Protein A chromatography-electrospray ionization mass spectrometry

Abstract

A method and system are provided for characterizing proteins using a chromatography system having a Protein A chromatography resin and an electrospray ionization mass spectrometer operated under non-denaturing conditions. TIFF2022517821000003.tif85131

Description

[Technical field]

[0001] Field The present invention relates generally to methods and systems for characterizing at least one protein using Protein A chromatography and electrospray mass spectrometry. [Background technology]

[0002] background Protein-based biopharmaceuticals have emerged as important drugs for the treatment of cancer, autoimmune diseases, infectious diseases, and cardiometabolic disorders and represent one of the fastest growing product segments in the pharmaceutical industry.

[0003] Protein-based biopharmaceuticals must meet very high purity standards. There are multiple process-related and product-related impurities found in biopharmaceuticals. These impurities do not have properties comparable to those of the desired product in terms of activity, efficacy, and safety. One example is protein post-translational modifications (PTMs), which greatly affect the protein properties relevant for therapeutic applications. These impurities may exhibit different mechanisms of action and potential toxicity or immunogenicity compared to the product. Furthermore, they may have lower stability than the product, which translates into a higher risk of aggregation and immunogenicity. Despite recent advances, the challenge of developing purity test methods for the quantitative evaluation of such impurities remains. In addition, a key challenge in analytical method development for bispecific antibodies is that the method must accurately and reproducibly detect impurities present at levels of 2% or less relative to the primary target species. It is therefore important to monitor and characterize such impurities during the various stages of drug development and manufacturing. Despite the importance of impurities for biological function, their study on a large scale has been hindered by the lack of suitable methods.

[0004] Analytical methods for purity testing must demonstrate sufficient accuracy and sensitivity to detect and quantify the desired product and impurities. Evaluation of impurities such as PTMs in antibodies and homodimers in bispecific antibodies can be challenging due to the similar structural and physicochemical properties between such impurities and the desired product. Direct analysis of such impurities requires isolation of the desired product in quantities large enough for testing, which is undesirable and possible only in selected cases.

[0005] Thus, there is a long felt need in the art for methods and / or systems for identifying and quantifying protein-impurities and / or desired products in protein-based biopharmaceuticals. Summary of the Invention

[0006] overview The growth in the development, production, and sale of protein-based biopharmaceuticals has resulted in an increased demand for characterization of active pharmaceutical ingredients and the presence of impurities in the biopharmaceuticals.

[0007] SUMMARY OF THE DISCLOSURE The exemplary embodiments disclosed herein fulfill the aforementioned needs by providing a method and system for characterizing at least one protein using Protein A chromatography and electrospray mass spectrometry.

[0008] The present disclosure provides, at least in part, a method for identifying proteins in a sample.In an exemplary embodiment, the method for identifying proteins includes contacting a sample containing proteins with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin with a mobile phase to obtain an eluate containing proteins, and identifying proteins in the eluate using an electrospray ionization mass spectrometer.

[0009] In one aspect of this embodiment, a method for identifying proteins in a sample can include contacting the sample with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin with a mobile phase to obtain an eluent comprising proteins, and identifying proteins in the eluent using an electrospray ionization mass spectrometer, wherein the chromatography system is coupled to the electrospray ionization mass spectrometer.

[0010] In one aspect of this embodiment, a method for identifying proteins in a sample can include contacting the sample with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin using a mobile phase to obtain an eluent comprising proteins, and identifying proteins in the eluent using a native electrospray ionization mass spectrometer, wherein the chromatography system is coupled to a native electrospray ionization mass spectrometer.

[0011] In one aspect of this embodiment, a method for identifying a protein in a sample may include contacting about 10 μg to about 100 μg of the sample with a chromatography system having a Protein A chromatography resin.

[0012] In one aspect of this embodiment, a method for identifying a protein in a sample may include washing a Protein A chromatography resin using a mobile phase that can be compatible with an electrospray ionization mass spectrometer.

