WO2014121031A1 - Caractérisation de mélanges d'anticorps par spectrométrie de masse - Google Patents

Caractérisation de mélanges d'anticorps par spectrométrie de masse Download PDF

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Publication number
WO2014121031A1
WO2014121031A1 PCT/US2014/014074 US2014014074W WO2014121031A1 WO 2014121031 A1 WO2014121031 A1 WO 2014121031A1 US 2014014074 W US2014014074 W US 2014014074W WO 2014121031 A1 WO2014121031 A1 WO 2014121031A1
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Prior art keywords
antibody
mass
antibodies
mixture
mass spectrometry
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PCT/US2014/014074
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English (en)
Inventor
Vincent W. Coljee
Sergey SHULGA-MORSKOY
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Excelimmune, Inc.
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Publication of WO2014121031A1 publication Critical patent/WO2014121031A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • Multi-component therapies based on recombinant antibodies represent promising new drugs for the treatment of various diseases and disorders including infectious diseases, cancer, neurological disorders, inflammation and immune disorders, and cardiovascular diseases because they simultaneously target multiple epitopes and, therefore, decrease the selective pressure for the development of resistant strains.
  • the effectiveness of these therapies is derived, in part, through the heterogeneity of their individual components. This heterogeneity, however, present obstacles to the thorough characterization of individual antibodies in the multi-component mixtures. Thorough characterization is necessary to document and ensure standardized production, as well as to ensure safe production and therapeutic use.
  • MS mass spectrometry
  • Bottom-up analysis starts typically with an enzymatic, or alternatively chemical, digestion of the intact protein into smaller peptides followed by separation and analysis of these peptides.
  • Top-down analysis performs MS analysis directly on the level of the intact protein.
  • Bottom-up analysis currently is the technology of choice for rapid protein identification and quantification of large numbers of proteins because of the relatively easy handling of peptides. Peptide separation can easily be achieved on a chromatographic level. However, there are intrinsic limitations in this technique which arise from the so-called "protein inference problem".
  • Bottom-up MS analysis identifies the peptides, not the proteins. The proteins from which these peptides originate are assigned solely based on statistics. This approach also means that mutations in or modifications on the protein sequence can be assigned only if they are explicitly observed. No statement is possible on non-observed deviations from the expected sequence.
  • Embodiments of the present invention are based on the insight that the limitations of bottom-up MS analysis are particularly acute if applied to mixtures of antibodies (i.e., polyclonal antibody compositions).
  • the present invention provides effective methods for accurate and comprehensive characterization of mixtures of intact or reduced antibodies based on top-down approaches of mass spectrometry analysis. Characterizations provided by embodiments of the invention include intact mass, amino acid sequence, and post- translational modification including glycosylation form distribution.
  • the present invention is based on the surprising discovery that heterogeneous mixtures of antibody populations can be characterized by mass spectrometry without any digestion step or pre-treatment of the antibodies. This allows for an improved, simpler, more efficient, less expensive and more accurate way to analyze antibody populations through mass spectrometry.
  • Embodiments of the present invention require reliable mass spectrometry techniques with superior resolution and mass accuracy, optionally paired with chromatography (e.g., HPLC). Mass
  • the spectrometers useful in the present invention can include a triple quadrupole, an Orbitrap type MS, an ESI-TOF, a Q-TOF, a TOF, an ion trap or any other instrument of suitable sensitivity and mass resolution.
  • a Q Exactive Orbitrap MS system with appropriate data analysis is used.
  • the present invention provides a method for characterizing an antibody mixture including a step of subjecting the antibody mixture to mass spectrometry analysis, wherein the antibody mixture comprises a plurality of distinct monoclonal antibody populations.
  • the antibody mixture comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 distinct monoclonal antibody populations.
  • an inventive method according to the invention further comprises evaluation of the distribution of molecular masses, post-translational
  • the evaluation step comprises determining the molecular mass of each individual monoclonal antibody population. In some embodiments, the evaluation step comprises determining the charge distribution and/or glycosylation pattern of each individual monoclonal antibody population. In some embodiments, the evaluation step comprises determining the amino acid sequence of the light chain and/or heavy chain of each individual monoclonal antibody population.
  • the evaluation step comprises determining the percentage of each individual monoclonal antibody population within the antibody mixture. In some embodiments, the evaluation step comprises determining the ratio(s) between distinct monoclonal antibody populations. In some embodiments, the evaluation step comprises determining the presence and/or amount of aggregated form of monoclonal antibodies (e.g., high molecular weight species). In some embodiments, the evaluation step comprises determining the purity of antibody mixture based on the presence and/or amount of aggregated form of monoclonal antibodies (e.g., high molecular weight species).
  • each individual monoclonal antibody population comprises intact antibodies, F(ab')2, F(ab)2, Fab', Fab, ScFvs, diabodies, triabodies and/or tetrabodies.
  • each individual monoclonal antibody population comprises intact antibodies (e.g., IgGl).
  • the mass spectrometry analysis involves using a three- dimentional quadrupole ion trap mass spectrometer, a linear quadrupole ion trap mass spectrometer, or an Orbitrap mass spectrometer. In some embodiments, the mass
  • an inventive method according to the present invention further includes a step of determining if the antibody mixture satisfies a pre-determined quality standard by comparing the mass spectrometry analysis result to a control.
  • the present invention provides a method of manufacturing an antibody mixture comprising cultivating a polyclonal cell population comprising a plurality of distinct sub-populations, each of which expresses a distinct monoclonal antibody population, to produce an antibody mixture; and characterizing the quality of the antibody mixture using a method described herein.
  • the characterizing step is conducted before releasing a lot.
  • a method according to the invention further includes a step of adjusting a manufacturing condition based on the characterization of the antibody mixture.
  • pre-treatment of the antibody sample is not required before analysis.
  • pre-treatment comprises denaturing the antibodies.
  • pre-treatment comprises reduction of the antibody sample.
  • pre-treatment comprises digestion of the antibody sample.
  • digestion comprises enzymatic proteolysis.
  • the present invention also provides an antibody mixture manufactured using a method described herein.
  • an antibody refers to a polypeptide that specifically binds to an epitope or antigen.
  • an antibody is a polypeptide whose amino acid sequence includes elements characteristic of an antibody -binding region (e.g., an antibody light chain or variable region or one or more complementarity determining regions ("CDRs") thereof, or an antibody heavy chain or variable region or one more CDRs thereof, optionally in presence of one or more framework regions).
  • an antibody is or comprises a full-length antibody.
  • an antibody is less than full-length but includes at least one binding site (comprising at least one, and preferably at least two sequences with structure of known antibody “variable regions”).
  • the term “antibody” encompasses any protein having a binding domain, which is homologous or largely homologous to an immunoglobulin-binding domain.
  • an included “antibody” encompasses polypeptides having a binding domain that shows at least 99% identity with an immunoglobulin binding domain.
  • an "antibody” is any protein having a binding domain that shows at least 70%, 80%, 85%, 90%, or 95% identity with an immunoglobulin binding domain, for example a reference immunoglobulin binding domain.
  • an included “antibody” may have an amino acid sequence identical to that of an antibody that is found in a natural source.
  • antibodies may be prepared by any available means including, for example, isolation from a natural source, recombinant production in or with a host system, chemical synthesis, etc., or combinations thereof.
  • An antibody may be monoclonal or polyclonal, mono-specific or bi-specific.
  • An antibody may be a member of any
  • immunoglobulin class including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • Antibodies may be chimeric or humanized mouse monoclonal antibodies.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • an antibody may be a human antibody or recombinant human antibody.
  • the term "antibody” includes any derivative of an antibody that possesses the ability to bind to an epitope of interest.
  • the "antibody” is an antibody fragment that retains at least a significant portion of the full-length antibody's specific binding ability.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
  • an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages.
  • Antigen refers to a molecule or entity to which an antibody binds.
  • an antigen is or comprises a polypeptide or portion thereof.
  • an antigen is a portion of an infectious agent that is recognized by antibodies.
  • an antigen is an agent that elicits an immune response; and/or (ii) an agent that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody (e.g., produced by a B cell) when exposed or administered to an organism.
  • an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism.
  • a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism species.
  • an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a target organism species.
  • an antigen binds to an antibody and/or T cell receptor, and may or may not induce a particular physiological response in an organism.
  • an antigen may bind to an antibody and/or to a T cell receptor in vitro, whether or not such an interaction occurs in vivo.
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer [in some embodiments other than a biologic polymer (e.g., other than a nucleic acid or amino acid polymer)] etc.
  • an antigen is or comprises a polypeptide.
  • an antigen is or comprises a glycan.
  • an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source).
  • antigens utilized in accordance with the present invention are provided in a crude form.
  • an antigen is or comprises a recombinant antigen.
