Patent Grant
12319735
The present invention relates generally to formulations of therapeutic antibodies, and their use in treating various disorders.
Antibodies may differ somewhat in the amino acid sequence of their constant domains, or in their framework sequences within the variable domains, but they typically differ most dramatically in the CDR sequences. Even antibodies binding to the same protein, the same polypeptide, or even potentially the same epitope may comprise entirely different CDR sequences. Therapeutic antibodies for use in human beings can also be obtained from human germline antibody sequence or from non-human (e.g. rodent) germline antibody sequences, such as in humanized antibodies, leading to yet further diversity in potential sequences. These sequence differences may result in potentially different stabilities in solution and different responsiveness to solution parameters. In addition, small changes in the arrangement of amino acids or changes in one or a few amino acid residues can result in dramatically different antibody stability and susceptibility to sequence-specific degradation pathways. As a consequence, it is not possible at present to predict the solution conditions necessary to optimize antibody stability. Each antibody must be studied individually to determine the optimum solution formulation. Bhambhani et al. (2012) J. Pharm. Sci. 101:1120.
Antibodies are also fairly large proteins (150,000 Da), for example as compared with other therapeutic proteins such as hormones and cytokines. Antibody drugs must be stable during storage to ensure efficacy and consistent dosing, so it is critical that whatever formulation is chosen supports desirable properties, such as high concentration, clarity and acceptable viscosity, and that also maintains these properties and drug efficacy over an acceptably long shelf-life under typical storage conditions.
LAG3 (CD223) is a cell surface molecule expressed on activated T cells (Huard et al. Immunogenetics 39:213-217, 1994), NK cells (Triebel et al. J Exp Med 171:1393-1405, 1990), B cells (Kisielow et al. Eur J Immunol 35:2081-2088, 2005), and plasmacytoid dendritic cells (Workman et al. J Immunol 182:1885-1891, 2009) that plays an important role in the function of these lymphocyte subsets. In addition, the interaction between LAG3 and its major ligand, Class II MHC, is thought to play a role in modulating dendritic cell function (Andreae et al. J Immunol 168:3874-3880, 2002). Recent preclinical studies have documented a role for LAG-3 in CD8 T-cell exhaustion (Blackburn et al. Nat Immunol 10:29-37, 2009).
As with chronic viral infection, tumor antigen-specific CD4+ and CD8+ T cells display impaired effector function and an exhausted phenotype characterized by decreased production of pro-inflammatory cytokines and hyporesponsiveness to antigenic re-stimulation. This is mediated by cell extrinsic mechanisms, such as regulatory T-cells (Treg), and cell intrinsic mechanisms, such as inhibitory molecules that are upregulated on exhausted, tumor-infiltrating lymphocytes (TIL). These inhibitory mechanisms represent a formidable barrier to effective antitumor immunity.
LAG- is expressed on tolerized TILs suggesting that they contribute to tumor-mediated immune suppression. Inhibition of LAG3 may lead to enhanced activation of antigen-specific T cells from which a therapeutic benefit may be gained.
PD-1 is recognized as an important molecule in immune regulation and the maintenance of peripheral tolerance. PD-1 is moderately expressed on naive T, B and NKT cells and upregulated by T/B cell receptor signaling on lymphocytes, monocytes and myeloid cells. Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers arising in various tissues. In large sample sets of e.g. ovarian, renal, colorectal, pancreatic, liver cancers and melanoma, it was shown that PD-L1 expression correlated with poor prognosis and reduced overall survival irrespective of subsequent treatment. Similarly, PD-1 expression on tumor infiltrating lymphocytes was found to mark dysfunctional T cells in breast cancer and melanoma and to correlate with poor prognosis in renal cancer. Thus, it has been proposed that PD-L1 expressing tumor cells interact with PD-1 expressing T cells to attenuate T cell activation and evasion of immune surveillance, thereby contributing to an impaired immune response against the tumor.
Several monoclonal antibodies that inhibit the interaction between PD-1 and one or both of its ligands PD-L1 and PD-L2 are FDA approved for treating cancer. It has been proposed that the efficacy of such antibodies might be enhanced if administered in combination with other approved or experimental cancer therapies, e.g., radiation, surgery, chemotherapeutic agents, targeted therapies, agents that inhibit other signaling pathways that are disregulated in tumors, and other immune enhancing agents.
As a consequence, the need exists for stable co-formulations of an anti-LAG3 antibody and an anti-PD-1 antibody. Such stable formulations will preferably exhibit stability over months to years under conditions typical for storage of drugs for self-administration, i.e. at refrigerator temperature in a syringe, resulting in a long shelf-life for the corresponding drug product.
The invention provides co-formulations of anti-LAG3 antibodies or antigen binding fragments thereof and anti-PD-1 antibodies or antigen binding fragments thereof. In one embodiment, the formulation comprises: about 16-22 mg/mL of an anti-LAG3 antibody or antigen binding fragment thereof; about 3-7 mg/mL of an anti-PD-1 antibody or antigen binding fragment thereof; about 30-120 mg/mL of a non-reducing disaccharide; about 0.02-2.0 mg/mL polysorbate 80 or polysorbate 20; a buffer at pH about 4.5-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof, wherein the anti-LAG3 antibody or antigen-binding fragment thereof comprises a variable light chain region comprising CDRL1 of SEQ ID NO: 39, CDRL2 of SEQ ID NO: 40, and CDRL3 of SEQ ID NO: 41, and a variable heavy chain region comprising CDRH1 of SEQ ID NO: 42, CDRH2 of SEQ ID NO: 59, and CDRH3 of SEQ ID NO: 44, and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable light chain region comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a variable heavy chain region comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8. In one embodiment, the formulation further comprises about 5-15 or about 5-10 mM L-methionine. Surprisingly, the anti-LAG3/anti-PD-1 co-formulations show better stability than the individual antibody formulations. The formulations can be lyophilized for reconstitution or in liquid form.
In another aspect of the invention, the formulation comprises: about 3-300 mg/mL of an anti-LAG3 antibody or antigen-binding fragment thereof and about 3-300 mg/mL of an anti-PD-1 antibody or antigen-binding fragment thereof at a molar ratio of 4:1 to 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), one or more of an excipient selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2, and FeCl2, at a total excipient concentration of about 10-1000 mM, and a buffer at pH about 5-8.
The present invention also provides a method of treating cancer or infection comprising administering the reconstituted or liquid formulation (solution formulation) to a subject in need thereof. In further embodiments, the formulation is used in treating chronic infection. Also contemplated is the use of the solution or lyophilized formulation in the manufacture of a medicament for treating cancer or infection.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise. Unless otherwise indicated, the proteins and subjects referred to herein are human proteins and human subjects, rather than another species.
As used herein, unless otherwise indicated, “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments.
A “Fab fragment” is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.
An “Fc” region contains two heavy chain fragments comprising the CH3 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
A “Fab′ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′) 2 molecule.
A “F(ab′)2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′) 2 fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab′)2 fragment” can be the product of pepsin cleavage of an antibody.
The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen-binding. 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 (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 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). As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. The residue numbering above relates to the Kabat numbering system and does not necessarily correspond in detail to the sequence numbering in the accompanying Sequence Listing.
“Proliferative activity” encompasses an activity that promotes, that is necessary for, or that is specifically associated with, e.g., normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.
The terms “cancer”, “tumor”, “cancerous”, and “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 including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, myeloma (such as multiple myeloma), salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer.
As cancerous cells grow and multiply, they form a mass of cancerous tissue, that is a tumor, which invades and destroys normal adjacent tissues. Malignant tumors are cancer. Malignant tumors usually can be removed, but they may grow back. Cells from malignant tumors can invade and damage nearby tissues and organs. Also, cancer cells can break away from a malignant tumor and enter the bloodstream or lymphatic system, which is the way cancer cells spread from the primary tumor (i.e., the original cancer) to form new tumors in other organs. The spread of cancer in the body is called metastasis (What You Need to Know About Cancer—an Overview, NIH Publication No. 00-1566; posted Sep. 26, 2000, updated Sep. 16, 2002 (2002)).
As used herein, the term “solid tumor” refers to an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).
As used herein, the term “carcinomas” refers to cancers of epithelial cells, which are cells that cover the surface of the body, produce hormones, and make up glands. Examples of carcinomas are cancers of the skin, lung, colon, stomach, breast, prostate and thyroid gland.
As used herein, an “aqueous” pharmaceutical composition is a composition suitable for pharmaceutical use, wherein the aqueous carrier is sterile water for injection. A composition suitable for pharmaceutical use may be sterile, homogeneous and/or isotonic. In certain embodiments, the aqueous pharmaceutical compositions of the invention are suitable for parenteral administration to a human subject. In a specific embodiment, the aqueous pharmaceutical compositions of the invention are suitable for intravenous and/or subcutaneous administration.
The term “about”, when modifying the quantity (e.g., mM, or M) of a substance or composition, the percentage (v/v or w/v) of a formulation component, the pH of a solution/formulation, or the value of a parameter characterizing a step in a method, or the like refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition; through instrumental error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like. In certain embodiments, “about” can mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.
As used herein, “x % (w/v)” is equivalent to x g/100 ml (for example, 5% w/v equals 50 mg/ml).
The term “buffer” encompasses those agents which maintain the solution pH in an acceptable range in the liquid formulation, prior to lyophilization and/or after reconstitution and may include but not limited to succinate (sodium or potassium), histidine, acetate, phosphate (sodium or potassium), Tris (tris (hydroxymethyl) aminomethane), diethanolamine, citrate (sodium) and the like.
“Co-formulated” or “co-formulation” or “coformulation” or “coformulated” as used herein refers to at least two different antibodies or antigen binding fragments thereof which are formulated together and stored as a combined product in a single vial or vessel (for example an injection device) rather than being formulated and stored individually and then mixed before administration or separately administered. In one embodiment, the co-formulation contains two different antibodies or antigen binding fragments thereof.
“Glycol” refers to an alkyl with two hydroxyl groups.
“Sugar alcohol” refers to polyols derived from a sugar and have the general formula HOCH2(CHOH)nCH2OH, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Examples include but are not limited to mannitol, sorbitol, erythritol, xylitol and glycerol.
As used herein “polyol” includes a glycol and a sugar alcohol.
The terms “lyophilization,” “lyophilized,” and “freeze-dried” refer to a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in pre-lyophilized formulations to enhance stability of the lyophilized product upon storage.
“Non-reducing sugar” is a sugar not capable of acting as a reducing agent because it does not contain or cannot be converted to contain a free aldehyde group or a free ketone group. Examples of non-reducing sugars include but are not limited to dissacharrides such as sucrose and trehalose.
The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the active ingredients to be effective, and which contains no additional components which are toxic to the subjects to which the formulation would be administered.
“Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
“Reconstitution time” is the time that is required to rehydrate a lyophilized formulation with a solution to a particle-free clarified solution.
A “stable” formulation is one in which the protein therein essentially retains 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 at a selected temperature for a selected time period. For example, in one embodiment, a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2-8° C.) for at least 12 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2-8° C.) for at least 18 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27° C.) for at least 3 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27° C.) for at least 6 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27° C.) for at least 12 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27° C.) for at least 18 months. The criteria for stability for an antibody formulation are as follows. Typically, no more than 10%, preferably 5%, of antibody monomer is degraded as measured by SEC-HPLC. Typically, the formulation is colorless, or clear to slightly opalescent by visual analysis. Typically, the concentration, pH and osmolality of the formulation have no more than +/−10% change. Potency is typically within 60-140%, preferably 80-120% of the control or reference. Typically, no more than 10%, preferably 5% of clipping of the antibody is observed, i.e., % low molecular weight species as determined, for example, by HP-SEC. Typically, or no more than 10%, preferably 5% of aggregation of the antibody is formed, i.e. % high molecular weight species as determined, for example, by HP-SEC.
“Surfactant” is a surface active agent that is amphipathic in nature.
An antibody “retains its physical stability” in a pharmaceutical formulation if it shows no significant increase of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering. The changes of protein conformation can be evaluated by fluorescence spectroscopy, which determines the protein tertiary structure, and by FTIR spectroscopy, which determines the protein secondary structure.
An antibody “retains its chemical stability” in a pharmaceutical formulation, if it shows no significant chemical alteration. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the protein chemical structure include hydrolysis or clipping (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as by peptide mapping in conjunction with mass spectroscopy or MALDI/TOF/MS), deamidation (evaluated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement), and isomerization (evaluated by measuring the isoaspartic acid content, peptide mapping, etc.).
An antibody “retains its biological activity” in a pharmaceutical formulation, if the biological activity of the antibody at a given time frame is within a predetermined range of biological activity exhibited at the time the formulation was prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay. In one embodiment, the biological activity of stable antibody formulation within 12 months is within 60-140% of the reference.
The term “isotonic” means that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure about 270-328 mOsm. Slightly hypotonic pressure is 250-269 and slightly hypertonic pressure is 328-350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or ice-freezing type osmometer.
A “reconstituted” formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration, (e.g. parenteral administration), and may optionally be suitable for subcutaneous administration.
“Total excipient concentration” refers to the sum of the molar concentrations of the referenced ionizable excipients (for example, histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2, and FeCl2), not accounting for concentrations of excipients referenced as buffer species.
When a range of pH values is recited, such as “a pH between pH 5.0 and 6.0,” the range is intended to be inclusive of the recited values. The pH is typically measured at 25° C. using standard glass bulb pH meter. As used herein, a solution comprising “histidine buffer at pH X” refers to a solution at pH X and comprising the histidine buffer, i.e. the pH is intended to refer to the pH of the solution.
Analytical Methods
Analytical methods suitable for evaluating the product stability include size exclusion chromatography (SEC), dynamic light scattering test (DLS), differential scanning calorimetery (DSC), iso-asp quantification, potency, UV at 350 nm, UV spectroscopy, and FTIR. SEC (I Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986)) measures percent monomer in the product and gives information of the amount of soluble aggregates. DSC (Pharm. Res., 15:200 (1998); Pharm. Res., 9:109 (1982)) gives information of protein denaturation temperature and glass transition temperature. DLS (American Lab., November (1991)) measures mean diffusion coefficient, and gives information of the amount of soluble and insoluble aggregates. UV at 340 nm measures scattered light intensity at 340 nm and gives information about the amounts of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278 nm and gives information of protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45:231 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); J. Pharm. Scien., 87:1069 (1998)) measures IR spectrum in the amide one region, and gives information of protein secondary structure.
The iso-asp content in the samples is measured using the Isoquant Isoaspartate Detection System (Promega). The kit uses the enzyme Protein Isoaspartyl Methyltransferase (PIMT) to specifically detect the presence of isoaspartic acid residues in a target protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to isoaspartic acid at the .alpha.-carboxyl position, generating S-adenosyl-L-homocysteine (SAH) in the process. This is a relatively small molecule, and can usually be isolated and quantitated by reverse phase HPLC using the SAH HPLC standards provided in the kit.
The potency or bioidentity of an antibody can be measured by its ability to bind to its antigen. The specific binding of an antibody to its antigen can be quantitated by any method known to those skilled in the art, for example, an immunoassay, such as ELISA (enzyme-linked immunosorbant assay).
Anti-LAG3 Antibodies
The CDR amino acid residues are highly variable between different antibodies, and may originate from human germline sequences (in the case of fully human antibodies), or from non-human (e.g. rodent) germline sequences. The framework regions can also differ significantly from antibody to antibody. The constant regions will differ depending on whether the selected antibody has a lambda (λ) or kappa (κ) light chain, and depending on the class (or isotype) of the antibody (IgA, IgD, IgE, IgG, or IgM) and subclass (e.g. IgG1, IgG2, IgG3, IgG4).
The LAG3 antibodies exemplified below have CDR sequences derived from non-human (in this case mouse) germline sequences, or human germline sequences. The germline sequences comprise the sequence repertoire from which an antibody's CDR sequences are derived, aside from somatic hypermutation derived changes, and as a consequence it would be expected that CDRs obtained starting with a mouse germline would systematically differ from those starting from a human germline. Use of human germline sequences is often justified on the basis that CDR sequences from human germlines will be less immunogenic in humans than those derived from other species, reflecting the underlying belief that CDRs will systematically differ depending on their species of origin. Although the increase in CDR diversity increases the likelihood of finding antibodies with desired properties, such as high affinity, it further magnifies the difficulties in developing a stable solution formulation of the resulting antibody.
Even antibodies that bind to the same antigen can differ dramatically in sequence, and are not necessarily any more closely related in sequence than antibodies to entirely separate antigens. Based on the low sequence similarity, the chemical properties of the antibodies, and thus their susceptibility to degradation, cannot be presumed to be similar despite their shared target.
As discussed above, antibodies are large, highly complex polypeptide complexes subject to various forms of degradation and instability in solution. The diversity of sequence, and thus structure, of antibodies gives rise to wide range of chemical properties. Aside from the obvious sequence-specific differences in antigen binding specificity, antibodies exhibit varying susceptibility to various degradative pathways, aggregation, and precipitation. Amino acid side chains differ in the presence or absence of reactive groups, such as carboxy-(D,E), amino-(K), amide-(N,Q), hydroxyl-(S,T,Y), sulfhydryl-(C), thioether-(M) groups, as well as potentially chemically reactive sites on histidine, phenylalanine and proline residues. Amino acid side chains directly involved in antigen binding interactions are obvious candidates for inactivation by side chain modification, but degradation at other positions can also affect such factors as steric orientation of the CDRs (e.g. changes in framework residues), effector function (e.g. changes in Fc region—see, e.g., Liu et al. (2008) Biochemistry 47:5088), or self-association/aggregation.
Antibodies are subject to any number of potential degradation pathways. Oxidation of methionione residues in antibodies, particularly in CDRs, can be a problem if it disrupts antigen binding. Presta (2005) J. Allergy Clin. Immunol. 116: 731; Lam et al. (1997) J. Pharm. Sci. 86:1250. Other potential degradative pathways include asparagine deamidation (Harris et al. (2001) Chromatogr., B 752:233; Vlasak et al. (2009) Anal. Biochem. 392:145) tryptophan oxidation (Wei et al. (2007) Anal. Chem. 79:2797), cysteinylation (Banks et al. (2008) J. Pharm. Sci. 97:775), glycation (Brady et al. (2007) Anal. Chem. 79:9403), pyroglutamate formation (Yu et al. (2006) J. Pharm. Biomed. Anal. 42:455), disulfide shuffling (Liu et al. (2008) J. Biol. Chem. 283:29266), and hydrolysis (Davagnino et al. (1995) J. Immunol. Methods 185:177). Discussed in Ionescu & Vlasak (2010) Anal. Chem. 82:3198. See also Liu et al. (2008) J. Pharm. Sci. 97:2426. Some potential degradation pathways depend not only on the presence of a specific amino acid residue, but also the surrounding sequence. Deamidation and isoaspartate formation can arise from a spontaneous intramolecular rearrangement of the peptide bond following (C-terminal to) N or D residues, with N-G and D-G sequences being particularly susceptible. Reissner & Aswad (2003) CMLS Cell. Mol. Life Sci. 60:1281.
