternative embodiments, the PEG is PEG 2,500 (i.e., having an average molecular weight of 2500), PEG 3,000, PEG 4,000, PEG 5,000, PEG 6,000, PEG 7,000, PEG 8,000, PEG 9,000, PEG 10,000, PEG 12,000, PEG 14000, PEG 15,000, PEG 1600, PEG 1800, PEG 20,000, PEG 22,000, PEG 24,000, PEG 25,000, PEG 26,000, PEG 28,000, PEG 30,000, PEG 32,000, PEG 34,000, or PEG 35,000.

[0185] The amount of PEG in the crystallization solution is about 2% to about 40% w / v; however, one of skill in the art will recognize that the methods of the present application vary the amount of PEG using PEGs of different molecular weights. In some embodiments, the PEG is present in the crystallization solution in an amount of about 5% to about 15% w / v. In alternative embodiments, the PEG is present in the crystallization solution in an amount of about 10% to about 30% w / v. In further embodiments, the PEG is present in the crystallization solution in an amount of about 5% to about 35% w / v, about 5% to about 30% w / v, about 5% to about 25% w / v, about 5% to about 10% w / v, about 10% to about 40% w / v, about 5% to about 35% w / v, about 10% to about 30% w / v, about 10% to about 25% w / v, about 10% to about 20% w / v, about 10% to about 15% w / v, about 15% to about 40% w / v, about 15% to about 35% w / v, about 15% to about 30% w / v, about 15% to about 25% w / v, about 15% to about 20% w / v, about 20% to about 40% w / v, about 20% to about 35% w / v, about 20% to about 30% w / v, about 20% to about 25% w / v, about 25% to about 40% w / v, about 25% to about 35% w / v, about 25% to about 30% w / v, about 30% to about 40% w / v, or about 30% to about 35% w / v.

[0186] In the methods of the present application, the crystallization solution is prepared by combining: (1) an aqueous buffered solution comprising an anti-PD-1 mAb (i.e., pembrolizumab or a pembrolizumab variant), (2) PEG, and (3) an additive, as described herein; wherein the components of the crystallization solution can be added in any order. In embodiments of the present application, the aqueous buffered solution comprising an anti-PD-1 mAb has a pH of about 6.0 to about 8.8. In further embodiments, the pH is about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, or about 8.8. In further embodiments, the pH of the aqueous buffered solution comprising an anti-PD-1 mAb is about 5.0 to about 6.0. In additional embodiments, the pH is about 6.8 to about 8.4.

[0187] In still further embodiments, the aqueous buffered solution comprising the anti-PD-1 mAb has a pH of about 6.2 to about 8.8, about 6.2 to about 8.6, about 6.2 to about 8.4, about 6.2 to about 8.2, about 6.2 to about 8.0, about 6.2 to about 7.8, about 6.2 to about 7.6, about 6.2 to about 7.4, about 6.2 to about 7.2, about 6.2 to about 7.0, about 6.2 to about 6.8, about 6.2 to about 6.6, about 6.2 to about 6.4, about 6.4 to about 8.8, about 6.4 to about 8.6, about 6.4 to about 8.4, about 6.4 to about 8.2, about 6.4 to about 8.0, about 6.4 to about 7.8, about 6.4 to about 7.6, about 6.4 to about 7.4, about 6.4 to about 7.2, about 6.4 to about 7.0, about 6.4 to about 6.8, about 6.4 to about 6.6, about 6.6 to about 8.8, about 6.6 to about 8.6, about 6.6 to about 8.4, about 6.6 to about 8.2, about 6.6 to about 8.0, about 6.6 to about 7.8, about 6.6 to about 7.6, about 6.6 to about 7.4, about 6.6 to about 7.2, about 6.6 to about 7.0, about 6.6 to about 6.8, about 6.8 to about 8.8, about 6.8 to about 8.6, about 6.8 to about 8.4, about 6.8 to about 8.2, about 6.8 to about 8.0, about 6.8 to about 7.8, about 6.8 to about 7.6, about 6.8 to about 7.4, about 6.8 to about 7.2, about 6.8 to about 7.0, about 7.0 to about 8.8, about 7.0 to about 8.6, about 7.0 to about 8.4, about 7.0 to about 8.2, about 7.0 to about 8.0, about 7.0 to about 7.8, about 7.0 to about 7.6, about 7.0 to about 7.4, about 7.0 to about 7.2, about 7.2 to about 8.8, about 7.2 to about 8.6, about 7.2 to about 8.4, about 7.2 to about 8.2, about 7.2 to about 8.0, about 7.2 to about 7.8, about 7.2 to about 7.6, about 7.2 to about 7.4, about 7.4 to about 8.8, about 7.4 to about 8.6, about 7.4 to about 8.4, about 7.4 to about 8.2, about 7.4 to about 8.0, about 7.4 to about 7.8, about 7.4 to about 7.6, about 7.6 to about 8.8, about 7.6 to about 8.6, about 7.6 to about 8.4, about 7.6 to about 8.2, about 7.6 to about 8.0, about 7.6 to about 7.8, about 7.8 to about 8.8, about 7.8 to about 8.6, about 7.8 to about 8.4, about 7.8 to about 8.2, or about 7.8 to about 8.0.

[0188] In particular embodiments of any of the methods herein, the aqueous buffered solution comprising the mAb further comprises a histidine buffer at a pH of about 5.0 to about 6.0. In particular embodiments, the aqueous buffered solution comprising the mAb further comprises a 20 mM histidine buffer at a pH of 5.4.

[0189] In certain embodiments of the methods of the application, the pH of the crystallization solution and the amount of PEG present in the solution are selected from the group consisting of:

[0190] a) the pH of the crystallization solution is about 6.0, and the amount of PEG is about 2% to about 4% w / v,

[0191] b) the pH of the crystallization solution is about 6.4, and the amount of PEG is about 2% to about 6% w / v,

[0192] c) the pH of the crystallization solution is about 6.8 to 8.4, and the amount of PEG is about 6% to about 12% w / v, and

[0193] d) the pH of the crystallization solution is about 8.8, and the amount of PEG is about 10% to 12% w / v.

[0194] In certain embodiments of the above methods, the PEG is PEG 3350.

[0195] In embodiments of the methods of the application, the solution concentration of the anti-PD-1 mAb in the crystallization solution is about 5 mg / mL to about 50 mg / mL. In further embodiments, the solution concentration of the anti-PD-1 mAb in the crystallization solution is about 5 mg / mL to about 45 mg / mL, about 5 mg / mL to about 40 mg / mL, about 5 mg / mL to about 35 mg / mL, about 5 mg / mL to about 30 mg / mL, about 5 mg / mL to about 25 mg / mL, about 5 mg / mL to about 20 mg / mL, about 5 mg / mL to about 15 mg / mL, about 5 mg / mL to about 10 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 45 mg / mL, about 10 mg / mL to about 40 mg / mL, about 10 mg / mL to about 35 mg / mL, about 10 mg / mL to about 30 mg / mL, about 10 mg / mL to about 25 mg / mL, about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 15 mg / mL, about 15 mg / mL to about 50 mg / mL, about 15 mg / mL to about 45 mg / mL, about 15 mg / mL to about 40 mg / mL, about 15 mg / mL to about 35 mg / mL, about 15 mg / mL to about 30 mg / mL, about 15 mg / mL to about 25 mg / mL, about 15 mg / mL to about 20 mg / mL, about 20 mg / mL to about 50 mg / mL, about 20 mg / mL to about 45 mg / mL, about 20 mg / mL to about 40 mg / mL, about 20 mg / mL to about 35 mg / mL, about 20 mg / mL to about 30 mg / mL, about 20 mg / mL to about 25 mg / mL, about 25 mg / mL to about 50 mg / mL, about 25 mg / mL to about 45 mg / mL, about 25 mg / mL to about 40 mg / mL, about 25 mg / mL to about 35 mg / mL, about 25 mg / mL to about 30 mg / mL, about 30 mg / mL to about 50 mg / mL, about 30 mg / mL to about 45 mg / mL, about 30 mg / mL to about 40 mg / mL, about 30 mg / mL to about 35 mg / mL, about 35 mg / mL to about 50 mg / mL, about 35 mg / mL to about 45 mg / mL, about 35 mg / mL to about 40 mg / mL, about 40 mg / mL to about 50 mg / mL, or about 40 mg / mL to about 45 mg / mL.

[0196] In certain embodiments of any of the methods of the application, the crystallization solution further comprises about 25 mM to about 250 mM HEPES buffer. In some embodiments, the crystallization solution further comprises about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 125 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 175 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 225 mM, about 230 mM, about 240 mM, about 245 mM, or about 250 mM HEPES buffer.

[0197] In other embodiments of the methods of the application, the crystallization solution further comprises any of the above specified amounts of Tris buffer (i.e., in place of HEPES buffer). In alternative embodiments, the crystallization solution further comprises PIPES, MOPS, TES, DIPSO, MOBS, or TAPSO buffer.

[0198] After mixing (1) the aqueous buffered solution comprising the anti-PD-1 mAb, (2) the PEG, and (3) the additive, the crystallization solution is incubated at a temperature of about 2 °C to about 37 °C for a length of time sufficient to form crystals. In certain embodiments, the incubation temperature of the crystallization solution is about 18 °C to about 25 °C. In yet other embodiments, the incubation temperature of the crystallization solution is about 2 °C to about 35 °C, about 2 °C to about 30 °C, about 2 °C to about 25 °C, about 2 °C to about 20 °C, about 2 °C to about 15 °C, about 2 °C to about 10 °C, about 5 °C to about 37 °C, about 5 °C to about 35 °C, about 5 °C to about 30 °C, about 5 °C to about 25 °C, about 5 °C to about 20 °C, about 5 °C to about 15 °C, about 5 °C to about 10 °C, about 10 °C to about 37 °C, about 10 °C to about 35 °C, about 10 °C to about 30 °C, about 10 °C to about 25 °C, about 10 °C to about 20 °C, about 10 °C to about 15 °C, about 15 °C to about 37 °C, about 15 °C to about 35 °C, about 15 °C to about 30 °C, about 15 °C to about 25 °C, about 15 °C to about 20 °C, about 20 °C to about 37 °C, about 20 °C to about 35 °C, about 20 °C to about 30 °C, about 20 °C to about 25 °C, about 25 °C to about 37 °C, about 25 °C to about 35 °C, about 25 °C to about 30 °C, about 30 °C to about 37 °C, or about 30 °C to about 35 °C.

[0199] In further embodiments, the crystallization solution is heated to about 50 °C (at which temperature it remains in solution) and then cooled, wherein the crystallization solution only crystallizes upon cooling to about 37 °C or lower.

[0200] In still further embodiments, the crystallization solution is heated to about 50 °C and then cooled to a temperature of about 18 °C to about 25 °C or to a temperature of about 25 °C or less.

[0201] In further embodiments, the crystallization solution is heated to about 50 °C and then cooled to a temperature of about 4 °C.

[0202] In particular embodiments of the methods of the application, the incubation temperature is raised from about 4 °C to about 10-40 °C

[0203] In any of the methods herein, the crystallization solution is incubated for a period of time sufficient for crystals to form. For example, crystal formation can be detected by visual inspection or SONICC TM imaging. In particular embodiments, the crystallization solution is incubated for about 15 minutes or more. In some embodiments, the crystallization solution is incubated for about 2 hours or more. In some embodiments, the crystallization solution is incubated overnight. In some embodiments, the crystallization solution is incubated for 18 hours or more. In particular embodiments, the crystallization solution is incubated for about 30 minutes or more, about 1 hour or more, about 3 hours or more, about 4 hours or more, about 5 hours or more, about 6 hours or more, about 7 hours or more, about 8 hours or more, about 9 hours or more, about 10 hours or more, about 11 hours or more, about 12 hours or more, about 13 hours or more, about 14 hours or more, about 15 hours or more, about 16 hours or more, about 17 hours or more, about 20 hours or more, or about 24 hours or more. In further embodiments, the crystallization solution is incubated for about 2 days, 3 days, 4 days, 5 days, 1 week, 10 days, 2 weeks, 15 days, 3 weeks, or more than 3 weeks.

[0204] In particular embodiments of any of the methods described herein, the crystallization solution is rotated or agitated during incubation.