  • Characteristic portion As used herein, the term a "characteristic portion" of a substance, in the broadest sense, is one that shares some degree of sequence or structural identity with respect to the whole substance. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance.
  • a "characteristic portion" of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids.
  • a characteristic portion of a substance is one that, in addition to the sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance; epitope-binding specificity is one example.
  • a characteristic portion may be biologically active.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3 ' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • a biological molecule may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).
  • Functional equivalent or derivative denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
  • a functional derivative or equivalent may be a natural derivative or is prepared synthetically.
  • Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
  • the substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.
  • the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
  • isolated cell refers to a cell not contained in a multi-cellular organism.
  • pharmaceutically acceptable refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Polypeptide refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified.
  • Protein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.
  • Specific binding refers to an interaction (typically non-covalent) between a target entity (e.g., a target protein or polypeptide) and a binding agent (e.g., an antibody, such as a provided antibody).
  • a target entity e.g., a target protein or polypeptide
  • a binding agent e.g., an antibody, such as a provided antibody.
  • an interaction is considered to be “specific” if it is favored in the presence of alternative interactions.
  • an interaction is typically dependent upon the presence of a particular structural feature of the target molecule such as an antigenic determinant or epitope recognized by the binding molecule.
  • an antibody is specific for epitope A
  • the presence of a polypeptide containing epitope A or the presence of free unlabeled A in a reaction containing both free labeled A and the antibody thereto will reduce the amount of labeled A that binds to the antibody.
  • specificity need not be absolute.
  • numerous antibodies cross-react with other epitopes in addition to those present in the target molecule. Such cross-reactivity may be acceptable depending upon the application for which the antibody is to be used.
  • Specificity may be evaluated in the context of additional factors such as the affinity of the binding molecule for the target molecule versus the affinity of the binding molecule for other targets (e.g., competitors).
  • a binding molecule exhibits a high affinity for a target molecule that it is desired to detect and low affinity for non-target molecules, the antibody will likely be an acceptable reagent for immunodiagnostic purposes.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Figure 1 shows exemplary MS spectra for antibody mixture having three antibodies
  • Figure 2 depicts exemplary results illustrating deconvoluted molecular mass for each antibody in the mixture having three antibodies (i.e., 42.11.D4, 26.3.E2, and 5.6.H9).
  • A Exemplary protein deconvolution spectra obtained using ReSpect algorithm and separately showing the molecular mass for each of the three antibodies in the mixture (shown by arrows).
  • B Table showing exemplary results obtained using ReSpect algorithm.
  • Figure 3 depicts exemplary spectrum of the light chain for antibodies in the mixture having three antibodies (i.e., 42.11.D4, 26.3.E2, and 5.6.H9).
  • A Exemplary raw spectrum for the MS analysis of the light chain of the antibodies in the mixture with three antibodies.
  • B Exemplary protein deconvolution spectra obtained using ReSpect algorithm and showing molecular mass of the light chain of antibodies in the mixture (shown by ovals).
  • Figure 4 shows exemplary MS spectra for antibody mixture having five antibodies
  • Figure 5 depicts exemplary results illustrating deconvoluted molecular mass for each antibody in the mixture having five antibodies (i.e., 42.1 1.D4, 26.3. E2, 5.6.H9, 5.55.D2, and 42.18. E12).
  • A Exemplary protein deconvolution spectra obtained using ReSpect algorithm and separately showing the molecular mass for each of the five antibodies in the mixture (shown by ovals).
  • B Table showing exemplary results obtained using ReSpect algorithm.
  • Figure 6 depicts exemplary spectrum of the light chain for antibodies in the mixture having five antibodies (i.e., 42.11.D4, 26.3.E2, 5.6.H9, 5.55.D2, and 42.18.E12).
  • A Exemplary raw spectrum for the MS analysis of the light chain of the antibodies in the mixture with five antibodies.
  • B Exemplary protein deconvolution spectra obtained using ReSpect algorithm and showing molecular mass of the light chain of antibodies in the mixture (shown by ovals).
  • Figure 7 depicts the exemplary spectra for quantitation of an intact antibody (antibody
  • Figure 8 depicts processing of the quantitation for an intact antibody using a bench- top Q Extractive Quadrupole Orbitrap Mass Spectrometer.
  • A Depicts an exemplary screenshot for the processing method performed on antibody 5.55.D2 using a bench-top Q Extractive Quadrupole Orbitrap Mass Spectrometer.
  • B Depicts a calibration curve for antibody 5.55.D2 between concentrations 12.5 ng and 500 ng.
  • C Table showing exemplary quantitation results for an intact antibody at a concentration range of 12.5 ng to 500 ng.
  • Figure 9 depicts exemplary zoomed-in images of the raw spectra for isotopically resolved light chains for four separate antibodies carried in a top-down characterization.
  • A Depicts the spectrum for intact 26.3.E2 light chain.
  • B Depicts the spectrum for intact 5.6.H9 light chain.
  • C Depicts the spectrum for intact 42.18.E12 light chain.
  • D Depicts the spectrum for intact 5.55.D2 light chain.
  • Figure 10 depicts the results of light chain characterization of antibody 26.3. E2 using the top-down approach.
  • A Exemplary raw spectrum of the MS analysis of the light chain antibody 26.3. E2.
  • B Depicts the location of post-translational modifications based upon observed and theoretical masses obtained for the light chain.
  • Figure 11 depicts the results of light chain characterization of antibody 5.6.H9 using the top-down approach.
  • A Exemplary raw spectrum of the MS analysis of the light chain antibody 5.6.H9.
  • B Depicts the location of post-translational modifications based upon observed and theoretical masses obtained for the light chain.
  • Figure 12 depicts the results of light chain characterization of antibody 42.18.E12 using the top-down approach.
  • A Exemplary raw spectrum of the MS analysis of the light chain antibody 42.18.E12.
  • B Depicts the location of post-translational modifications based upon observed and theoretical masses obtained for the light chain.
  • Figure 13 depicts the results of light chain characterization of antibody 5.55.D2 using the top-down approach.
  • A Exemplary raw spectrum of the MS analysis of the light chain antibody 5.55.D2.
  • B Depicts the location of post-translational modifications based upon observed and theoretical masses obtained for the light chain.
  • the present invention provides, among other things, a method for characterizing an antibody mixture including a step of subjecting the antibody mixture to mass spectrometry analysis, wherein the antibody mixture comprises a plurality of distinct monoclonal antibody populations.
  • the present invention also provides methods for characterizing antibody mixtures through mass spectrometry without any treatment or digestion of the mixture prior to spectrometric analysis. Characterization can include detections of micro-heterogeneity among antibodies including post-translational modifications such as glycosylation.
  • Particular embodiments of the invention comprise various combinations of liquid chromatography, mass spectrometry and data processing (i.e., protein deconvolution) to characterize, identify and quantify complex antibody mixtures.
  • Antibody Mixtures i.e., protein deconvolution
  • Antibody mixtures for use in embodiments of the invention may include any immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives of antibodies that maintain specific binding ability may also be included.
  • Antibody mixtures as described herein may contain any protein having a binding domain which is homologous or largely homologous to an immunoglobulin- binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced.
  • An antibody in the mixtures may be monoclonal or polyclonal.
  • the antibody mixtures comprises mixtures of monoclonal antibodies directed to a particular antigen or for treatment of a particular disease.
  • Antibody mixtures may comprise members any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • an antibody may be a member of the IgG immunoglobulin class.
  • the antibodies for analysis can be composed of one or more different antibody subclasses or isotypes, such as human isotypes IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, or isotypes from other species such as murine isotypes IgGl, IgG2a, IgG2b, IgG3, and IgA.
  • Antibody mixtures as described herein may comprise antibody fragments or characteristic portions of an antibody, which are used interchangeably and refer to any derivative of an antibody which is less than full-length.
  • antibody fragments in antibody mixtures of some embodiments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
  • Such fragments may be produced by any means.
  • an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence.
  • an antibody fragment may be wholly or partially synthetically produced.
  • An antibody fragment may optionally comprise a single chain antibody fragment.
  • an antibody fragment may comprise multiple chains which are linked together, for example, by disulfide linkages.
  • An antibody fragment may optionally comprise a multimolecular complex.
  • an antibody mixture refers to a composition that contains a plurality of distinct antibody (e.g., monoclonal antibody) populations.
  • An antibody mixture may contain any number of distinct monoclonal antibody populations.
  • an antibody mixture may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more distinct monoclonal antibody populations.
  • An antibody mixture may comprise polyclonal antibodies.
  • an antibody mixture comprises one or more human or humanized antibodies.
  • the antibody mixtures comprises one or more bispecific antibodies.
  • an antibody mixture may be capable of binding one, two, three, four, five or more different epitopes.
  • the antibody mixture comprises two or more antibodies that share a common light chain.