Antibodies are also subject to sequence-dependent non-enzymatic fragmentation during storage. Vlasak & Ionescu (2011) mAbs 3:253. The presence of reactive side chains, such as D, G, S, T, C or N can result in intramolecular cleavage reactions that sever the polypeptide backbone. Such sequence specific hydrolysis reactions are typically dependent on pH. Id. Antibodies may also undergo sequence-dependent aggregation, for example when CDRs include high numbers of hydrophobic residues. Perchiacca et al. (2012) Prot. Eng. Des. Selection 25:591. Aggregation is particularly problematic for antibodies that need to be formulated at high concentrations for subcutaneous administration, and has even led some to modify the antibody sequence by adding charged residues to increase solubility. Id.
Mirroring the diversity of potential sequence-specific stability issues with antibodies, potential antibody formulations are also diverse. The sequence variability of the antibody leads to chemical heterogeneity of the resulting antibodies, which results in a wide range of potential degradation pathways. Formulations may vary, for example, in antibody concentration, buffer, pH, presence or absence of surfactant, presence or absence of tonicifying agents (ionic or nonionic), presence or absence of molecular crowding agent. Commercially available therapeutic antibodies are marketed in a wide range of solution formulations, in phosphate buffer (e.g. adalimumab), phosphate/glycine buffer (e.g. basilixumab), Tris buffer (e.g. ipilimumab), histidine (e.g. ustekinumab), sodium citrate (e.g. rituximab); and from pH 4.7 (e.g. certolizumab) and pH 5.2 (e.g. adalimumab) to pH 7.0-7.4 (e.g. cetuximab). They are also available in formulations optionally containing disodium edetate (e.g. alemtuzumab), mannitol (e.g. ipilimumab), sorbitol (e.g. golimumab), sucrose (e.g. ustekinumab), sodium chloride (e.g. rituximab), potassium chloride (e.g. alemtuzumab), and trehalose (e.g. ranibizumab); all with and without polysorbate-80, ranging from 0.001% (e.g. abcixmab) to 0.1% (e.g. adalimumab).
Biological Activity of Humanized Anti-LAG3 and Anti-PD-1 Antibodies
Formulations of the present invention include anti-LAG3 antibodies and fragments thereof and anti-PD-1 antibodies and fragments thereof that are biologically active when reconstituted or in liquid formulation.
Exemplary anti-LAG3 antibodies are provided below (disclosed in WO 2016/028672, incorporated herein by reference in its entirety):
As used herein, an “Ab6 variant” means a monoclonal antibody which comprises heavy chain and light chain sequences that are substantially identical to those in Ab6 (as described below and in WO2016028672, incorporated by reference in its entirety), except for having three, two or one conservative amino acid substitutions at positions that are located outside of the light chain CDRs and six, five, four, three, two or one conservative amino acid substitutions that are located outside of the heavy chain CDRs, e.g, the variant positions are located in the FR regions or the constant region, and optionally has a deletion of the C-terminal lysine residue of the heavy chain. In other words, Ab6 and a Ab6 variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at no more than three or six other positions in their full length light and heavy chain sequences, respectively. An Ab6 variant is substantially the same as Ab6 with respect to the following properties: binding affinity to human LAG3 and ability to block the binding of human LAG3 to human MHC Class II.
The present invention provides formulations of anti-LAG3 antibodies, which comprises two identical light chains with the sequence of SEQ ID NO: 35 and two identical heavy chains with the sequence of SEQ ID NO:36, 45, 48, 51, 54, 57, 60, 63 or 66. The present invention also provides formulations of anti-LAG3 antibodies, which comprises two identical light chains with the sequence of SEQ ID NO: 35 and two identical heavy chains with the sequence of SEQ ID NO:57.
The present invention provides formulations of an anti-LAG3 antibody or antigen binding fragment that comprises a light chain variable region sequence of SEQ ID NO: 37 and a heavy chain variable region sequence of SEQ ID NO: 38, 46, 49, 52, 55, 58, 61, 64 or 67. The present invention also provides formulations of an anti-LAG3 antibody or antigen binding fragment that comprises a light chain variable region sequence of SEQ ID NO: 37 and a heavy chain variable region sequence of SEQ ID NO: 58. The present invention also provides formulations of an anti-LAG3 antibody or antigen binding fragment comprising a light chain variable region CDRL1 sequence of SEQ ID NO: 39, CDRL2 sequence of SEQ ID NO: 40, and CDRL3 sequence of SEQ ID NO: 41, and a heavy chain variable region CDRH1 sequence of SEQ ID NO: 42, CDRH2 sequence of SEQ ID NO: 43, 47, 50, 53, 56, 59, 62, 65 or 68, and CDRH3 sequence of SEQ ID NO: 44. The present invention also provides formulations of an anti-LAG3 antibody or antigen binding fragment comprising a light chain variable region CDRL1 sequence of SEQ ID NO: 39, CDRL2 sequence of SEQ ID NO: 40, and CDRL3 sequence of SEQ ID NO: 41, and a heavy chain variable region CDRH1 sequence of SEQ ID NO: 42, CDRH2 sequence of SEQ ID NO: 59, and CDRH3 sequence of SEQ ID NO: 44.
Other anti-LAG3 antibodies that could be included in the formulation include BMS-986016 disclosed in WO2014008218; IMP731, and IMP701. Therefore, the present invention provides formulations of an anti-LAG3 antibody or antigen binding fragment that comprises a light chain variable region sequence of SEQ ID NO: 69 and a heavy chain variable region sequence of SEQ ID NO: 70. The present invention also provides formulations of an anti-LAG3 antibody or antigen binding fragment comprising a light chain variable region CDRL1 sequence of SEQ ID NO: 71, CDRL2 sequence of SEQ ID NO: 72, and CDRL3 sequence of SEQ ID NO: 73, and a heavy chain variable region CDRH1 sequence of SEQ ID NO: 74, CDRH2 sequence of SEQ ID NO: 75, and CDRH3 sequence of SEQ ID NO: 76.
The formulation further comprises an anti-PD-1 antibody or antigen binding fragment as exemplified below.
As used herein, a “pembrolizumab variant” means a monoclonal antibody which comprises heavy chain and light chain sequences that are substantially identical to those in pembrolizumab, except for having three, two or one conservative amino acid substitutions at positions that are located outside of the light chain CDRs and six, five, four, three, two or one conservative amino acid substitutions that are located outside of the heavy chain CDRs, e.g, the variant positions are located in the FR regions or the constant region, and optionally has a deletion of the C-terminal lysine residue of the heavy chain. In other words, pembrolizumab and a pembrolizumab variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at no more than three or six other positions in their full length light and heavy chain sequences, respectively. A pembrolizumab variant is substantially the same as pembrolizumab with respect to the following properties: binding affinity to PD-1 and ability to block the binding of each of PD-L1 and PD-L2 to PD-1.
In another aspect of the invention, the formulation comprises an anti-LAG3 antibody or antigen binding fragment comprising a light chain variable region sequence of SEQ ID NO: 37 and a heavy chain variable region sequence of SEQ ID NO: 58; and an anti-PD-1 antibody or antigen binding fragment comprising a light chain variable region sequence of SEQ ID NO: 4 and a heavy chain variable region sequence of SEQ ID NO: 9. In another embodiment, the formulation comprises an anti-LAG3 antibody comprising a light chain sequence of SEQ ID NO: 35 and a heavy chain sequence of SEQ ID NO: 57; and an anti-PD-1 antibody comprising a light chain sequence of SEQ ID NO: 5 and a heavy chain sequence of SEQ ID NO: 10. The present invention also provides formulations of anti-LAG3 antibodies or antigen binding fragments thereof comprising a light chain CDRL1 sequence of SEQ ID NO: 39, CDRL2 sequence of SEQ ID NO: 40 and CDRL3 sequence of SEQ ID NO: 41, and a heavy chain CDRH1 sequence of SEQ ID NO: 42, CDRH2 sequence of SEQ ID NO: 59, and CDRH3 sequence of SEQ ID NO: 44; and an anti-PD-1 antibody comprising a light chain CDRL1 sequence of SEQ ID NO: 1, CDRL2 sequence of SEQ ID NO: 2, and CDRL3 sequence of SEQ ID NO: 3, and a heavy chain CDRH1 sequence of SEQ ID NO: 6, CDRH2 sequence of SEQ ID NO: 7, and CDRH3 sequence of SEQ ID NO: 8. In some formulations of the invention, the anti-LAG3 antibody is Ab6 or an Ab6 variant.
In a further aspect of the present invention, the formulations comprise an anti-LAG3 antibody or antigen binding fragment that comprises a light chain variable region sequence of SEQ ID NO: 69 and a heavy chain variable region sequence of SEQ ID NO: 70, and an anti-PD-1 antibody or antigen binding fragment that comprises a light chain variable region sequence of SEQ ID NO: 14 and a heavy chain variable region sequence of SEQ ID NO: 19. The present invention also provides formulations of an anti-LAG3 antibody or antigen binding fragment comprising a light chain variable region CDRL1 sequence of SEQ ID NO: 71, CDRL2 sequence of SEQ ID NO: 72, and CDRL3 sequence of SEQ ID NO: 73, and a heavy chain variable region CDRH1 sequence of SEQ ID NO: 74, CDRH2 sequence of SEQ ID NO: 75, and CDRH3 sequence of SEQ ID NO: 76, and an anti-PD-1 antibody or antigen binding fragment comprising a light chain variable region CDRL1 sequence of SEQ ID NO: 11, CDRL2 sequence of SEQ ID NO: 12, and CDRL3 sequence of SEQ ID NO: 13, and a heavy chain variable region CDRH1 sequence of SEQ ID NO: 16, CDRH2 sequence of SEQ ID NO: 17, and CDRH3 sequence of SEQ ID NO: 18.
Antibody or antigen binding fragments of the formulation can comprise a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises SEQ ID NO:4 or a variant of SEQ ID NO:4, and the heavy chain variable region comprises SEQ ID NO:9 or a variant of SEQ ID NO:9. In further embodiments, the light chain variable region comprises SEQ ID NO:14 or a variant of SEQ ID NO:14, and the heavy chain variable region comprises SEQ ID NO:19 or a variant of SEQ ID NO:19. In further embodiments, the heavy chain variable region comprises SEQ ID NO:27 or a variant of SEQ ID NO:27 and the light chain variable region comprises SEQ ID NO:28 or a variant of SEQ ID NO:28, SEQ ID NO:29 or a variant of SEQ ID NO:29, or SEQ ID NO:30 or a variant of SEQ ID NO:30. In such embodiments, a variant light chain or heavy chain variable region sequence is identical to the reference sequence except having one, two, three, four or five amino acid substitutions. In some embodiments, the substitutions are in the framework region (i.e., outside of the CDRs). In some embodiments, one, two, three, four or five of the amino acid substitutions are conservative substitutions.
In another embodiment, the formulations of the invention comprise an antibody or antigen binding fragment that has a VL domain and/or a VH domain with at least 95%, 90%, 85%, 80%, 75% or 50% sequence homology to one of the VL domains or VH domains described above, and exhibits specific binding to PD-1 or LAG3. In another embodiment, the antibody or antigen binding fragment of the formulations of the invention comprises VL and VH domains having up to 1, 2, 3, 4, or 5 or more amino acid substitutions, and exhibits specific binding to PD-1 or LAG3.
In embodiments of the invention, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:5 and a heavy chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:10. In alternative embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:15 and a heavy chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:20. In further embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:32 and a heavy chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:31. In additional embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:33 and a heavy chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:31. In yet additional embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:34 and a heavy chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:31. In some formulations of the invention, the anti-PD-1 antibody is pembrolizumab or a pembrolizumab variant.
Ordinarily, amino acid sequence variants of the anti-PD-1 or anti-LAG3 antibodies and antigen binding fragments of the invention will have an amino acid sequence having at least 75% amino acid sequence identity with the amino acid sequence of a reference antibody or antigen binding fragment (e.g. heavy chain, light chain, VH, VL, framework or humanized sequence), more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most 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 or anti-LAG3 residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.
In one embodiment, the ratio of anti-LAG3 antibody to anti-PD-1 antibody in the formulation is 1:1, 1:2 or 1:3. In another embodiment, the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody in the formulation is 1:1, 2:1, 3:1, 3.5:1, 4:1, 5:1 or 6:1. In one embodiment, the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody in the formulation is 4:1. In another embodiment, the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody in the formulation is 5:1.
Formulations
In some aspects of the invention, the formulations of the invention minimize the formation of antibody aggregates (high molecular weight species) and particulates, improve colloidal stability, minimize fragmentation (low molecular weight species), or insure that the antibody maintains its biological activity over time. In one aspect, the formulation comprises: about 3-300 mg/mL of an anti-LAG3 antibody or antigen-binding fragment thereof and about 3-300 mg/mL of an anti-PD-1 antibody or antigen-binding fragment thereof at a molar ratio of 4:1 to 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), one or more of an excipient selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2, and FeCl2, at a total excipient concentration of about 10-1000 mM, and a buffer at pH about 5-8. In one embodiment, the anti-LAG3 antibody or antigen-binding fragment thereof and anti-PD-1 antibody or antigen-binding fragment thereof have a molar ratio of 4:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof). In another embodiment, the anti-LAG3 antibody or antigen-binding fragment thereof and anti-PD-1 antibody or antigen-binding fragment thereof have a molar ratio of 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof). In another aspect, the formulation comprises: about 4-200 mg/mL of an anti-LAG3 antibody or antigen-binding fragment thereof and about 4-200 mg/ml of an anti-PD-1 antibody or antigen-binding fragment thereof. In one embodiment, one or more of an excipient selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2, and FeCl2, is at a total excipient concentration of about 25-250 mM. In another embodiment, one or more of an excipient selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2, and FeCl2, is at a total excipient concentration of about 40-250 mM.
In one aspect, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of about 15-250 mM. In one aspect, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of about 25-250 mM. In another embodiment, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of about 40-150 mM. In another embodiment, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of about 40-100 mM. In another embodiment, the excipient is L-arginine or a pharmaceutically acceptable salt thereof at a concentration of about 70 mM. In another embodiment, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of about 70-150 mM. Examples of pharmaceutically acceptable salts of arginine (L or D form) include but are not limited to L-arginine-hydrochloride and L-arginine succinate. In other aspects of the foregoing embodiments, the formulation further comprises a non-ionic surfactant, sugar or polyol, or glutamine, glycine, proline, or methionine.
In another aspect, the excipients are NaCl and arginine or a pharmaceutically acceptable salt thereof with a total excipient concentration of about 25-250 mM. In a further embodiment, the excipients are NaCl and arginine or a pharmaceutically acceptable salt thereof with a total excipient concentration of about 70-100 mM. In one embodiment, the NaCl to arginine concentration ratio is 1:1. In another embodiment, the NaCl concentration is about 35 mM and the arginine concentration is about 35 mM. In another embodiment, the NaCl concentration is about 50 mM and the arginine concentration is about 50 mM.
In a further aspect, the excipient is NaCl, KCl or LiCl at about 40-150 mM. In a further embodiment, the excipient is NaCl, KCl or LiCl at about 40-100 mM. In a further embodiment, the excipient is NaCl, KCl or LiCl at about 70-130 mM. In a further embodiment, the excipient is NaCl, KCl or LiCl at about 70-100 mM. In a further embodiment, the excipient is NaCl at about 70 mM. In other aspects of the foregoing embodiments, the formulation further comprises a non-ionic surfactant.
In a further aspect, the excipient is L-histidine at about 25-200 mM. In a further embodiment, the L-histidine is at about 50-200 mM. In yet a further embodiment, the L-histidine is at about 40-100 mM.
In a further aspect, the excipient is L-glutamine, L-glycine, L-proline or L-methionine, or a combination thereof at about 25-200 mM. In a further embodiment, the excipient is at about 50-200 mM. In yet a further embodiment, the excipient is at about 40-100 mM. In yet a further embodiment, the excipient is at about 70 mM.
In one embodiment, the excipient is L-glutamine, L-glycine, L-aspartate, or a combination thereof at about 25-200 mM. In another embodiment, the excipient is at about 20-50 mM. In a further embodiment, the excipient is at about 20 mM. In yet a further embodiment, the excipient is at about 40-100 mM. In yet a further embodiment, the excipient is at about 70 mM. In another embodiment, the excipients are about 20 mM L-aspartate and about 50 mM L-glycine. In another embodiment, the excipients are about 20 mM L-glutamine and about 50 mM L-glycine.
In one embodiment, the co-formulated composition has a buffer having a neutral or slightly acidic pH (pH 4.5-8), and arginine or a pharmaceutically acceptable salt thereof. In one embodiment, a buffer of pH about 5.5-6.5 is used in the composition. In one embodiment, a buffer of pH about 4.5-6.5 is used in the composition. In another embodiment, a buffer of pH about 5.5-6.0 is used in the composition. In a further embodiment, a buffer of pH about 5.0-6.0 is used in the composition. The buffer can have a concentration of about 5-1000 mM. In another embodiment, the buffer can have a concentration of about 5-150 mM. In a further embodiment, the buffer can have a concentration of about 5-300 mM. In a further embodiment, the buffer has a concentration of about 1-300 mM. In a another embodiment, the buffer can have a concentration of about 1-30 mM. In yet a further embodiment, the buffer can have a concentration of 5-30 mM. In yet a further embodiment, the buffer can have a concentration of about 5-20 mM. In yet a further embodiment, the buffer can have a concentration of about 8-12 mM. In one embodiment, the buffer is histidine, acetate or citrate. A preferred buffer contains about 10 mM histidine, acetate or citrate.
In one embodiment, the formulation comprises about 3-300 mg/mL of an anti-LAG3 antibody or antigen-binding fragment thereof and 3-300 mg/mL of an anti-PD-1 antibody or antigen-binding fragment thereof at a molar ratio of 4:1 to 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), sugar or polyol; a non-ionic surfactant, a histidine buffer or acetate buffer at pH about 4.5-8, about 10-1000 mM arginine or a pharmaceutically acceptable salt thereof and optionally methionine (L or D form), EDTA, DTPA, tryptophan (L or D form) or pyridoxine. In another embodiment, the formulation comprises about 4-250 mg/mL of an anti-LAG3 antibody or antigen-binding fragment thereof and about 4-250 mg/mL of an anti-PD-1 antibody or antigen-binding fragment thereof at a molar ratio of 4:1 to 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), a sugar or polyol; a non-ionic surfactant, about 50-500 mM histidine buffer at pH about 5-8, about 10-1000 mM salt of monovalent cations selected from NaCl, KCl and LiCl or salt of polyvalent cations selected from CaCl2, MgCl2, ZnCl2, FeCl2 and FeCl3, optionally about 10-1000 mM arginine or a pharmaceutically acceptable salt thereof and optionally methionine (D or L form), EDTA, DTPA, tryptophan and Pyridoxine. In another aspect, the formulation comprises: about 4-200 mg/mL of an anti-LAG3 antibody or antigen-binding fragment thereof and about 4-200 mg/ml of an anti-PD-1 antibody or antigen-binding fragment thereof. In one embodiment, the anti-LAG3 antibody or antigen-binding fragment thereof and anti-PD-1 antibody or antigen-binding fragment thereof has a molar ratio of 4:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof). In another embodiment, the anti-LAG3 antibody or antigen-binding fragment thereof and anti-PD-1 antibody or antigen-binding fragment thereof has a molar ratio of 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof).