[0205] Various methods of protein crystallization are known. Giege et al. (1994) Acta Crystallogr. D50:339; McPherson (1990) Eur. J. Biochem. 189:1. Such techniques include hanging-drop vapor diffusion (McPherson (1976) J. Biol. Chem. 251:6300), sitting-drop vapor diffusion, micro-batch, and dialysis.

[0206] Suspension and sitting drop vapor diffusion requires equilibrating a droplet containing purified protein, buffer, and precipitant with a larger reservoir containing a similar buffer and a higher concentration of precipitant. Initially, the droplet of protein solution contains a concentration of precipitant that is insufficient for crystallization, but as water evaporates from the droplet and is transferred to the reservoir, the concentration of precipitant increases to optimal levels for crystallization. As the system is in equilibrium, these optimal conditions are maintained until crystallization is complete. The difference between suspension and sitting drop methods is the vertical orientation of the droplet of protein solution within the system.

[0207] In the micro-batch method, the polypeptide is mixed with a precipitant to achieve supersaturation, and the vessel is sealed and left undisturbed until crystals appear.

[0208] In the dialysis method, the polypeptide is retained on one side of a dialysis membrane in contact with a solution containing a precipitant. Equilibration across the membrane increases the concentration of precipitant, resulting in the polypeptide reaching a supersaturation level.

[0209] Some of these techniques are used to prepare the pembrolizumab crystals of the present application, as described in more detail in the Examples.

[0210] In particular embodiments of any of the methods described herein, the crystallization solution is produced by vapor diffusion or batch crystallization.

[0211] In particular embodiments of any of the methods described herein for producing a crystal anti-PD-1 monoclonal antibody, the method further comprises the step of seeding the crystallization solution with a crystal of the anti-PD-1 mAb prior to or during the incubation step.

[0212] The anti-PD-1 mAb crystals can be analyzed by various methods to examine or characterize their physical properties, such as crystal size, shape, surface morphology, total surface area, and porosity. These analytical techniques include, for example, electron diffraction and solid-state nuclear magnetic resonance (ssNMR), optical microscopy, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and various light scattering techniques. Additionally, the biological activity and / or biophysical properties of the anti-PD-1 mAb in the crystals of the present application can be analyzed by "re-dissolving" or dissolving the antibody crystals in a buffer suitable for the desired analytical technique. For example, the dissolved anti-PD-1 mAb can be analyzed by one or more of ELISA, size exclusion chromatography, SDS PAGE, and dynamic light scattering.

[0213] IV. Anti-PD-1 crystalline antibody suspensions and compositions

[0214] In one aspect, the present application provides an isolated crystal formed by any of the methods of the present application, i.e., any of the methods described herein for producing a crystal of an anti-PD-1 mAb.

[0215] The present application also relates to an isolated crystal of pembrolizumab comprising a complex with caffeine, wherein the crystal is characterized by a solid state NMR13C spectrum showing peaks at about 182.16, 181.54, 179.99, 109.36, 108.23, 103.58, 76.88, and 76.04 ppm. a = b = g = 90°.

[0216] In one embodiment, the present application provides a crystal of pembrolizumab comprising a polypeptide, wherein the polypeptide is characterized by a structure coordinate comprising a root mean square deviation (RMSD) of conserved residue backbone atoms of less than about 2.0 Angstroms when superimposed on the backbone atoms described by the structure coordinates of Table 7.

[0217] In some embodiments, the crystal of pembrolizumab or the crystal of pembrolizumab variant of the present application has a particle size of about 0.5 to 200 microns after harvesting. In particular embodiments, the anti-PD-1 mAb crystal, e.g., the crystal of pembrolizumab, is homogenized after crystallization, resulting in a particle size of about 0.5 to about 50 microns after homogenization.

[0218] In one embodiment, the present application relates to a crystal of pembrolizumab comprising a complex with caffeine, characterized by a solid state NMR13C spectrum showing peaks at about 182.16, 181.54, 179.99, 109.36, 108.23, 103.58, 76.88, and 76.04 ppm. 13 In another embodiment, a crystal of pembrolizumab comprising a complex with caffeine is provided, characterized by a solid state NMR13C spectrum showing peaks at about 183.07, 182.16, 181.54, 180.55, 179.99, 110.70, 110.15, 109.36, 108.23, 103.58, 101.49, 99.75, 98.56, 76.88, 76.04, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26, and 11.13 ppm. 13 In another embodiment, a crystal of pembrolizumab is characterized by a solid state NMR13C spectrum as shown in Figure 10A 13

[0219] ​​In another aspect, the present application relates to pharmaceutical compositions comprising the novel anti-PD-1 crystals of the present application (i.e., novel pembrolizumab crystals or pembrolizumab variant crystals) and a pharmaceutically acceptable carrier. To prepare the pharmaceutical compositions, the anti-PD-1 mAb crystals of the present application or the anti-PD-1 mAb dissolved from such crystals are mixed with at least one pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984). The anti-PD-1 mAb crystals used in the pharmaceutical compositions of the present application need not have any particular diffractive properties, so long as the biological activity and stability of the antibody are maintained within the desired range.

[0220] In some embodiments, the excipient is added directly to the crystallization solution during or after the crystallization process. In other embodiments, the crystals are first harvested from the crystallization solution, washed by suspension in a stabilizing solution, harvested from the stabilizing solution, and then suspended in a liquid solution comprising the excipient. The composition of the liquid can be any pharmaceutically acceptable medium, and can include, for example, aqueous solutions and water-in-oil mixtures.

[0221] Pharmaceutical compositions of the crystals in solid form can be prepared by drying a liquid suspension comprising the crystals and the desired excipient, for example by flowing nitrogen, air, or an inert gas over the crystals, by air drying, vacuum drying, or freeze-drying. The moisture content in the final product is typically less than 10%, 7%, 5%, or 3% (by weight).

[0222] Pharmaceutical compositions comprising pembrolizumab that has been dissolved from the liquid suspension or dried solid of pembrolizumab crystals can be prepared by adding the desired amount of crystals to a pharmaceutically acceptable dissolution buffer and incubating at 4°C until the crystals are dissolved. In one embodiment, the dissolution buffer comprises 10 mM histidine, pH 5.6, 0.02% polysorbate 80 w / v, and up to 4% sucrose w / v. In one embodiment, any particulates in the resulting composition are removed prior to administration, for example by centrifugation or filtration.

[0223] In particular embodiments, the pharmaceutical composition is a crystal suspension and the concentration of the anti-PD-1 mAb is about 5-400 mg / mL. In further embodiments, the concentration of the anti-PD-1 mAb is >75 mg / mL, >100 mg / mL, >125 mg / mL, >150 mg / mL, >175 mg / mL, >200 mg / mL, >225 mg / mL, >250 mg / mL, >275 mg / mL, >300 mg / mL, >325 mg / mL, or >350 mg / mL.

[0224] In particular embodiments, the pharmaceutical composition of the present application further comprises about 5 mM to about 50 mM of a buffering agent. In some embodiments, the amount of the buffering agent is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0225] In particular embodiments, the pharmaceutical composition of the present application further comprises about 0.01% to about 0.10% w / v of a non-ionic surfactant. In some embodiments, the amount of the non-ionic surfactant is about 0.01% to about 0.05% w / v, about 0.01% to about 0.04% w / v, 0.02% to about 0.05% w / v, or 0.02% to about 0.04% w / v. In further embodiments, the pharmaceutical composition of the present application does not comprise any surfactant.

[0226] V. Methods of use

[0227] In one aspect, the present application relates to a method of treating cancer in a patient in need thereof, the method comprising administering to the subject an effective amount of (1) an anti-PD-1 mAb crystal of the present application; i.e., a crystal of pembrolizumab or a crystal of a pembrolizumab variant prepared by the methods described herein, or (2) a composition comprising an anti-PD-1 mAb crystal of the present application and a pharmaceutically acceptable carrier to the patient. In some embodiments of the present application, the pembrolizumab crystal is dissolved in solution (e.g., formulated into an aqueous formulation) prior to administration to the patient. In particular embodiments of the method, the composition is administered to the subject by intravenous administration. In other embodiments, the composition is administered to the subject by subcutaneous administration.

[0228] In some embodiments of the treatment method of the present application, the dose of the anti-PD-1 mAb is 200 mg, which is administered to the patient about every 3 weeks. In alternative embodiments, the dose of the crystal monoclonal antibody is 400 mg, which is administered to the patient about every 6 weeks.

[0229] In some embodiments of the application, the pembrolizumab crystal, the pembrolizumab variant crystal, or the composition comprising the pembrolizumab crystal or the pembrolizumab variant crystal is administered to the patient once every three weeks for 12 weeks or more. In other embodiments, the crystal or composition of the application is administered to the patient once every three weeks for 15 weeks or more, 18 weeks or more, 21 weeks or more, 24 weeks or more, 27 weeks or more, 30 weeks or more, 33 weeks or more, 36 weeks or more, 39 weeks or more, 42 weeks or more, 45 weeks or more, 48 weeks or more, 51 weeks or more, 54 weeks or more, 57 weeks or more, 60 weeks or more, 63 weeks or more, 66 weeks or more, 69 weeks or more, 72 weeks or more, 75 weeks or more, 78 weeks or more, 81 weeks or more, 84 weeks or more, 87 weeks or more, or 90 weeks or more.

[0230] In other embodiments of the application, the pembrolizumab crystal, the pembrolizumab variant crystal, or the composition comprising the pembrolizumab crystal or the pembrolizumab variant crystal is administered to the patient once every six weeks for 12 weeks or more. In other embodiments, the crystal or composition of the application is administered to the patient once every six weeks for 18 weeks or more, 24 weeks or more, 30 weeks or more, 36 weeks or more, 42 weeks or more, 48 weeks or more, 54 weeks or more, 60 weeks or more, 66 weeks or more, 72 weeks or more, 78 weeks or more, 84 weeks or more, 90 weeks or more, 96 weeks or more, 102 weeks or more, 108 weeks or more, 114 weeks or more, 120 weeks or more, 126 weeks or more, or 132 weeks or more.

[0231] In a first embodiment (Embodiment El), the application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of a pembrolizumab crystal of the application.

[0232] In a second embodiment (Embodiment E2), the application includes a method of treating melanoma in a human patient comprising administering to the patient an effective amount of a pembrolizumab crystal of the application.

[0233] In a sub-embodiment of Embodiment E2, the melanoma is unresectable or metastatic.

[0234] In a further sub-embodiment of Embodiment E2, the melanoma is adjuvant melanoma. In particular embodiments, the melanoma is resected Stage III melanoma.

[0235] In a third embodiment (Embodiment E3), the present application includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0236] In a sub-embodiment of Embodiment E3, the NSCLC is squamous. In an alternative embodiment, the NSCLC is non-squamous.

[0237] In a sub-embodiment of Embodiment E3, the method further comprises administering carboplatin-paclitaxel or nab-paclitaxel to the patient.

[0238] In a sub-embodiment of Embodiment E3 (Embodiment E3-A), the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) > 50%)] and has not previously received platinum-containing chemotherapy.

[0239] In another sub-embodiment of Embodiment E3 (Embodiment E3-B), the patient has a tumor with PD-L1 expression (TPS > 1%) and has previously received platinum-containing chemotherapy. In a particular embodiment of Embodiment E3-B, the patient has disease progression on or following platinum-containing chemotherapy.

[0240] In certain embodiments of Embodiment E3, the patient has a tumor with PD-L1 expression (TPS > 1%) and has not previously received platinum-containing chemotherapy.

[0241] In certain embodiments of Embodiment E3 (including Embodiments E3-A and E3-B), the PD-L1 TPS is measured by a FDA-approved test assay.

[0242] In certain embodiments of Embodiment E3 (including Embodiments E3-A and E3-B), the patient's tumor is free of EGFR or ALK genomic abnormalities.

[0243] In certain embodiments of Embodiment E3 (including Embodiments E3-A and E3-B), the patient's tumor has EGFR or ALK genomic abnormalities prior to receiving the anti-PD-1 antibody or antigen-binding fragment thereof, and has disease progression on or following treatment for the EGFR or ALK abnormality.

[0244] In a fourth embodiment (Embodiment E4), the present application includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient comprising: (1) administering to the patient an effective amount of the pembrolizumab crystals of the present application, and (2) administering to the patient pemetrexed and carboplatin. In sub-embodiments of Embodiment E4, the patient has not previously been treated with an anti-cancer therapeutic prior to commencing the combination treatment regimen with the pembrolizumab crystals of the present application in combination with pemetrexed and carboplatin.

