Methods for quantitating individual antibodies from a mixture

Abstract

To provide methods of quantitating the amount of an antibody molecule from a mixture comprising two or more antibody molecules.SOLUTION: The method comprises: separating each of the two or more antibody molecules from the mixture by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantitating an amount of each antibody molecule, the molecular weight of each antibody molecule being within 15 kDa of any other antibody molecule in the mixture and either each antibody molecule being different from another antibody molecule in the mixture by more than about 0.25 unit on the Kyte-Doolittle hydropathy scale, or each of the antibody molecules when run alone on HIC-HPLC elutes at distinct run time with little overlap from the other antibody molecules in the mixture, or both.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to the field of assays for co-formulation of therapeutic antibodies. [Background technology]

[0002] Administration of multiple monoclonal antibodies (mAbs) to a patient, rather than a single one, may improve the patient's diagnostic or therapeutic indication and efficacy. These mAbs may be co-formulated into a single pharmaceutical product (DP) and the DP administered to the patient.

[0003] Regulatory agencies are seeking methods to quantify individual mAbs in combined drug substances (cFDS) that are mixed into a DP, or the DP itself. Developing a method to separate two or more antibody molecules and measure the concentration of each mAb is difficult because the antibody molecules may have similar molecular weights, protein structures, and charge characteristics. Summary of the Invention

[0004] The present disclosure includes a method of quantifying the amount of an antibody from a mixture comprising a plurality of antibodies. In some embodiments, the method may include, inter alia, separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantifying the amount of each antibody in the mixture, wherein the molecular weight of each antibody in the mixture is within 15 kDa of the molecular weight of other antibodies in the mixture, the surface hydrophobicity of each antibody in the mixture differs from the surface hydrophobicity of another antibody in the mixture by more than about 0.25 units on the Kyte & Doolittle hydrophobicity index, or each antibody in the mixture elutes at a different analysis time than another antibody in the mixture when analyzed individually by HIC-HPLC, or both.

[0005] In some embodiments, the surface hydrophobicity of each antibody in the mixture differs from the surface hydrophobicity of each other antibody in the mixture by about 0.5 to about 1.0 units on the Kyte and Dolittle hydrophobicity index. In further embodiments, the surface hydrophobicity of each antibody in the mixture is determined by calculation of surface hydrophobicity based on protein structure or structural model, rapid screening for solubility in ammonium sulfate or PEG8000, or rapid screening for molecular interactions by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS).

[0006] In additional embodiments, a first antibody in the mixture elutes at a first analysis time during HIC-HPLC analysis and a second antibody in the mixture elutes at a second analysis time during HIC-HPLC analysis, and the first and second analysis times do not overlap. In further embodiments, the first antibody in the mixture and the second antibody in the mixture have at least 90% homologous protein sequences, the first antibody and the second antibody have at least 90% homologous protein structures as determined by their protein sequences, or the first antibody and the second antibody have isoelectric points (pI) within about 0.6 of each other as determined by their protein sequences.

[0007] In some embodiments, the plurality of antibodies comprises three antibodies. In further embodiments, one or more antibodies in the mixture are monoclonal antibodies. In further embodiments, one or more antibodies in the mixture are human monoclonal antibodies. In other embodiments, two or more antibodies in the mixture are of the same isotype. In some embodiments, two or more antibodies in the mixture are variants of each other. In further embodiments, two or more antibodies in the mixture bind to the same antigen.

[0008] In some embodiments, the mixture is a co-formulated composition. In additional embodiments, the combination composition is configured to treat MERS in a human patient. In a further embodiment, the combination composition is configured to treat Ebola in a human patient. In a further embodiment, the combination composition is configured to treat macular degeneration in a human patient. In a further embodiment, two or more antibodies in the combination composition are configured to treat an infectious disease in a human patient. In some embodiments, the combination composition is comprised in a pharmaceutical product.

[0009] In some embodiments, the HIC-HPLC is performed in a buffer of about pH 5.0 to about pH 7.0. In further embodiments, the method further comprises generating a chromatograph from the HIC-HPLC, where for elution of each antibody in the mixture, the chromatograph shows a peak that does not overlap with other peaks in the chromatograph.

[0010] The present disclosure also includes a method of quantifying the amount of an antibody from a mixture containing a plurality of antibodies, the method including: separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC), wherein the molecular weight of each antibody in the mixture is within 15 kDa of the molecular weight of each other antibody in the mixture; quantifying the amount of each antibody in the mixture; and generating a chromatograph from the HIC-HPLC, wherein for the elution of each antibody in the mixture, the chromatograph exhibits a peak that does not overlap with other peaks in the chromatograph. In some embodiments, one or more of the plurality of antibodies is a human monoclonal antibody. In further embodiments, the surface hydrophobicity of each antibody in the mixture differs from the surface hydrophobicity of another antibody in the mixture by more than about 0.25 units on the Kyte and Dolittle hydrophobicity index, or each antibody in the mixture elutes at a different analysis time than another antibody in the mixture when analyzed individually by HIC-HPLC, or both.