  • the antibody mixture comprises two or more antibodies that share a common heavy chain.
  • Mixtures of different antibodies can target different epitopes of the same antigen or different antigens involved in the same disease resulting in a synergistic effect with increased efficacy and potency.
  • the co-expression of mixtures of recombinant monoclonal antibodies have improved pharmacodynamic properties and greater cost reductions.
  • the present invention may be used to detect modifications to antibodies such as post-translational modifications including glycosylation. Additional modifications that can be detected on antibodies are: disulfide pairings, N- and C- terminal modifications such as pyroGlu, Lys and Gly clippings.
  • the present invention can be used to characterize glycosylated antibodies through the use of mass spectrometry without prior digestion or pre-treatment.
  • the methods of the present invention can be used to characterize glycosylated antibodies without proteolytic digestion through the use of mass spectrometers such as ion trap and Orbitrap MS.
  • the present invention may be used to detect other modifications including but not limited to isomerization, oxidation, deamidation, and N- and C- terminal variants.
  • the present invention further relates to methods for characterizing populations of different antibody species in recombinant polyclonal compositions.
  • the methods of the present invention are useful for quantitative analysis and can be used, for example, to analyze consistency between antibody mixes as well as to assess the compositional stability during manufacturing runs and to determine whether a certain mix meets certain standards.
  • the methods of the present invention can also be used to select for clones to create a polyclonal cell bank comprising clones that generate specific preferred antibodies.
  • the invention provides for methods for detecting variations between different populations of antibodies in recombinant polyclonal antibody compositions.
  • the present invention relates to methods for simultaneously analyzing the in vivo clearance of individual antibodies constituting a recombinant polyclonal
  • the methods of the present invention can also be used to characterize the polyclonality in a therapeutic/drug product for treatment. In some embodiments, the methods of the present invention are used to quantitate one or more recombinant antibodies in in-process samples.
  • polyclonal antibodies may be produced by injecting a host animal such as rabbit, rat, goat, mouse or other animal with an immunogen of choice. The sera are extracted from the host animal and are screened to obtain polyclonal antibodies which are specific to the immunogen. Methods of screening for polyclonal antibodies are well known to those of ordinary skill in the art such as those disclosed in Harlow & Lane, ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY: 1988), incorporated herein by reference.
  • specific monoclonal antibodies may be separately produced by conventional recombinant or hybridoma technologies, purified, and then combined into an antibody mixture.
  • recombinant antibodies may be produced by cloning cDNA or genomic DNA encoding the immunoglobulin light and heavy chains of the desired antibody from a hybridoma or immune cell (e.g., plasma cells or B cells) that encodes, expresses or produces an antibody of interest.
  • the cDNA or genomic DNA encoding those polypeptides is then inserted into recombinant expression vectors so that both genes are operatively linked to their own regulatory sequences, for control of transcription and translation.
  • the expression vector and the regulatory sequences for control of expression are chosen to be compatible for expression in the selected host cell.
  • both the heavy and light chain genes are inserted into the same expression vector so that expression of both is operatively linked.
  • Prokaryotic or eukaryotic cells may be used as expression hosts. Expression in eukaryotic host cells (e.g., yeast cells, CHO cells, HEK-293 cells) is preferred because such cells are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibody.
  • an antibody mixture may be produced by cultivating a polyclonal cell population comprising a plurality of distinct sub-populations, each of which expresses a distinct monoclonal antibody population.
  • an antibody mixture may be produced by cultivating a stable polyclonal cell population expressing multiple recombinant antibodies without requiring clonal selection (i.e., the use of a selection marker to select an individual cell clone— the progenitor cell— that is then propagated as an individual cell line).
  • stable cell populations expressing multiple recombinant antibodies can be generated from large numbers of individual subpopulations transformed with vectors encoding distinct antibody or components of an antibody (e.g., light chain and/or heavy chain of an antibody).
  • a polyclonal cell population can be generated by first generating individual cell populations that are stable and then mixing the stable individual cell populations. Stable polyclonal cell populations can be generated when sufficient numbers of individual cells are used to produce a cell population. It is contemplated herein that when cell populations with greater than 10,000 individual cells are mixed, the combined cell population yields a stable polyclonal cell population to produce a desired antibody mixture.
  • polyclonal antibody mixtures are produced through development of a mixture of monoclonal antibodies through a single production platform (such as a single batch or single production vessel) followed by structural/functional characterization of the mixture.
  • Recombinant polyclonal antibody compositions can be obtained from a single polyclonal cell culture at different time points during cultivation.
  • Recombinant polyclonal antibody compositions can also be obtained from different polyclonal cell cultures at different time points. The use of single production runs to generate antibody mixtures reduces costs and saves production time.
  • individual recombinant monoclonal antibody producer cell lines are generated (Rasmussen et al. BIOTECHNOL. LETT., 2007 29:845-852). These cell lines can be collectively drawn upon as a library stock. Selected cell lines can then be mixed to create a polyclonal cell culture used for generation of different target specific recombinant polyclonal antibodies.
  • mammalian cells are a preferred expression host for recombinant antibody production. Use of mammalian cells allows for proper protein folding, assembly, and post-translational modifications such as glycosylation.
  • Mammalian production cells for generating recombinant polyclonal antibodies include, but is not limited to, Chinese hamster ovary cells lines (CHO) but also mouse myeloma cell lines (SP2/0 and NSO) and human cell lines (embryonic kidney cells (HEK-293) and retinal cells (Per.C6)).
  • the polyclonality of the resulting antibody mixture can be assessed using assays specific for the variable region as a complement to the mass spectrometry techniques of the present invention.
  • stable cell populations are obtained by transforming a population of host cells using preferential integration of at least one vector comprising at least one copy of a nucleic acid sequence encoding an antibody or a component thereof into the host cell genomic DNA to generate large populations of individual transformed cells.
  • preferential integration can be used to produce cell populations expressing multiple recombinant antibodies. Exemplary vectors for preferential integration are described in in U.S. Patent No. 8,617,881, which is incorporated by reference herein in its entirety.
  • cell line refers to a population of cells derived from a single progenitor cell. Persons skilled in the art would understand that because cells contained in a given cell line are derived from a single progenitor cell that each of the cells in the cell line, at least initially, share the same genomic characteristics (see, e.g., Poste et ah, PNAS 1981 78:6226-6230).
  • a cell line is typically generated by selecting for an individual progenitor cell clone (e.g., using a selection marker, e.g., an antibiotic selection marker, e.g., neomycin, blasticidin, puromycin, or hygromycin). The individual progenitor cell clone is then cultured to expand the cell line capable of producing a gene product (e.g., antibody) of interest.
  • a selection marker e.g., an antibiotic selection marker, e.g., neomycin, blasticidin, puromycin, or hygromycin.
  • cell population refers to a population of cells that share a common genetic background, but are not genetically identical.
  • a DNA fragment of interest is integrated into a plurality of cells, where the integration site in each cell may be at one or more locations in the genome, creating cells in the population that are not genetically identical.
  • a cell population differs from a cell line because the cell population is not derived from an individual protein cell and/or is not comprised of cells with the same genetic content.
  • the cell populations described herein there is no selection process to identify a single progenitor cell followed by clonal expansion of the single progenitor cell. Rather the cell populations described herein are a mixture of cells where each cell in the mixture may have the DNA integrated in one or more locations in the genome. Stability of the cell population expressing a single recombinant polypeptide is achieved by population dynamics.
  • the cell population, cell mixture or transformed cell populations described herein may be a polyclonal, e.g., it expresses multiple recombinant antibodies.
  • a polyclonal cell population more than one DNA fragment of interest is integrated into a plurality of cells, wherein the integration site in each cell may be at one or more locations in the genome, creating cells that are not genetically identical.
  • a polyclonal cell population as described herein may be generated by mixing two or more monoclonal cell populations.
  • a polyclonal cell population maybe generated by a bulk transformation procedure wherein a plurality of DNA fragments of interest is integrated into a plurality of host cells (e.g., one DNA fragment of interest per host cell).
  • a polyclonal cell population as described herein does not require a selection process to identify individual progenitor cells followed by clonal expansion of individual cell lines (e.g., a polyclonal cell population is not a mixture of individual cell lines each derived from an individual progenitor cell). Stability of the polyclonal cell population is achieved by population dynamics.
  • transformation refers to any method for introducing foreign
  • transformation is a broad term that includes methods for introducing foreign DNA into a cell including transfection, infection, transduction or fusion of a donor cell and an acceptor cell.
  • antibody mixtures are produced using random integration of antibody-encoding nucleic acid vectors into host cell genomes.
  • random integration refers to the process of integrating a DNA fragment of interest (e.g., a partial or complete DNA encoding a protein of interest) into the genome of a cell, where the fragment of DNA can be integrated in any part of that genome with equal probability.