[0245] In particular embodiments of Embodiments E3 and E4 (including sub-embodiments thereof), the patient has non-squamous non-small cell lung cancer.

[0246] In sub-embodiments of Embodiment E4, the pemetrexed is administered to the patient in an amount of 500 mg / m 2 of the patient.

[0247] In sub-embodiments of Embodiment E4, the pemetrexed is administered to the patient via intravenous infusion every 21 days. In particular embodiments, the infusion time is about 10 minutes.

[0248] In sub-embodiments of Embodiment E4 (Embodiment E4-A), the present application further includes administering to the patient about 400 pg to about 1000 pg of folic acid once a day, starting about 7 days prior to the administration of pemetrexed to the patient and continuing until about 21 days after the patient is administered the last dose of pemetrexed. In certain embodiments, the folic acid is administered orally.

[0249] In sub-embodiments of Embodiments E4 and E4-A (Embodiment E4-B), the present application further includes administering to the patient about 1 mg of vitamin B 12 twice a day, about 1 week prior to the first administration of pemetrexed and about every 3 cycles of pemetrexed administration (i.e., about every 9 weeks). In certain embodiments, the vitamin B 12 is administered intramuscularly.

[0250] In sub-embodiments of Embodiments E4, E4-A, and E4-B (Embodiment E4-C), the present application further includes administering to the patient about 4 mg of dexamethasone twice a day, the day prior to, the day of, and the day after pemetrexed administration. In certain embodiments, the dexamethasone is administered orally.

[0251] In a fifth embodiment (Embodiment E5), the present application includes a method of treating recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0252] In certain sub-embodiments of Embodiment E5, the patient has not previously been treated with platinum-containing chemotherapy and the patient’s tumor expresses PD-L1 (combined positive score (CPS) > 20).

[0253] In certain sub-embodiments of embodiment E5, the patient has relapsed or metastatic HNSCC.

[0254] In sub-embodiments of embodiment E5, the patient has previously received platinum-containing chemotherapy treatment. In certain embodiments, the patient has disease progression on or after platinum-containing chemotherapy.

[0255] In a sixth embodiment (embodiment E6), the present application includes a method of treating a human patient for refractory classical Hodgkin’s lymphoma (cHL), comprising administering to the patient an effective amount of the present application’s pembrolizumab crystals.

[0256] In a seventh embodiment (embodiment E7), the present application includes a method of treating a human patient for classical Hodgkin’s lymphoma (cHL), comprising administering to the patient an effective amount of the present application’s pembrolizumab crystals, wherein the patient has relapsed after 3 or more lines of treatment for cHL.

[0257] In sub-embodiments of embodiments E6 and E7, the patient is an adult patient.

[0258] In alternative sub-embodiments of embodiments E6 and E7, the patient is a pediatric patient.

[0259] In an eighth embodiment (embodiment E8), the present application includes a method of treating a human patient for locally advanced or metastatic urothelial carcinoma, comprising administering to the patient an effective amount of the present application’s pembrolizumab crystals.

[0260] In sub-embodiments of embodiment E8, the patient is not eligible to receive cisplatin-containing chemotherapy.

[0261] In sub-embodiments of embodiment E8, the patient has a tumor that expresses PD-L1. In some embodiments, the level of PD-L1 expression is characterized by a CPS > 10.

[0262] In sub-embodiments of embodiment E8, the patient has disease progression during or after platinum-containing chemotherapy or within 12 months after neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

[0263] In a ninth embodiment (embodiment E9), the present application includes a method of treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient solid tumors in a human patient, comprising administering to the patient an effective amount of the present application’s pembrolizumab crystals.

[0264] In sub-embodiments of embodiment E9, the patient has disease progression after a prior anti-cancer treatment.

[0265] In a tenth embodiment (Embodiment E10), the present application includes a method of treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient colorectal cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystal of the present application.

[0266] In a sub-embodiment of Embodiment E10, the patient has disease progression after prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

[0267] In an eleventh embodiment (Embodiment E11), the present application includes a method of treating recurrent locally advanced or metastatic gastric cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystal of the present application.

[0268] In a twelfth embodiment (Embodiment E12), the present application includes a method of treating recurrent locally advanced or metastatic gastroesophageal junction adenocarcinoma in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystal of the present application.

[0269] In a sub-embodiment of Embodiments E11 and E12, the patient’s tumor expresses PD-L1 [combined positive score (CPS) > 1].

[0270] In a sub-embodiment of Embodiments E11 and E12, the patient has disease progression while on or after two or more prior lines of treatment comprising chemotherapy with a fluoropyrimidine and a platinum-containing.

[0271] In a sub-embodiment of Embodiments E11 and E12, the patient has disease progression while on or after two or more prior lines of treatment comprising HER2 / neu-targeted therapy.

[0272] In a thirteenth embodiment (Embodiment E13), the present application includes a method of treating cervical cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystal of the present application.

[0273] In a sub-embodiment of Embodiment E13, the patient has recurrent or metastatic cervical cancer.

[0274] In a further sub-embodiment of Embodiment E13, the patient has disease progression while on or after chemotherapy.

[0275] In another sub-embodiment of Embodiment E13, the patient has a tumor that expresses PD-L1 [CPS > 1].

[0276] In a fourteenth embodiment (Embodiment E14), the present application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application, wherein the patient has a cancer selected from the group consisting of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, Merkel cell carcinoma, and salivary gland cancer.

[0277] In a fifteenth embodiment (Embodiment E15), the present application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application, wherein the patient has small cell lung cancer.

[0278] In sub-embodiments of Embodiment E15, the patient has metastatic SCLC. In certain sub-embodiments, the patient has previously received platinum-based chemotherapy (disease progression on or after platinum-based chemotherapy) and at least one other prior line of treatment. In certain sub-embodiments, the patient has disease progression on or after platinum-based chemotherapy and at least one other prior line of treatment.

[0279] In a sixteenth embodiment (Embodiment E16), the present application includes a method of treating non-Hodgkin’s lymphoma in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0280] In sub-embodiments of Embodiment E16, the non-Hodgkin’s lymphoma is mediastinal large B-cell lymphoma. In some embodiments, the non-Hodgkin’s lymphoma is refractory primary mediastinal large B-cell lymphoma (PMBCL). In other embodiments, the patient has PMBCL and relapsed after 2 or more prior lines of treatment.

[0281] In a seventeenth embodiment (Embodiment E17), the present application includes a method of treating breast cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0282] In sub-embodiments of Embodiment E17, the breast cancer is triple-negative breast cancer.

[0283] In sub-embodiments of Embodiment E17, the breast cancer is ER+ / HER2- breast cancer.

[0284] In an eighteenth embodiment (Embodiment E18), the present application includes a method of treating nasopharyngeal cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0285] In a nineteenth embodiment (Embodiment E19), the present application includes a method of treating thyroid cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0286] In a twentieth embodiment (Embodiment E20), the present application includes a method of treating salivary gland cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0287] In a twenty-first embodiment (Embodiment E21), the present application includes a method of treating Merkel cell carcinoma (MCC) in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application. In a sub-embodiment, the MCC is relapsed, locally advanced, or metastatic.

[0288] In a twenty-second embodiment (Embodiment E22), the present application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application, wherein the cancer is selected from the group consisting of: melanoma, non-small cell lung cancer, relapsed or refractory classical Hodgkin’s lymphoma, head and neck squamous cell carcinoma, cervical cancer, urothelial cancer, esophageal cancer, gastric cancer, primary mediastinal large B-cell lymphoma, and hepatocellular carcinoma.

[0289] In a twenty-third embodiment (Embodiment E23), the present application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application, wherein the cancer is a heme malignancy.

[0290] In a sub-embodiment of Embodiment E23, the heme malignancy is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin’s lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin’s lymphoma (NHL), and small lymphocytic lymphoma (SLL).

[0291] In a twenty-fourth embodiment (Embodiment E24), the present application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application, wherein the patient has a tumor with a high mutational burden.

[0292] In a twenty-sixth embodiment (Embodiment E26), the present application includes a method of treating hepatocellular carcinoma in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application. In sub-embodiments of Embodiment E26, the patient has previously received treatment with sorafenib.

[0293] In a twenty-seventh embodiment (Embodiment E27), the present application includes a method of treating renal cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application. In sub-embodiments of Embodiment E27, the renal cancer is clear cell renal cell carcinoma.

[0294] In a twenty-eighth embodiment (Embodiment E28), the present application includes a method of treating esophageal cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application. In sub-embodiments of Embodiment E28, the esophageal cancer is recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus. In further sub-embodiments, the patient has disease progression after one or more lines of systemic therapy. In further sub-embodiments, the patient’s tumor expresses PD-L1 [combined positive score (CPS) > 10].

[0295] In a twenty-ninth embodiment (Embodiment E29), the present application includes a method of treating ovarian cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0296] In a thirtieth embodiment (Embodiment E30), the present application includes a method of treating colorectal cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application.

[0297] In a thirty-first embodiment (Embodiment E31), the present application includes a method of treating cancer in a human patient comprising administering to the patient an effective amount of the pembrolizumab crystals of the present application, wherein the cancer is selected from the group consisting of: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, endometrial cancer, hepatocellular carcinoma, Merkel cell carcinoma, glioblastoma, glioma, and other neoplastic malignancies.

[0298] In any of the methods of the application described herein, the "pembrolizumab crystal of the application" or "anti-PD-1 crystal mAb of the application" can be any pembrolizumab crystal or pembrolizumab variant crystal of the application (i.e., a crystal described herein or prepared by a method described herein), or a composition comprising a pembrolizumab crystal or pembrolizumab variant crystal of the application, as described in Section II (entitled "Pembrolizumab Crystals of the Invention") of the Invention Detail herein or as described in Section IV (entitled "Anti-PD-1 Crystal mAbs of the Invention") of the Invention Detail herein. Anti-PD-1 antibodies for use in the methods of the invention Anti-PD-1 antibody crystalline antibody suspensions and compositions

[0299] Malignancies that show improved disease-free and overall survival associated with the presence of tumor infiltrating lymphocytes in biopsies or surgical material, e.g., melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer, and ovarian cancer, are encompassed in the methods and treatments described herein. These cancer subtypes are known to be susceptible to immune control by T lymphocytes. In addition, refractory or recurrent malignancies that can be inhibited in their growth using the antibodies described herein are also included.

[0300] In some embodiments, the compositions of the application are administered to a subject having a cancer characterized by elevated expression of PD-L1 and / or PD-L2 in a test tissue sample, including: ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, and melanoma. Additional cancers that can benefit from treatment with the compositions of the application include those associated with persistent viral infections, e.g., human immunodeficiency virus, hepatitis A, B, C viruses, Epstein-Barr virus, human papilloma viruses known to be causally related to, e.g., Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0301] Additional aspects include methods of using the anti-PD-1 mAb crystals or pharmaceutical compositions of the application to treat patients having, suspected of having, or at risk of having an infectious or contagious disease. Accordingly, the application provides methods of treating a chronic infection in a mammalian subject, comprising administering to the subject an effective amount of an anti-PD-1 crystal mAb of the application or a composition comprising an anti-PD-1 crystal mAb of the application. In some particular embodiments of the method, the composition is administered to the subject by intravenous administration. In other embodiments, the composition is administered to the subject by subcutaneous administration.

[0302] ​​In this regard, the compositions of the present application can be used alone or in combination with vaccines to stimulate an immune response to pathogens, toxins, and self-antigens. The compositions of the present application can be used to stimulate an immune response against viruses that are infectious to humans, including but not limited to: human immunodeficiency virus, hepatitis A, B, and C viruses, Epstein-Barr virus, human cytomegalovirus, human papillomavirus, and herpes viruses. The compositions of the present application comprising an antagonist anti-PD-1 antibody or antibody fragment can be used to stimulate an immune response against infection by bacterial or fungal parasites and other pathogens. Viral infections of hepatitis B and C and HIV are specifically contemplated as chronic viral infections.

[0303] The anti-PD-1 mAb crystals and compositions of the present application can be administered to a patient in combination with one or more "additional therapeutic agents." The additional therapeutic agent can be a biologic therapeutic agent (including but not limited to antibodies against VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS), a growth inhibitory agent, an immunogenic agent (e.g., attenuated cancer cells, tumor antigens, antigen presenting cells (e.g., dendritic cells primed with tumor-derived antigens or nucleic acids), immunostimulatory cytokines (e.g., IL-2, IFN alpha 2, GM-CSF), and cells transfected with a gene encoding an immunostimulatory cytokine (e.g., but not limited to GM-CSF).