[0011] In accordance with these and other aspects of the present disclosure, numerous other aspects and embodiments are provided. Other features and aspects of the present disclosure will become more fully apparent from the following detailed description and the appended claims. [Brief description of the drawings]

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various examples and, together with the description, serve to explain the principles of the present disclosure. Any feature (e.g., device, method, etc.) of an embodiment or example described herein may be combined with any other embodiment or example and are included in the present disclosure. [Figure 1] 1 is an exemplary chromatograph of a HIC-HPLC analysis of a combination drug that includes an anti-Ebola mAb. [Diagram 2] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Diagram 3] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Figure 4] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Diagram 5] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Figure 6] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Figure 7] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Figure 8] 1 is an exemplary chromatograph of HIC-HPLC analysis of a combination drug that includes an anti-MERS mAb. [Figure 9] 1 is an exemplary chromatograph of a HIC-HPLC analysis of a combination drug that includes an anti-Ebola mAb. [Figure 10] A plot of the linearity of HIC-HPLC, plotting peak area versus amount of individual anti-Ebola mAb assays. [Figure 11] 1 is a range plot, plotting percent recovery of mAb versus amount of individual anti-Ebola mAb assays. [Figure 12] 13 is a plot of the accuracy of HIC-HPLC using different mixture sample lots, plotting percent recovery of mAb versus amount of individual anti-Ebola mAb assays. [Figure 13] 13 is a plot of the accuracy of HIC-HPLC using different ratios of individual anti-Ebola mAbs, plotting the percent recovery of mAbs versus the amount of individual anti-Ebola mAb assayed. [Figure 14] Plot of storage stability of mAbs in cFDS, plotting the amount of individual anti-Ebola mAbs against storage time in months. [Figure 15] 1 shows an exemplary chromatograph of a cation exchange ultra-high pressure liquid chromatography (CEX-UPLC) analysis of a combination drug that includes an anti-Ebola mAb. [Figure 16] A CEX-UPLC linearity plot plotting peak area versus amount of individual anti-Ebola mAbs. [Figure 17] 1 shows an exemplary chromatograph of a size-exclusion ultra-high pressure liquid chromatography (SE-UPLC) analysis of a combination drug that includes an anti-Ebola mAb. [Figure 18] 1 shows an exemplary imaging capillary isoelectric focusing (iCIEF) profile of a combination drug that includes an anti-Ebola mAb. [Figure 19] 1 shows an exemplary chromatograph of a reversed-phase ultra-high pressure liquid chromatography (RP-UPLC) analysis of a combination drug that includes an anti-Ebola mAb. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The term "antibody" may be used interchangeably with the term "immunoglobulin". Briefly, it may refer to the whole antibody, including two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes, including IgM, IgG, IgA, IgD, and IgE antibodies. The term "antibody" may include, for example, polyclonal antibodies, monoclonal antibodies (mAbs), chimerized or chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies, and fully human antibodies. Antibodies may be generated or derived from any of a variety of mammalian species, such as, for example, humans, non-human primates (e.g., orangutans, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies may be purified or recombinant antibodies. Antibodies may also be artificial or antibody-like proteins (e.g., fusion proteins) that contain at least one immunoglobulin domain. The artificial protein or antibody-like protein may also be a bispecific or trispecific antibody, or a dimeric, trimeric or multimeric antibody, or a bispecific antibody, DVD-Ig, CODV-Ig, Affibody®, or Nanobody®.

[0014] The terms "antibody variant," "antibody variant," and the like refer to an antibody that differs from another antibody in that the variant antibody is a deletion, insertion, and / or substitution variant of the other antibody.

[0015] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs may contain amino acid residues (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation) that are not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in human subjects.

[0016] As used herein, the terms "treat", "treating" or "treatment" refer to the reduction or amelioration of the severity of at least one symptom or sign of a disease or condition by administering a combination of two or more antibodies to a subject in need of treatment. The term includes the inhibition of disease progression. The term also includes a positive prognosis of the disease.

[0017] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the onset of a disease or condition, or any symptoms or signs of that disease or condition, upon administration of a combination of two or more antibodies.

[0018] The terms "for example" and "such as" and their grammatical equivalents are understood to be followed by the phrase "and without limitation" unless expressly stated otherwise. As used herein, the term "about" and the symbol "~" are meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about," unless expressly stated otherwise, regardless of whether that term is explicitly used. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0020] Antibody molecules, such as monoclonal antibody molecules, can be coformulated to treat one or more diseases or conditions in a patient, including a human patient. The terms "patient" and "subject" are used interchangeably herein.

[0021] A co-formulated drug product (DP) may contain a combined drug substance (cFDS) (also referred to herein as a combined drug product) containing two or more (e.g., three) human monoclonal antibody (mAb) molecules. The cFDS is prepared by mixing purified mAbs in a predetermined ratio. Regulatory agencies require a method to quantify each individual mAb in the cFDS.

[0022] The mAb molecules in the combination may be similar to each other; they may be immunoglobulins of approximately the same molecular weight (eg, about 145 kDa), such as IgG1, with similar protein structure and charge characteristics.

[0023] The methods disclosed herein for quantifying similar mAb molecules in a combination are accurate, precise and reproducible, suitable for use in a quality control environment, do not use expensive equipment and do not require laborious sample preparation.

[0024] method The present disclosure provides a method for quantifying the amount of antibody molecules from a mixture containing two or more antibody molecules. The method may include separating each of the two or more antibody molecules from the mixture by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantifying the amount of each antibody molecule, where each antibody molecule has a molecular weight within 15 kDa of other antibody molecules in the mixture, each antibody molecule differs from another antibody molecule in the mixture by more than about 0.25 units on the Kyte and Dolittle hydrophobicity index (see, e.g., Kyte and Dolittle, J. Mol. Biol., vol. 157, pp. 105-132 (1982)) or, when analyzed alone by HIC-HPLC, each of the antibody molecules elutes at a different analysis time with little overlap with other antibody molecules in the mixture, or both. In certain embodiments, for example, the relative hydrophobicity of each antibody molecule differs from each other antibody molecule in the mixture by about 0.5 to about 1.0 units on the Kyte and Dolittle hydrophobicity index. In other particular embodiments, when analyzed alone by HIC-HPLC, each of the antibody molecules elutes at a different analysis time with little overlap with another antibody molecule in the mixture. In particular embodiments, the mixture comprises multiple antibody molecules (e.g., 2, 3, 4, or 5 antibody molecules).