  • random integration refers to a transformation procedure where nothing is done to guide the expression construct to a predetermined position in a host cell genome.
  • random integration refers to an integration process which is performed naturally by the host cell machinery without the aid of extraneously added sequences or enzymes that affect the natural integration site (H. Wurtetle, Gene Therapy, 2003, 10: 1791-1799).
  • recombinant antibodies are produced by site-specific integration of nucleic acid vectors encoding the antibodies into genomes of host cells.
  • site-specific integration refers to the process of integrating a DNA fragment of interest (e.g., a partial or complete DNA encoding a protein of interest) into the genome of a cell, where the DNA fragment of interest is targeted to a specific sequence in that genome.
  • the specific target sequence can occur naturally in the genome or may be engineered into the genome of the host cell (e.g., engineering of a FRT-site into the genome of a cell for Flp recombinase mediated integration).
  • site-specific integration is the use of Flp recombinase to target integration of a DNA fragment of interest to a specific site in a host cell genome.
  • the specific site of integration occurs at a DNA sequence known as the FRT site.
  • FLP is an exception among integrases in that it is highly specific to FRT sites.
  • Site specific integration of DNA to the FRT site can be achieved by including FRT site DNA sequence in the DNA fragment for targeted integration.
  • FRT sites do not occur naturally in most genomes, typically, a FRT site must be integrated into the genome of a host cell of interest before introduction of the DNA fragment of interest.
  • recombinant antibodies are produced by preferential integration of nucleic acid vectors encoding the antibodies into genomes of host cells.
  • the term "preferential integration" as used herein refers to the process of integrating a DNA fragment of interest (e.g., a partial or complete DNA encoding a protein of interest) into the genome of a cell, where the fragment of DNA is targeted to a predetermined region of the genome (but not to a single defined site, e.g., a FRT site).
  • preferential integration is not a random integration event because there is an increased probability that the DNA of interest will integrate into a defined region or specific site in the host cell genome.
  • Preferential integration also differs from the site- specific integration because there is variability in the integration site.
  • Examples of preferential integration include integration using an AAV system by the
  • ITR inverted terminal repeat
  • RTS rep targeting sequence
  • AAVS l site located on chromosome 19 with an efficiency of -10%, with the remaining 90% spread over other RTS sites across the human genome (as described in e.g., Smith RH, GENE THERAPY, 2008, 15:817-822 and Huser et al, PLoS PATHOG, 6(7): el000985.
  • preferential integration examples include use of a retroviral vector system, wherein the retroviral vector integrates into open chromatin domains which encompass about 5% of the total genome; use of a phage integrase ⁇ C3 lor lambda integrase, which carries out recombination between the attP site and the attB site (A.C.
  • the methods and systems of the present invention comprise liquid chromatography (LC) in combination with mass spectrometry and data processing (i.e., protein deconvolution) to characterize, identify and quantify complex antibody mixtures.
  • LC may be used for one or more of desalting, separation of antibody mixtures and/or separation of light and heavy chains.
  • the antibody mixture is not subjected to enzymatic digestion before or after chromatographic separation.
  • the present invention comprises quantitative liquid chromatography tandem mass spectrometry (LC/MS/MS).
  • two-dimensional or tandem LC is used.
  • Particular embodiments of the invention comprise additional steps of liquid-liquid extractions, dialysis, sample dilution, and/or sample dehydration steps prior to analysis by mass spectrometry.
  • chromatography refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.
  • liquid chromatography means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • Liquid chromatography includes reverse phase liquid chromatography (RPLC), high performance liquid
  • HPLC high performance liquid chromatography
  • FPLC fast performance liquid chromatography
  • LPLC low pressure liquid chromatography
  • HTLC high turbulence liquid chromatography
  • HPLC high performance liquid chromatography
  • FPLC fast performance/protein liquid chromatography
  • LPLC low pressure liquid chromatography
  • mixtures of intact antibodies or individual antibodies may be reduced prior to chromatographic separation.
  • samples may be incubated for an appropriate time (e.g., about one hour) under appropriate conditions (e.g., temperature of about 60 °C) in 6 M guanidine-HCL containing 5 mM DDT.
  • appropriate conditions e.g., temperature of about 60 °C
  • mixtures of intact antibodies or individual antibodies may be reduced by treatment with urea prior to chromatographic separation.
  • the chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation).
  • the medium may include minute particles.
  • the particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties such as the biomarker analytes quantified in the experiments herein.
  • One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface.
  • Alkyl bonded surfaces may include C-4, C- 8, or C- 18 bonded alkyl groups.
  • the chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample.
  • a sample may be applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port.
  • Different solvent modes may be selected for eluting different analytes of interest.
  • liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode.
  • the column is heated. In particular embodiments, the column is heated to about 80 °C during analysis.
  • Embodiments of the invention are not limited by the type or number of LC steps used before mass spectrometry. Two or more of the LC techniques described herein may be combined before mass spectrometry (i.e., LC/LC/MS). Additional protein purification techniques may be employed prior to application of the antibody mixture to the LC column.
  • RP-HPLC reversed phase HPLC
  • the stationary phase is a silica which has been surface-modified with RMe 2 SiCl, where R is a straight chain alkyl group such as Ci 8 H 37 or CsHn.
  • Retention times can be increased by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase.
  • retention time can be decreased by adding more organic solvent to the eluent. For example, retention can be decreased by adding a less polar solvent
  • analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure.
  • analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO- or -NH3+ in their structure) are less retained as they are better integrated into water.
  • Such interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention.
  • mobile phase surface tension organizational strength in eluent structure
  • other mobile phase modifiers can affect analyte retention.
  • the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca.
  • buffering agent such as sodium phosphate
  • Buffers serve multiple purposes: control of pH, neutralize the charge on the silica surface of the stationary phase and act as ion pairing agents to neutralize analyte charge.
  • Ammonium formate may be used to improve detection of certain analytes by the formation of analyte-ammonium adducts.
  • a volatile organic acid such as acetic acid, or most commonly formic acid (FA), may be added to the mobile.
  • Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but may be used in some embodiments as it a fairly strong organic acid and can be effective in improving retention of analytes such as carboxylic acids.
  • Trifluoroacetic acid Those of skill in the art will appreciate that the effects of acids and buffers vary by application but generally improve chromatographic resolution.
  • the mobile phase comprises 0.1-0.5% formic acid (FA). In particular embodiments, the mobile phase comprises 0.1% FA. In some embodiments, the mobile phase comprises acetonitrile. In some embodiments, the mobile phase comprises acetonitrile and FA. In specific embodiments, the mobile phase comprises 0.1% FA and acetonitrile. In additional embodiments, the mobile phase further comprises 0.1-0.5% trifluoroacetic acid (TFA).
  • FFA formic acid
  • the mobile phase comprises 0.1% FA.
  • the mobile phase comprises acetonitrile.
  • the mobile phase comprises acetonitrile and FA. In specific embodiments, the mobile phase comprises 0.1% FA and acetonitrile. In additional embodiments, the mobile phase further comprises 0.1-0.5% trifluoroacetic acid (TFA).
  • FFA trifluoroacetic acid
  • the flow rate of the mobile phase is between 50-500 ⁇ . In particular embodiments, the flow rate is about 60 ⁇ 7 ⁇ , about 100 ⁇ 7 ⁇ , about 150 ⁇ 7 ⁇ , about 200 ⁇ 7 ⁇ , about 250 ⁇ / ⁇ , about 300 ⁇ 7 ⁇ , about 350 ⁇ 7 ⁇ , about 400 ⁇ / ⁇ , about 450 ⁇ / ⁇ , or about 500 ⁇ / ⁇ .
  • Size exclusion chromatography may be applied to separate antibody mixtures under native conditions. It has been routinely used for the characterization and quality control of monoclonal antibody therapeutics in the pharmaceutical industry (Analysis of Reduced Monoclonal Antibodies Using Size Exclusion Chromatography Coupled with Mass Spectrometry, J. AM. SOC. MASS SPECTROM. 20, 2258-2264 (2009)). However, with normal SEC salt containing mobile phases, direct mass spectrometry analysis of antibody fragments is not feasible. Thus, volatile mobile phases should be optimized for the separation of antibodies. In particular embodiments, TFA concentrations are reduced to lower than 0.05%. In particular embodiments, the acetonitrile concentration 10-30%. In specific embodiments, the mobile phase comprises 0.02% TFA, 1% FA and 20% acetonitrile.
  • Ion-exchange chromatography employs anion-exchange and cation-exchange.
  • Ion-exchange chromatography relies on the affinity of a substance for the exchanger, which affinity depends on both the electrical properties of the material and the relative affinity of other charged substances in the solvent. Bound material can be eluted by changing the pH, thus altering the charge of the material, or by adding competing materials, of which salts are but one example.