The formulation may include about 1-100 uM, about 1-30 uM, about 1-20 uM, about 10 uM-30 uM DTPA or EDTA. The formulation may also include about 1-30 mM L-methionine. In one embodiment, the formulation may also include about 1-20 mM L-methionine. The formulation may also include about 5-15 mM L-methionine. The formulation may also include about 5-15 mM L-methionine. The formulation may also include about 5-20 mM L-methionine. The formulation may also include about 10 mM, or at least about 10 mM L-methionine. Sometimes nitrogen overlay (blanketing, for example only 5% or 10% residual 02 upon nitrogen overlay) is used during production steps and/or prior to vial closure, to stabilize antibody against oxidation.
In another aspect of the invention, the formulation further comprises a sugar, polyol, or a non-ionic surfactant, or a combination thereof. In one embodiment, the sugar is selected from the group consisting of glucose, sucrose, trehalose and lactose or a combination thereof. In one embodiment, the sugar is a disaccharide such as sucrose, trehalose and maltose. In one embodiment, the sugar is a non-reducing sugar. In another embodiment, the sugar is a non-reducing disaccharide such as sucrose or trehalose, or a combination thereof. In one embodiment, the sugar is at a concentration of about 10-200 mg/ml. In another embodiment, the sugar is at a concentration of about 30-120 mg/ml. In another embodiment, the sugar is at a concentration of about 30-80 mg/ml. In a further embodiment, the sugar is at a concentration of about 50-90 mg/ml.
In one embodiment, the polyol is selected from the group consisting of mannitol, sorbitol, glycerol and polyethylene glycol. In another embodiment, the polyol is a sugar alcohol. In one embodiment, the sugar and polyol are selected from the group consisting of sucrose, trehalose, sorbitol, glycerol and polyethylene glycol. In a further embodiment, the polyol is a glycol. In one embodiment, the glycol is selected from the group consisting of ethylene glycol, propylene glycol and polyethylene glycol. In one embodiment, the polyol is at a concentration of about 10-200 mg/ml. In another embodiment, the polyol is at a concentration of about 10-50 mg/ml. In a further embodiment, the polyol is at a concentration of about 5-30 mg/ml.
In one embodiment, the formulation comprises about 10-250 mg/ml of sucrose or trehalose. In another embodiment, the formulation comprises about 20-200 mg/ml of sucrose or trehalose. In a further embodiment, the formulation comprises about 50-80 mg/ml of sucrose or trehalose. In a further embodiment, the formulation comprises about 30-80 mg/ml of sucrose or trehalose. In another embodiment, the formulation comprises about 50-90 mg/ml of sucrose or trehalose. In yet a further embodiment, the formulation comprises about 70-80 mg/ml of sucrose or trehalose. In yet a further embodiment, the formulation comprises at least about 50 mg/ml of sucrose or trehalose. In another embodiment, the formulation comprises about 20-200 mg/ml of sorbitol, PEG400 or glycerol. In a further embodiment, the formulation comprises about 20-50 mg/ml of sorbitol, PEG400 or glycerol.
In one embodiment, the non-ionic surfactant is selected from the group consisting of a polysorbate and a poloxamer. In yet another embodiment, the surfactant is selected from the group consisting of Tween80® (polysorbate 80), Tween20® (polysorbate 20), PluronicF88®, Pluoronic F-127®, PluronicF68®, Triton X-100®. In a preferred embodiment, the surfactant is polysorbate 20 or polysorbate 80, and the sugar is sucrose or trehalose. The polysorbate 80 or polysorbate 20 surfactant may be present in the formulation in an amount from about 0.005 to about 1 mg/ml. The polysorbate 80 or polysorbate 20 surfactant may be present in the formulation in an amount from about 0.02 to about 2 mg/ml. The polysorbate 80 or polysorbate 20 surfactant may be present in the formulation in an amount from about 0.05 to about 1 mg/ml. The polysorbate 80 or polysorbate 20 surfactant may be present in the formulation in an amount from about 0.1 to about 0.5 mg/ml. In another embodiment, the polysorbate 80 or polysorbate 20 surfactant may be present in the formulation in an amount from about at least about 0.005 mg/ml. The polysorbate 80 or polysorbate 20 surfactant may also be present in the formulation in an amount from about at least about 0.1 mg/ml. The polysorbate 80 surfactant may be present in the formulation in an amount from about 0.2 mg/ml.
In other aspects of the above formulations, at 50° C., the % High Molecular Weight (HMW) is less than 5% in the co-formulated anti-LAG3 antibody and anti-PD-1 antibody formulation after 10-days as measured by size exclusion chromatography.
The invention provides the following embodiments:
1. A formulation comprising:
about 16-22 mg/mL of an anti-LAG3 antibody or antigen binding fragment thereof about 3-7 mg/mL of an anti-PD-1 antibody or antigen binding fragment thereof about 30-120 mg/mL of a non-reducing disaccharide; about 0.02-2.0 mg/mL polysorbate 80 or polysorbate 20; a buffer at pH about 4.5-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof, wherein the anti-LAG3 antibody or antigen-binding fragment thereof comprises a variable light chain region comprising CDRL1 of SEQ ID NO: 39, CDRL2 of SEQ ID NO: 40, and CDRL3 of SEQ ID NO: 41, and a variable heavy chain region comprising CDRH1 of SEQ ID NO: 42, CDRH2 of SEQ ID NO: 59, and CDRH3 of SEQ ID NO: 44, and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable light chain region comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a variable heavy chain region comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8.
2. The formulation of embodiment 1 comprising about 18-22
mg/mL of the anti-LAG3 antibody; about 4-7 mg/mL of the anti-PD-1 antibody; about 50-90 mg/mL sucrose or trehalose; about 0.05-1.0 mg/mL polysorbate 80 or polysorbate 20; about 3-30 mM histidine buffer at pH about 5.0-6.5; and about 40-100 mM L-arginine or a pharmaceutically acceptable salt thereof.
3. The formulation of embodiment 1 comprising about 18-20
mg/mL of the anti-LAG3 antibody; about 4-7 mg/mL of the anti-PD-1 antibody; about 50-60 mg/mL sucrose or trehalose; about 0.05-1.0 mg/mL polysorbate 80 or polysorbate 20; about 8-12 mM histidine buffer at pH about 5.0-6.5; and about 40-70 mM L-arginine or a pharmaceutically acceptable salt thereof.
4. The formulation of any one of embodiments 1 to 3, further comprising about 5-15 mM L-methionine.
5. The formulation of any one of embodiments 1 to 3, further comprising about 5-10 mM L-methionine.
6. The formulation of embodiment 1 comprising about 18.75
mg/mL of the anti-LAG3 antibody; about 6.25 mg/mL of the anti-PD-1 antibody; about 55 mg/mL sucrose; about 0.2 mg/mL polysorbate 80; about 10 mM histidine buffer at pH about 5.8; and about 52.5 mM L-arginine or a pharmaceutically acceptable salt thereof, and further comprising about 7.5 mM L-methionine.
7. The formulation of embodiment 1 comprising about 20
mg/mL of the anti-LAG3 antibody; about 5 mg/mL of the anti-PD-1 antibody; about 54 mg/mL sucrose; about 0.2 mg/mL polysorbate 80; about 10 mM histidine buffer at pH about 5.8; and about 56 mM L-arginine or a pharmaceutically acceptable salt thereof, and further comprising about 8 mM L-methionine.
8. The formulation of embodiment 1 comprising about 20.83
mg/mL of the anti-LAG3 antibody; about 4.17 mg/mL of the anti-PD-1 antibody; about 53 mg/mL sucrose; about 0.2 mg/mL polysorbate 80; about 10 mM histidine buffer at pH about 5.8; and about 58.3 mM L-arginine or a pharmaceutically acceptable salt thereof, and further comprising about 8.3 mM L-methionine.
9. The formulation of embodiment 1 comprising about 18.02
mg/mL of the anti-LAG3 antibody; about 4.505 mg/mL of the anti-PD-1 antibody; about 50 mg/mL sucrose; about 0.2 mg/mL polysorbate 80; about 10 mM histidine buffer at pH about 5.8; and about 70 mM L-arginine or a pharmaceutically acceptable salt thereof; and further comprising about 10 mM L-methionine.
The following embodiments are also aspects of the invention:
Lyophilized formulations of therapeutic proteins provide several advantages. Lyophilized formulations in general offer better chemical stability than solution formulations, and thus increased shelf life. A lyophilized formulation may also be reconstituted at different concentrations depending on clinical factors, such as route of administration or dosing. For example, a lyophilized formulation may be reconstituted at a high concentration (i.e. in a small volume) if necessary for subcutaneous administration, or at a lower concentration if administered intravenously. High concentrations may also be necessary if high dosing is required for a particular subject, particularly if administered subcutaneously where injection volume must be minimized. Subcutaneous administration of antibody drugs enables self-administration. Self-administration avoids the time and expense associated with visits to a medical facility for administration, e.g., intravenously. Subcutaneous delivery is limited by the volume of solution that can be practically delivered at an injection site in a single injection, which is generally about 1 to 1.5 mL. Such limitation often requires solution of relatively high concentration to deliver desired amount of the drug. Subcutaneous self-administration is typically accomplished using a pre-filled syringe or autoinjector filled with a liquid solution formulation of the drug, rather than a lyophilized form, to avoid the need for the patient to re-suspend the drug prior to injection.
Typically the lyophilized formulation is prepared in anticipation of reconstitution at high concentration of drug product (DP), i.e. in anticipation of reconstitution in a low volume of liquid. Subsequent dilution with water or isotonic buffer can then readily be used to dilute the DP to a lower concentration. Typically, excipients are included in a lyophilized formulation of the present invention at levels that will result in a roughly isotonic formulation when reconstituted at high DP concentration, e.g. for subcutaneous administration. Reconstitution in a larger volume of water to generate a lower DP concentration will necessarily reduce the tonicity of the reconstituted solution, but such reduction may be of little significance during non-subcutaneous, e.g. intravenous administration as admixture with isotonic solution (0.9% sodium chloride, USP or 5% dextrose solution, USP). If isotonicity is desired at lower DP concentration, the lyophilized powder may be reconstituted in the standard low volume of water and then further diluted with isotonic diluent, such as 0.9% sodium chloride.
The lyophilized formulations of the present invention are formed by lyophilization (freeze-drying) of a pre-lyophilization solution. Freeze-drying is accomplished by freezing the formulation and subsequently subliming water at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about −30 to −25° C. (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of formulation to be lyophilized will dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hrs). A secondary drying may be carried out at about 0-40° C., depending primarily on the type and size of container and the type of protein employed. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours. Typically, the moisture content of a lyophilized formulation is less than about 5%, and preferably less than about 3%. The pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.
In some instances, it may be desirable to lyophilize the protein formulation in the container in which reconstitution of the protein is to be carried out in order to avoid a transfer step. The container in this instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial.
The lyophilized formulations of the present invention are reconstituted prior to administration. The protein may be reconstituted at a concentration of about 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or about 100 mg/mL or higher concentrations such as about 150 mg/mL, 200 mg/mL, 250 mg/mL, or 300 mg/mL up to about 500 mg/mL. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof concentration after reconstitution is about 4-7 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof concentration after reconstitution is about 3-7 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof concentration after reconstitution is about 4 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof concentration after reconstitution is about 5 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof concentration after reconstitution is about 6 mg/ml. In one embodiment, the anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 18-22 mg/ml. In one embodiment, the anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 20 mg/ml. In one embodiment, the anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 16-22 mg/ml. In one embodiment, the anti-PD-1 or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 3-300 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 4-250 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 150-250 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 180-220 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 50-150 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 50 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof or anti-LAG3 antibody or antigen binding fragment thereof concentration after reconstitution is about 25 mg/ml. High protein concentrations are particularly useful where subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous administration, lower concentrations of the protein may be desired (e.g. from about 5-25 mg/mL).
Reconstitution generally takes place at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
In one embodiment of the present invention, the anti-LAG3 antibody (or antigen binding fragment thereof) and anti-PD-1 antibody (or antigen binding fragment thereof) are co-formulated as a lyophilized powder for intravenous administration. In another embodiment of the present invention, the co-formulated product is a lyophilized powder for subcutaneous administration. In certain embodiments, the antibody (or antigen binding fragment thereof) is provided at about 100-1200 mg/vial, and is reconstituted with sterile water for injection prior to use. In other embodiments, the antibody (or antigen binding fragment thereof) is provided at about 200 mg/vial, and is reconstituted with sterile water for injection prior to use. In one embodiment, the target pH of the reconstituted formulation is about 6.0. In various embodiments, the lyophilized formulation of the present invention enables reconstitution of the anti-LAG3 antibody or anti-PD-1 antibody, or antigen binding fragments thereof, to concentrations, such as about 5, 10, 20, 25, 30, 40, 50, 60, 75, 100, 150, 200, 250 or more mg/mL. In other embodiments, the anti-LAG3 antibody or anti-PD-1 antibody, or antigen binding fragments thereof, concentration after reconstitution is about 3-300, 10-300, 20-250, 150-250, 180-220, 20-200, 40-100, or 50-150 mg/ml. In other embodiments, the anti-LAG3 antibody or anti-PD-1 antibody, or antigen binding fragments thereof, concentration pre-lyophilization is about 3-300, 10-300, 150-250, 180-220, 10-100, 10-50, or 25-50 mg/ml.
In other embodiments, the lyophilized formulation of the anti-LAG3 antibody or antigen binding fragment, and anti-PD-1 antibody or antigen binding fragment, is defined in terms of the reconstituted solution generated from the lyophilized formulation. Reconstituted solutions may comprise antibody, or antigen-binding fragment thereof, at concentrations of about 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg/mL or higher concentrations such as 150 mg/mL, 200 mg/mL, 250 mg/mL, or up to about 300 mg/mL. In one embodiment, the reconstituted formulation may comprise about 3-300 mg/mL of the antibody, or antigen-binding fragment thereof. In another embodiment, the reconstituted formulation may comprise about 10-200 mg/mL of the antibody, or antigen-binding fragment thereof. In another embodiment, the reconstituted formulation may comprise about 10-100 mg/mL of the antibody, or antigen-binding fragment thereof. In another embodiment, the reconstituted formulation may comprise about 10-60 or about 15-50 mg/mL of the antibody or antigen-binding fragment thereof. In another embodiment, the reconstituted formulation may comprise about 10-25 mg/mL of the antibody or antigen-binding fragment thereof. In a preferred embodiment, the reconstituted formulation may comprise about 20-30 or 25 mg/mL of the antibody or antigen-binding fragment thereof
Liquid Formulation
A liquid antibody formulation can be made by taking the drug substance which is in, for example, in an aqueous pharmaceutical formulation and buffer exchanging it into the desired buffer as the last step of the purification process. There is no lyophilization step in this embodiment. The drug substance in the final buffer is concentrated to a desired concentration.
Excipients such as stabilizers and surfactants are added to the drug substance and it is diluted using the appropriate buffer to final protein concentration. The final formulated drug substance is filtered using 0.22 μm filters and filled into a final container (e.g. glass vials). The formulation may be stored in a vial, and delivered through an injection device or vessel.
In another aspect of the invention, for the liquid co-formulated formulation comprising the anti-PD-1 antibody and anti-LAG3 antibody (or antigen binding fragments thereof), the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 3-300 mg/ml. In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 4-250 mg/ml. In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 40-100 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 10-60 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 20-30 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 10-30 mg/mL. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has the concentration of about 15-50 mg/ml. In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is at a concentration of about 10-100 mg/mL. In yet a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is at a concentration of about 20-30 or 25 mg/mL. In yet a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is at a concentration of about 16-22 mg/mL. In yet a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is at a concentration of about 18-22 mg/mL. In yet a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is at a concentration of about 20 mg/mL.
In another aspect of the invention, the anti-PD-1 antibody or antigen binding fragment thereof in the liquid formulation has the concentration of about 3-300 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at concentration of about 4-250 mg/ml. In another embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 40-100 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 10-60 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 20-30 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 10-30 mg/mL. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 15-50 mg/ml. In another embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 10-100 mg/mL. In another embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at a concentration of about 20-30 or 25 mg/mL. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at concentration of about 3-7 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is at concentration of about 4-7 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof has a concentration of about 4 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof has a concentration of about 5 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof has a concentration of about 6 mg/ml.
In one embodiment, the liquid formulation comprises a buffer at pH about 4.5-8, 5.0-6.5, 5.5-6.5, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2 and arginine or a pharmaceutically acceptable salt thereof. In one embodiment, the liquid formulation comprises a buffer at pH about 5-8. In one embodiment, the liquid formulation comprises a buffer at pH about 5.0-6.5. In one embodiment, the liquid formulation comprises a buffer at pH about 5.0-6.0. In other embodiments, the buffer is histidine. In another embodiment, the buffer is citrate or acetate. In a further embodiment, the liquid formulation comprises an acetate buffer at pH about 5-8, 5.0-6.5, 5.5-6.5, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2 and arginine or a pharmaceutically acceptable salt thereof.
The liquid antibody formulation of this invention is suitable for parenteral administration such as intravenous, intramuscular, intraperitoneal, or subcutaneous injection; particularly suitable for subcutaneous injection.
Dosing and Administration
Toxicity is a consideration in selecting the proper dosing of a therapeutic agent, such as a humanized anti-LAG3 or anti-PD-1 antibody (or antigen binding fragments thereof). Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with an immunosuppressive agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio of LD50 to ED50. Antibodies exhibiting high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
Suitable routes of administration may, for example, include parenteral delivery, including intramuscular, intradermal, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal. Drugs can be administered in a variety of conventional ways, such as intraperitoneal, parenteral, intraarterial or intravenous injection. Modes of administration in which the volume of solution must be limited (e.g. subcutaneous administration) require that a lyophilized formulation to enable reconstitution at high concentration.
Alternately, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into a pathogen-induced lesion characterized by immunopathology, often in a depot or sustained release formulation. Furthermore, one may administer the antibody in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, pathogen-induced lesion characterized by immunopathology. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
Selecting an administration regimen for a therapeutic 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 cells in the biological matrix. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. 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, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; 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 edition (November 2002).
Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. The appropriate dosage (“therapeutically effective amount”) of the protein will depend, for example, on the condition to be treated, the severity and course of the condition, whether the protein is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the protein, the type of protein used, and the discretion of the attending physician. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. The protein is suitably administered to the patient at one time or repeatedly. The protein may be administered alone or in conjunction with other drugs or therapies.