[0025] When two antibody molecules from a mixture are analyzed by HIC-HPLC, they have significant overlap in a chromatograph monitoring the elution profile of the antibody molecules by absorbance versus elution time, which indicates that the two antibody molecules elute at close times, resulting in insufficient separation in the column such that the antibody molecules are not purified (e.g., less than 50% purity). In such cases, the individual antibodies cannot be fully quantified due to the significant overlap. In some embodiments, when two antibody molecules from a mixture are analyzed by HIC-HPLC, they have little or no overlap in a chromatograph monitoring the elution profile of the antibody molecules by absorbance versus elution time, which indicates that the antibody molecules elute at times that are not close, resulting in a separation such that the antibody molecules are substantially purified, each of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0026] In some embodiments, the methods disclosed herein are used to quantify individual populations of antibodies, also referred to as "antibody molecules," from a mixture of antibodies that includes two or more distinct populations of antibodies, the distinct populations having at least one amino acid sequence difference between them. In some embodiments, a population of antibodies may have the same amino acid sequence as another population in the mixture of antibodies, but they may differ in post-translational modifications. In certain embodiments, the mixture is a combination composition such as or including a combined drug substance (cFDS). In certain embodiments, the mixture includes an excipient. In certain embodiments, the mixture may include sucrose and be a sucrose drug substance that includes mAb molecules. Each antibody in the antibody mixture will have a similar molecular weight within 1, 2, 3, 4, 5, 10, or 15 kilodaltons (kDa) of each other. In other aspects, the average molecular weight of all antibodies will be about 150 kDa. This method can also be used with two or more trap molecules that have similar molecular weights. In certain further embodiments, the combination composition includes a VEGF trap. (See, e.g., U.S. Pat. No. 9,265,827 and U.S. Patent Application Publication No. 14 / 943,490, which are incorporated by reference in their entireties.) The method may also be used to monitor the concentration of each of the mAbs in a mixture (e.g., a combination) when monitoring storage stability. In certain embodiments, the method is used to separate antibody molecules that cannot be separated by other chromatographic methods, such as reverse-phase high pressure liquid chromatography (HPLC) or ultra-high pressure liquid chromatography (UPLC).

[0027] Hydrophobic interaction chromatography (HIC) separates antibodies in a decreasing salt gradient based on differences in the surface hydrophobicity of the antibodies. Separation using HIC is based on reversible interactions between antibodies and hydrophobic ligands bound to the chromatographic matrix. While the hydrophobic amino acids of proteins and peptides are usually located away from the molecular surface, biomolecules have some exposed hydrophobic groups that allow interaction with hydrophobic ligands on the medium. The hydrophobic interactions are enhanced by buffers with high ionic strength.

[0028] Any suitable HIC-HPLC column may be employed in the methods disclosed herein, including, but not limited to, Dionex ProPac HIC-10, mAbPac HIC-10, mAbPac HIC-20, mAbPac HIC-Butyl (Dionex, Thermo Fisher Scientific, Sunnyvale, CA). The buffer may be any suitable buffer. In certain embodiments, the HIC-HPLC is performed in a buffer pH of about pH 5.0 to about pH 7.0, or about pH 6.0 to about pH 7.0, or about 6.5 to about 7.5. In certain embodiments, the columns are Dionex MabPac HIC-10 (100×4.6 mm, 5 μm, pore size 1000 A°) and ProPac HIC-10 (100×4.6 mm, 5 μm, pore size 300 A°).

[0029] A mixture containing two or more antibodies is analyzed by HIC-HPLC. The antibodies are eluted at different times (analysis times). The antibodies are monitored by measuring their absorbance in the UV spectrum, for example at 280 nm. A chromatograph can be used to monitor and record the analysis results. In a particular embodiment, the buffer used in HIC-HPLC is different gradients of 1 M ammonium sulfate (100% to 0%) in 100 mM phosphate buffer.

[0030] In a particular embodiment, the antibody molecules are quantified by comparing their absorbance at 280 nm to a standard curve. The standard curve may be constructed by determining the absorbance of known antibodies at known concentrations from HIC-HPLC analysis, for example at absorbance at 280 nm. The higher the absorbance (or "optical density"), the higher the protein concentration. Using these data for known concentrations of antibodies, a standard curve is constructed, plotting the concentration on the X-axis and the absorbance obtained from the chromatograph of the antibody's elution profile from the HIC-HPLC analysis on the Y-axis. A sample containing two or more antibodies at unknown concentrations containing an antibody from the standard curve is analyzed. To analyze the data, the antibodies are separated by HIC-HPLC. The antibody elution profile is displayed on a chromatograph, expressing the antibody concentration in terms of absorbance monitored over time by a spectrophotometer. The absorbance of each antibody, identified by its elution time (analysis time), is used to determine the corresponding absorbance value on the standard curve. The X-axis value corresponding to that point on the standard curve is the concentration of that antibody in the sample.

[0031] In some embodiments of the current disclosure, the methods for quantifying individual antibodies in a mixture disclosed herein may be useful when the relative surface hydrophobicity of each antibody in the mixture differs from each other antibody in the mixture by more than about 0.25 units, such as about 0.5 to about 1.0 units on the Kyte and Dolittle hydrophobicity index. The relative surface hydrophobicity of antibodies may be determined / estimated by several methods.