[0013] In some particular exemplary embodiments, a method for identifying a protein in a sample can include washing a Protein A chromatography resin using a mobile phase, which can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0014] In some particular exemplary embodiments, a method for identifying a protein in a sample may include washing a Protein A chromatography resin using a mobile phase comprising a total salt concentration of up to 600 mM.

[0015] In one aspect of this embodiment, a method for identifying a protein in a sample may include washing a Protein A chromatography resin using a mobile phase at a flow rate of 0.2 ml / min to 0.4 ml / min.

[0016] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin with additional functionality, where the protein can be an antibody.

[0017] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin, and the fragments can be degradation products of the antibody.

[0018] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin, and the fragment can be a variant of an antibody.

[0019] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A chromatography resin, wherein the protein is an impurity.

[0020] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin, wherein the protein is a bispecific antibody.

[0021] In one aspect of this embodiment, a method for identifying proteins in a sample can include identifying the proteins using a mass spectrometer, and the electrospray ionization mass spectrometer can be operated under non-denaturing conditions.

[0022] In one aspect of this embodiment, the method for identifying a protein in a sample can include identifying the protein using a mass spectrometer, and the electrospray ionization mass spectrometer can be a tandem mass spectrometer.

[0023] In one aspect of this embodiment, the method for identifying a protein in a sample can include identifying the protein using a mass spectrometer, and the electrospray ionization mass spectrometer can be a tandem mass spectrometer.

[0024] In one aspect of this embodiment, the method for identifying a protein in a sample can include identifying the protein using a mass spectrometer, and the electrospray ionization mass spectrometer can be a nano-electrospray ionization mass spectrometer.

[0025] In one aspect of this embodiment, a method for identifying a protein in a sample may include fluidly connecting a chromatography system to an electrospray ionization mass spectrometer using a splitter having at least two paths to couple the electrospray ionization mass spectrometer to the chromatography system.

[0026] In one aspect of this embodiment, a method for identifying a protein in a sample may include fluidly connecting a chromatography system to an ultraviolet detector using a splitter having at least two paths.

[0027] In one aspect of this embodiment, a method for identifying a protein in a sample may include fluidly connecting a chromatography system to an ultraviolet detector using a splitter having at least two paths.

[0028] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting a sample containing a protein with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin using a mobile phase to obtain an eluent, wherein the eluent can be introduced into an electrospray ionization mass spectrometer, and wherein the flow rate of the electrospray from the electrospray ionization is about 10 nL / min to about 50 nL / min.

[0029] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample containing the protein with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin using a mobile phase to obtain an eluent, wherein the eluent obtained from washing the Protein A chromatography resin is introduced into an electrospray ionization mass spectrometer, and wherein the spray voltage of the electrospray is about 0.8 kV to about 1.5 kV.

[0030] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting a sample, where the sample can include at least two proteins.

[0031] In one aspect of this embodiment, a method for identifying a protein in a sample can include contacting the sample, where the sample can be subjected to conditions selected from the group consisting of hydrogen peroxide, oxidation, heat, ultraviolet light, low temperature white light, or combinations thereof.

[0032] The present disclosure provides, at least in part, a method for quantifying protein in a sample.In an exemplary embodiment, the method for quantifying protein includes contacting a sample containing protein with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin with a mobile phase to obtain an eluate containing protein, and quantifying the protein in the eluate using an electrospray ionization mass spectrometer.

[0033] In one aspect of this embodiment, the method for quantifying proteins in a sample is also capable of quantifying the relative abundance of proteins in a sample.

[0034] In one aspect of this embodiment, a method for quantifying proteins in a sample can include contacting the sample with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin using a mobile phase to obtain an eluent comprising proteins, and quantifying proteins in the eluent using an electrospray ionization mass spectrometer, wherein the chromatography system is coupled to the electrospray ionization mass spectrometer.

[0035] In one aspect of this embodiment, a method for quantifying protein in a sample may include contacting about 10 μg to about 100 μg of the sample with a chromatography system having a Protein A chromatography resin.