Antibodies, or antibody fragments can be provided by continuous infusion, or by doses at intervals of, e.g., one day, 1-7 times per week, one week, two weeks, three weeks, monthly, bimonthly, etc. A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
In certain embodiments, the pharmaceutical formulations of the invention will be administered by intravenous (IV) infusion or injection.
In other embodiments, the pharmaceutical formulations of the invention will be administered by subcutaneous administration. Subcutaneous administration may performed by injected using a syringe, or using other injection devices (e.g. the Inject-Ease® device); injector pens; or needleless devices (e.g. MediJector and BioJector®).
Subcutaneous administration may be performed by injection using a syringe, an autoinjector, an injector pen or a needleless injection device. Intravenous injection may be performed after diluting the formulation with suitable commercial diluent such as saline solution or 5% dextrose in water.
Although the high concentration solution formulations of the present invention are particularly advantageous for uses requiring a high concentration of antibody, there is no reason that the formulations can't be used at lower concentrations in circumstances where high concentrations are not required or desirable. Lower concentrations of antibody may be useful for low dose subcutaneous administration, or in other modes of administration (such as intravenous administration) where the volume that can be delivered is substantially more than 1 ml. Such lower concentrations can include about 15, 10, 5, 2, 1 mg/ml or less.
Uses
The present invention provides lyophilized or liquid formulations of an anti-LAG3 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment for use in the treatment of cancer and infection.
Those skilled in the art will realize that the term “cancer” to be the name for diseases in which the body's cells become abnormal and divide without control. Cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. In one embodiment, the cancer is selected from colorectal cancer, gastric cancer and head and neck cancer.
Diffusion Interaction Parameter (kD) Measurement
The B22 in 10 mM Histidine pH 5.6 was found to be negative signifying the inherent property of the molecule to self-associate. The presence of 50 mM sodium chloride in 10 mM histidine pH 5.6 was found to increase diffusion interaction parameter (KD) or reduce self-interaction, improve relative solubility and reduce turbidity (OD350) of anti-LAG3 antibody Ab6 (SEQ ID NOs: 35 and 57, light and heavy chains) as seen in
Since anti-LAG3 antibody has been found to phase separate in buffers at lower ionic strength (10 mM). In order to assess the self-associating properties as well as the colloidal, physical, chemical as well as thermal stability of anti-LAG3 antibody in presence of three different charged species [L-arginine, L-histidine and sodium chloride (NaCl)] at different levels of concentration (mM), nine different formulations were prepared as listed in Table 3 below. Unformulated anti-LAG3 (˜37 mg/mL) in 10 mM L-histidine 70 mM L-arginine hydrochloride pH 5.8 was dialyzed against three 10 mM histidine pH 5.8 buffer solutions; each buffer solution containing 100 mM L-arginine, 100 mM sodium chloride or 100 mM L-histidine. Anti-LAG3 antibody was formulated at 25 mg/mL using dialyzates of respective formulations. The formulations containing 40 mM to 130 mM of L-arginine or sodium chloride were prepared by diluting respective anti-LAG3 antibody stock solution with L-histidine buffer at pH 5.8 and concentrating anti-LAG3 antibody to 25 mg/mL.
The diffusion interaction parameter (kD) of the nine formulations were assessed using dynamic light scattering (DLS) at 20° C. for five acquisitions. The interaction parameter (kD) was calculated from the slope and y-intercept of the plot of the recorded diffusion coefficient values (cm2/s) against series of diluted concentrations (mg/mL) of respective formulations. A positive diffusion interaction parameter (kD) is suggestive of repulsive interaction. With increasing concentration (>40 mM) of L-arginine, L-histidine or sodium chloride, anti-LAG3 antibody shows increase in kD suggesting reduction of molecular self-association (less molecular crowding). The effect is comparatively pronounced for L-arginine followed by sodium chloride and L-histidine in relative order (see
Relative Solubility Studies
Automated relative solubility screening of the nine formulations was assessed using polyethylene glycol (PEG)-induced precipitation requiring 10 mg/mL protein concentration. 40% (w/v) PEG 6000 was prepared in each buffer solution after which solutions of PEG-6000, 2%-36% (w/v) at various increments were prepared using JANUS G3 automated liquid handling system. A 10 mg/mL protein solution was added to the PEG solutions in a 96-well costar clear plate to obtain a final assay concentration of 1 mg/mL. The plate was equilibrated at room temperature overnight and transferred to Abgene PCR plate and spun for 4600 rpm for 30 min in order to force precipitate protein to the bottom of each well. The supernatent was transferred from each well to a fresh 96-well costar clear plate. The plate was read on SpectraMax M5 plate reader at 280 and 320 nm to determine protein loss due to precipitation during the overnight incubation. Absorbance (280-320) versus PEG concentration data was analyzed to determine % PEGmidpt.
Anti-LAG3 antibody shows improved relative solubility in presence of increasing concentrations of charged species such as L-arginine, L-histidine or sodium chloride (40 mM up to 100 mM) suggesting reduction in molecular crowding of anti-LAG3 antibody at those concentrations. See
Change in Charged Species Studies
The change in charged heterogeneity and isoelectric point (pI) of anti-LAG3 antibody in the presence of L-arginine, L-histidine or sodium chloride was assessed using ProteinSimple's capillary isoelectric focusing (cIEF) system. The samples were mixed with carrier ampholyte prior to injection into the capillary. By applying an electric field to the capillary, a pH gradient was created by the carrier ampholyte in the capillary and protein molecules migrated to a location in the capillary where the local pH value equaled isoelectric pH (pI) values. The detection of the separated proteins was achieved by taking a full scan of the entire capillary using the iCE systems (iCE3 from ProteinSimple). The last image taken by the instrument was used for data quantification. The area percentages of the resolved peaks are estimated by taking the area of the individual species divided by the total area of the protein. The pI value of the protein is estimated by linearly calibrating the distance between the two pI markers bracketing the protein. The operating parameters included autosampler temperature at 10° C.; fluorocarbon (FC) coated cartridge, detection wavelength of 280 nm, with focusing period of one minute at 1500 V. The nine formulations were transferred to a 96-well plate and were assessed for change in charged species (% acidic variants, % main peak and % basic variants) at initial time-point using cIEF. The remaining samples of the nine formulations were transferred to another 96-well plate, tightly sealed and placed for thermal stress for 10 days at 50° C. Upon stress, the change in charged species was re-assessed. The data in
Sodium chloride showed the least change in % acidic variants and % main peak for anti-LAG3 antibody formulation followed by L-arginine and L-histidine. Sodium chloride showed an improvement in chemical stability in the concentration range of 40 to 100 mM, especially at ≥70 mM concentration. L-arginine showed better chemical stability at 70 mM concentration whereas L-histidine showed better chemical stability up to 100 mM concentration.
In order to assess the self-associating properties as well as the colloidal stability of anti-LAG3 antibody in presence of L-arginine or sodium chloride (NaCl), twelve different formulations were prepared as listed in Table 4. Unformulated anti-LAG3 antibody (˜37 mg/mL) in 10 mM L-histidine 70 mM L-arginine hydrochloride pH 5.8 was dialyzed against four 10 mM histidine pH 5.8 buffer solutions; each buffer solution containing either 150 mM L-arginine, 150 mM sodium chloride or a mixture of 35 mM L-arginine and 35 mM sodium chloride or a mixture of 50 mM L-arginine and 50 mM sodium chloride. Anti-LAG3 antibody was formulated at 25 mg/mL using dialyzates of respective formulation. The formulations containing 40 mM to 130 mM of L-arginine or sodium chloride were prepared by diluting respective anti-LAG3 antibody stock solution with L-histidine buffer at pH 5.8 and concentrating anti-LAG3 antibody to 25 mg/mL.
Second Virial Coefficient (B22) Measurement
Second virial coefficient (B22) measurements for each of the twelve formulations were made at 5 mg/mL using dynamic light scattering (DLS). Automatic measurements were made at 20° C. using backscatter of 173°.
Positive second virial coefficient (B22) suggests repulsive interactions between protein molecules (lower crowding) in the formulation matrix. Both L-arginine and sodium chloride in concentrations greater than 40 mM appeared to be favorable in reducing molecular crowding. See
Turbidity (OD350) Measurement
In order to assess the colloidal stability of anti-LAG3 antibody in the formulation matrix, the turbidity (OD350) of the twelve formulations were assessed using ultraviolet (UV) absorbance spectrophotometer. The UV absorbances of the samples were measured in a 96-well co-star clear plate at 350 nm wavelength with pathcheck corrected for plate absorbance.
Anti-LAG3 antibody shows improved colloidal stability (OD350) with increasing concentrations of either L-arginine or sodium chloride with comparable values between the two. See
Viscosity Measurement
In order to assess the concentrateability of anti-LAG3 in different formulation matrix, the twelve anti-LAG3 antibody formulations listed in Table 4 were concentrated up to 60 mg/mL using an Eppendorf centrifuge at 3000 rpm at 15° C. The viscosities of the twelve formulations were measured at 20° C. using RheoSense VROC® Initium viscometer on a 96-well plate.
The viscosities of anti-LAG3 antibody at 60 mg/mL in presence of L-arginine or sodium chloride mixture were comparable in the range of 40 to 150 mM concentrations. The viscosities at 60 mg/mL in presence of equivalent ratio of L-arginine and sodium chloride (35:35 or 50:50) showed similar viscosity values. See
Osmolality Measurement
The osmolality of anti-LAG3 antibody was measured using Vapro Vapor Pressure 5520 Osmometer. The unit was calibrated with 100 mmol/kg, 290 mmol/kg and 1000 mmol/kg calibration standards prior to measurement.
The osmolalities of the twelve anti-LAG3 antibody formulations listed in Table 4 were found to be comparable in presence of either L-arginine or sodium chloride. The osmolalities in presence of equivalent ratio of L-arginine and sodium chloride (50:50) showed similar viscosity values whereas equivalent ratios of 35:35 showed lower osmolality values. See
In order to assess the stability of anti-LAG3 antibody in presence of charged species (salt and amino acids), ten formulations listed in Table 5 were prepared and screened for changes in physico-chemical properties of anti-LAG3 antibody by high throughput analysis. The formulations were appropriately sealed in 96-well plate and stressed at 50° C. for 10 days in a dry heat oven. The thermally stressed samples were also assessed for changes in physico-chemical properties of anti-LAG3 antibody. The 20 mM concentrations of L-aspartic acid or L-glutamic acid were selected based on their solubility limit.
Protocol for Turbidity (OD350)
The turbidity (OD350) of the nine formulations was assessed using ultraviolet (UV) absorbance spectrophotometer. The UV absorbances of the samples were measured in a 96-well co-star clear plate at 350 nm wavelength with pathcheck corrected for plate absorbance.
As seen in
UP-SEC
Purity of the sample was assessed by UP-SEC in which the percentage of monomer was determined, as well as the percentages of high molecular weight species (HMW) and late eluting peaks (LMW species). UP-SEC was performed on Acquity H class (DS) by diluting the samples to 1.0 mg/mL in mobile phase (100 mM phosphate, 100 mM sodium chloride, pH 7.0). The column temperature was maintained at 25±3° C. and the flow rate was maintained at 0.5 mL/min using an isocratic elution. The diluted samples were injected (1 μL) into a UPLC equipped with a Waters BEH200 column and a UV detector. Proteins in the sample were separated by size and detected by UV absorption at 214 nm.
As seen in
cIEF
The change in charged heterogeneity and isoelectric point (pI) of anti-LAG3 in the presence of L-arginine, L-histidine or sodium chloride was assessed using ProteinSimple's capillary isoelectric focusing (cIEF) system. The samples were mixed with carrier ampholyte prior to injection into the capillary. By applying an electric field to the capillary, a pH gradient was created by the carrier ampholyte in the capillary and protein molecules migrated to a location in the capillary where the local pH value equaled isoelectric pH (pI) values. The detection of the separated proteins was achieved by taking a full scan of the entire capillary using the iCE systems (iCE3 from ProteinSimple). The last image taken by the instrument was used for data quantification. The area percentages of the resolved peaks are estimated by taking the area of the individual species divided by the total area of the protein. The pI value of the protein is estimated by linearly calibrating the distance between the two pI markers bracketing the protein. The operating parameters included autosampler temperature at 10° C.; fluorocarbon (FC) coated cartridge, detection wavelength of 280 nm, with focusing period of one minute at 1500 V.
The data in
As seen in
DLS
The measure of the hydrodynamic diameter was performed using Wyatt's dynamic light scattering (DLS) instrument on a 96 well glass bottom plate. The sample was diluted to a protein concentration of 5 mg/mL and run on automatic mode using scattering detection of 158° at 20° C., run duration of 5 seconds for five measurements.
As seen in
In order to assess the stability of anti-LAG3 antibody Ab6 (25 mg/mL in 10 mM L-histidine 70 mM L-arginine hydrochloride or in 70 mM sodium chloride at pH 5.8) in the presence of different stabilizers such as sugars and polyols, eleven formulations were prepared as listed in Table 6.
Ultra Performance Size-Exclusion Chromatography (UP-SEC)
Purity of the sample was assessed by UP-SEC in which the percentage of monomer was determined, as well as the percentages of high molecular weight species (HMW) and late eluting peaks (LMW species). UP-SEC was performed on Waters Acquity UPLC system H-class Bio by diluting the samples to 1.0 mg/mL in mobile phase (100 mM phosphate, 100 mM sodium chloride, pH 7.0). The column temperature was maintained at 25±3° C. and the flow rate was maintained at 0.5 mL/min using an isocratic elution. The diluted samples were injected (5 μL) into a UPLC equipped with a Waters BEH200 column and a UV detector. Proteins in the sample were separated by size and detected by UV absorption at 214 nm.
As seen in
Change in Charged Species (cIEF)
The change in charged heterogeneity and isoelectric point (pI) of anti-LAG3 antibody in the presence of L-arginine, L-histidine or sodium chloride was assessed using ProteinSimple's capillary isoelectric focusing (cIEF) system. The samples were mixed with carrier ampholyte prior to injection into the capillary. By applying an electric field to the capillary, a pH gradient was created by the carrier ampholyte in the capillary and protein molecules migrated to a location in the capillary where the local pH value equaled isoelectric pH (pI) values. The detection of the separated proteins was achieved by taking a full scan of the entire capillary using the iCE systems (iCE3 from ProteinSimple). The last image taken by the instrument was used for data quantification. The area percentages of the resolved peaks are estimated by taking the area of the individual species divided by the total area of the protein. The pI value of the protein is estimated by linearly calibrating the distance between the two pI markers bracketing the protein. The operating parameters included autosampler temperature at 10° C.; fluorocarbon (FC) coated cartridge, detection wavelength of 280 nm, with focusing period of one minute at 1500 V.
The eleven formulations were filled in 2 mL sterile vials (2.0 mL fill), sealed and capped and visually inspected. The initial time point of the eleven formulations were stored at 2 to 8° C. (protected from light) and the samples meant for heat-stress were placed inverted in a container protected from light for 10 days at 50° C. in a dry heat oven. The data in
As shown in
DSC
The heat capacities (cp) in kcal/° C. of the eleven formulations of anti-LAG3 antibody listed in Table 6 were measured using differential scanning microcalorimetry (DSC) at 1 mg/mL. The Tm1, Tm2 and Tonset for the eleven formulations were determined from the plot of cp (cal/mol/° C.) versus temperature (° C.).
As seen in
In order to determine the optimal concentration of polysorbate 80 in the formulation matrix (25 mg/mL anti-LAG3 antibody in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, pH 5.8), eight different formulations were prepared, each containing polysorbate in the range of 0 mg/mL up to 1.0 mg/mL as noted in Table 7. The formulations were exposed to agitation shaking at 300 rpm up to 7 days. Two formulations consisted of placebos (0.1 mg/mL or 1.0 mg/mL polysorbate 80 in the same formulation matrix without anti-LAG3 antibody i.e., formulation #1 in Table 7).
Turbidity
In order to assess the colloidal stability of anti-LAG3 antibody in the formulation matrix containing different concentrations of polysorbate 80, the turbidity (OD350) of the eight formulations were assessed using ultraviolet (UV) absorbance spectrophotometer. The UV absorbances of the samples were measured in a 96-well co-star clear plate at 350 nm wavelength with pathcheck corrected for plate absorbance.
As seen in
UP-SEC
Purity of the sample was assessed by UP-SEC in which the percentage of monomer was determined, as well as the percentages of high molecular weight species (HMW) and late eluting peaks (LMW species). UP-SEC was performed on Waters Acquity Liquid Chromatography system by diluting the samples to 1.0 mg/mL in mobile phase (0.1M sodium phosphate monobasic monohydrate, 0.1 M sodium phosphate dibasic dihydrate, 0.1M L-arginine, pH 7.0). The diluted samples were injected (5 μL) into the liquid chromatography equipped with Protein BEH SEC column and a UV detector. Proteins in the sample were separated by size and detected by UV absorption at 214 nm.
As seen in
HP-IEX
In order to determine charge variants in anti-LAG3 antibody formulations, high performance ion exchange chromatography (HP-IEX) was employed. The analysis is performed using a Dionex MabPac® SCX-10, 10 μm 4×250 mm column and mobile phase gradient from 25 mM MES, 14 mM Tris, pH 6.25 to 25 mM MES, 22 mM Tris, 100 mM LiCl pH 6.85. UV detection is performed at 280 nm. This method also includes an optional stripping buffer (15 mM EDTA 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1) to improve the reliability and sustainability of the assay. The sample was prepared at 5 mg/mL with an injection volume of 10 μL.
As seen in
Anti-LAG3 antibody Ab6 Formulation A: 25 mg/mL anti-LAG3 antibody; 50 mg/mL sucrose; 0.2 mg/mL polysorbate 80; 10 mM histidine buffer; 70 mM L-Arginine-HCl was screened in the pH range of 5.3 to 6.4 considering target formulation pH of 5.8. As seen in
In order to determine the effect of antioxidant on anti-LAG3 antibody in the formulation (25 mg/mL anti-LAG3 antibody in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, pH 5.8), three different levels of L-methionine were evaluated in the formulation. Four different formulations were prepared as listed in Table 8, filled (2.2 mL) in a 2 mL Type 1 glass vial and sealed appropriately. The four formulations were exposed to 0.2 ICH, 0.5 ICH, and 1 ICH light stress (ultraviolet and cool white light or visible light). A dark control (covered in foil) for each of the four formulations (control) was also exposed up to 1 ICH light stress.
Turbidity
The turbidity (OD350) of the four formulations was assessed using ultraviolet (UV) absorbance spectrophotometer. The UV absorbances of the samples were measured in a 96-well co-star clear plate at 350 nm wavelength with pathcheck corrected for plate absorbance.