[0304] As described above, in some embodiments of the methods of the present application, the method further comprises administering an additional therapeutic agent. In particular embodiments, the additional therapeutic agent is an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-TIGIT antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In further embodiments, the additional therapeutic agent is dinaciclib. In still further embodiments, the additional therapeutic agent is a STING agonist. In still further embodiments, the additional therapeutic agent is a PARP inhibitor. In still further embodiments, the additional therapeutic agent is a polytyrosine kinase inhibitor. In additional embodiments, the additional therapeutic agent is a MEK inhibitor. In additional embodiments, the additional therapeutic agent is a CXCR2 antagonist. In additional embodiments, the additional therapeutic agent is navarixin. In additional embodiments, the additional therapeutic agent is olarparib. In additional embodiments, the additional therapeutic agent is selumetinib. In additional embodiments, the additional therapeutic agent is axitinib.

[0305] Suitable routes of administration of the additional therapeutic agent can include, for example, parenteral delivery, including intramuscular, subcutaneous, and intrathecal, direct intraventricular, intravenous, intraperitoneal. The drug can be administered by a variety of conventional routes, such as intraperitoneally, parenterally, intra-arterially, or intravenously.

[0306] The selection of the dosage of the additional therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cell, tissue, or organ in the individual being treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Thus, the dosage and frequency of administration of each additional therapeutic agent (e.g., a biologic or a chemotherapeutic agent) will depend, in part, on the particular therapeutic agent, the severity of the cancer being treated, and the patient characteristics. We provide guidance for selecting appropriate dosages of antibodies, cytokines, and small molecules. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 :1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th ed. (November 2002). The clinician can determine an appropriate dosage regimen, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend on, e.g., the patient's clinical history (e.g., previous treatments), the type and stage of the cancer being treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0307] Various literature references can be obtained to facilitate the selection of pharmaceutically acceptable carriers or excipients for additional therapeutic agents. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0308] In some embodiments, the additional therapeutic agent is administered by continuous infusion or at doses at intervals of, e.g., one day, 1-7 times per week, one week, two weeks, three weeks, one month, two months, etc. The preferred dosage regimen is one that involves the maximum dosage or dosage frequency that avoids serious adverse side effects. The total weekly dose is typically at least 0.05 μg / kg, 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg body weight, or more. See, e.g., Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (2003) Cancer Immunol. Immunother. 52:133-144. The required dosage of a small molecule therapeutic agent (e.g., a peptide mimetic, natural product, or organic chemical) is roughly the same as the required dosage of an antibody or polypeptide in moles per kilogram.

[0309] In certain embodiments, the administration includes administering to the subject escalating doses of 1.0, 3.0, and 10 mg / kg of the additional therapeutic agent over the course of treatment. The formulation can be a reconstituted liquid formulation, or it can be a liquid formulation that has not been previously lyophilized. The schedule can vary, and can continue for as long as a desired effect is obtained. In certain embodiments, the dose escalation will continue up to a dose of about 10 mg / kg. In certain embodiments, the subject has a histological or cytological diagnosis of melanoma or other form of solid tumor, and in certain cases, the subject can have non-measurable disease. In certain embodiments, the subject has received treatment with other chemotherapeutic agents, while in other embodiments, the subject has not received treatment.

[0310] In certain embodiments, the administration regimen includes administering a dose of about 0.005 mg / kg to about 10 mg / kg, with dose escalation within a patient. In certain embodiments, a dose of 5 mg / kg or 10 mg / kg is administered at intervals of every 3 weeks or every 2 weeks. In still further embodiments, a dose of 3 mg / kg is administered at three week intervals for patients with melanoma or other solid tumors. In these embodiments, the patient should have non-resectable disease; however, the patient can have previously undergone surgery.

[0311] In certain embodiments, a 30 minute IV infusion of any of the pharmaceutical formulations described herein is administered to a subject. In certain embodiments of escalating doses, the dosing interval between the first and second doses is about 28 days (± 1 day). In certain embodiments, the interval between the second and third doses is about 14 days (± 2 days). In certain embodiments, the dosing interval is about 14 days (± 2 days) for doses following the second dose.

[0312] Subcutaneous administration can be performed by injection using a syringe or using other injection devices (e.g. Devices); injection pens; or needle-free devices (e.g. MediJector and ) injections.

[0313] Embodiments of the application also include one or more anti-PD-1 mAb crystals of the application (e.g., a crystal pembrolizumab or a pembrolizumab variant) or a formulation comprising a crystal described herein or made by a method described herein, for use (i) for, (ii) as a medicament or composition for, or (iii) in the manufacture of a medicament for: (a) treatment of (e.g., a human body); (b) medicine; (c) inducing or increasing an anti-tumor immune response; (d) reducing the amount of one or more tumor markers in a patient; (e) stopping or delaying the growth of a tumor or a blood cancer; (f) stopping or delaying the progression of a PD-1 -associated disease; (g) stopping or delaying the progression of a cancer; (h) stabilizing a PD-1 -associated disease; (i) inhibiting the growth or survival of tumor cells; (j) eliminating or reducing the size of one or more cancerous lesions or tumors; (k) reducing the progression, onset, or severity of a PD-1 -associated disease; (1) reducing the severity or duration of clinical symptoms of a PD-1 -associated disease (e.g., a cancer); (m) prolonging survival of a patient relative to the expected survival time of a similar untreated patient; (n) inducing a complete or partial remission of a cancerous condition or other PD-1 -associated disease; (o) cancer treatment; or (p) treatment of an infection or infectious disease.

[0314] For purposes of describing and disclosing the methods and materials in connection with the present application, all publications mentioned herein are incorporated by reference.

[0315] Various embodiments of the application have been described herein with reference to the drawings, but it is to be understood that the application is not limited to these precise embodiments, and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope or spirit of the application defined by the appended claims.

[0316] Example 1

[0317] High-throughput crystallization screening of pembrolizumab

[0318] A variety of small molecule reagents were screened for their ability to promote crystallization of pembrolizumab, including amino acids, peptides, organic salts and acids, and biologically active small molecules (Hampton Research Silver Bullet Bio Screen from Hampton Research (catalog #HR2-088)). Solutions containing pembrolizumab (44 mg / mL) in 10 mM histidine, pH 5.6 were screened in 1536 unique crystallization plates (microbatch-under-oil) using 0.2 μΐ of pembrolizumab and 0.2 μΐ of screen solution (Luft et al., Journal of Structural Biology 142: 170-179 (2003)). The screen solutions can be broken down into three broad categories: (1) salts, buffers (36 salts at three concentrations combined with eight buffers); (2) PEG, salts, buffers (eight PEGs at two concentrations combined with 36 salts and eight buffers) and (3) PEG, Silver Bullets Bio reagents. The Silver Bullets Bio screen consists of 96 solutions in a high-throughput format from a single deep well module (Greiner 780261). Each reagent is a mixture of small molecules or macromolecular digests in 0.02 M HEPES sodium, pH 6.8 buffer. Each solution contains 2 to 20 small molecules. The Silver Bullets Bio screen was diluted 1:10 in 15% PEG 3350, 0.02 M HEPES, pH 6.8 as a precipitant. Experiments were performed at 4 °C, 20-22 °C, and 30 °C. Plate wells were microscopically monitored for crystallization formation over time.

[0319] After 1 month, several molecules were identified that induced crystallization of pembrolizumab. Crystals were visualized using SONICC TM imaging system (Formulatrix, Bedford, MA). Second order nonlinear imaging of chiral crystals (SONICC) is an imaging technique that visualizes protein crystals and finds and identifies protein crystals. Two techniques (second harmonic generation (SHG) technique that probes crystallinity and ultraviolet two-photon excited fluorescence (UV-TPEF) technique that is specific to proteinaceous samples) are combined to actively identify protein crystals. Crystals appear white against a completely black background, enabling identification of crystals even in dimly lit environments. SONICC also enables detection of very small crystals, or microcrystals, defined as microcrystals that are less than 1 μιη in at least one dimension.

[0320] One of the molecules identified at 30°C, ammonium dihydrogen phosphate, was also identified as a crystallization agent compatible with the previously developed high-salt process. See WO 2016 / 137850. In addition, a new crystallization agent as a mixture was identified that was different from the molecules used for the previous high-salt process. Table 3 below provides the mixtures that can be used to produce crystals and the temperatures at which the crystallization screens were positive for crystals.

[0321] Table 3. Crystallization screen results

[0322]

[0323]

[0324] Four molecules were identified for further study: caffeine, theophylline, 2’- deoxyguanosine-5’-monophosphate, and gibberellin A3. No crystals were observed at 4°C for any of the molecules tested in this screen. Figure 1 provides images of crystals formed with Silver Buller Bio A2 as the crystallization additive.

[0325] Example 2

[0326] Confirming crystallization agents using drop vapor diffusion

[0327] Drop vapor diffusion experiments (96-well - 3-drop Swissci plates) were performed to confirm the crystallization agents identified in Example 1. Antibody solutions containing 44 mg / mL of pembrolizumab were prepared in 10 mM histidine, pH 5.6. Several different mixtures containing 50 mM HEPES, pH 6.8, 12-15% w / v PEG 3350, and one additive (total volume 0.6 μΐ^) were prepared. The drop ratios were varied as follows: drop 1 : 0.4 μΐ^ mixture + 0.2 μΐ^ pembrolizumab, drop 2: 0.3 μΐ^ mixture + 0.3 μΐ^ pembrolizumab, and drop 3: 0.2 μΐ^ mixture + 0.4 μΐ^ pembrolizumab. The experiments were performed at 23°C. The plate wells were microscopically monitored for crystal formation over time.

[0328] The results confirmed that 0.1-0.18% caffeine alone, in the presence of 12-15% w / v PEG 3350 and 50 mM HEPES, pH 6.8, helped to crystallize pembrolizumab. 0.15% w / v caffeine and 0.15% w / v gibberellin A3, whether mixed together or independently, also effectively produced pembrolizumab crystals in the presence of 12-15% w / v PEG 3350, 50 mM HEPES, pH 6.8. Theophylline did not produce crystals at 0.15% w / v, but at higher concentrations of 0.25% and 0.30% w / v, was effective in crystallizing pembrolizumab in the presence of 12-15% w / v PEG 3350, 50 mM HEPES, pH 6.8. 0.15% w / v theophylline paired with other Silver Bullet reagents did produce crystals when mixed with 0.2% w / v 2’-deoxyguanosine 5’-monophosphate hydrogen sodium salt hydrate, 0.2% ethanolamine, 0.2% IPTG, 0.2% pyridoxal 5’-phosphate, 0.2% choline base solutions. See Figure 2. 50 mM HEPES buffer and 12-15% w / v PEG 3350 alone did not produce any crystals.

[0329] Example 3

[0330] Batch Crystallization of Pembrolizumab

[0331] The experiment was designed to determine the optimal micro-batch crystallization conditions for producing a suspension of pembrolizumab crystals with a uniform particle size distribution of 10-50 microns.

[0332] A stock solution of pembrolizumab in 10 mM histidine buffer, pH 5.6, was concentrated in a concentrator to achieve a final protein concentration of 44 mg / mL. The concentrated solution was diluted to 20 mg / mL pembrolizumab in histidine buffer. A 2.5% w / v caffeine solution was prepared by adding 1.25 g of caffeine (Sigma Catalog # C7731-250G) to 50 mL of 20 mM histidine, pH 5.4, and heating the resulting mixture to 40°C until the caffeine dissolved and a solution was formed.

[0333] 0.2% caffeine in 10 mM tris, pH 8.0 with mix 16% PEG 3350, 50 mM HEPES added to 20 mg / mL pembrolizumab solution (50 mM histidine buffer, pH 5.4) and pH changed at 0.1 intervals from 6.8-7.4. Batch crystallizations were set up in 1.5 mL in Eppendorf tubes with 200 μΐ, total volume at 1 : 1, 1 :2 and 1 :3 pembrolizumab: mix ratios. Tubes were placed on a rotating platform or stir plate. All experiments were performed at room temperature except for early batch plates at 4°C which did not produce crystals but were mostly precipitate.