[0036] In one aspect of this embodiment, a method for quantifying a protein in a sample may include washing a Protein A chromatography resin using a mobile phase that may be compatible with an electrospray ionization mass spectrometer.

[0037] In some particular exemplary embodiments, a method for quantifying protein in a sample can include washing a Protein A chromatography resin with a mobile phase, which can be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0038] In some particular exemplary embodiments, a method for quantifying protein in a sample may include washing a Protein A chromatography resin using a mobile phase comprising a total salt concentration of up to 600 mM.

[0039] In one aspect of this embodiment, a method for quantifying protein in a sample may include washing a Protein A chromatography resin using a mobile phase at a flow rate of 0.2 ml / min to 0.4 ml / min.

[0040] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin with additional functionality, where the protein can be an antibody.

[0041] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin, and the fragments can be degradation products of the antibody.

[0042] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin, and the fragment can be a variant of an antibody.

[0043] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A chromatography resin, where the protein can be an impurity.

[0044] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample with a chromatography system having a Protein A resin, where the protein can be a bispecific antibody.

[0045] In one aspect of this embodiment, a method for quantifying proteins in a sample can include identifying the proteins using a mass spectrometer, and the electrospray ionization mass spectrometer can be operated under non-denaturing conditions.

[0046] In one aspect of this embodiment, the method for quantifying proteins in a sample can include identifying the proteins using a mass spectrometer, and the electrospray ionization mass spectrometer can be a tandem mass spectrometer.

[0047] In one aspect of this embodiment, the method for quantifying proteins in a sample can include identifying the proteins using a mass spectrometer, and the electrospray ionization mass spectrometer can be a tandem mass spectrometer.

[0048] In one aspect of this embodiment, the method for quantifying proteins in a sample can include identifying the proteins using a mass spectrometer, and the electrospray ionization mass spectrometer can be a nano-electrospray ionization mass spectrometer.

[0049] In one aspect of this embodiment, a method for quantifying proteins in a sample may include fluidly connecting a chromatography system to an electrospray ionization mass spectrometer using a splitter having at least two paths for coupling the electrospray ionization mass spectrometer to the chromatography system.

[0050] In one aspect of this embodiment, a method for quantifying a protein in a sample may include fluidly connecting a chromatography system to an ultraviolet detector using a splitter having at least two paths.

[0051] In one aspect of this embodiment, a method for quantifying a protein in a sample may include fluidly connecting a chromatography system to an ultraviolet detector using a splitter having at least two paths.

[0052] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting a sample comprising a protein with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin using a mobile phase to obtain an eluent, wherein the eluent can be introduced into an electrospray ionization mass spectrometer, and wherein the flow rate of the electrospray from the electrospray ionization can be from about 10 nL / min to about 50 nL / min.

[0053] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample containing the protein with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin using a mobile phase to obtain an eluent, wherein the eluent obtained from washing the Protein A chromatography resin is introduced into an electrospray ionization mass spectrometer, and the spray voltage of the electrospray can be about 0.8 kV to about 1.5 kV.

[0054] In one aspect of this embodiment, a method for quantifying a protein in a sample can include contacting the sample, where the sample can include at least two proteins.

[0055] In one aspect of this embodiment, a method for quantifying protein in a sample can include contacting the sample, where the sample can be subjected to conditions selected from the group consisting of deglycosylation, oxidation, heat, ultraviolet light, low temperature white light, or combinations thereof.

[0056] The present disclosure provides, at least in part, a system that includes a chromatography column having a Protein A chromatography resin and an electrospray ionization mass spectrometer.

[0057] In one exemplary embodiment, the system may include an electrospray ionization mass spectrometer that can be coupled on-line to a chromatography column.

[0058] In one aspect of this embodiment, the system can include a chromatography column having a Protein A chromatography resin, the chromatography column capable of accepting a mobile phase and a sample comprising a protein.

[0059] In one aspect of this embodiment, the system may include a chromatography column that may be coupled to an ultraviolet detector using a splitter.