[0032] For example, HIC-HPLC may be used to determine the relative surface hydrophobicity of different antibody molecules. Each of the antibody molecules is analyzed separately by HIC-HPLC and the analysis time (elution time) of each antibody is determined.

[0033] In certain embodiments, differences in surface hydrophobicity between antibodies in a mixture are determined by one or more of the following methods: calculation of surface hydrophobicity based on protein structure or structural models, rapid screening for solubility in ammonium sulfate or PEG8000, and rapid screening for molecular interactions by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS).

[0034] Relative surface hydrophobicity may be calculated based on a known structure (e.g., based on a crystal structure or NMR structure) or a structural model of an antibody. Methods for such calculations are known in the art.

[0035] Relative surface hydrophobicity may be calculated / estimated by determining the solubility of antibody molecules by rapid screening methods for solubility in conditions of increasing hydrophobicity, such as in ammonium sulfate or PEG 8000. The more soluble the protein, the less hydrophobic it is. (See, e.g., Kramer et al., Biophysical Journal, Vol. 102, April, pp. 1907-1915 (2012)). Relative surface hydrophobicity may be calculated / estimated by rapid screening for molecular interactions by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). (See, e.g., Estep et al., mAbs, vol. 7, 3, pp. 553-561 (2015).) AC-SINS is an approach that involves coating gold nanoparticles with polyclonal anti-human antibodies, immobilizing human mAbs using these complexes, and assessing mAb self-interactions by measuring the plasmon wavelength of the antibody complex as a function of ammonium sulfate concentration.

[0036] In other specific embodiments, antibodies in a mixture that can be separated using the described methods have a similar size or overall charge as determined by one or more of the following methods: protein structure or structural modeling based on sequence and other known protein structures, calculated overall protein charge based on protein sequence, hydrophobicity calculated based on protein sequence, protein sequencing.

[0037] In certain embodiments, the antibodies in the mixture have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous protein structures or structural models based on sequence and other known protein structures. In certain embodiments, the antibodies in the mixture have calculated protein charge properties within 3, 4, or 5, 10, or 15 base units of each other, where the protein charge properties are calculated based on the protein sequences. In certain embodiments, two or more antibodies in the mixture have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity between their protein sequences.

[0038] In one embodiment, the method of quantifying antibodies in a mixture using HIC-HPLC as disclosed herein is less useful when there are large differences between antibody molecules calculated or predicted by protein structure, sequence-based structural models, protein sequence, and other known aspects of protein structure. When there are differences in the hydrophobicity profiles of antibodies, the method using HIC-HPLC may still facilitate separation of antibody species.

[0039] In another embodiment, the method of quantifying antibodies in a mixture using HIC-HPLC as disclosed herein is less useful when there are large differences between antibody molecules as calculated / estimated by the overall charge properties of the protein of each antibody molecule calculated based on the protein sequence of each antibody molecule. If there are differences in the hydrophobicity profiles of the antibodies, the method using HIC-HPLC may still facilitate separation of the antibody species.

[0040] In another embodiment, the method of quantifying antibodies in a mixture using HIC-HPLC as disclosed herein is not very useful when there are large differences between antibody molecules as calculated / estimated by examining protein charge or size. If there are differences in the hydrophobicity profiles of the antibodies, the method using HIC-HPLC may still facilitate the separation of antibody species.

[0041] In certain embodiments, one or more of the antibodies in the mixture are monoclonal antibodies. In certain embodiments, one or more of the monoclonal antibodies are human monoclonal antibodies. In certain embodiments, two or more of the antibody molecules are of the same isotype. In certain embodiments, two or more of the antibody molecules are variants of each other. In yet certain other embodiments, two or more of the antibody molecules bind to the same antigen. The mAb may be a whole antibody molecule.

[0042] In certain embodiments, the mixture is a combination composition. In certain further embodiments, the combination composition comprises two or more mAbs effective for treating Middle East Respiratory Syndrome (MERS) in a human patient. Antibodies against MERS coronavirus (MERS-CoV) are disclosed, for example, in US Patent Application Publication No. 14 / 717760 and WO 2015 / 179535 A1. In certain further embodiments, the combination composition comprises a mAb effective for treating Ebola hemorrhagic fever in a human patient. Antibodies against Ebola virus are disclosed, for example, in US Patent Application Publication No. 15 / 005334, filed January 25, 2016. In certain further embodiments, the combination composition comprises a mAb effective for treating macular degeneration in a human patient. In certain further embodiments, the combination composition comprises the mAbs alirocumab and evinacumab, as disclosed in US Provisional Patent Application No. 62 / 302907. In certain embodiments, the combination composition comprises a PD1 antibody and another immuno-oncology antibody product, such as a bispecific antibody. (See, e.g., the disclosures of PD-1 and CD3xCD20 in U.S. Provisional Application No. 62 / 270749, U.S. Patent Application Publication No. 15 / 386443, and U.S. Patent Application Publication No. 15 / 386453, and the disclosures of PD-1 and Lag3 in U.S. Provisional Application No. 62 / 365006 and U.S. Patent Application Publication No. 15 / 289032.) In certain embodiments, the combination composition comprises an anti-Zika virus antibody. (See, e.g., U.S. Provisional Application No. 62 / 363546.) In certain further embodiments, the combination composition comprises trevoglumab and activin A antibodies. (See, e.g., U.S. Patent No. 8,871,209.) In certain embodiments, two or more antibodies in the combination composition can treat an infectious disease in a human patient. The disclosures of each of the patent documents cited above in this paragraph are incorporated herein by reference.