[0334] For experiments performed at room temperature, crystals formed within the first day and continued to form for over 18 hours. A small amount of the crystallization solution was extracted for imaging on a batch plate. The observed optimal conditions for obtaining 10-50 micron single needle crystals were 15 mg / mL pembrolizumab, 0.20% caffeine, 14% PEG 3350, 50 mM HEPES and pH 7.3 at room temperature for 18 hours.

[0335] Example 4

[0336] Batch crystallization scale-up experiments (1 mL scale) - comparison of static and rotating methods

[0337] Two 1 mL batch crystallization experiments were set up by mixing 333 μΐ of 19.4 mg / mL pembrolizumab, 0.175% caffeine, 50 mM histidine, pH 5.5 (part A) with 666 μΐ of 50 mM HEPES, pH 7.7, 10.18% PEG 3350 (part B) in 1.5 mL eppendorf tubes. Preparation of part A solution: a 44 mg / mL pembrolizumab solution was diluted to 20 mg / mL with 50 mM histidine, pH 5.5. To 1.4 mL of the diluted solution was added 112 μΐ of 2.5% caffeine in 10 mM histidine, pH 5.5. The final composition of part A was 19.4 mg / mL pembrolizumab, 7% caffeine, 50 mM histidine, pH 5.5. Preparation of part B solution: a 50 mM HEPES, pH 7, 10.18% PEG 3350 solution was prepared using an Optimatrix maker liquid handling system.

[0338] One tube was incubated under static conditions and the other tube was placed on a Labnet MiniLab Roller H5500 at 30°C for 18 hours. Examination under 200x microscopy showed clusters of crystals under static conditions while for the rotating sample a uniform suspension of needle-shaped crystals (10-30 microns) was observed, indicating that rotation is a preferred step compared to static incubation.

[0339] Example 5

[0340] Pembrolizumab fractionation crystallization using the Caffeine / PEG 3350 process (10 mL scale batch)

[0341] A 20 mg / mL solution of pembrolizumab was prepared by diluting a 44 mg / mL stock solution of pembrolizumab with 20 mM Histidine buffer, pH 5.4 to a total volume of 3.33 mL. To this solution was added 6.66 mL of 13% PEG 3350, 50 mM HEPES, pH 7.7 and 20 mM Histidine buffer, pH 5.4 with 1.0 mL of 2.5% Caffeine. The final composition of the resulting solution was 6.7 mg / mL pembrolizumab, 9.8% PEG 3350, 45 mM HEPES, pH 7.7, 6.6 mM Histidine, 0.23% Caffeine. The solution was placed on a Labnet Mini LabRoller H5500 rotisserie at 30 °C at 24 RPM. The solution was initially clear, but turbidity was observed after 18 hours. The turbid suspension was microscopically examined and the formation of micro-needles was confirmed by microscopic examination at 200x. Micrographs of the derived crystals are seen in FIG. 1. Figure 3 .

[0342] The derived crystal suspension was further processed to remove non-crystallized pembrolizumab and excess Caffeine from the suspension and the crystallization yield was measured.

[0343] A 1 mL aliquot of the crystal suspension was centrifuged in a microcentrifuge at 3000 RPM for 3 minutes. The resulting pellet was re-suspended in 1 mL of 13% PEG 3350, 50 mM HEPES, pH 7.7 and the supernatant was labeled wash 1. The suspension was centrifuged in a microcentrifuge at 3000 RPM for 3 minutes. The resulting pellet was re-suspended in 1 mL of 13% PEG 3350, 50 mM HEPES, pH 7.7 and the supernatant was labeled wash 2. The suspension was centrifuged in a microcentrifuge at 3000 RPM for 3 minutes. The resulting pellet was re-dissolved in 1 mL of cold 20 mM Histidine buffer, pH 5.4. The pellet dissolved within 5 minutes.

[0344] Protein concentrations were determined using a nano drop spectrophotometer with an extinction coefficient of 1.4. Stock protein concentration was 78 mg / mL (twisted due to caffeine), wash 1 : 24 mg / mL (twisted due to caffeine), wash 2: 3.87 mg / mL (twisted due to caffeine), and final resolubilized crystals: 6.2 mg / ml (280:260 nm ratio of 0.52, same as starting pembrolizumab solution). Total yield was 94% based on protein assay.

[0345] Example 6

[0346] Temperature range 0-50 °C crystallinity screen

[0347] A solution of 44 mg / mL pembrolizumab in 20 mM histidine buffer, pH 5.4 (0.2 micron filtered) was prepared using sterile, pyrogen-free water.

[0348] A 2.5% caffeine, 20 mM histidine, pH 5.4 solution was prepared by adding 1.25 g caffeine (Sigma; lot SLBK4804V) to 50 mL 20 mM histidine (Sigma; H-8000), pH 5.4. The mixture was heated to 60 °C until the caffeine went into solution. The resulting solution was cooled to room temperature before use.

[0349] A 10.18% PEG 3350, 50 mM HEPES, pH 7.4 solution was prepared by adding 2.5 mL 1 M HEPES (1 M solution, pH 7.4; Hampton Research HR2-941-27), pH 7.4 and 10.2 mL 50% PEG 3350 to 37.3 mL sterile water for injection. The resulting solution was 0.2 micron filtered.

[0350] To 33 μΐ of a pembrolizumab solution (44 mg / mL) in 20 mM histidine buffer, pH 5.4 was added 66 μΐ of a 10.18% PEG 3350, 50 mM HEPES, pH 7.4 solution at room temperature. To the resulting solution was added 10 μΐ of a 2.5% caffeine, 20 mM histidine buffer, pH 5.4. The 1.45 mg of pembrolizumab, 6 mM histidine, pH 5.4, 6.1% PEG 3350, 30 mM HEPES, 0.23% caffeine (measured at pH 7.2) mixture (in solution) was incubated at 2 °C (wet ice) or 50 °C (water bath) for 18 hours. Crystals were observed by microscopy in the 2 °C sample. The 50 °C sample was clear for 18 hours and crystallized after cooling to room temperature over 1 hour.

[0351] In addition to SONICC TM The analysis shown below, of a 1 / 16 dilution of both samples in 10.18% PEG 3350, 50 mM HEPES, pH 7.4 solution, was performed using SONICC TM The analysis. Both experiments showed positive UV and SHG imaging, consistent with protein chiral crystals. See Figure 4.

[0352] Example 7

[0353] pH range crystallinity study

[0354] This study was designed to investigate the pH of the solution to determine which pH range was effective for producing crystals.

[0355] Using a Formulatrix Formulator TM Liquid handler, using 50 mM HEPES buffer on each row and 1-12% PEG 3350 in each column to dispense a grid of pH 6.0 to 8.8 into 96 well microbatch plates (Hampton HR267) with a final volume of 66 μΐ in each well. To each well was added 33 μΐ of a solution containing pembrolizumab (44 mg / mL) in 20 M Histidine buffer (pH 5.4) followed by 10 μΐ of 2.5% caffeine, 20 mM Histidine buffer (pH 5.4) at room temperature. The 1.45 mg of pembrolizumab, 6 mM Histidine pH 5.4, 0.23% caffeine plate components were mixed by 7X aspiration and dispense steps. The mixture (in solution) was incubated at 22 °C for 18 hours and analyzed using SONICC TM Analysis confirmed crystal formation.

[0356] Crystals were observed throughout the pH range from pH 6.0 to 8.8. At the lower pH range between 6.0 and 6.4, fewer crystals were observed than at the higher pH and a mixture of crystals and precipitate were observed. The best crystals based on size and quality were observed at pH 6.7-8.0. Crystals were observed above pH 8.0 but only when higher % PEG was used. Crystallinity was confirmed using SONICC imaging. See Table 4.

[0357] Table 4. Results of pH range study

[0358]

[0359]

[0360] Example 8

[0361] Crystallinity screening using various molecular weight PEGs

[0362] A 44 mg / mL solution of pembrolizumab in 20 mM histidine buffer, pH 5.4 (0.2 micron filtered) was prepared using sterile, pyrogen-free water.

[0363] A 2.5% caffeine, 20 mM histidine, pH 5.4 solution was prepared by adding 1.25 g caffeine (Sigma; Lot SLBK4804V) to 50 mL 20 mM histidine (Sigma; H-8000), pH 5.4, heated to 60 °C until dissolved. The solution was allowed to cool to room temperature prior to use.

[0364] A 10.18% PEG 3350, 50 mM HEPES, pH 7.4 solution was prepared by adding 2.5 mL of 1 M HEPES (1 M solution, pH 7.4; Hampton Research HR2-941-27), pH 7.4 and 10.2 mL of 50% PEG 3350 to 37.3 mL of sterile water for injection. The resulting solution was 0.2 micron filtered.

[0365] A Formulatrix Formulator TM A liquid handler, 1-12% linear gradient of PEG 200, 400, 3000, 3350, 8000, 10,000 and 20,000 and 50 mM HEPES, pH 7.2 was varied in each column and dispensed into a 96-well microbatch plate (Hampton HR267) to a final volume of 66 μΐ in each well. 33 μΐ of pembrolizumab in 20 mM histidine buffer, pH 5.4 (44 mg / mL) was added at room temperature followed by 10 μΐ of 2.5% caffeine, 20 mM histidine buffer, pH 5.4. The plate components were mixed by 7X aspiration and dispensing steps. The mixture (in solution) was incubated at 22 °C for 18 hours.

[0366] Crystals were microscopically observed in all rows except the PEG 200 and PEG 400 rows. SONICC TM The analysis was performed using aliquots in Whatman Fast Frame 4 slide well plates. All wells containing PEG molecules with molecular weights of 3,000 to 20,000 showed positive UV and SHG imaging consistent with protein chiral crystals. See Table 5.

[0367] Table 5. Crystallization screen of various molecular weight PEG molecules

[0368]

[0369] Example 9

[0370] Monoclonal antibody crystallization screen

[0371] This study was performed to determine if the above PEG / Caffeine conditions, which can be used to crystallize pembrolizumab, are also effective to crystallize other monoclonal antibodies.

[0372] Several human recombinant monoclonal antibodies (10-40 mg / mL) were screened in 1536 unique crystallization plates using the oil under microbatch method described in Luft et al. (Journal of Structural Biology 142 (2003) 170-179)) using 0.2 μΐ of monoclonal antibody (10-40 mg / mL) and 0.2 μΐ of precipitant solution (commercially available screens, including Silver Bullets Bio screens). Crystallization screens were performed at 4°C, room temperature, and 30°C. After 1 month, none of the screened monoclonal antibodies, except for pembrolizumab, crystallized at any of the tested temperatures under 0.16-0.2% caffeine, 12-15% PEG 3350, 0.05 M HEPES, pH 6.8, including the anti-PD-1 antibody nivolumab. A list of mAb targets and IgG types is provided in Table 6.

[0373] Table 6. Crystallization screen using different antibodies

[0374]

[0375]

[0376] Example 10

[0377] Preparation of pembrolizumab crystals suitable for X-ray diffraction analysis

[0378] The Hampton Research additive screen (HR2-138) consisting of 96 unique additives was set up in sitting drop vapor diffusion plates using base conditions of 12% PEG 3350, 0.1 M HEPES, pH 6.8, 0.2% caffeine (72 μΐ) with the addition of 10% of the additive screen (8 μΐ) to the stock solution. The stock solution was mixed using a Crystal Gryphon (Art Robbins Instruments, LLC, Sunnyvale, CA) and dispensed into 0.4, 0.3, and 0.2 μΙ single droplet wells 1-3 to a 3 well Intelli-plate 96. Pembrolizumab (20 mg / mL) was added to the 3 droplet single droplet wells at 0.2, 0.3, and 0.4 μΐ, respectively, resulting in 2:1, 1:1, and 1:2 stock solution to pembrolizumab droplet ratios for each supplemented stock solution. The plates were incubated at 14 °C. After 1 day, crystals appeared in many of the wells, with the wells containing dextran sulfate sodium as an additive (condition E3 of the Hampton additive screen) producing thicker needle-like crystals than the other additives.