[0060] In one aspect of this embodiment, the system can include a chromatography column that can be coupled to an ultraviolet detector using a splitter having at least three paths.

[0061] In one aspect of this embodiment, the system can include an electrospray ionization mass spectrometer capable of operating under non-denaturing conditions.

[0062] In one aspect of this embodiment, the system may include a nano-electrospray ionization mass spectrometer operable under non-denaturing conditions.

[0063] In one aspect of this embodiment, the system can include a chromatography column having a Protein A chromatography resin and an electrospray ionization mass spectrometer, and the system can identify the protein.

[0064] In one aspect of this embodiment, the system can include a chromatography column having a Protein A chromatography resin and an electrospray ionization mass spectrometer, and the system can rank the Protein A affinity of the monoclonal antibody variants.

[0065] In one aspect of this embodiment, the system can include a chromatography column having a Protein A chromatography resin and an electrospray ionization mass spectrometer, where the Protein A chromatography resin and the electrospray ionization mass spectrometer can be compatible with a mobile phase selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0066] In one aspect of this embodiment, the system can include a chromatography column having a Protein A chromatography resin, and the chromatography column can be washed using a mobile phase at a flow rate of 0.2 ml / min to 0.4 ml / min.

[0067] In one aspect of this embodiment, the system can rank the Protein A affinity of monoclonal antibody variants. [The present invention 1001] contacting a sample containing protein with a chromatography system having a Protein A chromatography resin; washing the Protein A chromatography resin with a mobile phase to obtain an eluate comprising the protein; and identifying said proteins in said eluate using an electrospray ionization mass spectrometer operated under non-denaturing conditions. 2. A method for identifying at least one protein comprising: [The present invention 1002] 1001. The method of claim 1001, wherein said electrospray ionization mass spectrometer is coupled online to said chromatography system having said Protein A chromatography resin. [The present invention 1003] 1001. The method of claim 1001, wherein the electrospray ionization mass spectrometer is a nanoelectrospray ionization mass spectrometer. [The present invention 1004] 1001. The method of claim 1001, wherein a splitter having at least two paths is used to couple said electrospray ionization mass spectrometer to said chromatography system having said Protein A chromatography resin. [The present invention 1005] 1001. The method of claim 1001, wherein a splitter having at least two paths is used to connect an ultraviolet detector to said chromatography system having said Protein A chromatography resin. [The present invention 1006] The method of any one of claims 1 to 10, wherein the mobile phase used to wash the size exclusion chromatography resin comprises ammonium acetate. [The present invention 1007] The method of any one of claims 1 to 10, wherein the mobile phase used to wash the size exclusion chromatography resin comprises a volatile salt. [The present invention 1008] 1001. The method of claim 10, wherein said mobile phase used to wash said Protein A chromatography resin has a flow rate of about 0.2 ml / min to about 0.4 ml / min. [The present invention 1009] 1001. The method of claim 1001, wherein the amount of said sample containing said protein that is contacted with said chromatography system is from about 10 μg to about 100 μg. [The present invention 1010] The method of the present invention 1001, wherein the eluent obtained from washing the Protein A chromatography resin is introduced into the electrospray ionization mass spectrometer, and the flow rate of electrospray from the electrospray ionization is about 10 nL / min to about 50 nL / min. [The present invention 1011] 1001. The method of the present invention, wherein said eluate obtained from washing of Protein A chromatography resin is introduced into said electrospray ionization mass spectrometer, and the spray voltage of electrospray is about 0.8 kV to about 1.5 kV. [The present invention 1012] The method of any one of claims 1 to 10, wherein said protein is a monoclonal antibody. [The present invention 1013] The method of claim 10, wherein the protein is a product associated impurity. [The present invention 1014] The method of any one of claims 1 to 10, wherein said protein is a bispecific antibody. [The present invention 1015] The method of claim 10, wherein the protein is an impurity. [The present invention 1016] The method of claim 10, wherein the protein is a monoclonal antibody variant. [The present invention 1017] 1001. The method of claim 1001, wherein the sample comprises at least two proteins. [The present invention 1018] 1001. The method of claim 1001, wherein the protein has a post-translational modification. [The present invention 1019] 1001. The method of claim 1001, wherein said sample is subjected to conditions selected from the group consisting of deglycosylation, oxidation, heat, ultraviolet light, low temperature white light, or a combination thereof. [The present invention 1020] a chromatography column having a Protein A chromatography resin, the chromatography column being capable of accepting a mobile phase and a sample comprising a protein; and an electrospray ionization mass spectrometer capable of being coupled on-line to said chromatography column and capable of being operated under non-denaturing conditions; Including, the system. [The present invention 1021] The system of claim 1020, wherein the chromatography column is further connectable to an ultraviolet detector using a splitter having at least three paths. [The present invention 1022] The system of the present invention 1020, wherein the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer. [The present invention 1023] The system of the present invention 1020, which is capable of identifying the protein. [The present invention 1024] The system of the present invention 1020 capable of ranking the Protein A affinity of monoclonal antibody variants. [The present invention 1025] contacting a sample containing protein with a chromatography system having a Protein A chromatography resin; washing the Protein A chromatography resin with a mobile phase to obtain an eluate comprising the protein; and identifying said proteins in said eluate using an electrospray ionization mass spectrometer operated under non-denaturing conditions. 23. A method for characterizing at least one protein, comprising: [Brief description of the drawings]