As seen in
UP-SEC
Purity of the sample was assessed by UP-SEC in which the percentage of monomer was determined, as well as the percentages of high molecular weight species (HMW) and late eluting peaks (LMW species). UP-SEC was performed on UPLC acquity H class system by diluting the samples to 1.0 mg/mL in mobile phase (100 mM phosphate and 100 mM sodium chloride, pH 7.0). The diluted samples were injected (5 μL) into the liquid chromatography equipped with Protein BEH SEC column and a UV detector, flow-rate of 0.5 mL/min. Proteins in the sample were separated by size and detected by UV absorption at 214 nm and 280 nm.
As seen in
HP-IEX
In order to determine charge variants in anti-LAG3 antibody formulations, high performance ion exchange chromatography (HP-IEX) was employed. The analysis is performed using a Dionex MabPac® SCX-10, 10 μm 4×250 mm column and mobile phase gradient from 25 mM MES, 14 mM Tris, pH 6.25 to 25 mM MES, 22 mM Tris, 100 mM LiCl pH 6.85. UV detection is performed at 280 nm. This method also includes an optional stripping buffer (15 mM EDTA 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1) to improve the reliability and sustainability of the assay. The sample was prepared at 5 mg/mL with an injection volume of 10 μL and flow-rate of 0.5 to 1.0 mL/min.
As seen in
Reduced Peptide Mapping
The changes in oxidation level of the oxidative post translational modifications of anti-LAG3 antibody were assessed using reduced peptide mapping. Reduced peptide mapping was performed on Waters Acquity H Bio Class system with mobile phase A (0.1% Trifluoroacetic acid in LC/MS grade water), mobile phase B (0.1% Trifluoroacetic acid in LC/MS grade acetonitrile). The injection volume is 50 μL equipped with HALO Peptide ES-C18 column with flow-rate of 0.2 mL/min and detection absorbance of 214 nm. The mass spectrometry consisted of capillary 3.0, sample cone of 30, source temperature of 120° C., cone gas 30, desolvation gas, m/z range of 100-200, MS collected from 2 to 110 min. The samples were reduced and alkylated with appropriate reagents prior to column run. A blank (non-sample) digestion was performed to identify non-sample related peaks eluting in the region of interest.
As seen in
In order to assess the high concentration (200 mg/mL) feasibility of anti-LAG3 antibody in three different buffers at pH 5.8 (histidine, acetate, and citrate; each containing 70 mM L-arginine hydrochloride) and of the formulation containing L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 in the presence of different stabilizers, nine formulations were prepared as listed in Table 9. Each of the nine formulations were filled in 96-well plates and sealed appropriately. The formulations were stressed at 50° C. for 10 days in a dry heat oven. Analysis was performed for the initial and stressed samples.
Turbidity
The turbidity (OD350) of the nine formulations was assessed using ultraviolet (UV) absorbance spectrophotometer. The UV absorbances of the samples were measured in a 96-well co-star clear plate at 350 nm wavelength with pathcheck corrected for plate absorbance.
As seen in
UP-SEC
Purity of the sample was assessed by UP-SEC in which the percentage of monomer was determined, as well as the percentages of high molecular weight species (HMW) and late eluting peaks (LMW species). UP-SEC was performed on Acquity H class (DS) by diluting the samples to 1.0 mg/mL in mobile phase (100 mM phosphate, 100 mM sodium chloride, pH 7.0). The column temperature was maintained at 25±3° C. and the flow rate was maintained at 0.5 mL/min using an isocratic elution. The diluted samples were injected (5 μL) into a UPLC equipped with a Waters BEH200 column and a UV detector. Proteins in the sample were separated by size and detected by UV absorption at 214 nm.
As seen in
Change in Charged Species (cIEF)
The change in charged heterogeneity and isoelectric point (pI) of anti-LAG3 antibody in the presence of L-arginine, L-histidine or sodium chloride was assessed using ProteinSimple's capillary isoelectric focusing (cIEF) system. The samples were mixed with carrier ampholyte prior to injection into the capillary. By applying an electric field to the capillary, a pH gradient was created by the carrier ampholyte in the capillary and protein molecules migrated to a location in the capillary where the local pH value equaled isoelectric pH (pI) values. The detection of the separated proteins was achieved by taking a full scan of the entire capillary using the iCE systems (iCE3 from ProteinSimple). The last image taken by the instrument was used for data quantification. The area percentages of the resolved peaks are estimated by taking the area of the individual species divided by the total area of the protein. The pI value of the protein is estimated by linearly calibrating the distance between the two pI markers bracketing the protein. The samples were prepared at 5 mg/mL and the operating parameters included autosampler temperature at 10° C.; fluorocarbon (FC) coated cartridge, detection wavelength of 280 nm, with focusing period of one minute at 1500 V.
The chemical stability of 200 mg/mL anti-LAG3 antibody was comparable in 10 mM L-histidine as well as 10 mM citrate buffer in comparison to 10 mM acetate buffer in presence of 70 mM L-arginine hydrochloride at pH 5.8. 5% (w/v) glycerol was effective in reducing change in charged species (% acidic and basic variants) followed by 5% (w/v) sucrose (% basic variants). The stabilizing effect of amino acids i.e., 70 mM L-glutamine, 70 mM L-glycine, 70 mM proline and 70 mM L-methionine were comparable.
Co-formulations of the anti-PD-1 antibody (MK-3475, pembrolizumab, heavy chain SEQ ID NO: 10, and light chain SEQ ID NO: 5) and anti-LAG3 antibody Ab6 (heavy chain SEQ ID NO: 57, and light chain SEQ ID NO: 35) were prepared as in Table 10.
Thermal Stability Study
Thermal stability studies were conducted using 1.0 mL liquid formulations of F1-F6 in 2 mL vials with 13 mm serum stopper at up to 12 weeks at 5° C. (ambient humidity), 25° C. (60% humidity), and 40° C. (75% relative humidity) storage conditions. Stability samples were assessed by turbidity and Mixed-mode chromatography (MMC).
Mixed-Mode Chromatography
Mixed-mode chromatography enabled separation of individual antibodies (anti-LAG3 antibody and anti-PD-1 antibody) in co-formulations and also enabled monitoring anti-LAG3 antibody aggregates and anti-PD-1 aggregates and oxidation in co-formulations. In MMC, percentage of monomer for each mAb was determined by the main peak area of each mAb. For anti-LAG3 antibody, the percentages of high molecular weight (aggregates) and low molecular weight species (fragments) were calculated. For anti-PD-1 antibody, the percentages of high molecular weight (aggregates) and low molecular weight species (fragments) as well as the oxidation species (Ox1 and Ox2) were calculated based on individual peak area corresponding to each species. Mixed-mode chromatography was performed by diluting the samples to 1.0 mg/mL in mobile phase (PBS, pH7.4). The column temperature was maintained at 25° C. and the flow rate was maintained at 0.5 mL/min using an isocratic elution. The diluted samples were injected (15 μL) into HPLC equipped with a customized Sepax Zenix SEC-300 column. Different components in the sample were separated by both size and hydrophobicity and detected by UV absorption at 280 nm.
Turbidity Measurement
Turbidity analysis was performed on the stability samples using UV-visible spectroscopy on SpectrMax M5 Plate reader. Absorbance was measured at 350 nm and 500 nm. Turbidity was calculated by subtracting absorbance at 500 nm from absorbance at 350 nm.
Co-formulations (F3, F5 and F6) showed similar or better stability than individual formulations (
Anti-LAG3 antibody Ab6 and Pembrolizumab (MK-3475) drug substance (DS) were mixed at the following ratios, 1:1 (12D), 3:1 (12E), 4:1 (12F) and 5:1 (12 G). Formulation 12A and 12C are Ab6 single entity (SE) and MK-3475 single entity (SE) controls, respectively, in their original composition. Formulation 12B is Control MK-3475 SE in excipient composition of 12A. 12 F-12H are co-formulation with Ab6 to MK-3475 ratio of 4:1, with (12H) and without diluent (12 F). Diluent was added to the formulation composition to restore the Ab6 composition (with 70 mM Arginine concentration).
The formulations were filtered via 0.22 μm cellulose acetate membrane filter. The filtrate for each formulation was filled into 2 R vials with a 2.2 mL fill/vial. The formulations were staged on stability for 10 days at 5° C., 25° C. and 40° C. The stability of the formulations were analyzed by checking quality attributes including turbidity, sub-visible particulates total protein concentration, aggregation, oxidation for MK-3475, purity and charge profile. Formulations 12A, 12B, 12 F and 12H were mainly assessed for stability.
Turbidity and Total Protein Concentration
Turbidity was measured using Spectramax UV Absorbance Spectrophotometer (MRK0406661). 200 μL samples were filled in 96-well Co-star clear plate, absorbance was measured at 350 nm and 500 nm and path check corrected with plate absorbance of 0.025 and 0.020, respectively.
Concentration was measured using SoloVPE Variable Pathlength Extension (MRK0416083) using Quick Slope. A weighted ratio-specific average extinction coefficient of (1.42 for MK-3475 and 1.45 for Ab6) and a wavelength of 280 nm was used for to measure the total protein concentration.
All samples were clear, colorless and free of any visible particles. There was no impact of Ab6:MK-3475 ratio on turbidity. All prototypes with Ab6 dose range of 200-1000 mg in the co-formulation showed similar turbidity at all stability conditions. The composition of the matrix does not impact turbidity.
Aggregation Analysis by UP-SEC
UP-Size Exclusion Chromatography (UP-SEC) was used to assess total protein aggregation and identification of high molecular weight (HMW), low molecular weight (LMW) and total monomer content. Both the samples and reference material were diluted to 5 mg/mL and 200 μL of the diluted sample was used. The mobile phase consisted of 50 mM NaPhosphate, 450 mM Arginine HCl, pH 7.0. An isocratic flow rate of 0.5 mL/min, a detection wavelength of 280 nm, injection volume of 6 μL and column temperature of 30° C. was used. For each test sample the area percent for the Monomer (Mon), HMW species and LMW species (formerly known as Late Eluting) peaks was calculated.
% Monomer=PMon×100÷ΣP(all peaks)
% HMW species=PHMW×100±ΣP(all peaks)
% LMW species=PLMW×100±ΣP(all peaks)
PMon, PHMW, PLMW are areas of the individual peaks and ΣP is the sum of all the peak areas (excluding artifact peaks, peaks appearing in the void volume, and buffer related peaks).
Formulation #12B with MK-3475 alone shows the highest amount of High Molecular Weight (HMW) species (See
Particle Size Analysis by Micro-Flow Imaging (MFI)
Sub-visible particulates in the range of >1 micron, >2 micron, >5 micron, >10 micron, >25 micron, >50 microns was analyzed using MFI by pooling enough samples to analyze a minimum of 3 replicates using a Brightwell Micro-Flow Imaging system.
The particle count was higher for high concentrations formulations (single entity and co-formulation) compared to low concentration formulations (12 D and 12E). Significant increase in count for all particle sizes was observed for Ab6 single entity (12A) at 50° C. over 10 days. Similarly, co-formulations with higher protein content (12 F, 12 G and 12H) showed higher counts for all particle sizes at all storage conditions compared to formulations 12 D and 12E. However, the ≥10 μm and ≥25 μm particle count was significantly lower than that observed for Ab6 single entity (12A) at 50° C.
Hydrophobic Interaction Chromatography (HIC) for Quantification of MK-3475 Oxidation in the Co Formulation
HIC was used to assess the identification and quantitation of all the oxidation products of MK-3475 in the co-formulation. The column used was Tosoh Phenyl-5PW 10 μm 7.5×75 mm and a column temperature of 30° C. was used. The samples were diluted to yield a total protein concentration of 5 mg/mL with a total sample volume of at least 2004. A gradient method using a combination of Mobile phase A: 2% ACN in 5 mM Na3(PO4), pH 7.0 and Mobile phase B: 2% ACN in 400 mM ammonium sulfate, 5 mM Na3(PO4), pH 6.9 and injection volume of 10 μL. A detection wavelength of 280 and 214 nm was used and results were reported only at 280 nm. For result reporting, the relative % area for pre peak 3 equals the % of Pre-peak 3. Pre peaks 1 and 2 are summed to equal the % of Pre-peak 1+2.
Ab6/MK-3475 co-formulation (12H) shows the highest extent of oxidation of the Fab (Met 105) in MK-3475. The (12 F) composition shows a lower extent of oxidation.
Ion Exchange Chromatography (IEX) for Assessing Charge Distribution of Individual mAbs in the Co-Formulation
Ion exchange chromatography (IEX) was used to assess the identification and quantitation of all the charged and the main species in each of the mAbs of the co-formulation. Each of the samples are diluted to yield a total protein amount of 50 μg total protein in a maximum injection volume of 125 pt. A Dionnex MabPac SCX-10 10 um, 4×250 mm column using a column temperature of 35° C. was used. The mobile phase used was a combination of (A): 5 mM MES, 14 mM Tris, pH 6.25 (B): 20 mM Na3(PO4), 95 mM NaCl, pH 8.0, 4% Acetonitrile (ACN) and (C): 15 mM EDTA, 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1. A gradient method with a total run time of 70 minutes was used with a detection wavelength of 280 nm. The acidic, basic and main species of each of the mAb in the co-formulation were identified and quantified using this method. Overall, as temperature increases, the charge profile varies and the highest change is observed at 50° C. (see Table 15). No measurable difference was observed in main peak or acidic and basic variants after 10 days at 5° C., 40° C. and 50° C. for 800:200 co-formulation prototypes with (12H) and without diluent (12 F).
Purity Analysis by Capillary Electrophoresis-SDS (CE-SDS)
mAb purity analysis was performed using non-reduced CE-SDS. Samples were prepared as per the internal method protocols and incubated at 70° C. for 10 minutes on a heat block. The samples were then equilibrated to ambient temperature, centrifuged at 10,000 g and 95 uL of the sample was used for analysis.
The above table shows that there is no impact of Ab6:MK-3475 ratio on purity.
Ab6A injection is a sterile, preservative-free solution that requires dilution for intravenous infusion. Ab6A is a fixed dose combination of anti-LAG3 antibody Ab6 and anti-PD-1 antibody MK-3475 (pembrolizumab, heavy chain SEQ ID NO: 10, and light chain SEQ ID NO: 5), each single-use vial contains 40 mg of Ab6 and 10 mg of MK-3475 in a 2.0 mL fill. The drug product composition is 20.0 mg/mL Ab6, 5.0 mg/mL MK-3475 in 0.56 mg/mL L-histidine, 1.35 mg/mL L-histidine monohydrochloride monohydrate, 11.80 mg/mL L-arginine hydrochloride, 1.19 mg/mL L-methionine, 54.0 mg/mL sucrose, 0.20 mg/mL polysorbate 80 at pH 5.8 (Formulation 12 F in Example 8). The recommended storage condition is 5° C.±3° C. (2-8° C.) protected from light.
Three months of stability data for Ab6A drug product in glass vials remains essentially unchanged when stored at the recommended storage condition of 5° C. (5° C.±3° C.). There were slight changes observed in several of the attributes, up to three months when stored at the accelerated condition of 25° C. (25° C., 60% RH (Relative Humidity). The changes at the stressed condition of 40° C. (40° C., 75% RH) are more substantial than those at 25° C. The following assays remain essentially unchanged through the three month time-point at all temperature conditions (including the stressed condition of 40° C., 75% RH): appearance and visible particles, clarity and degree of opalescence, color, potency, protein concentration, pH, and particulate matter. The available stability data support the use of this product when stored at 5° C.±3° C. with a 12 month shelf life.
The below summarizes three months of stability data for Ab6A drug product at the storage condition of 5° C.±3° C. (inverted), at the accelerated condition of 25° C. (25° C.±2° C., 60% relative humidity, inverted), and at the stressed condition of 40° C. (40° C.±2° C., 75% relative humidity, inverted) per ICH guidelines.
Appearance and Visible Particles
The appearance and visible particles were performed using a light box equipped with white-light source for samples in a clear vial. The test results for appearance and visible particles for drug product Ab6A under all conditions evaluated to date is “Liquid essentially free from visible particles”. The observations do not change as a function of storage condition or time.
Clarity and Degree of Opalescence
Clarity and degree of opalescence was measured using HACH™ 2100AN Turbidimeter. HACH™ StablCal reference solutions (<0.1, 1.0, 3.0, 6.0, 10.0, 18.0, and 30.0 NTU)) and purified water (as blank) were used for the system suitability test (SST) before sample analyses. There was no change to the clarity and degree of opalescence for drug product Ab6A at the Initial time point up to the 3 month time point across all conditions.
Charge Variants by HP-IEX
High performance-Ion Exchange Chromatography (HP-IEX) was performed on a Waters e2695 separation module with a 2489 UV Vis detector using a Dionex MabPac® SCX-10, 10 um 4×250 mm, part #074625. Mobile phase A contains 20 mM MES, 14 mM Tris (pH 6.25). Mobile phase B contains 20 mM NaPO4, 96 mM NaCl, 4% Acetonitrile (pH 8.0) and mobile C contains 15 mM EDTA, 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1. Column and sample temperatures were set at 35° C. and 4° C. respectively. A total run of 70 min with gradient setting at a flow rate of 0.5 mL/min with UV detection at 280 nm was performed. For each molecule chromatograms (Ab6 and MK-3475), relative percent area was calculated for 5 peaks (acidic 2, acidic 1, main, basic1 and basic), and combined to have only three categories (acidic variants, main and basic variants) for each antibody. In addition, % of each group for each antibody is normalized so that the total peak % is equal to 100% for each antibody as calculated. The MK-3475 and Ab6 acidic and basic peaks are separated by an ion exchange column and elute either earlier (acidic variants) or later (basic variants) relative to the main peak.
The HP-IEX method is a combination method that separates the charge variants for both Ab6 and MK-3475. For both the Ab6 (
The main species for both Ab6 and MK-3475 at 5° C. show no change over 3 months (
The basic variants for Ab6 show no change at both the 5° C. and 25° C. condition up to 3 months (
Purity by UP-SEC
Size exclusion chromatography is a separation technique based on size. Ab6 and MK-3475 monomers are similar in size, thus the monomers co-elute. Sufficient separation of the HMW species from the co-eluting monomers is achieved and hence, the method is suitable to determine the purity of Ab6A Drug Product (DP). Ab6A DP contains low levels of high molecular weight (HMW) species resolved by UP-SEC. Low molecular weight (LMW) are usually detected but at low levels. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using Waters H-Class Acquity UPLC with HALO C4 UHPLC COLUMN, 2.1×75 mm. The mobile phases were Water with 0.1% (v/v) Trifluoroacetic acid (mobile phase A) and Acetonitrile with 0.1% (v/v) Trifluoroacetic Acid (mobile phase B). The reference standards of MK-3475 and Ab6 were prepared to 4 mg/mL with water and used to generate the standard curve and all samples were diluted to 4 mg/mL and injected 5 uL for measurement. The column and autosampler were maintained at 75° C. and 4° C. respectively. UV detection was performed at 280 nm and Water Empower software was used for data analysis. The concentrations of MK-3475 and Ab6 in general can be based on the standard curve established in the measurement.