[0379] Prior to collecting data, crystals were harvested at room temperature and transferred to a cryoprotectant solution made from a precipitant mixture enhanced with 20% ethylene glycol. After about 20 seconds of soaking in this cryoprotectant solution, the crystals were hooked using a cryoloop and frozen in liquid nitrogen. The frozen crystals were then loaded onto a diffractometer equipped with a nitrogen-cooled stream at the SER-CAT beamline of the Advanced Photon Source (APS) at Argonne National Laboratory (Argonne, IL, USA). X-ray diffraction was collected using a Rayonix MX300 HS detector. The pembrolizumab crystals were fully characterized using the following conditions: 12% PEG 3350, 0.1 M HEPES pH 6.8, 0.2% caffeine, 3% dextran sulfate sodium, 20 mg / mL pembrolizumab, 1:1 ratio (0.3 μΐ pembrolizumab / 0.3 μΐ 12% PEG 3350, 0.1 M HEPES pH 6.8, 0.2% caffeine, 3% dextran sulfate sodium). Data were integrated and scaled using the autoPROC program (Global Phasing) set to integrate using XDS, confirm space group using POINTLESS, scale using AIMLESS, anisotropy analysis and conversion to amplitudes using STARANISO. Micrographs of the crystals are seen in FIG. 1. Figure 5 .

[0380] Characteristics and data collection statistics for the PEG / caffeine crystals are provided below:

[0381]

[0382] The packing analysis using the MATTHEWS program indicated that the asymmetric unit contains half of the antibody and the other half was generated by applying 2-fold axis symmetry to the crystal. The crystal structure was solved using the molecular replacement program MOLREP with PDB entry 5DK3 as the search model. The search was performed by successively finding each rigid body part, keeping the antibody part that was positioned as fixed coordinates. The parts were positioned in the following order: VL and VH, CL and CH1, CH2, CH3. Optimization was performed using the autoBUSTER program as part of the Global Phasing package. Pictures of the antibody are shown in Figure 6A . Figure 6B An enlarged view showing the interaction of the caffeine with its environment in the crystal is given.

[0383] The complete structure information and characterization of the pembrolizumab crystal is given in Table 7.

[0384]

[0385] Table 7. Three-dimensional crystal coordinates of the caffeine-pembrolizumab complex.

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[0390]

[0391]

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[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568] The crystal has a distinct quaternary structure compared to the pembrolizumab crystal prepared using the aforementioned high salt process. See WO 2016 / 137850. The caffeine binding site found in this pembrolizumab crystal is new compared to the pembrolizumab antibody structure determined using crystals grown using the prior high salt process.

[0569] Example 11

[0570] Batch crystallization process (175 mL scale)

[0571] A 42.7 mg / mL solution of pembrolizumab in 20 mM histidine buffer, pH 5.4 (0.2 micron filtered) was prepared using a sterile pyrogen-free water solution. A 10.18% PEG 3350 (50% solution Rigaku Item # 108058), 50 mM HEPES (1 M solution, pH 7.4 Hampton Research HR2-941-27), pH 7.0 solution (400 mL) was prepared by adding 20 mL of 1 M HEPES, pH 7.4 and 81.6 mL of 50% PEG 3350 to 298.4 mL of sterile water for injection (Hospira RI-4469). The resulting solution was 0.2 micron filtered and stored at room temperature.

[0572] A 2.5% caffeine (Sigma Lot# SLBK4804V), 20 mM Histidine, pH 5.4 solution was prepared by adding 1.25 g of caffeine to 50 mL of 20 mM Histidine, pH 5.4. The solution was heated to 60 °C until the caffeine went into solution. The solution was cooled to room temperature before use.

[0573] In 50 mL polypropylene centrifuge tubes (Fisherbrand TM Sterile Cat# 05-539-8) at room temperature, 13.32 mL of pembrozekumab (42.7 mg / mL) solution in 20 mM Histidine buffer, pH 5.4, and 26.4 mL of a solution containing 10.18% PEG 3350, 50 mM HEPES, pH 7.0 were added. Then 4 mL of a solution containing 2.5% caffeine, 20 mM Histidine buffer, pH 5.4 was added. This process was repeated for a total of four 50 mL centrifuge tubes.

[0574] The tubes (in solution) were placed on a Labnet rotisserie (Cat# H5600) and rotated at room temperature. Visible turbidity was observed after 15 minutes. Rotation of the batch was continued at room temperature for 2 hours. Formulatrix SONICC TM Analysis verified the degree of crystallinity. Figure 7 shows representative analysis using the SONICC TM imaging system.

[0575] The 50 mL tubes were centrifuged at 2300 RPM for 10 minutes at room temperature in a Beckman Coulter Allegra X-15R centrifuge. The resulting supernatant was decanted off. The resulting pellet in each 50 mL conical tube was resuspended with 40 mL of 10% PEG 3350, 50 mM HEPES, and pH 7.0 buffer. This process was repeated. The resulting pellet was analyzed by SONICC TMThe resulting precipitate was analyzed for crystallinity. The concentration of pembrolizumab was measured by dissolution in a cold phosphate buffered saline solution (PBS); weight: volume, 100 mg suspension: 1 mL PBS. Using a micro-UV spectrophotometer, a 19.5 mg measurement of A280 in 10 mL of PBS solution gave a final concentration of 195 mg / mL (9.4 mL), with a total yield of 84%. Further dilution was performed to prepare 175 and 150 mg / mL suspensions using 10% PEG 3350, 50 mM HEPES, and pH 7.0 buffer.

[0576] This experiment demonstrates that the crystallization process is scalable and reproducible, with a high yield of crystal suspension obtained within 2 hours at room temperature. The results also demonstrate that the pembrolizumab crystal suspension can be concentrated to high concentrations.

[0577] Example 12

[0578] Characterization of the Pembrolizumab Crystal Suspension

[0579] The pembrolizumab crystal suspension prepared in Example 11 was characterized by measuring particle size, dynamic viscosity, and injectability, as described below.

[0580] Particle Size Analysis

[0581] A Horiba LA-960 was used to measure the average particle size. The Horiba LA-960 combines modern sizing technology with optimization that allows measurement of 10 nanometer to 5 millimeter suspension samples. The core theory of laser diffraction is that particles scatter light at angles determined by the size of the particle. Larger particles scatter at smaller angles, while smaller particles scatter at wide angles. A collection of particles will produce a pattern of scattered light defined by intensity and angle, which can be converted to a particle size distribution result. The sample was diluted 1:10 with 10% PEG 3350, 50 mM HEPES, pH 7.0 buffer. The average particle size was 4.4 microns.

[0582] Dynamic Viscosity Measurement

[0583] The Rheosense m-VROC instrument uses the Hagen-Poiseuille equation to derive viscosity from pressure drop. Shear sweeps were performed in the range of 1,500-95,000 (1 / s) to measure dynamic viscosity. The viscosity of the 200 mg / mL formulation was measured using a BD Hypak 1 ml pre-filled syringe with either a 27 gauge regular wall (RW) or 29 gauge thin wall (TW) with a ½" needle and plotted against different shear rates. Figure 8AViscosity versus shear rate data is provided. For the 200 mg / mL crystal suspension sample, the viscosity at room temperature was 26 cP at a shear rate of about 2000 sec-1. As the shear rate was further increased to 80000 sec-1and 180000 sec-1, a concomitant decrease in viscosity was observed. The viscosity decreased from 18 cP to 12 cP, which is within the acceptable range for high concentration injection products such as monoclonal antibodies. The unexpected shear thinning behavior in this crystal suspension formulation can be used to facilitate injection of the drug product from a device such as a syringe or auto-injector.

[0584] Injectability measurements

[0585] Preliminary injection force feasibility tests were performed on the 200 mg / mL crystal pembrolizumab suspension in a variety of 1 mL plastic and glass syringes and needles. See Table 8.

[0586] Table 8. Injection force of 200 mg / mL crystal pembrolizumab suspension

[0587]

[0588] Syringe injection force is the force required to dispense the contents of a syringe at a fixed rate. This force is typically measured using a tensile / compression tester (i.e., INSTRON). A tensile / compression tester (INSTRON, Norwood, MA) was used to measure the injection force of pembrolizumab crystal suspensions filled in 1-mL polycarbonate syringes at 200, 175, and 150 mg / mL at an injection rate of 120 mm / min (175 and 200 mg / mL) or 225 mm / min (150 mg / mL). The injection force of the three different concentrations of pembrolizumab suspensions is provided in Table 9, and the injection force of samples filled in glass syringes is shown in Table 10. Figure 8B Figure 9

[0589] The results show that the break-loose and glide forces are higher (7.08 N and 4.52-5.12 N, respectively) for the 200 mg / mL suspension, while the break-loose and glide forces are lower (3.96 N and 3.57-3.97 N, respectively) for the lower concentration (175 and 150 mg / mL) samples. See Table 8. The injection force varies depending on the material type of the syringe barrel (plastic or glass), the injection rate, the needle size (27 or 29G), and the needle thickness (thin-walled or regular-walled). Figure 8B Figure 9 . The injection force of plastic syringes (<8.5 N) is lower compared to glass syringes (>12 N). See Table 8. Figure 8B .

[0590] ​​​For 200 mg / mL suspensions, higher injection rates require higher injection forces. See Table 8 and Figure 9 For example, in a BD Hypak 1 mL glass syringe, for an injection rate of 133.86 mm / min, the injection force was 12.1 N, compared to 16.2 N injection force for an injection rate of 300 mm / min. The observed injection forces (6.36-18.41 N) are within an acceptable range for subcutaneous injection of 200 mg / mL pembrolizumab crystal suspensions. Overall, these data indicate that for injection of 200 mg / mL pembrolizumab crystal suspensions, a 27 or 29G stainless steel needle, made of polycarbonate plastic or glass, thin or regular wall, can be used at an injection rate of 133.86 to 300 mm / min, using an injection force that is acceptable for subcutaneous injection.

[0591] Example 13

[0592] High Performance Ion Exchange Chromatography Analysis

[0593] Materials

[0594] A 44 mg / mL solution of pembrolizumab in 20 mM Histidine buffer, pH 5.4 (0.2 micron filtered) was prepared using a sterile, pyrogen-free aqueous solution.

[0595] A solution of 10.18% PEG 3350, 50 mM HEPES, pH 7.4 was prepared by adding 2.5 ml of 1 M HEPES (Hampton Research HR2-941-27), pH 7.4 and 10.2 ml of 50% PEG 3350 (50% solution; Rigaku Item # 108058) to 37.3 mL of sterile water for injection (Hospira RI-4469). The resulting solution was 0.2 micron filtered.

[0596] A solution of 2.5% caffeine, 20 mM Histidine, pH 5.4 was prepared by adding 1.25 g of caffeine (Sigma; Lot SLBK4804V) to 50 mL of 20 mM Histidine (Sigma; H-8000), pH 5.4. The solution was heated to 60 °C until the caffeine went into solution. The solution was cooled to room temperature before use.

[0597] Batch Crystallization Method (1 mL)

[0598] To 333 μΐ of pembrolizumab (44 mg / mL) in 20 mM histidine buffer, pH 5.4, was added 666 μΐ of 10.18% PEG 3350, 50 mM HEPES, pH 7.2 at room temperature. To the resulting solution was added 100 μΐ of 2.5% caffeine, 20 mM histidine buffer, pH 5.4. The mixture (in solution) was placed on a Labnet rotisserie at room temperature. Visible turbidity was observed after 15 minutes. The batch was continued to rotate for 2 hours at room temperature. Crystals were observed based on microscopic examination.

[0599] The crystal suspension was centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at room temperature. The supernatant was removed. The pellet was resuspended in 1 mL of 10.18% PEG 3350, 50 mM HEPES, and pH 7.2, centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at room temperature. The wash was removed. The pellet was resolubilized in 1 mL of PBS at 4°C for 8 minutes and centrifuged at 3000 RPM for 3 minutes in a microcentrifuge at 4°C.

[0600] HP-IEX method

[0601] High performance ion exchange chromatography (HP-IEX) was used to assess the charge distribution of the crystallized pembrolizumab versus the non-crystallized material. The ion exchange HPLC method was performed using a Dionex ProPac WCX-10 column and a UV detector at 280 nm. The samples were diluted in purified water and injected at 80 g for analysis. Different charge variants were eluted using a gradient of the following mobile phases (Mobile Phase A: 24 mM MES, pH 6, 4% acetonitrile (v / v); Mobile Phase B: 20 mM phosphate, 95 mM NaCl, pH 8, 4% acetonitrile (v / v)). The % area of the main peak (representing non-degraded pembrolizumab) as well as the different charge variants of the pembrolizumab starting material and solubilized pembrolizumab crystals are provided in Table 9. The results indicate that the % charge variants of pembrolizumab in the crystal suspension were similar to the starting material in aqueous solution.