[0043] The anti-MERS mAb in the combination may, for example, bind to the spike protein of MERS-CoV (e.g., the spike protein of MERS-CoV isolate EMC / 2012). The epitope of the spike protein may be within the receptor binding domain of the spike protein (e.g., amino acids selected from amino acids 367-606 of GenBank Accession No. AFS88936.1). The anti-MERS mAb in the combination composition may be a fully human monoclonal antibody that binds to MERS-CoV spike protein, interacts with one or more amino acid residues within the receptor binding domain of MERS-CoV spike protein selected from amino acid residues 367-606 of GenBank Accession Number AFS88936.1, binds to MERS-CoV spike protein with a dissociation constant (Ko) of less than 18.5 nM as measured by a surface plasmon resonance assay, or blocks binding of MERS-CoV spike protein to dipeptidyl peptidase 4 (DPP4) by greater than 90%.

[0044] A combination composition may be any composition that contains two or more antibodies directed to the same or different targets, and is effective to treat the same or different diseases or conditions in a patient, including a human patient.

[0045] Pharmaceutical Compositions and Formulations A composition containing two or more antibody molecules may be formulated as a pharmaceutical composition (e.g., DP) for administration to a subject. A pharmaceutical composition may contain, for example, three antibody molecules. Any suitable pharmaceutical composition and formulation, as well as suitable methods for formulation and suitable routes and suitable sites of administration, are within the scope of the present invention. Also, any suitable dosage and frequency of administration are contemplated, unless otherwise specified.

[0046] The mAbs in the combination are purified by methods known in the art prior to co-administration. The combination of two or more antibodies may be any suitable combination. The pharmaceutical composition / combination may contain a pharma- ceutically acceptable carrier (i.e., excipient). "Pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, diluents, glidants, and the like. The composition may contain a pharma- ceutically acceptable salt, for example, an acid addition salt or a base addition salt (see, for example, Berge et al., J Pharm Sci, vol. 66, pp. 1-19 (1977)). The composition may contain sucrose or may be coated, if appropriate.

[0047] In certain embodiments, the protein composition can be stabilized and formulated as a solution, microemulsion, dispersion, liposome, lyophilized cake, solid, etc. Sterile injectable solutions can be prepared by incorporating two or more mAbs in a suitable solvent with one or a combination of the above-listed ingredients in the required amount, as needed, followed by filtration sterilization. In general, dispersions are prepared by incorporating two or more mAb molecules into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as monostearate salts and gelatin, in the composition.

[0048] Working Example In order that the present invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0049] The anti-Ebola mAbs in the combination used in Examples 1 and 3-7 are all fully human IgG1 monoclonal antibodies. The three mAbs (mAb A, mAb B, and mAb C) have similar molecular weights (e.g., about 145 kDa), protein structures, and charge characteristics (e.g., pI difference of about 0.6 or less as determined by protein sequence). The isoelectric point (pI) of mAb A is determined to be 9.0, the pI of mAb B is determined to be 8.5, and the pI of mAb C is determined to be 9.1.

[0050] The anti-MERS mAb in the combination used in Example 2 is a whole, fully human anti-Ebola mAb. The two mAbs have similar molecular weights (e.g., within about 15 kDa of each other) and charge characteristics. (See U.S. Patent Application Publication No. 2015 / 0337029, WO 2015 / 179535 A1, and U.S. Patent Application Publication No. 14 / 717760, the disclosures of each of which are incorporated herein by reference.) Example 1 Using HIC-HPLC method, three anti-Ebola monoclonal antibodies (mAb1, mAb2, and mAb3) with similar molecular weight, protein structure, and charge characteristics are quantified from the combination by first separating the three mAbs from the combination and then quantifying the antibodies individually.

[0051] A Dionex ProPac HIC-10 column, catalog number 063655 (Dionex, Thermo Fisher Scientific, Sunnyvale, Calif.), 4.6×100 mm, is used.

[0052] Preparation of mobile phase The mobile phases include mobile phase A and mobile phase B. Mobile phase A contains 1 M ammonium phosphate and 100 mM phosphoric acid at a pH of 7.0. Preparation of mobile phase A includes dissolving 13.8 g sodium phosphate monobasic monohydrate (NaH2PO4.H2O) and 132.1 g ammonium phosphate in 800 mL MilliQ, adjusting the pH to 7.0 with 50% NaOH, bringing the volume to 1000 mL, and filtering the solution through a 0.22 μM filter. Mobile phase B contains 100 mM phosphate at a pH of 7.0. Preparation of mobile phase B involves dissolving 13.8 g of sodium phosphate monobasic monohydrate (NaH2PO4.H20) in 900 mL of MilliQ, adjusting the pH to 7.0 with 50% NaOH, bringing the volume to 1000 mL, and filtering the solution through a 0.22 μM filter.

[0053] HIC-HPLC method The HIC-HPLC runs the column at a flow rate of 0.5 mL / min. The column temperature is maintained at 30° C., and the combined sample temperature is 5° C. The column dwell time is 40 min. The 280 nm UV absorbance of the effluent is monitored using a UV detector. Table 1 below shows the mix of mobile phase A and mobile phase B as a percentage of the mobile phase composition gradient introduced over the column run time. JPEG2024173983000002.jpg48170

[0054] Calibration with 3 individual mAbs To calibrate the results for the three individual mAbs, known amounts of each mAb in FDS are analyzed. FDS with known concentrations of each mAb are prepared and the concentrations are measured by either reverse phase (RP) chromatography or Solo VPE® spectroscopy (e.g., See Technologies). The known FDS for each mAb are as follows: JPEG2024173983000003.jpg49170

[0055] A control sample for the calibration sequence is also prepared. The control sequence has a concentration of 50.31 mg / mL as measured by Solo VPE® spectroscopy (e.g., See Technologies, Inc.). One injection of the control sample is 12.0 μL (603.72 μg).