[0068] [Figure 1] Spectra obtained from normal and native electrospray ionization mass spectrometry are shown. [Diagram 2] 1 shows a Protein A chromatography-native electrospray mass spectrometry system according to an exemplary embodiment. [Diagram 3] 1 shows a Protein A chromatography-native nano-electrospray mass spectrometry system according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description Recombinantly produced products may contain size variants (e.g., aggregates, fragments, degradation products, etc.) generated during production and storage. Because aggregates and fragments can affect immunogenicity and potency, their levels are typically monitored during lot release, stability, and characterization (Amy S. Rosenberg, Effects of protein aggregates: An immunologic perspective, 8 THE AAPS JOURNAL (2006)).

[0070] Monoclonal antibodies (mAbs) have emerged as one of the most important classes of biopharmaceuticals, although the development of these molecules has been long and challenging. Sensitive and high-throughput analytical methods that improve process understanding and provide mechanistic insights for process improvement remain critical in the biopharmaceutical industry for the controlled production and purification of high-quality therapeutic mAbs.

[0071] Protein A chromatography is one of the most important steps in therapeutic mAb purification. Even more so for the purification of bispecific mAbs using a strategy that relies on the different Protein A binding affinities between the bispecific product and the monospecific impurities (Andrew D. Tustian et al., Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity, 8 MABS 828-838 (2016)). However, the mobile phase from the Protein A chromatography column cannot be directly injected into the mass spectrometer, and additional steps including mobile phase modification are required.

[0072] Given the limitations of existing methods, a rapid online approach combining affinity-based chromatography with native mass spectrometry was developed to assess the attenuation of Protein A binding interactions with mAb variants induced by modifications at the intact level. This method was then used to investigate the impact of different modifications on Protein A purification of therapeutic and bispecific mAbs.

[0073] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials are described below. All publications mentioned are incorporated herein by reference.

[0074] The term "a" should be understood to mean "at least one." The terms "about" and "approximately" should be understood to allow for standard variations understood by one of ordinary skill in the art. When ranges are stated, the endpoints are included.

[0075] Biopharmaceuticals are required to exhibit high levels of potency, purity, and low levels of structural heterogeneity. Structural heterogeneity often affects the biological activity and efficacy of the drug. Therefore, characterization and quantification of therapeutic proteins and / or impurities is important in drug development. Protein structural heterogeneity can result from post-translational modifications as well as inherent chemical modifications during production and storage conditions. Proteins produced in the biotechnology industry require complementary separation techniques to both purify the target protein and accurately capture the quality of the final product. The complexity of the product precludes the use of simple one-dimensional separation strategies.