[0602] Table 9. IEX analysis of pembrolizumab crystal suspension versus non-crystallized starting material

[0603]

[0604]

[0605] Example 14

[0606] Pembrolizumab competition binding ELISA

[0607] Preparation of crystal suspension for bioassay analysis

[0608] To 333 μΐ of pembrolizumab (44 mg / mL) in 20 mM Histidine buffer, pH 5.4, was added 666 μΐ of 10.18% PEG 3350, 50 mM HEPES, pH 7.2 at room temperature. To the resulting solution was added 100 μΐ of 2.5% caffeine, 20 mM Histidine buffer, pH 5.4. The mixture (in solution) was incubated at 30 °C for 1 month. Crystals were observed based on microscopic visual inspection.

[0609] The crystal suspension was centrifuged in a microfuge at 3000 RPM for 3 minutes at room temperature. The supernatant was removed. The pellet was resuspended in 1 mL of 10.18% PEG 3350, 50 mM HEPES, pH 7.2 and centrifuged in a microfuge at 3000 RPM for 3 minutes at room temperature. The wash was removed. The pellet was resolubilized in 1 mL of PBS at 4 °C for 8 minutes and centrifuged in a microfuge at 3000 RPM for 3 minutes at 4 °C. A protein concentration of 6.061 mg / mL was determined based on a Nano drop 280 nm reading; the total volume was 1 mL. The sample was used for bioassay analysis, as described below.

[0610] Competitive binding ELISA

[0611] The pembrolizumab competitive binding ELISA assesses the ability of pembrolizumab to compete with PD-L1 (PD-1 ligand) for binding to PD-1 / Fc immobilized on an ELISA plate. A sample of non-crystallized pembrolizumab (“reference”) was used as a reference material to test the potency of a crystallized pembrolizumab suspension (“test sample”) prepared by the method described above. The reference and test samples at 4.5 μg / mL were serially 2-fold diluted in PBS pH 6.5, 1% BSA and mixed with an equal volume of 600 ng / mL rhPD-L1 / Fc chimera (“PD-L1”, Bio-techne, R&D Systems (Catalog # 156-B7), Minneapolis, MN) before being transferred to an ELISA plate. The final concentrations of the assay components were 2.25 μg / mL (reference and test sample) and 300 ng / mL (PD-L1). The level of PD-L1 bound to PD-1 / Fc was detected by biotinylated anti-PD-L1 (Bio-techne, R&D Systems (Catalog # BAF156)), followed by peroxidase-conjugated streptavidin and chemiluminescent substrate. Luminescence was measured using a microplate reader and the resulting inhibition response curves were analyzed using 4-PL curve fitting software (SoftMax Pro).

[0612] The IC50values generated from this assay are a measure of the ability of pembrolizumab to inhibit PD-L1 binding to PD-1 / Fc. The biological potency of the crystalline samples is expressed as % relative potency to a reference material of pembrolizumab. The geometric mean of the relative potency of multiple replicates (N=3) of the same sample is reported, along with the geometric standard deviation (% GSD) and 95% confidence interval. The results show that the dissolved crystalline samples have a relative potency of 95% compared to the reference (non-crystalline) pembrolizumab. See Table 10.

[0613] Table 10. Relative Potency of Pembrolizumab Crystals

[0614]

[0615] Example 15

[0616] Laboratory Batch Crystallization Method - Non-cleanroom Conditions

[0617] A 42.7 mg / mL solution of pembrolizumab (Lot W15-MK3475P-081) in 20 mM Histidine buffer, pH 5.4 was prepared using sterile pyrogen-free water solution (0.2 micron filtered).

[0618] A 10.18% PEG 3350 (50% solution Rigaku Item # 10805850) mM HEPES, pH 7.0 (400 mL) solution was prepared by adding 20 mL of 1 M HEPES (Hampton Research HR2-941-27), pH 7.4 and 81.6 mL of 50% PEG 3350 to 298.4 mL of sterile water. The resulting solution was 0.2 micron filtered and stored at room temperature.

[0619] A 2.5% caffeine, 20 mM Histidine, pH 5.4 solution was prepared by adding 1.25 g of caffeine (Sigma Lot SLBK4804V) to 50 mL of 20 mM Histidine (Sigma H-8000), pH 5.4. The resulting solution was heated to 60 °C until the caffeine went into solution. The solution was cooled to room temperature prior to use.

[0620] Four milliliter tubes of crystallization solution were prepared (43.72 mL scale). To 13.32 mL of pembrozumab (42.7 mg / mL) in 20 M Histidine buffer, pH 5.4 was added 26.4 mL of 10.18% PEG 3350, 50 mM HEPES, pH 7.0 at room temperature. To the resulting solution in a 50 mL tube was added 4 mL of 2.5% caffeine, 20 mM Histidine buffer, pH 5.4. The mixture (in solution) was placed on a Labnet rotisserie at room temperature. Visible turbidity was observed after 15 minutes. The batch was rotated for an additional 2 hours at room temperature. SONICC analysis was performed and confirmed crystallinity.

[0621] The 50 mL conical tubes were centrifuged in a Beckman Coulter Allegra X-15R centrifuge at 2600 RPM at room temperature for 10 minutes each. The supernatant was decanted. The pellet in each 50 mL conical tube was re-suspended in 40 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. The process of centrifugation, decanting, and re-suspending was repeated. For the final centrifugation step, the bottles were centrifuged in a Beckman Coulter Allegra X-15R centrifuge at 3500 RPM for 20 minutes at room temperature. The supernatant was decanted. The protein concentration was 216 mg / mL as measured by A280 reading at a weight:volume, 1:10. The final volume was 9.7 mL (92% yield). Particle size analysis measured using a Horiba particle size analyzer was 1.3 microns average particle size.

[0622] Example 16

[0623] Batch Crystallization (43.72 mL scale) - Cleanroom conditions

[0624] A 2.5% caffeine, 20 mM Histidine, pH 5.4 solution was prepared by adding 1.25 g of caffeine (Sigma Lot # SLBK4804V) to 50 mL of 20 mM Histidine (Sigma H-8000), pH 5.4. The mixture was heated to 60 °C until the caffeine went into solution. The solution was allowed to cool to room temperature and sterile filtered prior to use.

[0625] A 10.18% PEG 3350, 50 mM HEPES, pH 7.0 solution (400 mL) was prepared by adding 20 mL of 1 M HEPES (Hampton Research HR2-941-27), pH 7.4 and 81.6 mL of 50% PEG 3350 (Rigaku Item # 108058) to 298.4 mL of sterile water. The resulting solution was 0.2 micron filtered and stored at room temperature.

[0626] A 42.7 mg / mL pembrolizumab solution in 20 mM histidine buffer, pH 5.4 (0.2 micron filtered) was prepared using sterile, pyrogen-free water.

[0627] To 13.32 mL of sterile filtered pembrolizumab (42.7 mg / mL) in 20 M histidine buffer, pH 5.4 (4 x 50 mL tubes) was added 26.4 mL of 10.18% PEG 3350, 50 mM HEPES, pH 7.0 at room temperature. To this solution was added 4 mL of 2.5% caffeine, 20 mM histidine buffer, pH 5.4 in a 50 mL sterile conical tube in a clean room. After mixing, the component concentrations of the crystallization solution were 6 mM histidine, 6% PEG 3350, 30 mM HEPES, 0.23% caffeine, 2.28 g pembrolizumab in pH 6.8 in 4 x 50 mL conical tubes.

[0628] The mixture (in solution) was placed on a Labnet rotisserie at room temperature. Visible turbidity was observed after 15 minutes. The 4 x 50 mL sterile conical tubes were spun for 2 hours at room temperature. SONICC TM Analysis was performed and the degree of crystallinity was confirmed.

[0629] Four 50 mL sterile conical tubes were centrifuged at 2600 RPM for 10 minutes each in a Beckman Coulter Allegra X-15R centrifuge at room temperature. The supernatant was decanted. The pellet was resuspended; each 50 mL conical tube in 40 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. This process was repeated. For the final centrifugation step, the conical tubes were centrifuged at 3500 RPM for 20 minutes in a Beckman Coulter Allegra X-15R centrifuge at room temperature. The supernatant was decanted. The protein concentration was 231 mg / mL as measured by weight:volume 1 :10 A280. The sample was diluted to 0.8 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. The protein concentration was 200.3 mg / mL final concentration as measured by weight:volume 1 :10 A280. The final volume was 8.4 mL (74% yield). The protein concentration was 192.5 mg / mL (1.2 mM) and the caffeine concentration was 0.5 mg / mL (2.5 mM) as measured using the RPLC method. The average particle size was 1.3 microns as measured using a Horiba particle size analyzer.

[0630] Example 17

[0631] Batch dialysis crystallization method

[0632] Four 50 mL sterile conical tubes were centrifuged at 2600 RPM for 10 minutes each in a Beckman Coulter Allegra X-15R centrifuge at room temperature. The supernatant was decanted. The pellet was resuspended; each 50 mL conical tube in 40 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. This process was repeated. For the final centrifugation step, the conical tubes were centrifuged at 3500 RPM for 20 minutes in a Beckman Coulter Allegra X-15R centrifuge at room temperature. The supernatant was decanted. The protein concentration was 231 mg / mL as measured by weight:volume 1 :10 A280. The sample was diluted to 0.8 mL of 10% PEG 3350, 50 mM HEPES, pH 7.0. The protein concentration was 200.3 mg / mL final concentration as measured by weight:volume 1 :10 A280. The final volume was 8.4 mL (74% yield). The protein concentration was 192.5 mg / mL (1.2 mM) and the caffeine concentration was 0.5 mg / mL (2.5 mM) as measured using the RPLC method. The average particle size was 1.3 microns as measured using a Horiba particle size analyzer.

[0633] A slight haze was observed after 3 hours, and the haze increased significantly after 18 hours. An aliquot was analyzed by SONICC to characterize and confirm crystal formation. The resulting suspension was centrifuged in a microfuge at 3000 RPM for 3 minutes. The resulting pellet was washed with 1 mL of 50 mM HEPES, pH 6.8, 10% PEG 3350, and centrifuged again in a microfuge at 3000 RPM for 3 minutes. The resulting pellet was dissolved in 10 mL of normal PBS (5 minutes at room temperature). The final A280 reading was 1.6 mg / mL (16 mg total protein). The total yield was 91% (17.6 mg of starting total mAb content). This experiment demonstrated that the crystallization method using dialysis can produce a crystal pembrolizumab suspension at room temperature in 18 hours with high yield.

[0634] Example 18

[0635] Pharmacokinetic study of pembrolizumab crystal formulation

[0636] A PK comparability study was performed in male Wistar Han rats using the pembrolizumab crystal formulation. The dose of pembrolizumab was 50 mg / kg for all groups. Pembrolizumab at 20 mg / mL in a liquid IV formulation (7% sucrose, 0.02% polysorbate 80, 10 mM histidine, pH 5.5 (Group 1)) and a liquid SC formulation (7% sucrose, 0.02% polysorbate 80, 10 mM histidine, pH 5.5, 10 mM methionine (Group 2)) were included as bioavailability control groups (Group 1 and 2 each N=3).

[0637] The pembrolizumab crystal suspension was prepared as described in Example 16 and used for subcutaneous dosing of the pembrolizumab crystal formulation containing the concentrations listed in Table 11 for 20 mg / mL (Group 3, N=4), 40 mg / mL (Group 4, N=4), and 100 mg / mL (Group 5, N=4) pembrolizumab and 50 mM HEPES buffer, pH 7.0 and 10.18% PEG 3350. To ensure accurate dosing for each group, the BD Hypak 2.25 mL prefilled syringes for each group described in Table 11 were accurately filled using weight / density measurements (1 g / mL).

[0638] Table 11. Crystal formulation groups tested in rat study

[0639] Group Dose Concentration (mg / mL) Syringe Fill* (g) 3 50 mpk 20 1.1 4 50 mpk 40 0.7 5 50 mpk 100 0.5

[0640] *0.1 g dead space in each syringe due to backfilling

[0641] A prescribed weight of the crystal suspension was added to BD Hypak™ 2.25 mL pre-filled glass syringes using a sterile 10 mL positive displacement pipette for each group. An air vent tool was used to reach the liquid surface of the suspension within each filled syringe with the plunger cap. A total of six syringes were prepared per group.