[0056] Table 2 below lists the calibrator samples used for each of the mAb FDS. JPEG2024173983000004.jpg126170

[0057] A calibration sequence is run during which one injection of a control sample is introduced at the beginning of each sample set (e.g., a set of mAb1 FDS injections, a set of mAb2 FDS injections, or a set of mAb3 FDS injections), after every 20-24 injections of mAb FDS, and at the end of each injection sequence.

[0058] During one calibration sequence, one injection of known amounts of each mAb is made. During another calibration sequence, four injections of known amounts of each mAb are made to determine the reproducibility of the injections.

[0059] Prepare a standard calibration curve for each mAb. The calibration curve for each mAb is shown in R 2 ≧0.999, with variability of ≦1% for replicate injections of the same amount of each mAb. Control Specifications A control HIC-HPLC analysis of each known mAb FDS is performed. The percent recovery of each known mAb FDS is calculated by measuring the concentration by HIC-HPLC and then dividing the concentration measured by HIC-HPLC by the known mAb concentration in Table 2. The percent recovery is calculated to be 90-110% of the known concentration for all three known mAb FDS.

[0060] Unknown sample A sample mixture with a total of 600 μg of mAb (e.g., 12 μL of 50 mg / mL mAb sample) is run on the HIC-HPLC. For example, 12 μL of the mixture with a total of 600 μg of mAb contains the equivalent of three injections of 200 μg / injection of each mAb. The individual mAbs in the mixture are mAb1, mAb2, and mAb3.

[0061] Duplicate analyses of samples were performed and the mean concentration (mg / mL) of each mAb in the combination and the ratio of each mAb to the total amount of mAb are reported. Figure 1 is a chromatograph showing the HIC-HPLC analysis of this combination. Each mAb is plotted as a separate line, and the cFDS is plotted as a whole line. The three antibodies are well separated by HIC-HPLC, with little overlapping peaks.

[0062] Example 2 Using multiple HIC-HPLC methods, two anti-MERS monoclonal antibodies with similar molecular weight and charge characteristics are quantified from the combination by first separating the two mAbs from the combination and then quantifying them individually.

[0063] mAb Drug Substance Two mAb (mAb1 and mAb2) drug substances are combined: mAb1 drug substance contains 52.3 mg / ml mAb1 and 10 mM His at pH 5.5 (Regeneron Pharmaceuticals, Tarrytown, NY). mAb2 drug substance contains 40.4 mg / ml mAb2 and 10 mM His at pH 6.0 with 5% (w / v) sucrose (Regeneron Pharmaceuticals, Tarrytown, NY).

[0064] HIC-HPLC method Seven HIC-HPLC methods are performed. For each method, 50 μg (mAb1 and mAb2) of sample is loaded. The effluent is monitored using UV detection at 280 nm. The mobile phase uses two solvents in various ratios. Solvent A is 1 M (NH4)SO4 20 mM acetate at pH 5.5 and solvent B is 20 mM acetate at pH 5.5.

[0065] 1. Method 1 JPEG2024173983000005.jpg48170

[0066] Figure 2 shows the chromatograph of this analysis. The two antibodies are somewhat separated but have considerable overlap. 2. Method 2 0.5% per minute JPEG2024173983000006.jpg54170

[0067] Figure 3 shows the chromatograph of this analysis. The two antibodies are somewhat separated but have considerable overlap. 3. Method 3 0.2% per minute JPEG2024173983000007.jpg52170

[0068] Figure 4 shows the chromatograph of this analysis. The two antibodies are somewhat separated but have considerable overlap. 4. Method 4: 2% per minute JPEG2024173983000008.jpg54170

[0069] Figure 5 shows the chromatograph of this analysis: the two antibodies are somewhat separated, but still have some overlap. 5. Method 5: 1% per minute JPEG2024173983000009.jpg53170

[0070] Figure 6 shows the chromatograph of this analysis. The two antibodies are somewhat separated, but with some overlap. 6. Method 6: 1% per minute JPEG2024173983000010.jpg54170

[0071] Figure 7 shows the chromatograph of this analysis. The two antibodies are somewhat separated, but with some overlap. 7. Method 7 0.5% per minute JPEG2024173983000011.jpg54170

[0072] Figure 8 shows the chromatograph of this analysis. The two antibodies are somewhat separated, but with some overlap. Example 3 An HIC separation of a combination of three anti-Ebola mAbs with similar molecular weight, protein structure, and charge characteristics will be developed and evaluated on an Agilent 1100 HPLC system.

[0073] The HIC-HPLC method uses a Dionex ProPac HIC-10 column with a binary mobile phase (mobile phase A (100 mM phosphate, 1 M ammonium sulfate, pH 7.0) and mobile phase B (100 mM phosphate, pH 7.0)) at a flow rate of 0.5 mL / min. The HIC method is qualified using a five-point calibration curve for each mAb. Accuracy (recovery) is determined by comparing the measured concentrations to the theoretical concentration of each mAb. The column is run using a gradient of 600 to 100 mM ammonium sulfate.

[0074] Figure 9 shows the chromatograph of the HIC-HPLC analysis. The three antibodies are well separated as can be seen by the three distinct peaks, with little or no overlap.