UP-SEC for Ab6A showed no change at the 5° C. condition for high molecular weight species and only a slight increase at 25° C. with a corresponding slight decrease in monomer over 3 months on stability (
Non-Reduced (NR) CE-SDS and Reduced (R) CE-SDS
Non-Reduced (NR) CE-SDS and Reduced (R) CE-SDS analyses utilizes Beckman PA800 Plus CE instrument and bare fused silica capillary (total length of 30.2 cm and inner diameter of 50 μm) with a 100×200 μm aperture. Samples were prepared using IgG Purity/Heterogeneity Assay Kit and treated before analyzing on the instrument according to the perspective protocols, where under reducing conditions, the mAb samples were denatured in the presence of 1.0% SDS and reduced using 5% β-mercaptoethanol and under non-reducing conditions, the mAb samples were denatured in the presence of 1.0% SDS and treated with N-Ethylmaleimide (NEM) followed by subjection to heating for 10 min at 70° C. Separation was conducted at 15 kV for 40 Minutes and UV detection was performed at 220 nm and Water Empower software was used for data analysis. For NR-CE-SDS, intact IgG (purity) and total low molecular weight impurities were reported as percentage. For R-CE-SDS, total purity (Heavy chain+light chain) and total impurities were reported as percentage.
Non-reduced CE-SDS was run for Ab6A drug product up to 3 months on stability. No changes were observed at the 5° C. condition (
Reduced CE-SDS was run for Ab6A drug product up to 3 months on stability. No changes were observed at the 5° C. condition (
Particulate Matter
Subvisible particulate characterization was performed using the Brightwell Micro-Flow Imaging system. Each sample was pooled from multiple containers (minimum of three) to 50 mL polypropylene tubes by pouring gently and allowing the tubes to sit undisturbed under ambient conditions for 30 minutes prior to testing. Sub-visible particulates in the range of >1 micron, >2 micron, >5 micron, >10 micron, >25 micron, >50 microns was analyzed by applying a standard filter in the instrument software MVSS (version 2-R5.0.0.43.5864).
Particulate Matter data by HIAC was calculated for Ab6A drug product. At the 5° C., 25° C., and 40° C. conditions, the results were well below the acceptance criteria of ≤6000 particles per container for ≥10 μm and ≤600 particles per container for ≥25 μm from the Initial to 3 months). At the ≥2 μm and ≥5 μm particle sizes, which are report results, across all conditions up to 3 months no major trends in the data were observed.
HP-HIC MK-3475 Oxidation
Hydrophobic Interaction Chromatography HPLC (HP-HIC) was performed on an Agilent 1260 system using Tosoh column Phenyl-5PW 10 μm, 7.5×75 mm (PN 05753). The mobile phases were 2.0% Acetonitrile in 5 mM sodium phosphate at pH7.0 (mobile phase A) and 2.0% Acetonitrile in 400 mM ammonium sulfate and 5 mM sodium phosphate at pH 6.9 (mobile phase B). The flow rate was 0.5 mL/min. The column and autosampler were maintained at 30° C. and 4° C. respectively. Samples were diluted to 5 mg/mL with Milli-Q water, 10 μL was loaded onto the column (total injection amount 50 μg). Detection wavelengths were set at 280 nm for analysis. The relative % area for pre peak 3 is reported. Pre peaks 1 and 2 are summed to equal the % of pre-peak 1+2. Also, the relative % areas for post peaks 1 and 2 are summed to equal % hydrophobic variants.
The HP-HIC assay was run to assess the oxidation of MK-3475 in Ab6A drug product. There was no change in the total pre-peaks at 5° C. (
Turbidity A350
Turbidity was measured using Spectramax UV Absorbance Spectrophotometer. 200 μL samples were filled in 96-well Co-star clear plate, absorbance was measured at 350 nm and 500 nm and path check corrected with plate absorbance of 0.025 and 0.020, respectively.
Turbidity A350 was assessed for Ab6A drug product. No noteworthy changes were determined at the 5° C. condition up to 3 months on stability (
Tyrosine Assay
Ab6 and MK-3475 were mixed in the presence of 10 mM histidine, 56 mM arginine, 54.0 mg/mL sucrose, 0.20 mg/mL polysorbate 80, at pH 5.8. Three solutions were prepared with protein ratios of MK-3475:Ab60:1, 1:0, 1:1, where the Ab6, MK-3475 concentrations and Ab6/MK-3475 concentrations are 20 mg/mL, 20 mg/ml and 10 mg/ml, respectively. The fluorescent results obtained for the single protein formulations and the co-formulation are comparable only if the total protein concentration is the same across the samples. Solutions are incubated at room temperature over a period of 72 hours and aliquots are taken at regular intervals and subjected to oxidation followed by a fluorogenic labeling reaction.
The samples taken at pre-specified timepoints are mixed with 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) and incubated at 37° C. for 3 hours in the dark to oxidize the proteins. This reaction is quenched by the addition of methionine. The following fluorogenic reaction consist of mixing the oxidized protein samples with (2-aminomethyl)-benzene-sulfonic acid (ABS) in the presence of a mild oxidant (K3Fe(CN)6).
AAPH oxidation reaction yields the formation of 3,4-dihydroxyphenylalanine (DOPA) at tyrosine residues. The fluorogenic labeling reaction permits the specific tagging of DOPA products. The final fluorophore consists of a benzoxazole group in resonance with the benzene moiety of ABS molecule. Such fluorophore allows to monitor the formation of DOPA by fluorescence at λem=490 nm after excitation of the final samples at λex=360 nm.
If protein aggregation occurs in the sample storage up to 72 hours, a decay of the fluorescence signal is expected (
FRET Assay
A widely used fluorescence technique to study bi-molecular interactions is FRET (Forster resonant energy transfer), which utilizes the non-radiative (dipole-dipole) energy transfer from a fluorescent donor to an acceptor that can take place only when the two fluorophores are situated at distances <10 nm. In the case of two proteins labeled with donor and acceptor tags, this implies that FRET occurs only if and when the two proteins interact with each other.
Purified Ab6 and MK-3475 proteins were derivatized with fluorescent dyes Dylight-488® and Dylight-596®, respectively. Dylight-488® or Dylight-596® are amine-reactive dyes, which react preferentially with the primary amine of lysine residues. Fluorescently labeled Ab6 and MK-3475 are named Ab6-488, and MK-3475-596, where the labels 488 and 596 refer to the fluorescent dyes Dylight-488® and Dylight-596®, respectively. Ab6-488 and MK-3475-596 were mixed in the presence of 10 mM histidine, 54.0 mg/mL sucrose, at pH 5.8, in the absence or presence of i) arginine (56 mM) and/or ii) polysorbate 80 (0.20 mg/mL). MK-3475-596/Ab6-488 ratios are 1:4, 1:1, and 4:1. Due to the sensitivity of the assay, the molar concentration of the fluorogenic tagged proteins is either 3.2 uM or 800 nM depending on the protein ratio. FRET fluorescence signals are recorded for 72 hours at λem=620 nm (λex=488 nm) (
If protein interaction occurs, we expect to see a growth of the fluorescence signal as a function of time (
In view of data from the Tyrosine assay and the FRET assay, when a co-formulation of MK-3475 and Ab6 is prepared in the presence of 10 mM histidine, 54.0 mg/mL sucrose, 56 mM arginine, and 0.20 mg/mL polysorbate 80 at pH 5.8, the stability of the co-formulation is improved in comparison to the single antibody formulations for the same matrix of excipients. For the 1:1 and 1:4 co-formulations, based on the Tyrosine assay and the FRET assay, respectively, it is unlikely that the co-formulation will drive protein-protein interactions, therefore, there is no increase in protein aggregation.
Comparison of Ab6A and MK-3475 formulation samples, thermally stressed at 25° C. and 40° C. in the same formulation matrix, shows that the level of M105 oxidation of MK-3475 in the Ab6A composition is lower in comparison to MK-3475 alone (see
The present application is the 371 national phase application of International Application No. PCT/US2019/059954, filed Nov. 6, 2019, which claims the benefit of U.S. Provisional Application No. 62/756,678, filed Nov. 7, 2018, hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/059954 | 11/6/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2020/097139 | 5/14/2020 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4401820 | Chibata et al. | Aug 1983 | A |
| 4816567 | Cabilly et al. | Mar 1989 | A |
| 5262296 | Ogawa et al. | Nov 1993 | A |
| 5762905 | Burton et al. | Jun 1998 | A |
| 6171586 | Lam et al. | Jan 2001 | B1 |
| 6267958 | Andya et al. | Jul 2001 | B1 |
| 6329511 | Vasquez et al. | Dec 2001 | B1 |
| 6818216 | Young et al. | Nov 2004 | B2 |
| 6875432 | Liu et al. | Apr 2005 | B2 |
| 7247707 | Besman et al. | Jul 2007 | B2 |
| 7364736 | Boyle et al. | Apr 2008 | B2 |
| 7374762 | Amphlett et al. | May 2008 | B2 |
| 7375193 | Baca et al. | May 2008 | B2 |
| 7563869 | Honjo et al. | Jul 2009 | B2 |
| 7592004 | Kaisheva et al. | Sep 2009 | B2 |
| 7615213 | Kasaian et al. | Nov 2009 | B2 |
| 7635473 | Warne et al. | Dec 2009 | B2 |
| 7662384 | Ramakrishnan et al. | Feb 2010 | B2 |
| 7666413 | Liu et al. | Feb 2010 | B2 |
| 7691379 | Allan et al. | Apr 2010 | B2 |
| 7705132 | Rehder et al. | Apr 2010 | B2 |
| 7740842 | Arvinte et al. | Jun 2010 | B2 |
| 7833525 | Shenoy et al. | Nov 2010 | B2 |
| 7959922 | Bakker et al. | Jun 2011 | B2 |
| 7960516 | Matheus et al. | Jun 2011 | B2 |
| 7993645 | Benson et al. | Aug 2011 | B2 |
| 7998477 | Yakovlevsky et al. | Aug 2011 | B2 |
| 8034906 | Borhani et al. | Oct 2011 | B2 |
| 8067547 | Ewert et al. | Nov 2011 | B2 |
| 8142776 | Liu et al. | Mar 2012 | B2 |
| 8168760 | Borhani et al. | May 2012 | B2 |
| 8216583 | Kruase et al. | Jul 2012 | B2 |
| 8221759 | Pilkington et al. | Jul 2012 | B2 |
| 8263080 | Katsikis et al. | Sep 2012 | B2 |
| 8293883 | Presta | Oct 2012 | B2 |
| 8354509 | Carven et al. | Jan 2013 | B2 |
| 8568720 | Morichika et al. | Oct 2013 | B2 |
| 8580297 | Essler et al. | Nov 2013 | B2 |
| 8703126 | Liu et al. | Apr 2014 | B2 |
| 8747847 | Rotem-Yehudar et al. | Jun 2014 | B2 |
| 8933075 | Wang et al. | Jan 2015 | B2 |
| 8952136 | Carven et al. | Feb 2015 | B2 |
| 9220776 | Sharma et al. | Dec 2015 | B2 |
| 9278131 | Dauty et al. | Mar 2016 | B2 |
| 9592297 | Xiang et al. | Mar 2017 | B2 |
| 9605051 | Soane et al. | Mar 2017 | B2 |
| 9713641 | Hicklin et al. | Jul 2017 | B2 |
| 9782470 | Bhambhani et al. | Oct 2017 | B2 |
| 9926371 | Liu et al. | Mar 2018 | B2 |
| 10072072 | Vora et al. | Sep 2018 | B2 |
| 10188730 | Liang | Jan 2019 | B2 |
| 10444520 | Dholakia et al. | Oct 2019 | B2 |
| 10787518 | Bernett et al. | Sep 2020 | B2 |
| 10918736 | Kim et al. | Feb 2021 | B2 |
| 11633476 | Sharma et al. | Apr 2023 | B2 |
| 20030138417 | Kaisheva et al. | Jul 2003 | A1 |
| 20040091490 | Johnson et al. | May 2004 | A1 |
| 20050101770 | Presta | May 2005 | A1 |
| 20050175986 | Gross et al. | Aug 2005 | A1 |
| 20060029599 | Kaisheva et al. | Feb 2006 | A1 |
| 20060057702 | Rosenthal et al. | Mar 2006 | A1 |
| 20060088523 | Andya et al. | Apr 2006 | A1 |
| 20060210557 | Luisi et al. | Sep 2006 | A1 |
| 20060210567 | Collins et al. | Sep 2006 | A1 |
| 20060246004 | Adams et al. | Nov 2006 | A1 |
| 20060286103 | Kolhe et al. | Dec 2006 | A1 |
| 20070009526 | Benson et al. | Jan 2007 | A1 |
| 20070009541 | Amphlett et al. | Jan 2007 | A1 |
| 20070048315 | Presta | Mar 2007 | A1 |
| 20070053900 | Liu et al. | Mar 2007 | A1 |
| 20070059803 | Oppmann et al. | Mar 2007 | A1 |
| 20070065437 | Elson et al. | Mar 2007 | A1 |
| 20070184050 | Ishikawa et al. | Aug 2007 | A1 |
| 20070190047 | Brych et al. | Aug 2007 | A1 |
| 20080003220 | Gokarn | Jan 2008 | A1 |
| 20080050375 | Davies et al. | Feb 2008 | A1 |
| 20080057070 | Long et al. | Mar 2008 | A1 |
| 20080112953 | Mcauley et al. | May 2008 | A1 |
| 20080124326 | Rehder et al. | May 2008 | A1 |
| 20080152658 | Dagan et al. | Jun 2008 | A1 |
| 20080213282 | Jacob | Sep 2008 | A1 |
| 20080248048 | Fish et al. | Oct 2008 | A1 |
| 20080254026 | Long et al. | Oct 2008 | A1 |
| 20080286270 | Oliver et al. | Nov 2008 | A1 |
| 20080311119 | Maloney et al. | Dec 2008 | A1 |
| 20090042315 | Li et al. | Feb 2009 | A1 |
| 20090060906 | Barry et al. | Mar 2009 | A1 |
| 20090130119 | Abate et al. | May 2009 | A1 |
| 20090162352 | Adler et al. | Jun 2009 | A1 |
| 20090181027 | Dal Monte et al. | Jul 2009 | A1 |
| 20090208492 | O'Connor et al. | Aug 2009 | A1 |
| 20090217401 | Korman et al. | Aug 2009 | A1 |
| 20090285802 | Igawa et al. | Nov 2009 | A1 |
| 20090291076 | Morichika et al. | Nov 2009 | A1 |
| 20090304706 | Lu et al. | Dec 2009 | A1 |
| 20090311253 | Ghayur et al. | Dec 2009 | A1 |
| 20100021461 | Burke et al. | Jan 2010 | A1 |
| 20100137213 | Fernandez et al. | Jun 2010 | A1 |
| 20100209434 | Bishop et al. | Aug 2010 | A1 |
| 20100209437 | Elson et al. | Aug 2010 | A1 |
| 20100226928 | Dani | Sep 2010 | A1 |
| 20100266617 | Carven et al. | Oct 2010 | A1 |
| 20100272731 | Presta et al. | Oct 2010 | A1 |
| 20100278822 | Fraunhofer et al. | Nov 2010 | A1 |
| 20100286038 | Antochshuk et al. | Nov 2010 | A1 |
| 20100303827 | Sharma et al. | Dec 2010 | A1 |
| 20100316638 | Gurny et al. | Dec 2010 | A1 |
| 20110014203 | Nilsson et al. | Jan 2011 | A1 |
| 20110059079 | Babuka et al. | Mar 2011 | A1 |
| 20110060290 | Bonk et al. | Mar 2011 | A1 |
| 20110086038 | Hope et al. | Apr 2011 | A1 |
| 20110123550 | Shibayama et al. | May 2011 | A1 |
| 20110226650 | Gokarn et al. | Sep 2011 | A1 |
| 20110229490 | Li et al. | Sep 2011 | A1 |
| 20110256135 | Fraunhofer et al. | Oct 2011 | A1 |
| 20110300135 | Lobo et al. | Dec 2011 | A1 |
| 20110318343 | Kaisheva et al. | Dec 2011 | A1 |
| 20120039876 | Oliver et al. | Feb 2012 | A1 |
| 20120076784 | Matheus et al. | Mar 2012 | A1 |
| 20120128687 | Adler et al. | May 2012 | A1 |
| 20120148576 | Sharma et al. | Jun 2012 | A1 |
| 20120183531 | Lucas et al. | Jul 2012 | A1 |
| 20120231972 | Golyshin et al. | Sep 2012 | A1 |
| 20130022625 | Igawa et al. | Jan 2013 | A1 |
| 20130058958 | Bowen et al. | Mar 2013 | A1 |
| 20130108651 | Carven et al. | May 2013 | A1 |
| 20130186797 | Walsh | Jul 2013 | A1 |
| 20140044708 | Dauty et al. | Feb 2014 | A1 |
| 20140044727 | Monck et al. | Feb 2014 | A1 |
| 20140178401 | Nabozny et al. | Jun 2014 | A1 |
| 20140206845 | Kameoka et al. | Jul 2014 | A1 |
| 20140227250 | Li et al. | Aug 2014 | A1 |
| 20140234296 | Sharma et al. | Aug 2014 | A1 |
| 20140314714 | Honjo et al. | Oct 2014 | A1 |
| 20140348841 | Schebye et al. | Nov 2014 | A1 |
| 20150071936 | Mendiratta et al. | Mar 2015 | A1 |
| 20150086537 | Adler et al. | Mar 2015 | A1 |
| 20150086559 | Mueller et al. | Mar 2015 | A1 |
| 20150100030 | Dix et al. | Apr 2015 | A1 |
| 20150110783 | Lu et al. | Apr 2015 | A1 |
| 20150258209 | Benz et al. | Sep 2015 | A1 |
| 20150290325 | Kashi et al. | Oct 2015 | A1 |
| 20150307606 | Basarkar et al. | Oct 2015 | A1 |
| 20150359900 | Wang et al. | Dec 2015 | A1 |
| 20160022814 | Petit et al. | Jan 2016 | A1 |
| 20160045615 | Li et al. | Feb 2016 | A1 |
| 20160090419 | Morichika et al. | Mar 2016 | A1 |
| 20160166685 | Cheung et al. | Jun 2016 | A1 |
| 20160176963 | Maurer et al. | Jun 2016 | A1 |
| 20160222116 | Korman | Aug 2016 | A1 |
| 20160289315 | Mirza et al. | Oct 2016 | A1 |
| 20160355589 | Williams et al. | Dec 2016 | A1 |
| 20170051039 | Gombotz et al. | Feb 2017 | A1 |
| 20170056347 | Glick et al. | Mar 2017 | A1 |
| 20170089914 | Loo et al. | Mar 2017 | A1 |
| 20170097333 | Bhagwat et al. | Apr 2017 | A1 |
| 20170210792 | Mason et al. | Jul 2017 | A1 |
| 20170210812 | Wong et al. | Jul 2017 | A1 |
| 20170216433 | Li et al. | Aug 2017 | A1 |
| 20170218069 | Rosengren et al. | Aug 2017 | A1 |
| 20170360929 | Sinha et al. | Dec 2017 | A1 |
| 20180044419 | Rosengren et al. | Feb 2018 | A9 |