[0642] To form a control for potential caffeine effects, the study included a formulation of pembrolizumab without caffeine (50 mM HEPES, pH 6.8, 10% PEG 3350, Group 6) and a formulation of pembrolizumab without PEG (50 mM HEPES, pH 6.8, caffeine, Group 7).

[0643] Blood was collected at 0.5 hours, 3 hours, 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 216 hours, 336 hours, 408 hours, and 504 hours post-dose and serum was prepared from 0.3 mL of post-dose whole blood. Pembrolizumab in serum was measured by MSD (Meso Scale Discovery) immunoassay. PK parameters were calculated with Phoenix PK software 64.6.3. Bioavailability (F) was calculated based on AUC of the liquid formulation, Group IV (F = AUC of SC / AUC of IV * 100%). Injection sites were monitored throughout the study.

[0644] The results showed that the tested pembrolizumab SC liquid formulations achieved similar bioavailability to the 20 mg / mL crystal formulation at 20 mg / mL. See Table 12. The results also showed that the highest concentration (C max ), exposure (area under the curve, AUC), and bioavailability (F) increased in a concentration-dependent manner: the higher the concentration, the higher the C max , AUC, and F. The time to reach the highest concentration in serum (T max ) was shorter for the highest concentration crystal formulation compared to the lower concentrations, indicating a fast absorption rate (Ka) for the highest concentration crystal formulation.

[0645] Table 12. Bioavailability of liquid and crystal formulations

[0646]

[0647] Example 19

[0648] Solid-state NMR characterization of pembrolizumab crystal suspensions

[0649] Solid-state NMR spectra were obtained on a Bruker Avance III HD 400 MHz spectrometer equipped with a 4.0 mm H / F / X magic angle spinning (MAS) probe and a Bruker Avance III 500 MHz spectrometer equipped with a 4.0 mm H / C / N MAS probe. For experiments on the 400 MHz spectrometer, the probe was tuned to triple resonance C / H / F; for experiments on the 500 MHz spectrometer, the probe was tuned to triple resonance C / H / N. The MAS frequency for all experiments was 12 kHz. The sample temperature was controlled at 10 °C on the 400 MHz spectrometer and at 21 °C on the 500 MHz spectrometer. On the 400 MHz spectrometer, 13C cross-polarization (CP) MAS spectra were collected during acquisition with 1H dipolar decoupling at 90.9 kHz and a CP contact time of 1 ms and a recycle delay of 2 s. On the 500 MHz spectrometer, 13C CPMAS spectra were collected during acquisition with 1H dipolar decoupling at 71.4 kHz and a CP contact time of 1 ms and a recycle delay of 2 s. On the 500 MHz spectrometer, 15N CP MAS spectra were collected during acquisition with 1H dipolar decoupling at 71.4 kHz and a CP contact time of 2.5 ms and a recycle delay of 2 s. 13 C (carbon-13) experiment, the probe was tuned to triple resonance C / H / F; for experiments on the 500 MHz spectrometer, 13 C (carbon-13) and 15 N (nitrogen-15) experiment, the probe was tuned to triple resonance C / N / H. The MAS frequency for all experiments was 12 kHz. The sample temperature was controlled at 10 °C on the 400 MHz spectrometer and at 21 °C on the 500 MHz spectrometer. On the 400 MHz spectrometer, 13C cross-polarization (CP) MAS spectra were collected during acquisition with 1H dipolar decoupling at 90.9 kHz and a CP contact time of 1 ms and a recycle delay of 2 s. On the 500 MHz spectrometer, 13C CPMAS spectra were collected during acquisition with 1H dipolar decoupling at 71.4 kHz and a CP contact time of 1 ms and a recycle delay of 2 s. On the 500 MHz spectrometer, 15N CP MAS spectra were collected during acquisition with 1H dipolar decoupling at 71.4 kHz and a CP contact time of 2.5 ms and a recycle delay of 2 s. 13 C (carbon-13) experiment, the probe was tuned to triple resonance C / H / F; for experiments on the 500 MHz spectrometer, 13 C (carbon-13) experiment, the probe was tuned to triple resonance C / H / F; for experiments on the 500 MHz spectrometer, 15 C (carbon-13) experiment, the probe was tuned to triple resonance C / H / F; for experiments on the 500 MHz spectrometer, 13 C chemical shift reference of the carbonyl carbon of glycine (a-form) at 176.45 ppm. For the purposes of solid-state NMR, the term“about” means ± 0.1 ppm. 13 C signal. For the purposes of solid-state NMR, the term“about” means ± 0.1 ppm.

[0650] Pembrolizumab-Caffeine crystals were measured using the above described solid-state 13 C 400 MHz NMR equipment and procedures. Specifically, crystalline pembrolizumab was prepared using the method described in Example 11. 13C (carbon-13) CPMAS NMR spectra of the pembrolizumab-caffeine crystals were obtained. 13 C (carbon-13) CPMAS NMR spectra. Figure 10A and Figure 10B show the full spectrum and some enlarged regions, respectively. Characteristic peaks of the pembrolizumab-caffeine crystals were observed at about 183.07, 182.16, 181.54, 180.55, 179.99, 110.70, 110.15, 109.36, 108.23, 103.58, 101.49, 99.75, 98.56, 76.88, 76.04, 74.97, 74.41, 73.52, 72.69, 13.85, 13.27, 12.26, and 11.13 ppm.

[0651] The solid state 13 C and 15 N 500 MHz NMR equipment and procedures were used to measure the 13C and 1,3- 13 C and 1,3- 15 N isotopically labeled caffeine prepared pembro-caffeine crystals. The pembro-caffeine crystals were obtained 13 C (carbon-13) and 15 N (nitrogen-15) CP MAS NMR spectra. Figure 11A and Figure 11B showing resolved 13 C and 15 N caffeine peaks are shown in the expanded spectral regions. Characteristic chemical shift differences in the 13 C and 15 N caffeine peaks were observed between the pembro-caffeine crystals and the caffeine-only crystals at about 1.69 ppm and 0.92 ppm, respectively.

Claims

1. A method for producing crystallized anti-PD-1 monoclonal antibody (mAb), comprising: a) Mixing: i. An aqueous buffer solution containing 5 mg / mL to 80 mg / mL of the mAb, wherein the anti-PD-1 mAb is pembrolizumab. ii. Polyethylene glycol (PEG), wherein the average molecular weight of the PEG is 2,500 to 35,000, and iii. Selected additives from the following: caffeine, theophylline, theophylline and 2'-deoxyguanosine-5'-monophosphate, and bioactive gibberellin A3, wherein when theophylline is used, it is present in the final crystallization solution in an amount of 0.25% w / v to 0.30% w / v; To form a crystallization solution having a pH of 6.0 to 8.8 and containing 2% to 40% by weight / volume (w / v) PEG and 0.1% to 0.30% by weight (w / v) additives; b) Incubating the crystallization solution for a period of time sufficient to form crystals, wherein the crystallization solution is incubated at an incubation temperature of 18°C ​​to 25°C; and c) Optionally, the crystal anti-PD-1 mAb is harvested from the solution.

2. The method of claim 1, wherein the aqueous buffer solution comprising the mAb further comprises a histidine buffer at pH 5.0-6.

0.

3. The method according to claim 1 or 2, wherein the PEG and the additive are mixed together to form a precipitant solution before being mixed with the aqueous buffer solution containing the mAb.

4. The method according to claim 1 or 2, wherein the aqueous buffer solution comprising the mAb is mixed with the PEG prior to mixing with the additive.

5. The method according to claim 1 or 2, wherein the aqueous buffer solution comprising the mAb is mixed with the additive prior to mixing with the PEG.

6. The method according to claim 1, wherein the additive is gibberellin A3 or a pharmaceutically acceptable salt thereof.

7. The method of claim 1, wherein the additive is 0.15% to 0.30% w / v caffeine or 0.25% to 0.30% w / v theophylline.

8. The method of claim 1, wherein the additive is caffeine, and the crystallization solution further comprises 1% to 10% w / v sodium dextran sulfate.

9. The method according to claim 8, wherein the amount of sodium dextran sulfate is 5% w / v.

10. The method according to claim 1 or 2, wherein the PEG is present in the crystallization solution in an amount of 5% to 15% w / v.

11. The method according to claim 1 or 2, wherein the PEG is present in the crystallization solution in an amount of 10% to 30% w / v.

12. The method according to claim 1, wherein the PEG is PEG 3350.

13. The method of claim 12, wherein the pH of the crystallization solution and the amount of PEG present in the crystallization solution are selected from the group consisting of: a) The pH of the crystallization solution is 6.0, and the amount of PEG is 2-4% w / v. b) The pH of the crystallization solution is 6.4, and the amount of PEG is 2-6% w / v. c) The pH of the crystallization solution is 6.8-8.4, and the amount of PEG is 6-12% w / v. d) The pH of the crystallization solution is 8.8, and the amount of PEG is 10-12% w / v.

14. The method according to claim 1 or 2, wherein the crystallization solution is heated to 50°C and then cooled to 37°C or lower.

15. The method of claim 14, wherein the crystallization solution is cooled to a temperature of 18°C ​​to 25°C.

16. The method of claim 14, wherein the crystallization solution is cooled to a temperature of 4°C.

17. The method of claim 14, wherein the incubation temperature is increased from 4°C to 10-40°C.

18. The method according to claim 1 or 2, wherein the crystallization solution is incubated for 15 minutes or longer.

19. The method of claim 18, wherein the crystallization solution is incubated for 2 hours or longer.

20. The method according to claim 1 or 2, wherein the crystallization solution is rotated or stirred during incubation.

21. The method according to claim 1 or 2, wherein the concentration of the anti-PD-1 mAb in the crystallization solution is from 5 mg / mL to 50 mg / mL.

22. The method according to claim 1 or 2, wherein the crystallization solution is generated by vapor diffusion, batch crystallization or dialysis.

23. The method of claim 1 or 2, wherein the crystallization solution further comprises 25 mM to 250 mM HEPES buffer.

24. The method of claim 23, wherein the crystallization solution comprises 50 mM HEPES buffer.

25. The method according to claim 1 or 2, further comprising the step of seeding the crystallization solution with the crystals of the anti-PD-1mAb.

26. The method according to claim 1 or 2, further comprising the step of homogenizing the crystal anti-PD-1mAb.

27. A crystal comprising a separated pembrolizumab complexed with caffeine, wherein the crystal is characterized by space group P2221, as measured using a Rayonix MX300 HS detector on a SER-CAT beamline goniometer and confirmed using POINTLESS. α = β = γ = 90°.

28. The crystal of claim 27, comprising a polypeptide, wherein the polypeptide is characterized in that, when superimposed on the backbone atoms described by the structural coordinates in Table 7, it comprises structural coordinates of conserved residue backbone atoms with a root mean square deviation (RMSD) of less than 2.0 Å.

29. The crystal according to claim 27 or 28, wherein the particle size of the crystal is 0.5 to 50 micrometers.

30. A crystal pembrolizumab comprising pembrolizumab complexed with caffeine, characterized in that... Solid-state NMR as shown in Figure 10A 13 C spectrum.

31. A composition comprising the crystal of any one of claims 27-29 or the crystal of claim 30, and a pharmaceutically acceptable carrier.

32. The composition according to claim 31, wherein the composition is a crystal suspension and the concentration of the anti-PD-1 mAb is 5-400 mg / mL.

33. The composition according to claim 31, wherein the concentration of the anti-PD-1 mAb is ≥75 mg / mL.

34. The composition according to any one of claims 31-33, further comprising a buffer of 5 mM to 20 mM.

35. The composition according to any one of claims 31-32, further comprising 0.01% to 0.10% w / v of a nonionic surfactant.

36. The composition according to any one of claims 31-33, further comprising a second active pharmaceutical ingredient (API).

37. The composition of claim 36, wherein the second API is a small molecule or a biological agent.

38. Use of the crystal of any one of claims 27-29, the crystal pembrolizumab of claim 30, or the composition of any one of claims 31-37 in the preparation of a medicament for treating cancer in human patients, wherein said cancer is selected from: melanoma, non-small cell lung cancer, small cell lung cancer, Hodgkin's lymphoma, head and neck cancer, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, esophageal cancer, renal cancer, endometrial cancer, hepatocellular carcinoma, Merkel cell carcinoma, and cervical cancer.

39. The use according to claim 38, wherein the crystal or the composition is administered intravenously or subcutaneously to the patient.

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