[0076] As used herein, the term "protein" includes any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those of skill in the art. A protein can include one or more polypeptides to form a single functional biomolecule. Proteins can include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another exemplary embodiment, proteins can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems such as insect bacculovirus systems, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For an illustrative review of biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," (Biotechnol. Genet. Eng. Rev. (2012) 147-75). In some exemplary embodiments, the proteins include modifications, additions, and other covalently attached moieties.These modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels and other dyes, etc. Proteins can be classified based on composition and solubility, and thus may include simple proteins, such as globular proteins and fibrous proteins; complex proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0077] In some exemplary embodiments, the protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, or a combination thereof.

[0078] As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region comprises a C H1 , C H2 , and C H3 Each light chain comprises three domains: a light chain variable region (herein referred to as LCVR or V L The light chain constant region contains one domain (C.sub.L1). H Area and V L The region can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various exemplary embodiments, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term "antibody" as used herein also includes antigen-binding fragments of an intact antibody molecule. As used herein, terms such as "antigen-binding portion" of an antibody and "antigen-binding fragment" of an antibody include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolysis or recombinant genetic engineering techniques (including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains). Such DNA is known and / or readily available, for example from commercial DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, form cysteine ​​residues, modify, add or delete amino acids, etc.

[0079] An "antibody fragment" as used herein includes a portion of an intact antibody, such as, for example, an antigen-binding region or a variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are linked by a peptide linker. Antibody fragments can be produced by various means. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of an intact antibody and / or they can be produced recombinantly from a gene encoding a partial antibody sequence. Alternatively, or in addition, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally comprise a single chain antibody fragment. Alternatively, or in addition, the antibody fragment may comprise multiple chains linked together, for example, by sulfide bonds. The antibody fragment may optionally comprise a multimolecular complex.

[0080] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be produced using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0081] The term "Fc fusion protein" as used herein includes parts or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not fused in their native state. The preparation of fusion proteins including certain heterologous polypeptides fused to various portions of an antibody-derived polypeptide (including the Fc domain) is described, for example, in Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. A "receptor Fc fusion protein" includes one or more of the extracellular domains of one or more receptors bound to an Fc site, which in some embodiments includes the CH2 and CH3 domains of an immunoglobulin followed by a hinge region. In some embodiments, the Fc fusion protein comprises two or more separate receptor chains that bind to a single or multiple ligands. For example, the Fc fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which comprises an IL-1 RAcP ligand binding domain fused to an IL-1R1 extracellular domain fused to an Fc of hIgG1; see US Pat. No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept, which comprises an Ig domain 2 of the VEGF receptor Flt1 fused to an Ig domain 3 of the VEGF receptor Flk1 fused to an Fc of hIgG1; see US Pat. Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

[0082] In some exemplary embodiments, the protein may be an impurity. In some particular exemplary embodiments, the protein may be a product-associated impurity.

[0083] The term "impurities" as used herein may include any undesired proteins present in a protein biopharmaceutical. Impurities may include process and product-related impurities. Impurities may further be of known structure, partially characterized, or unidentified. Process-related impurities may originate from the manufacturing process and may include three major categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from the host organism and nucleic acids (host cell genome, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical treatment reagents (e.g., cyanogen bromide, guanidine, oxidizing agents, and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables. Product-associated impurities (e.g., precursors, certain degradation products) may be molecular variants arising during manufacture and / or storage that do not have properties comparable to those of the desired product in terms of activity, efficacy, and safety. Such variants may require significant efforts for separation and characterization to identify the type of modification. Product-associated impurities include truncated forms, modified forms, and aggregates. Cleaved forms are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modified forms include, but are not limited to, deamidation, isomerization, mismatched S-S bonds, oxidation, or altered complex forms (e.g., glycosylation, phosphorylation). Modified forms may also include any post-translationally modified forms. Aggregates include dimers and higher order multimers of the desired product.(Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products, ICH August 1999, USDept. of Health and Humans Services).