| 20180237524 | Reichert et al. | Aug 2018 | A1 |
| 20180339045 | Li et al. | Nov 2018 | A1 |
| 20190010231 | Rothe et al. | Jan 2019 | A1 |
| 20200055938 | Desai et al. | Feb 2020 | A1 |
| 20200147213 | Sharma et al. | May 2020 | A1 |
| 20200206350 | Chu et al. | Jul 2020 | A1 |
| 20200262922 | Bhattacharya et al. | Aug 2020 | A1 |
| 20200354453 | De et al. | Nov 2020 | A1 |
| 20210155913 | Park | May 2021 | A1 |
| 20210317215 | Reichert et al. | Oct 2021 | A1 |
| 20210380694 | Forrest, Jr. et al. | Dec 2021 | A1 |
| 20220002410 | Antochshuk et al. | Jan 2022 | A1 |
| 20220089738 | Krishnamachari et al. | Mar 2022 | A1 |
| Number | Date | Country |
|---|---|---|
| 2010200784 | Mar 2010 | AU |
| 2476934 | Sep 2003 | CA |
| 2918888 | Jan 2015 | CA |
| 1801123 | Jun 2007 | EP |
| 2116265 | Nov 2009 | EP |
| 2238985 | Aug 2012 | EP |
| 2275119 | Sep 2013 | EP |
| 3117837 | Jun 2017 | EP |
| 2589691 | Jul 2016 | RU |
| 1989011297 | Nov 1989 | WO |
| 199704801 | Feb 1997 | WO |
| 2000053631 | Sep 2000 | WO |
| 2001018051 | Mar 2001 | WO |
| 2001030393 | Mar 2001 | WO |
| 2002072636 | Sep 2002 | WO |
| 2003009817 | Feb 2003 | WO |
| 2003039485 | May 2003 | WO |
| 2003086310 | Oct 2003 | WO |
| 2004007520 | Jan 2004 | WO |
| 2004018312 | Mar 2004 | WO |
| 2004055164 | Jul 2004 | WO |
| 2004056875 | Jul 2004 | WO |
| 2004071517 | Aug 2004 | WO |
| 2004081190 | Sep 2004 | WO |
| 2005120571 | Dec 2005 | WO |
| 2006121168 | Nov 2006 | WO |
| 2007019232 | Feb 2007 | WO |
| 2007024846 | Mar 2007 | WO |
| 2007092772 | Aug 2007 | WO |
| 2007110339 | Oct 2007 | WO |
| 2007124299 | Nov 2007 | WO |
| 2007147019 | Dec 2007 | WO |
| 2008076321 | Jun 2008 | WO |
| 2008079290 | Jul 2008 | WO |
| 2008086395 | Jul 2008 | WO |
| 2008103473 | Aug 2008 | WO |
| 2008121301 | Oct 2008 | WO |
| 2008153610 | Dec 2008 | WO |
| 2008156712 | Dec 2008 | WO |
| 2008157409 | Dec 2008 | WO |
| 2009009407 | Jan 2009 | WO |
| 2009043933 | Apr 2009 | WO |
| 2009084659 | Jul 2009 | WO |
| 2009120684 | Oct 2009 | WO |
| 2009126688 | Oct 2009 | WO |
| 2010032220 | Mar 2010 | WO |
| 2010062372 | Jun 2010 | WO |
| 2010069858 | Jun 2010 | WO |
| 2010102241 | Sep 2010 | WO |
| 2010129469 | Nov 2010 | WO |
| 2010148337 | Dec 2010 | WO |
| 2011012637 | Feb 2011 | WO |
| 2011017070 | Feb 2011 | WO |
| 2011024862 | Mar 2011 | WO |
| 2011029892 | Mar 2011 | WO |
| 2011056772 | May 2011 | WO |
| 2011080209 | Jul 2011 | WO |
| 2011089062 | Jul 2011 | WO |
| 2011139718 | Nov 2011 | WO |
| 2012010799 | Jan 2012 | WO |
| 2012018538 | Feb 2012 | WO |
| 2012047954 | Apr 2012 | WO |
| 2012076670 | Jun 2012 | WO |
| 2012135035 | Oct 2012 | WO |
| 2012165917 | Dec 2012 | WO |
| 2013016648 | Jan 2013 | WO |
| 2013063468 | May 2013 | WO |
| 2014004436 | Jan 2014 | WO |
| 2014036076 | Mar 2014 | WO |
| 2015011199 | Jan 2015 | WO |
| 2015038777 | Mar 2015 | WO |
| 2015038782 | Mar 2015 | WO |
| 2015038811 | Mar 2015 | WO |
| 2015038818 | Mar 2015 | WO |
| 2015042246 | Mar 2015 | WO |
| 2016015675 | Feb 2016 | WO |
| 2016024228 | Feb 2016 | WO |
| 2016028656 | Feb 2016 | WO |
| WO-2016028672 | Feb 2016 | WO |
| 2016035006 | Mar 2016 | WO |
| 2016100882 | Jun 2016 | WO |
| 2016118654 | Jul 2016 | WO |
| 2016140717 | Sep 2016 | WO |
| 2016153839 | Sep 2016 | WO |
| 2016168716 | Oct 2016 | WO |
| 2016176504 | Nov 2016 | WO |
| 2016196173 | Dec 2016 | WO |
| 2016200782 | Dec 2016 | WO |
| 2017015560 | Jan 2017 | WO |
| 2017030823 | Feb 2017 | WO |
| 2017037203 | Mar 2017 | WO |
| 2017040864 | Mar 2017 | WO |
| 2017048824 | Mar 2017 | WO |
| 2017054646 | Apr 2017 | WO |
| 2017055547 | Apr 2017 | WO |
| 2017079150 | May 2017 | WO |
| 2017112621 | Jun 2017 | WO |
| 2017198741 | Nov 2017 | WO |
| 2018091729 | May 2018 | WO |
| 2018116198 | Jun 2018 | WO |
| 2018158332 | Sep 2018 | WO |
| 2018160722 | Sep 2018 | WO |
| 2018183928 | Oct 2018 | WO |
| 2018187057 | Oct 2018 | WO |
| 2018204343 | Nov 2018 | WO |
| 2018204368 | Nov 2018 | WO |
| 2018204374 | Nov 2018 | WO |
| 2018204405 | Nov 2018 | WO |
| 2018222718 | Dec 2018 | WO |
| 2018222722 | Dec 2018 | WO |
| 2020022791 | Jan 2020 | WO |
| 2020096917 | May 2020 | WO |
| 2020097141 | May 2020 | WO |
| Entry |
|---|
| FDA insert for KEYTRUDA (pembrolizumab). May 2017. (Year: 2017). |
| Telikepalli et al. Structural Characterization of IgG1 mAb Aggregates and Particles Generated Under Various Stress Conditions. Journal of Pharmaceutical Sciences. 2014. 103(3): 796-809. (Year: 2014). |
| Agarkhed, Meera et al., Effect of Polysorbate 80 Concentration on Thermal and Photostability of a Monoclonal Antibody, AAPS Pharm Sci Tech, 14(1), 1-9, 2013. |
| Bittner, Beate et al., Subcutaneous Administration of Biotherapeutics: An Overview of Current Challenges and Opportunities, BioDrugs, 32, 425-440, 2018. |
| Cleland, Jeffrey L. et al., A Specific Molar Ratio of Stabilizer to Protein is Required for Storage Stability of a Lyophilized Monoclonal Antibody, Journal of Pharmaceutical Sciences, 90(3), 310-321, 2001. |
| Clinical Trials.gov: “NCT03656718 A Study of Subcutaneous Nivolumab Monotherapy With or Without Recombinant Human Hyaluronidase PH20 (rHuPH20)”. The document was first posted online Aug. 31, 2018 and updated inter alia on Feb. 7, 2020 and thus is prior art. Retrieved from Clinical Trials.gov archive. (16 pages). |
| Daugherty et al., Formulation and delivery issues for monoclonal antibody therapeutics, Advanced Drug Delivery Reviews, vol. 58, No. 5-6, pp. 686-706, 2006. |
| Disclosed Anonymously, “Biopharmaceutical Composition”, IP.com, IPCOM000259113D, pp. 1-735, https://ip.com/IPCOM/000259113, Jul. 12, 2019 (Dec. 7, 2019). |
| Hufnagel, Stephanie et al., Dry powders for inhalation containing monoclonal antibodies made by thin-film freeze-drying, International Journal of Pharmaceutics, 618, 1-12, 2022. |
| Larson, S. B. et al., Progress in the Development of an Alternative Approach to Macromolecular Crystallization, Crystal Growth and Design, 8(8), 3038-3052, 2008. |
| Morar-Mitrica, Sorina et al., Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration, mAbs, 7:4, 792-803, 2015. |
| Shpilberg, O. et al., Subcutaneous administration of rituximab (MabThera) and trastuzumab (Herceptin) using hyaluronidase, British Journal of Cancer, 109, 1556-1561, 2013. |
| Warne, N., Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, p. 1-17. |
| Wenquan et al., Pharmaceutics, Science and Technology Literature Press, N/A, 226-227, 2005, English translation. |
| Ahamed, Tangir, Phase Behavior of an Intact Monoclonal Antibody, Biochemical Journal, 2007, 610-619, 93. |
| Altschul, Stephen F., A Protein Alignment Scoring System Sensitive at All Evolutionary Distances, J Mol Evol, 1993, 290-300, 36. |
| Armstrong, NA, Sucrose, Handbook of Pharmaceutical Excipients, 2009, 703-707, 6th Edition. |
| Banks et al., Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody improves homogeneity, stability, and biological activity, J Pharm Sci, 2008, 775-790, 97(2). |
| Banks, Douglas D. et al., The Effect of Sucrose Hydrolysis on the Stability of Protein Therapeutics during Accelerated Formulation Studies, J. Pharm. Sci., 2009, 4501-4510, 98(12). |
| Basu et al., Protein crystals for the delivery of biopharmaceuticals, Expert Opinion on Biological Therapy, 2004, pp. 301-317, vol. 4(3). |
| Benlysta prescribing information, Mar. 2011. |
| Bhambhani, Akhilesh, Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions, Journal of Pharmaceutical Sciences, 2012, 1120-1135, vol. 101, No. 3. |
| Blank, Christian et al., PD-L1/B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor (TCR) Transgenic CD8+ T Cells, Cancer Research, 2004, 1140-1145, 64. |
| Borwankar, A.U. et al., Viscosity Reduction of a Concentrated Monoclonal Antibody with Arginine⋅HCI and Arginine⋅Glutamate, Ind. Eng. Chem. Res., 2016, 11225-11234, 55(43). |
| Bowman, Edward P. et al., Rationale and safety of anti-interleukin-23 and anti-interleukin-17A therapy, Curr Opin Infect Dis, 2006, 245-252, 19(3). |
| Byrn, Stephen, et al., Pharmaceutical Solids: A Strategic Approach to Regulatory Considerations, Pharmaceutical Research, 1995, p. 945-954, vol. 12, No. 7. |
| Caira, Crystalline Polymorphism of organic compounds, Topics in Current Chemistry, 1998, 163-208, 198. |
| Carpenter, John F. et al., Rational Design of Stable Lyophilized Protein Formulations: Some Practical Advice, Pharmaceutical Research, 1997, 969-975, 14(8). |
| Carpenter, John F., Application of infrared spectroscopy to development of stable lyophilized protein formations, European Journal of Pharmaceutics and Biopharmaceutics, 1998, 231-238, 45. |
| Chang, B.S. and Hershenson, S., Practical approaches to protein formulation development in “Rationale Design of stable protein formulations-theory and practice”, Kluwer Academic/Plenum Publishers, 2002, 1-25. |
| Chang, Byeong et al., Physical Instability in Peptide and Protein Pharmaceuticals, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 69-104, Chapter 3. |
| Chauhan, Veeren M., Advancements in the co-formulation of biologic therapeutics, Journal of Controlled Release, 2020, pp. 397-405, vol. 327. |
| Chauvin et al., TIGIT and PD-1 impair tumor antigen-specific CD8 T cells in melanoma patients, Journal of Clinical Investigation, 2015, pp. 2046-2058, vol. 125(5). |
| Chen, et al., Influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms, 2003, 1952-1960, 20(12), Pharm Res. |
| Connor, Robert J. et al., A Preclinical Investigation into the Effects of Aging on Dermal Hyaluronan Properties and Reconstitution Following Recombinant Human Hyaluronidase PH20 Administration, Dermatology and Therapy, 2020, 503-513, 10(3). |
| Cordoba et al., Non-enzymatic hinge region fragmentation of antibodies in solution, 2005, 115-121, 818(2), J Chromatogr B Analyt Technol Biomed Life Sci. |
| Costantino, Henry R., The Secondary Structure and Aggregation of Lyophilized Tetanus Toxoid, Journal of Pharmaceutical Sciences, 1996, 1290-1293, vol. 85, No. 12. |
| Cua, Daniel J. et al., TGF-beta, a ‘double agent’ in the immune pathology war, Nat. Immunol., 2006, 557-559, 7(6). |
| Cudney, R., Protein Crystallization and Dumb Luck, The Rigaku Journal, 1999, 1-7, vol. 16, No. 1. |
| Daugherty, Ann L. et al., Formulation and Delivery Issues for Monoclonal Antibody Therapeutics, Current Trends in Monoclonal Antibody Development and Manufacturing, 2010, 103-129, Chapter 8. |
| Davagnino, Juan et al., Acid hydrolysis of monoclonal antibodies, J. Immunol. Methods, 1995, 177-180, 185(2). |
| Davies et al., Structural Determinants of Unique Properties of Human IgG4-Fc, Journal of Molecular Biology, 2014, pp. 630-644, vol. 426(3). |
| Dayhoff, M.O., A Model of Evolutionary Change in Proteins, Atlas of Protein Sequence and Structure, 1978, 345-352, 22. |
| Dear et al., Contrasting the Influence of Cationic Amino Acids on the Viscosity and Stability of a Highly Concentrated Monoclonal Antibody, Pharm. Res., 2017, 193-207, vol. 34. |
| Dembo, Amir, Limit Distribution of Maximal Non-Aligned Two-Sequence Segmental Score, The Annals of Probability, 1994, 2022-2039, vol. 22, No. 4. |
| European Medicines Agency, European Public Assessment Report (EPAR) Avastin, Scientific Discussion. Jan. 24, 2006, pp. 1-61. |
| Falconer, Robert J. et al., Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients, J Chem Technol Biorechnol, 2011, 942-948, 86. |
| Falconer, Robert J., Advances in liquid formulations of parenteral therapeutic proteins, Biotechnology Advances, 2019, 1-9, 37(7):107412. |
| FDA label for Amjevita (Adalimumab), Sep. 2016, p. 1-61. |
| FDA label for Arzerra (Ofatumumab), Oct. 2009, p. 1-13. |
| FDA label for Avastin (Bevacizumab), Sep. 2011, p. 1-25. |
| FDA label for Bavencio (Avelumab), Mar. 2017, p. 1-20. |
| FDA label for Campath or Lemtrada (Alemtuzumab), Sep. 2014, p. 1-18. |
| FDA label for Cimzia (Certolizumab), Jan. 2017, p. 1-40. |
| FDA label for Drazalex (Daratumumab), Nov. 2016, p. 1-26. |
| FDA label for Humira (Adalimumab), Jan. 2008, p. 1-34. |
| FDA label for Kadcyla (Ado-Trastuzumab Emtansine), Aug. 29, 2013, p. 1-26. |
| FDA label for Mylotarg (Gemtuzumab Ozogamicin), Aug. 2005, p. 1-21. |
| FDA label for Opdivo (Nivolumab), Dec. 2017, p. 1-73. |
| FDA label for Praxbind (Idarucizumab), Oct. 2015, p. 1-10. |
| FDA label for Prolia (Denosumab), Sep. 2011, p. 1-20. |
| FDA label for Prostascint (Capromab Pendetide), Jun. 2012, p. 1-16. |
| FDA label for Protrazza (Necitumumab), Nov. 2015, p. 1-12. |
| FDA label for Raxibacumab, Dec. 2012, p. 1-14. |
| FDA label for Reopro (Abciximab), dated Nov. 4, 1997, p. 1-17. |
| FDA label for Repatha (Evolocumab), Aug. 2015, p. 1-34. |
| FDA label for Rituxan (Rituximab), Feb. 2010, p. 1-35. |
| FDA label for Simulect (Basiliximab), May 1998, p. 1-7. |
| FDA label for Soliris (Eculizumab), Sep. 2011, p. 1-24. |
| FDA label for Tysabri (Natalizumab), Jan. 2012, p. 1-32. |
| FDA label for Vectibix (Panitumumab), Jun. 2017, p. 1-31. |
| FDA label for Zevalin (Ibritumomab Tiuxetan), Sep. 2009, p. 1-11. |
| FDA label of Adcetris, Nov. 2014, pp. 1-19. |
| FDA label of Benlysta, Mar. 2012, pp. 1-22. |
| FDA label of Blincyto, Dec. 2014, pp. 1-24. |
| FDA label of Cinqair, Mar. 2016, pp. 1-16. |
| FDA label of Empliciti, Nov. 2015, pp. 1-22. |
| FDA label of Entyvio, May 2014, pp. 1-21. |
| FDA label of Erbitux, Jan. 2012, pp. 1-31. |
| FDA label of Fasenra, Nov. 2017, pp. 1-8. |
| FDA label of Ilaris, Mar. 2012, pp. 1-13. |
| FDA label of Kevzara, May 2017, pp. 1-45. |
| FDA label of Nucala, Nov. 2015, pp. 1-28. |
| FDA label of Ocrevus, Mar. 2017, pp. 1-18. |
| FDA label of Raptiva, Mar. 2009, pp. 1-36. |
| FDA label of Remicade, Feb. 2011, pp. 1-47. |
| FDA label of Siliq, Feb. 2017, pp. 1-22. |
| FDA label of Sylvant, 2014, pp. 1-16. |
| FDA label of Taltz Mar. 2016, pp. 1-25. |
| FDA label of Xolair, 2007, pp. 1-20. |
| FDA label of Yervoy, Oct. 2015, pp. 1-32. |
| FDA label of Zinbryta, May 2016, pp. 1-32. |
| FDA label of Zinplava, Oct. 2016, pp. 1-11. |
| Fukuda, Masakazu et al., Thermodynamic and Fluorescence Analyses to Determine Mechanisms of IgG1 Stabilization and Destabilization by Arginine, Pharm. Res., 2014, 992-1001, 31. |
| Garber, Ellen et al., A broad range of Fab stabilities within a host of therapeutic IgGs, Biochemical and Biophysical Research Communications, 2007, 751-757, 355. |
| Ghosh et al., Natalizumab for active Crohns disease, New England J. Med., 2003, pp. 24-32, 348. |
| Giege, et al., Crystallogenesis of Biological Macromolecules: Facts and Perspectives, Acta Cryst., 1994, pp. 339-350, D50. |
| Gikanga, Benson et al., Manufacturing of High-Concentration Monoclonal Antibody Formulations via Spray Drying—the Road to Manufacturing Scale, PDS J Pharm Sci and Tech, 2015, 59-73, 69. |
| Gizzi, Patrick et al., Molecular Tailored Histidine-Based Complexing Surfactants: From Micelles to Hydrogels, Eur. J. Org. Chem., 2009, 3953-3963, N/A. |
| Grillo, Adeolla O., Late Stage Formulation Development and Characterization of Biopharmaceuticals, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 161-171, Chapter 7. |
| Guo, Zheng et al., Structure-Activity Relationship for Hydrophobic Salts as Viscosity-Lowering Excipients for Concentrated Solutions of Monoclonal Antibodies, Pharm Res, 2012, 3102-3109, 29. |
| Harris et al., Comparison of the conformations of two intact monoclonal antibodies with hinges, Immunological Reviews, 1998, pp. 35-43, vol. 163. |
| Harris et al., Crystallization of Intact Monoclonal Antibodies, Proteins: Structure, Function, and Genetics, 1995, pp. 285-289, vol. 23, No. 2. |
| Harris, Reed J. et al., Identification of multiple sources of charge heterogeneity in a recombinant antibody, Journal of Chromatography B, 2001, 233-245, 752(2). |