[0075] Reproducibility and precision of HIC-HPLC Reproducibility is assessed by analyzing triplicate samples at 75%, 100%, 125%, and 150% of the target amount of cFDS (600 μg / cFDS injection). The relative standard deviation (RSD) of the reproducibility is determined to be ≤0.2%. JPEG2024173983000012.jpg59170

[0076] The repeatability study is repeated on different days using different lots of columns and different batches of mobile phase. The RSD of the intermediate precision is determined to be ≦1.7%. JPEG2024173983000013.jpg59170

[0077] Linearity of HIC-HPLC Linearity is determined by analyzing samples of cFDS samples in triplicate at nine stages (75-1200 μg cFDS, 25-400 μg individual mAbs). A plot of the peak area of ​​the individual mAbs against the injected amount (Figure 10) results in the following linear curve. From 75-300 μg, R 2 ≧0.999, 25-400μg, R 2 ≧0.98.

[0078] Scope of HIC-HPLC The ranges are those for which the method has acceptable precision (≤1.7%), accuracy (93–106% recovery), and linearity (R 2 The interval between the lowest and highest concentrations at which a mAb recovery of ≥ 0.999 can be demonstrated. The range has been determined to be 300-900 μg for cFDS samples (100-300 μg for individual mAbs). Figure 11 is a plot of mAb recovery versus amount of individual mAbs.

[0079] HIC-HPLC accuracy across different cFDS lots The accuracy of the method is assessed using data obtained from intra-day and inter-day precision (cFDS Lot #1), and the range study (cFDS Lot #2). Figure 12 is a plot of mAb recovery versus the amount of individual mAb. Recoveries are within 93-106% at 75%, 100%, 125%, and 150% of the target amount (600 μg total protein, 200 μg of each mAb). Accuracy (recovery) is determined to be between 93% and 106%.

[0080] Accuracy of HIC-HPLC across different ratios of individual mAbs in cFDS The cFDS are formulated by mixing the individual mAbs 1:1:1, 2:1:1, 1:2:1, and 1:1:2. Each cFDS is analyzed at 100% of the target amount (600 μg / injection, n=3). Figure 13 shows the mAb recoveries for the individual mAbs at different cFDS ratios. The recoveries are within 94-106% and the reproducibility is ≦0.3%. JPEG2024173983000014.jpg115170

[0081] Application of HIC-HPLC in combination drug development The use of HIC-HPLC to quantify each of the mAbs in a combination or DP may have various applications in combination development, for example, the concentration of each mAb in a combination or DP may be monitored in storage stability studies.

[0082] Figure 14 is a chart monitoring the storage stability of three mAbs in DP as a function of time (months). HIC-HPLC is used to quantify the amount, and therefore concentration, of each of the three mAbs at six time points over a one-year period. An exemplary set of data points from one time point in this study includes measuring the concentration of mAb A at 16.6 mg / mL, mAb B at 17.5 mg / mL, and mAb C at 17.1 mg / mL. In this case, the storage stability shows no apparent change in the concentration of each of the mAbs (≦4%) over the monitored period.

[0083] The HIC-HPLC method demonstrated acceptable reproducibility (≤0.2%), intermediate precision (≤1.7%), accuracy (93%–106% recovery), and linearity (R 2 ≧0.999) and can be used to determine the concentrations of each of the three mAbs in the cFDS and DP.

[0084] The HIC-HPLC method can be used as a release method to quantitate each of the three mAbs in the co-formulated DS and DP. This method can also be used to aid in formulation development.

[0085] Example 4 To develop and evaluate a cation exchange ultra-high pressure liquid chromatography (CEX-UPLC) separation of a combination of three anti-Ebola mAbs with similar molecular weight, protein structure, and charge characteristics.

[0086] The CEX-UPLC method uses a YMC-BioPro SP-F column with a mobile phase containing 200 mM MES buffer and a 10-120 mM NaCl gradient at pH 6.5. A six-point standard calibration curve is constructed for each of the three mAb molecules (mAb A, mAb B, mAb C). The linearity of each calibration curve is measured using a linear regression model with a linear regression analysis. 2 ≧0.99. The three mAb molecules are separated from the combination (i.e., mixture) using CEX-UPLC and a UPLC chromatograph is generated. The chromatograph shows separation of each mAb with some overlap between mAb A and mAb C.

[0087] The reproducibility of the method is assessed by analyzing the combination sample in triplicate at three different amounts (150 μg, 225 μg, and 300 μg). The accuracy (recovery) is determined by comparing the measured concentration of each sample to the theoretical value. The linear quantification range of each mAb is determined to be 50-100 μg for each antibody, except for mAb A, with R 2 ≥ 0.973 and accuracy of 75%-91%.

[0088] Figure 15 shows a chromatograph of the CEX-UPLC analysis of the separation of the three mAbs from the combination. The three antibodies are separated with little overlap between mAb A and mAb C.

[0089] CEX-UPLC reproducibility and accuracy Reproducibility is assessed by analyzing samples of cFDS in triplicate at five different percentages (25%-100%) of the target amount (600 μg / injection of cFDS). The RSD of reproducibility is determined to be between 3.7% and 21.4%. JPEG2024173983000015.jpg75170

[0090] Linearity of CEX-UPLC The data from the reproducibility study are used to evaluate the range and linearity of the CEX-UPLC method. Plots of the peak areas of the individual mAbs against the injected amount (Figure 16) generally yield linear curves for each mAb. However, because the RSDs of the mAbs are greater than 10%, the data point for mAb A at 100 μg is not statistically significant enough to conclude that there is a linear curve for mAb A extending out to 100 μg.