  • the principle of ion- exchange chromatography is that charged molecules adsorb to ion exchangers reversibly so that molecules can be bound or eluted by changing the ionic environment.
  • Separation on ion exchangers is usually accomplished in two stages: first, the substances to be separated are bound to the exchanger, using conditions that give stable and tight binding; then the column is eluted with buffers of different pH, ionic strength, or composition and the components of the buffer compete with the bound material for the binding sites.
  • An ion exchanger is usually a three-dimensional network or matrix that contains covalently-linked charge groups. If a group is negatively charged, it will exchange positive ions and is a cation exchanger.
  • a typical group used in cation exchangers is the sulfonic group, SO 3 — . If an H+ is bound to the group, the exchanger is said to be in the acid form; it can, for example, exchange on H+ for one Na+ or two H+ for one Ca 2+ .
  • the sulfonic acid group is a strongly acidic cation exchanger. Other commonly used groups are phenolic hydroxyl and carboxyl, both weakly acidic cation exchangers.
  • the charged group is positive— for example, a quaternary amino group— it is a strongly basic anion exchanger.
  • the most common weakly basic anion exchangers are aromatic or aliphatic amino groups.
  • the matrix can be made of various materials. Commonly used materials are dextran, cellulose, agarose and copolymers of styrene and vinylbenzene in which the divinylbenzene both cross-links the polystyrene strands and contains the charged groups.
  • ion-exchangers for use in particular embodiments of the invention include SP- Sephadex, CM-Sephadex, QAE-Sephadex, DEAE-Sephadex, CM-cellulose, P-cel, DEAE- cellulose, PEI-cellulose, DEAE(BND)-cellulose, PAB-cellulose, AG 50, AG 1 -Source 15Q, AG 501, Bio-Rex 70, Toso Haas TSK-Gel-Q-5PW, Bio-Rex 40 and AG-3.
  • the first choice to be made is whether the exchanger is to be anionic or cationic. If the materials to be bound to the column have a single charge (i.e., either plus or minus), the choice is clear. However, many substances (e.g., antibodies), carry both negative and positive charges and the net charge depends on the pH. In such cases, the primary factor is the stability of the substance at various pH values. Most proteins have a pH range of stability (i.e., in which they do not denature) in which they are either positively or negatively charged. Hence, if a protein is stable at pH values above the isoelectric point, an anion exchanger should be used; if stable at values below the isoelectric point, a cation exchanger is required.
  • strong and weak exchangers are also based on the effect of pH on charge and stability. For example, if a weakly ionized substance that requires very low or high pH for ionization is chromatographed, a strong ion exchanger is called for because it functions over the entire pH range. However, if the substance is labile, weak ion exchangers are preferable because strong exchangers are often capable of distorting a molecule so much that the molecule denatures. The pH at which the substance is stable must, of course, be matched to the narrow range of pH in which a particular weak exchanger is charged.
  • Weak ion exchangers are also excellent for the separation of molecules with a high charge from those with a small charge, because the weakly charged ions usually fail to bind. Weak exchangers also show greater resolution of substances if charge differences are very small. If a macromolecule has a very strong charge, it may be impossible to elute from a strong exchanger and a weak exchanger again may be preferable. In general, weak exchangers are more useful than strong exchangers.
  • the density of ionizable groups can be made several times greater than is possible with cellulose ion exchangers.
  • the increased charge density introduces an increased affinity so that adsorption can be carried out at higher ionic strengths.
  • these exchangers retain some of their molecular sieving properties so that sometimes molecular weight differences annul the distribution caused by the charge differences; the molecular sieving effect may also enhance the separation.
  • the cellulose ion exchangers have proved to be the most effective for purifying large molecules such as proteins and polynucleotides. This is because the matrix is fibrous, and hence all functional groups are on the surface and available to even the largest molecules. In many cases, however, beaded forms such as DEAE-Sephacel and DEAE-Biogel P are more useful because there is a better flow rate and the molecular sieving effect aids in separation.
  • Buffers themselves consist of ions, and therefore, they can also exchange, and the pH equilibrium can be affected.
  • the rule of buffers is adopted: use cationic buffers with anion exchangers and anionic buffers with cation exchangers. Because ionic strength is a factor in binding, a buffer should be chosen that has a high buffering capacity so that its ionic strength need not be too high. Furthermore, for best resolution, it has been generally found that the ionic conditions used to apply the sample to the column (starting conditions) should be near those used for eluting the column.
  • Some embodiments of the invention employ hydrophobic interaction chromatography (HIC), in which certain proteins are retained on affinity columns containing hydrophobic spacer arms. Hydrophobic adsorbents including octyl or phenyl groups may be used.
  • Hydrophobic interactions are strong at high solution ionic strength, as such samples being analyzed need not be desalted before application to the adsorbent. Elution is achieved by changing the pH or ionic strength or by modifying the dielectric constant of the eluant using, for instance, ethanediol.
  • cellulose derivatized with additional more hydroxyl groups is used to interact with proteins by hydrogen bonding. Samples may be applied to the matrix in a concentrated (over 50% saturated, >2M) solution of ammonium sulphate. Proteins may be eluted by diluting the ammonium sulphate. This introduces more water which competes with protein for the hydrogen bonding sites.
  • Affinity chromatography may be used in particular embodiments.
  • affinity chromatography techniques include immobilized metal affinity chromatography (IMAC), sulfated affinity chromatography, dye affinity chromatography, and heparin affinity.
  • the chromatographic medium may be prepared using one member of a binding pair, e.g., a receptor/ligand binding pair, or antibody/antigen binding pair (immunoaffinity chromatography).
  • affinity chromatography is used to selectively separate antibodies from a mixture by using protein A or G as a functionality of the stationary phase.
  • the LC separation is performed in-line with a mass spectrometry (MS) analysis.
  • MS mass spectrometry
  • the LC apparatus is directly connected to the mass spectrometer such that the mass spectrometer directly receives the separated product from the LC column.
  • Mass spectrometric techniques can fundamentally be divided into those starting from intact proteins (top-down) and those starting from peptides derived by chemical or, more commonly, enzymatic digestion (bottom up).
  • Digestion refers to any enzymatically or chemically induced proteolysis (i.e., the breakdown of proteins into smaller polypeptides or amino acids by, in general, the hydrolysis of the peptide bonds) of individual antibodies in an antibody mixtures.
  • Some aspects of the invention may utilize a bottom-up approach, in which proteins are enzymatically digested into smaller peptides using a protease such as trypsin.
  • proteins for analysis are enzymatically digested into smaller peptides using proteases such as trypsin.
  • proteases such as trypsin.
  • the digestion is carried out by first denaturing the proteins (with urea or guanidine HCl), reducing the disulfide bonds (with dithiothreitol or mercaptoethanol), alkylating the cytseines (through addition of iodoacetamide) and then digesting with a proteolytic enzyme (typically trypsin).
  • proteins are analyzed after a very limited proteolytic step prior to introduction into the MS instrument.
  • the methods disclosed herein do not require any pre-treatment (i.e., digestion or reduction) prior to mass spectrometric analysis. Stated another way, the methods of the present invention do not require the pre-treatment and/or digestion required for the bottom-up approach to mass spectrometry, regardless of how the distinct antibody populations in the antibody mixture are produced. By removing the steps of pre-treatment and digestion, the methods of the present invention increase efficiency and throughput, reduce cost, and are simpler and more accurate. Less sample handling is involved and the exact masses of all the antibodies of the mixture in their native state can be determined.
  • mass spectrometers useful in the present invention can include a triple quadrupole, an Orbitrap type MS, an ESI- TOF, a Q-TOF, a TOF, an ion trap or any other instrument of suitable sensitivity and mass resolution.
  • Mass spectrometry is an analytical technique used to produce spectra of the masses of the atoms or molecules within a sample.
  • the elemental signature of a sample, the masses of particles (and of molecules), and the chemical structures of molecules (such as peptides and other chemical compounds) can all be determined using the resultant MS spectra.
  • Mass spectrometry ionizes chemical compounds to then generate charged molecules or molecule fragments allowing for the measurement of the mass-to-charge (m/z) ratios.
  • a sample in a solid, liquid, or gaseous form
  • is ionized for example by bombarding it with electrons.
  • the ions are detected by a mechanism capable of detecting charged particles, typically an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio.
  • the atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic
  • a mass spectrometer typically includes an ion source, a mass analyzer that measures m/z of the ionized analytes, and a detector that registers the number of ions at each m/z value. The differences in masses of the fragments allows the mass analyzer to sort the ions by their mass-to-charge (m/z) ratio. The detector measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. Because mass analysis uses electromagnetic fields in a vacuum, typically molecules are first electrically charged and transferred into the gas phase. There is a wide variety of ionization techniques, depending on the phase (solid, liquid, gas) of the sample and the efficiency of various ionization mechanisms for the unknown species.