| He et al., Humanization and Pharmacokinetics of a Monoclonal Antibody with Specificity for both E- and P-Selectin, J. Immunol., 1998, pp. 1029-1035, 160. |
| Herold, Anti-CD3 Monoclonal Antibody in New-Onset Type 1 Diabetes Mellitus, New England Journal of Medicine, 2002, pp. 1692-1698, 346. |
| Humphrey, J.H. et al., International standard for hyaluronidase, Bulletin of the World Health Organization, 1957, 291-294, 16. |
| Ionescu, Roxana et al., Kinetics of Chemical Degradation in Monoclonal Antibodies: Relationship between Rates at the Molecular and Peptide Levels, Anal. Chem., 2010, 3198-3206, 82(8). |
| Zutsu, Ken-Ichi et al., Excipient crystallinity and its protein-structure—stabilizing effect during freeze-drying, Journal of Pharmacy and Pharmacology, 2002, 1033-1039, 54. |
| Jezek, Jan et al., Viscosity of concentrated therapeutic protein compositions, Advanced Drug Delivery Reviews, 2011, 1107-1117, 63. |
| Jones, Andrew J.S., Analysis of Polypeptides and proteins, Advanced Drug Delivery Reviews, 1993, 29-90, 10. |
| Jorgensen, Lene et al., Recent trends in stabilising peptides and proteins in pharmaceutical formulation—considerations in the choice of excipients, Expert Opinion on Drug Delivery, 2009, 1219-1230, 6(11). |
| Joshi, Sangeeta B. et al., An Empirical Phase Diagram/ High Throughput Screening Approach to the Characterization and Formulation of Biopharmaceuticals, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 173-205, Chapter 8. |
| Kaithamana, Shashi, Induction of Experimental Autoimmune Graves' Disease in BALB/c Mice, The Journal of Immunology, 1999, 5157-5164, 163. |
| Kang, Jichao et al., Rapid formulation development for monoclonal antibodies, Bio Process International, 2016, 40-45, 14(4). |
| Karagianni, A. et al., Pharmaceutical Cocrystals: New Solid Phase Modification Approaches for the Formulation of APIs, Pharmaceutics, 2018, 1-30, 10(1). |
| Keytruda (Merck & Co., Inc., Whitehouse Station, NJ USA; initial U.S. approval 2014, updated Sep. 2017) 49 pages. |
| Kheddo, Priscilla et al., The effect of arginine glutamate on the stability of monoclonal antibodies in solution, Int. J. Pharmaceutics, 2014, 126-133, 473. |
| Kohler et al., Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity, Nature, 1975, pp. 195-497, vol. 256. |
| Krishnan, Development of Formulations for Therapeutic Monoclonal Antibodies and Fc Fusion Proteins, Chugai Exhibit 2014, 2010, pp. 1-48. |
| Krishnan, Sampathkumar et al., Development of Formulations for Therapeutic Monoclonal Antibodies and Fc Fusion Proteins, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 383-427, Chapter 16. |
| Kuhn, Darl (Editor), Biopharmaceutical Composition, IP Com, IP Com, Inc., West Henrietta, NY, US, 2019, 723-726, N/A. |
| Kundrot, C.E., Which strategy for a protein crystallization project?, Cellular Molecular Life Science, 2004, 525-536, 61. |
| Lam, Xanthe M. et al., Antioxidants for Prevention of Methionine Oxidation in Recombinant Monoclonal Antibody HER2, J. Pharm. Sci., 1997, 1250-1255, 86(11). |
| Langrish, Claire L. et al., IL-12 and IL-23: master regulators of innate and adaptive immunity, Immunol. Rev., 2004, 96-105, 202. |
| Le Doussal et al., Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen-positive mouse B cells with monoclonal antibody conjugate cocktails, J. Immunol., 1991, pp. 169-175, 146. |
| Liang Wenquan et al., Pharmaceutics, Science and Technology Literature Press, 2005, 226-227, N/A. |
| Liu, Dingjiang et al., Structure and Stability Changes of Human IgG1 Fc as a Consequence of Methionine Oxidation, Biochemistry, 2008, 5088-5100, 47(18). |
| Liu, Hongcheng et al., Heterogeneity of Monoclonal Antibodies, J. Pharm. Sci., 2008, 2426-2447, 97(7). |
| Liu, Jun et al., Reversible Self-Association Increases the Viscosity of a Concentrated Monoclonal Antibody in Aqueous Solution, Journal of Pharmaceutical Sciences, 2005, 1928-1940, 94(9). |
| Liu, Y. Diana et al., Human IgG2 Antibody Disulfide Rearrangement in Vivo, J. Biol. Chem., 2008, 29266-29272, 283(43). |
| Mach, Henryk et al., Addressing new analytical challenges in protein formulation development, European Journal of Pharmaceutics and Biopharmaceutics, 2011, 196-207, 78. |
| Manning, Mark Cornell et al., Prediction of Protein Aggregation Propensities from Primary Sequence Information, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 329-347, Chapter 14. |
| Manzini, B. et al., Polymer-supported syntheses of oxo-crown ethers and derivatives containing a-amino-acid residues, Reactive & Functional Polymers, 2008, 1297-1306, 68(9). |
| McCoy et al., Phaser crystallographic software, Journal of Applied Crystallography, 2007, pp. 658-674, vol. 40. |
| McDermott, et al., PD-1 as a potential target in cancer therapy, Cancer Medicine, 2013, pp. 662-673, WO. |
| Menne, Kerstin M.L., A comparison of signal sequence prediction methods using a test set of signal peptides, Bioinformatics Applications Note, 2000, 741-742, 16. |
| Milgrom et al., Treatment of allergic asthma with monoclonal anti IgE antibody, New England Journal Med., 1999, pp. 1966-1973, 341. |
| Morissette, Sherry L. et al., High-throughput crystallization: polymorphs, salts, co-crystals and solvates of pharmaceutical solids, Advanced Drug Delivery Reviews, 2004, 275-300, 56. |
| Murakami, Monica S., Cell Cycle Regulation, Oncogenes, and Antineoplastic Drugs, The Molecular Basis of Cancer, 1995, 3-17, Chapter 1. |
| Nayar, Rajiv et al., Efficient Approaches to Formulation Development of Biopharmaceuticals, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 309-328, Chapter 13. |
| Nonappa, et al., Caffeine as a Gelator, Gels, 2016, 1-10, 2. |
| Ollmann Saphire et al., Crystal Structure of a Neutralizing Human IgG Against HIV-1: A Template for Vaccine Design, Science, 2001, pp. 1155-1159, vol. 293. |
| Pearlman, Rodney, Analysis of Protein Drugs, Peptide and Protein Drug Delivery, 1991, 247-301, Chapter 6. |
| Perchiacca, Joseph M. et al., Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions, Protein Engineering, Design & Selection, 2012, 591-601, 25 (10). |
| Perez-Ramirez, Bernardo et al., Preformulation Research: Assessing Protein Solution Behavior Early During Therapeutic Development, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 119-146, Chapter 5. |
| Poole, Raewyn M., Pembrolizumab: First Global Approval, Drugs, 2014, 1973-1981, 74(16). |
| Portielje, IL-12: a promising adjuvant for cancer vaccination, Cancer Immunol Immunother, 2003, 133-144, 52. |
| Presta, Leonard G. et al., Selection, design, and engineering of therapeutic antibodies, J. Allergy Clin. Immunol., 2005, 731-736, 116(4). |
| Presta, Leonard G., Engineering of therapeutic antibodies to minimize immunogenicity and optimize function, Advanced Drug Delivery Reviews, 2006, 640-656, 58. |
| Prestrelski, Steven J., Optimization of Lyophilization Conditions for Recombinant Human Interleukin-2 by Dried State Conformational Analysis Using Fourier-Transform Infrared Spectroscopy, Pharmaceutical Research, 1995, 1250-1259, vol. 12, No. 9. |
| Prolia prescribing information, Jun. 2010. |
| Qing, G. et al., Chiral Effect at Protein/Graphene Interface: A Bioinspired Perspective to Understand Amyloid Formation, Journal of the American Chemical Society, 2014, 10736-10742, 136(30). |
| Reich, Gabriele. Chapter 10: “Pharmaceutical Formulation and Clinical Application”. Chapter from the textbook “Handbook of Therapeutic Antibodies, vol. 1”, published by Wiley & Sons in 2007. |
| Reichert, et al., Monoclonal antibody successes in the clinic, Nature Biotechnology, 2005, pp,. 1073-1078, vol. 23. |
| Reissner, K. J. et al., Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?, Cell. Mol. Life Sci., 2003, 1281-1295, 60. |
| Remmele, Richard L., Interleukin-1 Receptor (IL-1R) Liquid Formulation Development Using Differential Scanning Calorimetry, Pharmaceutical Research, 1998, 200-208, vol. 15, No., 2. |
| Robak, Tadeusz, The emerging therapeutic role of antibody mixtures, Expert Opinion on Biological Therapy, 2013, 953-958, 13:7. |
| Rodrigues, M. et al., Pharmaceutical cocrystallization techniques. Advances and challenges, International Journal of Pharmaceutics, 2018, 404-420, 547(1-2). |
| Rustandi, Richard R. et al., Applications of CE SDS gel in development of biopharmaceutical-antibody-based products, Electrophoresis, 2008, 3612-3620, 29(17). |
| Sane, Samir U. et al., Raman Spectroscopic Characterization of Drying-Induced Structural Changes in a Therapeutic Antibody: Correlating Structural Changes with Long-Term Stability, Journal of Pharmaceutical Sciences, 2004, 1005-1018, 93(4). |
| Scapin et al., Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab, Nature Structural & Molecular Biology, 2015, pp. 953-958, vol. 22, No. 12. |
| Schermeyer, Marie-Therese et al., Characterization of highly concentrated antibody solution—A toolbox for the description of protein long-term solution stability, MABS, 2017, 1169-1185, 9(7). |
| Seifert, Tina et al., Chroman-4-one- and Chromone-Based Sirtuin 2 Inhibitors with Antiproliferative Properties in Cancer Cells, Journal of Medicinal Chemistry, 2014, 9870-9888, 57. |
| Shahrokh, Zahra, Approaches to Analysis of Aggregates and Demonstrating Mass Balance in Pharmaceutical Protein (Basic Fibroblast Growth Factor) Formulations, Journal of Pharmaceutical Sciences, 1994, 1645-1650, vol. 83, No. 12. |
| Sharma et al., Preparation, purification and crystallization of antibody Fabs and single-chain Fv domains, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1997, pp. 15-37, vol. 1. |
| Shire, Steven J. et al., Formulation and manufacturing of biologics, Current Opinion in Biotechnology, 2009, 708-714, 20. |
| Shire, Steven J. et al., High Concentration Antibody Formulations, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 349-381, Chapter 15. |
| Shire, Steven J., et al., Challenges in the Development of High Protein Concentration Formulations, Journal of Pharmaceutical Sciences, 2004, 1390-1402, 93(6). |
| Sigma-Aldrich, Co., Products for Life Science Research, 2001, 1-47, N/A. |
| Slamon et al., Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2, New England J. Med., 2001, pp. 783-792, 344. |
| Sluzky, Victoria, Chomatographic Methods for Quantitative Analysis of Native, Denatured, and Aggregated Basic Fibroblast Growth Factor in Solution Formulations, Pharmaceutical Research, 1994, 485-490, vol. 11, No. 4. |
| Study NCT01295827 posted in Feb. 2011 on ClinicalTrials.gov (see p. 6 “First Posted”), 14 pages. |
| Sule, S.V. et al., Solution pH That Minimizes Self-Association of Three Monoclonal Antibodies Is Strongly Dependent on lonic Strength, Mol. Pharmaceutics, 2012, 744-751, 9. |
| Sumit Goswami, Developments and Challenges for mAb-based Therapeutics, Antibodies, 2013, 452-500, 2. |
| Sworn statement of Chakravarthy Nachu Narasimhan, 2 pages. |
| Te Booy, Marcel, Evaluation of the Physical Stability of Freeze-Dried Sucrose-Containing Formulations by Differential Scanning Calorimetry, Pharmaceutical Research, 1992, 109-114, vol. 9, No. 1. |
| Tomar, Dheeraj S., Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development, mAbs, 2016, 216-228, vol. 8, No. 2. |
| Topalian et al., Survival, Durable Tumor Remission, and Long-Term Safety in Patients With Advanced Melanoma Receiving Nivolumab, Clinical Journal of Oncology, 2014, pp. 1020-1030, vol. 32, No. 10. |
| Topp, Elizabeth M. et al., Chemical Instability in Peptide and Protein Pharmaceuticals, Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals, 2010, 41-67, Chapter 2. |
| Tysabri prescribing information, Nov. 2004. |
| Uchiyama, Susumu, Liquid formulation for antibody drugs, Biochimica et Biophysica Acta, 2014, 2041-2052, 1844. |
| Usami, A., The effect of pH, hydrogen peroxide and temperature on the stability of human monoclonal antibody, Journal of Pharmaceutical and Biomedical Analysis, 1996, 1133-1140, 14. |
| Vermeer, Arnoldus W. P. et al., The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein, Biophysical Journal, 2000, 394-404, 78(1). |
| Vlasak, Josef et al., Fragmentation of monoclonal antibodies, MABS, 2011, 253-263, 3(3). |
| Vlasak, Josef et al., Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody, Anal. Biochem., 2009, 145-154, 392(2). |
| Von Heijne et al., A new method for predkting signal sequence cleavage sites, Nucleic Acids Res., 1986, pp. 4683-4690, 14. |
| Walily, El, Simultaneous determination of tenoxicam and 2-aminopyridine using derivative spectrophotometry and high-performance liquid chromatography, Journal of Pharmaceutical and Biomedical Analysis, 1997, 1923-1928, 15. |
| Wang et al., Antibody structure, instability, and formulation, J. Pharm. Sci., 2007, 1-26, 96(1). |
| Wang, B. et al., Amino acid endcapped poly(p-dioxanone): synthesis and crystallization, J Polym Res, 2013, 1-9, 20(4). |
| Wang, Instability, stabilization, and formulation of liquid protein pharmaceuticals, Int J Pharm, 1999, pp. 129-188, vol. 185, No. 2. |
| Wang, Shujing et al., Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies, Mol. Pharmaceutics, 2015, 4478-4487, 12. |
| Warne, Development of high concentration protein biopharmaceuticals: the use of platform approaches in formulation development, 2011, 208-212, 78(2), Eur J Pharm Biopharm. |
| Warne, Nicholas W., Formulation Development of Phase 1-2 Biopharmaceuticals: An Efficient and Timely Approach, John Wiley & Sons, Inc., 2010, 147-159, Chapter 6. |
| Weber, Patricia C., Overview of Protein Crystallization Methods, Methods in Enzymology, 1997, 13-22, 276. |
| Webster, Simon, Predicting Long-Term Storage Stability of Therapeutic Proteins, Pharmaceutical Technology, 2013, 1-7, 37(11). |
| Wei, Ziping et al., Identification of a Single Tryptophan Residue as Critical for Binding Activity in a Humanized Monoclonal Antibody against Respiratory Syncytial Virus, Anal. Chem., 2007, 2797-2805, 79(7). |
| Wiekowski, Maria T. et al., Ubiquitous Transgenic Expression of the IL-23 Subunit p19 Induces Multiorgan Inflammation, Runting, Infertility, and Premature Death, J. Immunol., 2001, 7563-7570, 166(12). |
| Wolchok et al., Nivolumab plus Ipilimumab in Advanced Melanoma, The New England Journal of Medicine, 2013, pp. 122-133, vol. 369(2). |
| Yang et al., A Randomized Trial of Bevacizumab, an Anti-Vascular Endothelial Growth Factor Antibody, for Metastatic Renal Cancer, New England Journal of Medicine, 2003, pp. 427-434, 349. |
| Yang, M. et al., Crystalline monoclonal antibodies for subcutaneous delivery, Proceedings of the the National Academy of Sciences, Jun. 10, 2003, 6934-6939, 100-12. |
| Yu, Lei et al., Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development, J. Pharm Biomed. Anal., 2006, 455-463, 42(4). |
| Yu, Lian, Amorphous pharmaceutical solids: preparation, characterization and stabilization, Advanced Drug Delivery Reviews, 2001, 27-42, 48. |
| Zang, Yuguo, Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale, PLoS One, 2011, 1-8, 6(9). |
| Zhang, J. et al., Synthesis and characterization of heterotelechelic poly(ethylene glycol)s with amino acid at one end and hydroxyl group at another end, Journal of Applied Polymer Science, 2008, 2432-2439, 110(4). |
| Zhou, Shuxia et al., Biotherapeutic Formulation Factors Affecting Metal Leachables from Stainless Steel Studied by Design of Experiments, AAPS PharmSciTech, 2012, 284-294, 13(1). |
| Daugherty et al., “Formulation and delivery issues for monoclonal antibody therapeutics”, Advance Drug Delivery Reviews 58,(2006), 686-705. |
| Co-pending U.S. Appl. No. 16/609,961, filed Oct. 31, 2019. |
| Co-pending U.S. Appl. No. 16/609,671, filed Oct. 30, 2019. |
| Co-pending U.S. Appl. No. 16/610,188, filed Nov. 1, 2019. |
| Number | Date | Country | |
|---|---|---|---|
| 20220002410 A1 | Jan 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62756678 | Nov 2018 | US |