[0091] Scope of CEX-UPLC For cFDS samples with 150–300 μg total antibody, reproducibility (RSD ≤ 10%), accuracy (> 80%), and linearity (R 2 ≥ 0.97) is shown. However, the range of CEX-UPLC is not as clearly shown as that of HIC-HPLC. JPEG2024173983000016.jpg103170

[0092] The CEX-UPLC method separates the three mAbs based on their charge characteristics with some overlap. The overlap means that manual integration of the method is required. The precision, accuracy, and range of CEX-UPLC are not as optimal as HIC-HPLC.

[0093] Example 5 Evaluating SE-UPLC of a combination of three anti-Ebola mAbs with similar molecular weights. SE-UPLC separates the mAbs by size. Waters Acquity A H UPLC system is used. Two Waters BEH200 SEC columns are linked in series. The mobile phase includes 10 mM phosphate buffer at pH 6.0 and 1 M perchlorate. Figure 17 shows the chromatogram of SE-UPLC. As shown, there is a significant overlap between the elution times of the three mAbs. Thus, SE-UPLC is a suboptimal method for separating the three anti-MERS mABs of similar molecular weight.

[0094] Example 6 iCIEF of a combination containing three anti-Ebola mAbs with similar molecular weight, protein structure, and charge characteristics is evaluated. iCIEF separates proteins by isoelectric point (pI). A ProteinSimple iCE3 charge variant analyzer is used. 4% pH 3-10 Pharmalyte® is used as the ampholyte and 2 M urea is used as the buffer. Figure 18 shows an iCIEF profile generated using these specifications. As shown, there is significant overlap between the elution times of mAb A and mAb C. Thus, iCIEF is a suboptimal method for separating the three anti-MERS mAbs.

[0095] Example 7 RP-UPLC of a combination of three anti-Ebola mAbs with similar molecular weight, protein structure, and charge characteristics is evaluated. RP-UPLC separates the mAbs by hydrophobicity. A Waters Acquity UPLC system is used. A ZORBAX 300SB-C8 column is used and the column is run at 80°C. The mobile phase contains 60-90% acetonitrile in 0.1% TFA. Figure 19 shows the chromatogram of RP-UPLC. As shown, there is significant overlap between the elution times of mAb A and mAb B. Thus, RP-UPLC is a suboptimal method for separating the three anti-MERS mAbs of similar molecular weight.

[0096] Embodiment The foregoing discussion discloses only exemplary embodiments of the present invention. Although the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Thus, while only certain features of the invention have been illustrated and described, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. A method for quantifying an amount of an antibody from a mixture containing a plurality of antibodies, the method comprising the steps of: separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC); generating a chromatograph from the HIC-HPLC; quantitating the amount of each antibody in the mixture; wherein for the elution of each antibody in the mixture, the chromatograph exhibits a peak that does not overlap with other peaks in the chromatograph, the molecular weight of each antibody in the mixture is within 15 kDa of the molecular weight of other antibodies in the mixture, and when analyzed individually by HIC-HPLC, each antibody in the mixture elutes at a different analysis time than another antibody in the mixture, and wherein the plurality of antibodies in the mixture is configured to treat an infectious disease in a human patient.

2. 2. The method of claim 1, wherein a first antibody in the mixture elutes at a first analysis time during HIC-HPLC analysis and a second antibody in the mixture elutes at a second analysis time during HIC-HPLC analysis, and wherein the first analysis time and the second analysis time do not overlap.

3. The method of claim 1 or 2, wherein the HIC-HPLC is carried out in a buffer of about pH 5.0 to about pH 7.

0.

4. The method of any one of claims 1 to 3, wherein the plurality of antibodies comprises three antibodies.

5. 5. The method of any one of claims 1 to 4, wherein the surface hydrophobicity of each antibody in the mixture is determined by one or more methods selected from the group consisting of calculated surface hydrophobicity based on protein structure or structural models, rapid screening for solubility in ammonium sulfate or PEG 8000, or rapid screening for molecular interactions by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS).

6. the first antibody in the mixture and the second antibody in the mixture have protein sequences that are at least 90% identical; or The method of any one of claims 1 to 5, wherein the first antibody and the second antibody have isoelectric points (pI) within about 0.6 of each other as determined by their protein sequences.

7. The method of claim 1 , wherein one or more of the antibodies in the mixture are monoclonal antibodies, in particular human monoclonal antibodies.

8. The method of any one of claims 1 to 7, wherein two or more of the antibodies in the mixture are of the same isotype, are variants of each other, and / or bind to the same antigen.

9. 9. The method of any one of claims 1 to 8, wherein the mixture is a combination composition, in particular the combination composition is configured to treat MERS in a human patient, Ebola in a human patient, and / or macular degeneration in a human patient.

10. The method of claim 9 , wherein the combination composition is included in a pharmaceutical product.

11. A method for quantifying an amount of an antibody from a mixture containing a plurality of antibodies, the method comprising the steps of: separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC), wherein the mixture is a combination composition configured to treat a viral infection in a human patient, and wherein a molecular weight of each antibody in the mixture is within 15 kDa of a molecular weight of each other antibody in the mixture; quantitating the amount of each antibody in the mixture; generating a chromatograph from said HIC-HPLC; wherein for elution of each antibody in the mixture, the chromatograph shows a peak that does not overlap with other peaks in the chromatograph.

12. The method of any one of claims 1 to 10, wherein each antibody in the mixture is directed against a spike protein of a virus.

13. 13. The method of claim 12, wherein the spike protein is an Ebola virus or coronavirus spike protein.

14. 12. The method of claim 11, wherein the viral infection is an Ebola virus infection, a coronavirus infection, or a Zika virus infection.

15. The method of claim 11 or 14, wherein the combination composition comprises two or three antibodies.

16. The method of claim 15, wherein the antibody is a human monoclonal antibody.

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