[0084] In some exemplary embodiments, the protein may be a post-translationally modified protein. In some specific exemplary embodiments, the post-translationally modified protein may be introduced by design or may be induced by a stressor. Non-limiting examples of stressors include hydrogen peroxide, oxidation, heat, ultraviolet light, low temperature white light, or combinations thereof.

[0085] The general term "post-translational modification" or "PTM" as used herein refers to covalent modifications that polypeptides undergo either during (co-translational modification) or after (post-translational modification) their ribosomal synthesis. PTMs are usually introduced by specific enzymes or enzymatic pathways. Many occur at the site of specific characteristic protein sequences (signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications always affect some features of protein structure or function (Walsh, G. "Proteins" (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853).Various post-translational modifications include, but are not limited to, truncation, N-terminal extension, proteolysis, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (the addition of an acetyl group, usually at the N-terminus of a protein), alkylation (the addition of an alkyl group, e.g., methyl, ethyl, propyl, usually at a lysine or arginine residue), methylation, adenylation, ADP-ribosylation, covalent cross-links within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (proline and lysine hydroxylation and carboxy-terminal amidation), vitamin K-dependent modifications (where vitamin K is the hydroxylation site of a protein that forms γ-carboxyglutamic acid (glu) residues), and the like. These include covalent attachment of a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, polyketide, nonribosomal peptide, and leucine biosynthesis, and phosphopantetheinylation (addition of a 4'-phosphopantetheinyl moiety from coenzyme A, as in the biosynthesis of fatty acids, polyketides, nonribosomal peptides, and leucine), phosphorylation (addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine), and sulfation (addition of a sulfate group, usually to a tyrosine residue). Post-translational modifications that change the chemical properties of amino acids include, but are not limited to, citrullination (conversion of arginine to citrulline by deimination) and deamidation (conversion of glutamine to glutamic acid or asparagine to aspartic acid).Post-translational modifications that involve structural changes include, but are not limited to, the formation of disulfide bridges (covalent bonding of two cysteine ​​amino acids) and proteolytic cleavage (breaking of a protein at a peptide bond).Specific post-translational modifications include the addition of other proteins or peptides, such as ISGylation (covalent attachment to ISG15 protein (interferon-activated gene)), SUMOylation (covalent attachment to SUMO protein (small ubiquitin-like modifier)), and ubiquitination (covalent attachment to protein ubiquitin). For a more detailed controlled nomenclature of PTMs collected by UniProt, see European Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics,EUROPEAN BIOINFORMATICS INSTITUTE DRS-DROSOMYCIN PRECURSOR-DROSOPHILA MELANOGASTER (FRUIT FLY)-DRS GENE&PROTEIN,http: / / www.uniprot.org / docs / ptmlist (last visited January 15, 2019).

[0086] In some exemplary embodiments, proteins can be identified using a system including a Protein A chromatography resin and a mass spectrometer.

[0087] As used herein, the term "chromatography" refers to a process by which a chemical mixture carried by a liquid or gas can be separated into multiple components as a result of the differential distribution of the chemicals as they flow around or over a liquid or solid stationary phase.

[0088] As used herein, the term "Protein A" refers to Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or recombinant techniques), and Protein A. H 2 / C H and variants thereof that retain the ability to bind to proteins having three domains. Non-limiting examples of commercial manufacturers of Protein A include Repligen, Pharmacia, and Fermatech.

[0089] Protein A is immobilized on a solid phase. By "solid phase" is meant a non-aqueous matrix to which Protein A can adhere. Solid phases of interest herein can include glass or silica surfaces. The solid phase can be a purification column or a discontinuous phase of discrete particles.