  • the ion source is the part of the mass spectrometer that ionizes the material under analysis.
  • the ions are then transported to the mass analyzer by electric or magnetic fields.
  • Ionization techniques are key to determining what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with liquid and solid biological samples include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization
  • MALDI Integrated liquid-chromatographic ESI-MS systems
  • ions can be produced using a variety of methods including, but not limited to, electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization ("MALDI”), surface enhanced laser desorption ionization (“SELDI”), photon ionization, electrospray ionization (ESI), and inductively coupled plasma.
  • MALDI matrix-assisted laser desorption ionization
  • SELDI surface enhanced laser desorption ionization
  • ESI electrospray ionization
  • inductively coupled plasma inductively coupled plasma.
  • ESI mass spectrometers are described in further detail in e.g., U.S. Pat. No. 6,673,253 (describing a method of fabricating integrated LC/ESI device and description of ESI mass spectrometry); U.S. Pat. No. 6,642,515 (describing a method and an apparatus for performing electrospray ionization mass spectrometric analysis); U.S. Pat. No. 6,627,883 (describing methods for analyzing samples in a dual ion trap mass spectrometer but provides background description of ESI-MS); U.S. Pat. No. 6,621,075 (describing a device for the delivery of multiple liquid sample streams to a mass spectrometer and provides further guidance of the knowledge of those of skill in the art regarding ESI-MS); and U.S. Pat. No. 6, 188,065
  • ESI is used as the ionization technique.
  • ESI allows the antibody ions to be extracted directly from solution, whose composition may be reasonably close to the "native" environment (e.g., pharmaceutically acceptable preparations) in terms of pH and ionic strength.
  • ESI induces ion formation from small droplets, once the ions are formed they are subject to collisions before entering the mass analyzer. These collisions can decluster aggregates, induce fragmentation, and change the charge states by removing protons.
  • the energy with which the ions enter the mass analyzer through the orifice can determine the amount of fragmentation that will take place. This energy can be adjusted by varying the electrospray declustering or fragmentation potential.
  • ESI-generated biopolymer ions are typically produced as multiply charged ions, and the extent of multiple charging is determined by the physical dimensions of the biopolymer molecule in solution (Kaltashov IA, and Abzalimov RR., "Do ionic charges in ESI MS provide useful information on macromolecular structure? '' J AM SOC MASS SPECTROM., 2008, 19: 1239 ⁇ 16). This feature of ESI MS allows it to be used as a means to probe protein higher order structure and detecting large-scale conformational transitions in solution.
  • ESI ESI
  • Multiple charging makes it possible to observe large proteins with mass analyzers that have a relatively small mass range.
  • observing multiple peaks for the same peptide allows one to make multiple molecular weight calculations from a single spectrum. These values can be averaged to obtain a more accurate molecular weight.
  • Another advantage of generating multiply charged ions with ESI is that multiply charged peptide ions tend to give more complete fragmentation spectra, which is extremely useful in distinguishing between similar species (i.e., various monoclonal antibodies) in a complex mixture (i.e., in a mixture of antibodies).
  • nanoelectrospray ionization is used for further increases in sensitivity.
  • ESI-MS Another important feature of ESI-MS is its ability to directly analyze compounds from aqueous or aqueous/organic solutions.
  • ESI is used with quadrupole or ion trap analyzers, which allows for MS analysis at relatively high LC flow rates (e.g., 0.5 ml/min) and high mass accuracy ( ⁇ 0.01%).
  • ESI is used as part of LC/MS or LC tandem mass spectrometry (LC/MS/MS) techniques.
  • Non-volatile electrolytes (which are present in all biopharmaceutical formulations) have a detrimental effect on the quality of ESI MS data even at relatively low concentrations and must be removed by either dialysis or ultrafiltration before the direct ESI MS
  • Sample preparation is generally achieved by dissolving the sample in a protic volatile solvent system that is relatively homogeneous and contains less than one millimolar concentration of salt, although higher concentrations may be used (as high as 100 mM with NH 4 Ac). However, some salts (alkali and alkaline) and phosphate buffers are more detrimental to signal. Thus, in some embodiments, the antibody mixture is transferred to a volatile electrolyte solution (e.g., ammonium acetate or ammonium bicarbonate).
  • a volatile electrolyte solution e.g., ammonium acetate or ammonium bicarbonate
  • MALDI matrix-assisted laser desorption ionization
  • MALDI refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay.
  • the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.
  • ICP Inductively coupled plasma
  • a plasma flame that is electrically neutral overall, but has a large fraction of its atoms ionized by high temperature, is used to atomize introduced sample molecules and to further strip the outer electrons from those atoms.
  • the plasma is usually generated from argon gas, since the first ionization energy of argon atoms is higher than the first of any other elements except He, O, F and Ne, but lower than the second ionization energy of all except the most electropositive metals.
  • the heating is achieved by a radio-frequency current passed through a coil surrounding the plasma.
  • Ion source techniques include glow discharge, field desorption (FD), fast atom bombardment (FAB), thermospray, desorption/ionization on silicon (DIOS), Direct Analysis in Real Time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), spark ionization and thermal ionization (TIMS).
  • FD field desorption
  • FAB fast atom bombardment
  • DIOS desorption/ionization on silicon
  • DART Direct Analysis in Real Time
  • APCI atmospheric pressure chemical ionization
  • SIMS secondary ion mass spectrometry
  • spark ionization spark ionization and thermal ionization
  • Ion attachment ionization is an ionization technique that allows for fragmentation free analysis.
  • Some embodiments of invention employ fragmentation analysis for top-down amino acid sequence analysis of individual antibodies or individual light or heavy chains present in complex antibody mixtures.
  • intact antibodies are broken into fragments in the gas phase.
  • collision-induced dissociation CID
  • CID collision-induced dissociation
  • analytes are collided in the gas phase with gas atoms or molecules (typically argon or nitrogen).
  • CID is used in combination with TOF or Orbitrap detection technology.
  • high energy CID is used with a unique spectrum multiplexing feature (msx HCD).
  • the multiplexing features refers to a data acquisition mode in which fragment ions produced from several individual HCD events, each on a precursor of a different charge state of an antibody, are detected together in an Orbitrap mass analyzer.
  • ECD electron-capture dissociation
  • ETD electron-transfer dissociation
  • ISD in-source decay fragmentation
  • ECD and ETD techniques are used in combination with ESI MS.
  • ETD is used to fragment antibodies with labile modifications (e.g., glycosylation), while preserving the modification intact on the on the antibody fragment.
  • ETD induces fragmentation all over the protein backbone, which allows top-down fragmentation of intact antibodies in the mixture.
  • ISD on MALDI allows fast straight- forward top-down sequence analysis of undigested proteins based on fragmentation of the entire protein chain caused by hydrogen radical transfer from the MALDI-ISD matrix.
  • the technique provides high mass accuracy and enables fast sequencing of terminal domains of monoclonal antibodies in a targeted way (Ayoub et al, mAbs 20135:5, 699-710).
  • the mass analyzer is typically the central component of a mass spectrometer.
  • Exemplary mass analyzers include ion trap, time-of- flight (TOF), quadrupole and Fourier transform ion cyclotron (FT-MS) analyzers. In some embodiments, these analyzers can be used alone or in tandem to exploit their strengths and minimize any weaknesses. In some embodiments, TOF-MS, or FT-MS is used to analyze an antibody mixture. These two types of instrument typically have wide mass range, and high mass accuracy.
  • the mass resolving power is the measure of the ability to distinguish two peaks of slightly different m/z.
  • the mass accuracy is the ratio of the m/z measurement error to the true m/z. Mass accuracy is usually measured in ppm or milli mass units.
  • the mass range is the range of m/z amenable to analysis by a given analyzer.
  • the linear dynamic range is the range over which ion signal is linear with analyte concentration.
  • Speed refers to the time frame of the experiment and ultimately is used to determine the number of spectra per unit time that can be generated.
  • TOF time-of-flight
  • a typical quadrupole mass analyzer includes four circular rods set parallel to each other, and is responsible for filtering sample ions based on their m/z.
  • a linear series of three quadrupoles can be used to make a triple quadrupole mass spectrometer.
  • the first and third quadrupoles typically act as mass filters, and the middle quadrupole typically acts as a collision cell.
  • a quadrupole ion trap can thus exist in linear and three-dimensional formats and refers to an ion trap that uses constant direct current (DC) and radio frequency (RF) oscillating alternate current (AC) electric fields to trap ions.
  • a combination of a quadrupole mass filter with a TOF analyzer may be used.
  • Quadrupole TOF instruments achieve peptide separation "in space", i.e., the ions are separated nearly instantaneously by passing through either the quadrupole section, in which only a chosen small mass range has stable trajectories, or by traversing the TOF section.
  • an Orbitrap mass analyzer is used.
  • the Orbitrap mass analyzer typically includes a small electrostatic device into which ion peaks are injected at high energies to orbit around a central, spindle-shaped electrode. Image current of the axial motion of the ions is picked up by the detector and its signal is Fourier Transformed (FT) to yield high resolution mass spectra.
  • FT Fourier Transformed
  • a hybrid quadrupole Orbitrap instrument is able to select ions virtually instantaneously due to the fast switching times of quadrupoles and it is able to fragment peptides in High Energy Collision (HCD) mode on a similarly fast time scale.
  • HCD High Energy Collision
  • a combination of a quadrupole mass filter with an Orbitrap analyzer can be used.
  • the detector which records either the charge induced or the current produced when an ion passes by or hits a surface.
  • the signal produced in the detector during the course of the scan versus where the instrument is in the scan will produce a mass spectrum, a record of ions as a function of m/Q.
  • Some type of electron multiplier is used, though other detectors including Faraday cups and ion-to-photon detectors are also used. Because the number of ions leaving the mass analyzer at a particular instant is typically quite small, considerable amplification is often necessary to get a signal. MicroChannel plate detectors are commonly used in modern commercial instruments.
  • the detector In FTMS and Orbitraps, the detector consists of a pair of metal surfaces within the mass analyzer/ion trap region which the ions only pass near as they oscillate. No direct current is produced, only a weak AC image current is produced in a circuit between the electrodes.
  • FTMS Fourier transform mass spectrometry
  • FTMS Fourier transform mass spectrometry
  • a detector such as an electron multiplier
  • the ions are injected into a Penning trap (a static electric/magnetic ion trap) where they effectively form part of a circuit.
  • Detectors at fixed positions in space measure the electrical signal of ions which pass near them over time, producing a periodic signal.
  • Ion cyclotron resonance is an older mass analysis technique similar to FTMS except that ions are detected with a traditional detector. Ions trapped in a Penning trap are excited by an RF electric field until they impact the wall of the trap, where the detector is located. Ions of different mass are resolved according to impact time.
  • Embodiments of the present invention include analysis of antibody mixtures without pre-treatment or enzymatic digestion of the sample using, for example, Orbitrap MS instruments.
  • Orbitrap MS can provide baseline separation of intact half (approximately 75 kDa) and full (approximately 150 kDa) monoclonal antibodies.
  • the antibody signal is condensed into a few ion signals in the mass spectrum, allowing for a simpler and more straightforward interpretation of the results and possibly reducing overlap between adjacent monoclonal antibody species.
  • the high resolving power of the Orbitrap MS allows differentiation between antibodies that are very close in mass. In some embodiments, the high resolving power of the Orbitrap MS can be attributed to more efficient desolvation and less adduct formation.
  • a Q Exactive Orbitrap instrument which couples a quadrupole mass filter to an Orbitrap analyzer.
  • the Q Exactive instrument features high ion currents because of an S-lens, and fast high-energy collision- induced dissociation peptide fragmentation because of parallel filling and detection modes.
  • the image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a higher energy collisional dissociation method.
  • Various features, settings and protocols for this instrument are described in
  • resolution is set at between 17,500 to 140,000 for full MS and 140,000 for top-down tandem MS.
  • the methods disclosed herein can characterize and differentiate between at least two different antibodies in an antibody mixture when the antibodies differ by no more than 0.01% of their total mass (e.g., resolving two monoclonal antibodies differing by 15 Da on a total mass of approximately 150 kDa). In some embodiments, the methods disclosed herein can characterize (i.e., identify and quantify) at least two antibodies in an antibody mixture when the antibodies differ by no more than 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10% of their total mass.
  • the methods disclosed herein can characterize (i.e., identify and quantify) at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen or more individual antibodies in an antibody mixture when the antibodies differ by no more than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.10% of their total mass.
  • the smallest difference between any two antibodies in the mixture is no more than about 10 Da, no more than about 15 Da, no more than about 20 Da, no more than about 25 Da, no more than about 30 Da, no more than about 35 Da, no more than about 40 Da, no more than about 45 Da, or no more than about 50 Da. In some embodiments, the smallest difference between any two antibodies in the antibody mixture is less than about 1.1 kilodalton, less than about 1.0 kilodalton, less than about 900 Da, less than about 800 Da, less than about 700 Da, less than about 600 Da, or less than about 500 Da.
  • the mass accuracy for a given antibody in an antibody mixture is less than about 10 ppm, less than about 9 ppm, less than about 8 ppm, less than about 7 ppm, or less than about 5 ppm.
  • Low mass error i.e., precision
  • the ppm mass error is less than about 5, less than about 5.5, less than about 6.0, less than about 6.5, less than about 7.0, less than about 7.5, less than about 8.0, less than about 8.5, less than about 9.0, less than about 9.5, or less than about 10.0 ppm.
  • Embodiments of the present invention also require consistency. In some
  • the mass difference is less than 10 ppm between instruments; e.g., less than 9 ppm, less than 8 ppm, less than 7 ppm, less than 6 ppm, less than 5 ppm, less than 4 ppm, or less 3 ppm, or less than 2 ppm per instrument.
  • ESI-tandem MS ESI-MS/MS is applied to provide amino acid sequence information of individual antibodies.
  • the individual antibodies are found within complex mixtures of antibodies.
  • the antibodies are reduced.
  • top-down MS/MS is applied using msx HCD.
  • an Orbitrap MS system is used. The combination of msx HCD and Orbitrap MS can provide improved throughput from spectrum multiplexing, as well as advanced signal processing to provide improved resolution and higher Orbitrap scan speeds.
  • amino acid sequence information of antibody light chains is provided. In some such embodiments, sequence coverage of over 40% is achieved, including the N- terminal variable region. In some such embodiments, sequence coverage of over 50%, over 60%, over 70%, over 80%, over 90% and up to 100% is achieved, including the N-terminal variable region. Is some embodiments, the mass error is less than 5 ppm (e.g., 5 ppm, 4 ppm, 3 ppm, etc.) for fragment ions.
  • the methods of the present invention combine mass spectrometry with spectroscopic methods to obtain secondary and tertiary structural information about antibodies.
  • Circular dichroism and fluorescence spectroscopy can be applied to the analysis of protein conformations in solution. Circular dichroism allows for the rapid evaluation of the secondary structure of proteins. Left and right circularly polarized light in the far-UV region is absorbed by the periodic repeated binding angles of the secondary structure elements to differing extents. Alpha-helical secondary structures have a stronger absorption of light compared to beta strands.
  • tandem mass spectrometry refers to two stages of mass analysis that are employed to examine selectively the fragmentation of particular species (e.g., antibodies) in a mixture. See, e.g., de Hoffmann, E., "Tandem Mass Spectrometry: a Primer," J. MASS
  • individual antibodies in an antibody may be characterized (i.e., identified and quantified) based on MS spectrum generated solely from the light chain (i.e., a light chain spectrum).
  • individual antibodies in an antibody may characterized (i.e., identified and quantified) based on MS spectrum generated solely from the heavy chain (i.e., a heavy chain spectrum).
  • individual antibodies in an antibody may be characterized based on MS spectrum generated from both the heavy and light chains.
  • An additional aspect of the present invention are methods to quantitate individual antibodies in a complex antibody mixture. In particular embodiments, this is done by calculating base chromatographs at different concentrations of a given antibody. In some embodiments, at least five, at least six, at least seven, at least eight or more concentrations are used to calculate the base peak chromatographs. In certain embodiments, the base peak chromatograph at each concentration is calculated in triplicate (i.e., 3 different injections). In certain embodiments, the concentrations used for generating the base peak chromatographs ranges between about 10.0 - 500 ng (e.g., base chromatographs are constructed at 12.5 ng, 25 ng, 50 ng, 100 ng, 125 ng, 200 ng, 250 ng and 500 ng). The base peak chromatographs are then used to construct a calibration curve based on peak area. The amount of a given antibody in a test sample can be extrapolated from the calibration curve by comparing the area of the test antibody's characteristic chromatographic peak.
  • Additional embodiments of the invention comprise top-down characterization of intact antibody light chains.
  • intact antibody light chain mass determination is done by MALDI-TOF, ESI-TOF, or ESI in conjunction with FT-based detection (either ICR or Orbitrap).
  • MALDI-TOF MALDI-TOF
  • ESI-TOF ESI-TOF
  • ESI FT-based detection
  • mass accuracy down to a few ppm can be achieved, corresponding to absolute errors of 0-2 Da for intact proteins.
  • resolution of 60,000 to 240,000 can be achieved.
  • the light chain mass measurement accuracy is less than 1.0 ppm, less than 0.9 ppm, less than 0.8 ppm, less than 0.7 ppm, less than 0.6 ppm, less than 0.5 ppm, less than 0.4 ppm or less than 0.3 ppm.
  • Top-down characterization of antibodies as described herein may also be used to identify post-translational modifications.
  • both ESI and MALDI may be applied to glycosylated protein analysis. Glycosylation is one of the most common forms of enzymatic post-translational modification.
  • the analysis begins with a glycoform profiling, where ESI MS or MALDI MS is used to obtain mass distribution of the glycoprotein.
  • Glycan release from glycoproteins can be carried out using peptide N-glycosidase F (PNGase F) and/or PNGase A for N-glycans and chemical methods (e.g., hydrozinolysis) for O-glycans.
  • PNGase F peptide N-glycosidase F
  • PNGase A PNGase A
  • chemical methods e.g., hydrozinolysis
  • Methods of tandem MS/MS, especially electron-based ion fragmentation methods such as electron capture dissociation, ECD, and electron transfer dissociation, ETD
  • ECD electron capture dissociation
  • ETD electron transfer dissociation
  • the high resolving power and mass accuracy of methods described herein allow glycosylation states of individual antibodies in an antibody mixture to be identified without deglycosylation. While a deglycosylated spectrum yields nicely resolved peaks, embodiments of the present invention permit differentiation between the antibody species with the glycans still attached.
  • Full MS spectra of intact or reduced antibodies may be analyzed using protein deconvolution software, which assists in characterization of antibodies from mass spectrometric data.
  • This software produces accurate results, even for low-abundance proteins, enabling detection of small protein modifications with mass shifts of just a few Da, such as when a few disulfide bonds are reduced.
  • protein deconvolution software simplifies or condenses the mass spectrum, making interpretation more
  • a typical protein deconvolution software employs two different deconvolution algorithms, each optimized for a different type of data, to ensure the highest quality results.
  • Two algorithms are Xtract and ReSpect, which are optimized for isotopically resolved and unresolved data, respectively.
  • ReSpect the protein deconvolution software deconvolutes isotopically unresolved data, across a wide mass range of up to 160,000 Da, compatible with fast chromatography.
  • Mass spectra for deconvolution are produced by averaging spectra across the most abundant portion of the elution profile for the antibody. The average spectra are
  • at least 6, at least 7, at least 8, at least 9 or at least 10 consecutive charge states from the input m/z spectrum are used to produce a deconvoluted peak.
  • data processing may be used to identify glycoforms by comparing the masses of antibodies to their expected masses inclusive of the various combinations of commonly found glycoforms.
  • top-down msc HCD spectra are analyzed using appropriate software (e.g., ProSightPC software) under a single protein mode with a fragment ion tolerance of 5 ppm.
  • appropriate software e.g., ProSightPC software
  • samples that were analyzed included five individual mAbs, a mixture of three of these antibodies, and a mixture of all five of these antibodies. These samples were desalted using a PLRP-S column (1.0 * 50 mm; 8 ⁇ particle size; 4000 A pore size) and a five minute gradient. Whole or intact antibodies were eluted from the column and analyzed using a bench-top Q Exactive Quadrupole Orbitrap mass spectrometer. Furthermore, top-down tandem mass spectrometry (MS/MS) was performed on light chains of the antibodies using high energy collision dissociation with a unique spectrum multiplexing feature (msx HCD) in the Q Exactive Quadrupole Orbitrap mass spectrometer.
  • msx HCD unique spectrum multiplexing feature
  • Each charge state in this spectra revealed baseline separated major glycosylated forms (glycoforms) of the mAbs, and the deconvulated spectra showed the expected molecular mass of all the major glycoforms for each mAb.
  • the MS spectra for antibody mixture having three antibodies showed complete separation of each individual antibody in the mixture ( Figure 1). This allowed the confirmation that the deconvoluted molecular mass for each antibody in the mixture was in agreement with what was measured for each of these antibodies individually ( Figure 2).
  • the isotopic peaks of the light chain were baseline resolved by using the 140 K resolution setting, which resulted in monoisotopic molecular mass determination with an error of less than 5 ppm ( Figures 3 and 6).
  • the amino acid sequence of the antibodies was obtained by applying top- down MS/MS analysis to the light chains of the antibodies using the msx HCD approach.
  • fragment ions produced from several individual HCD events, each on a precursor of a different charge state of the reduced mAb were detected together in the Orbitrap mass analyzer.
  • the results in this Example showed that precise mass measurement, and extensive and high confidence sequence information can be obtained for intact mAbs either individually or in a mixture.
  • Example 2. Mass spectrometry (MS)-based quantitation of whole or intact antibodies
  • FIG. 9A-9D depicts the raw mass spectra for the isolated resolved light chains of antibodies 26.3.E2; 5.6.H9; 42.18.E12; and 5.55.D2 respectively.
  • the light chain from each antibody was analyzed in an Orbitrap mass spectrometer in High Energy Collision mode (msx) using tandem mass spectrometry.

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Abstract

La présente invention concerne, entre autres, des procédés et des compositions pour la caractérisation de mélanges d'anticorps hétérogènes (c'est-à-dire, des populations d'anticorps polyclonales) à l'aide d'une spectrométrie de masse. La présente invention est basée, en partie, sur la surprenante découverte selon laquelle des mélanges d'anticorps peuvent être caractérisés à l'aide de techniques de spectrométrie de masse, sans digestion préalable ou prétraitement des anticorps.
PCT/US2014/014074 2013-01-31 2014-01-31 Caractérisation de mélanges d'anticorps par spectrométrie de masse WO2014121031A1 (fr)

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WO2015150446A1 (fr) * 2014-04-02 2015-10-08 F. Hoffmann-La Roche Ag Procédé de détection de mésappariement de chaînes légères d'anticorps multispécifiques
CN106164288A (zh) * 2014-04-02 2016-11-23 豪夫迈·罗氏有限公司 检测多特异性抗体轻链错配的方法
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US11604196B2 (en) 2014-04-04 2023-03-14 Mayo Foundation For Medical Education And Research Isotyping immunoglobulins using accurate molecular mass
US11209439B2 (en) 2015-09-24 2021-12-28 Mayo Foundation For Medical Education And Research Identification of immunoglobulin free light chains by mass spectrometry
WO2017144900A1 (fr) * 2016-02-25 2017-08-31 The Binding Site Group Limited Kit de spectrométrie de masse
CN108885218A (zh) * 2016-02-25 2018-11-23 拜恩顶赛集团有限公司 质谱法试剂盒
EP4123308A1 (fr) * 2016-02-25 2023-01-25 The Binding Site Group Limited Kit de spectrométrie de masse
US10955420B2 (en) 2016-09-07 2021-03-23 Mayo Foundation For Medical Education And Research Identification and monitoring of cleaved immunoglobulins by molecular mass
US11092597B2 (en) 2017-02-28 2021-08-17 Waters Technoligies Corporation Devices and methods for analyzing intact proteins, antibodies, antibody subunits, and antibody drug conjugates
CN110352354A (zh) * 2017-02-28 2019-10-18 沃特世科技公司 用于分析完整蛋白质、抗体、抗体亚基和抗体药物缀合物的装置和方法
WO2018158690A1 (fr) * 2017-02-28 2018-09-07 Waters Technologies Corporation Dispositifs et méthodes d'analyse de protéines intactes, d'anticorps, de sous-unités d'anticorps et de conjugués anticorps-médicament
CN110352354B (zh) * 2017-02-28 2024-05-10 沃特世科技公司 用于分析完整蛋白质、抗体、抗体亚基和抗体药物缀合物的装置和方法
US11946937B2 (en) 2017-09-13 2024-04-02 Mayo Foundation For Medical Education And Research Identification and monitoring of apoptosis inhibitor of macrophage
JP2021512284A (ja) * 2018-02-01 2021-05-13 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 治療用モノクローナル抗体の品質特性の定量化及びモデル化
JP7288449B2 (ja) 2018-02-01 2023-06-07 リジェネロン・ファーマシューティカルズ・インコーポレイテッド 治療用モノクローナル抗体の品質特性の定量化及びモデル化
JPWO2019155576A1 (ja) * 2018-02-08 2021-01-07 株式会社島津製作所 モノクローナル抗体の検出結果を向上する方法
JP7056675B2 (ja) 2018-02-08 2022-04-19 株式会社島津製作所 モノクローナル抗体の検出結果を向上する方法
WO2019155576A1 (fr) * 2018-02-08 2019-08-15 株式会社島津製作所 Procédé d'amélioration de résultats de détection d'anticorps monoclonaux
WO2021258114A1 (fr) * 2020-06-18 2021-12-23 Northwestern University Procédé de lecture directe d'immunoglobulines
WO2022060947A1 (fr) * 2020-09-16 2022-03-24 Novilytic, LLC Validation de processus spécifique de protéoglycanes

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