[0090] The term "mass spectrometer" as used herein includes devices that can identify specific molecular species and measure their exact mass. The term is intended to include any molecular detector in which a polypeptide or peptide can be eluted for detection and / or characterization. A mass spectrometer may include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to form gas phase ions. The atoms, molecules, or clusters of interest can be transferred to the gas phase and ionized simultaneously (similar to electrospray ionization). The choice of ion source is highly dependent on the application.

[0091] In some embodiments, the mass spectrometer is an electrospray mass spectrometer.

[0092] The term "electrospray ionization" or "ESI" as used herein refers to the process of spray ionization in which either positive or negative ions in a solution are transferred to the gas phase by formation and desolvation at atmospheric pressure of a stream of highly charged droplets obtained by applying a potential difference between an electrospray needle tip containing the solution and a counter electrode. There are typically three main steps in the generation of gas phase ions from electrolyte ions in solution. These are: (a) generation of charged droplets at the ES injection tip; (b) shrinkage of the charged droplets by solvent evaporation and repeated droplet collapse, resulting in the generation of small highly charged droplets capable of generating gas phase ions; and (c) a mechanism by which gas phase ions are generated from very small and highly charged droplets. Steps (a)-(c) typically occur in the atmospheric pressure region of the device.

[0093] The term "electrospray injection setup" as used herein refers to an electrospray ionization system that is compatible with a mass spectrometer used for mass analysis of proteins. In electrospray ionization, an electrospray needle has its orifice located near the entrance orifice of the spectrometer. A sample containing the protein of interest can be pumped through a syringe needle. An electric potential between the orifice of the syringe needle and an orifice leading to the mass spectrometer forms a spray of solution ("electrospray"). Electrospray can be performed at atmospheric pressure and delivers highly charged droplets of the solution. The electrospray injection setup can include an electrospray emitter, a nebulizer gas, and / or an ESI power supply. The setup can optionally be automated to perform sample aspiration, sample dispensing, sample delivery, and / or sample nebulization.

[0094] In some exemplary embodiments, the electrospray ionization mass spectrometer may be a nano-electrospray ionization mass spectrometer.

[0095] The term "nanoelectrospray" or "nanospray" as used herein refers to electrospray ionization at very low solvent flow rates, typically a few hundred nanoliters per minute or less of sample solution, often without the use of an external solvent supply. Electrospray injection setups that form nanoelectrosprays can use static nanoelectrospray emitters or dynamic nanoelectrospray emitters. Static nanoelectrospray emitters perform continuous analysis of small sample (analyte) solution volumes over an extended period of time. Dynamic nanoelectrospray emitters use a capillary column and solvent supply system to perform chromatographic separation of mixtures prior to analysis by a mass spectrometer.

[0096] The term "mass analyzer" as used herein includes devices capable of separating species, i.e. atoms, molecules, or clusters, according to their mass. Non-limiting examples of mass analyzers that can be used for fast protein sequence analysis are time-of-flight (TOF), magnetic / electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and accelerator mass spectrometry (AMS).

[0097] In some exemplary embodiments, mass spectrometry can be performed under non-denaturing conditions.

[0098] The term "non-denaturing conditions" or "native MS" or "native ESI-MS" as used herein may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see Review: Elisabetta Boeri Erba&Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015). Some of the differences between native ESI and regular ESI are shown in Table 1 and Figure 1 (Hao Zhang et al., Native mass spectrometry of photosynthetic pigment-protein complexes, 587 FEBS Letters 1012-1020 (2013)).

[0099] [Table 1]

[0100] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer.

[0101] As used herein, the term "tandem mass spectrometry" includes techniques in which structural information about sample molecules is obtained by using multiple stages of mass selection and mass separation. The prerequisite is that the sample molecules can be transferred into the gas phase and ionized intact, and that they can be induced to break down in some predictable and controllable manner after the initial mass selection step. Multistage MS / MS, or MS n The first step is to extract the precursor ion (MS 2016) as long as meaningful information can be obtained or the fragment ion signal is detectable. 2 ) were selected and isolated to fragment the primary fragment ions (MS 3 ) is isolated and fragmented into secondary fragment ions (MS 4 ) and so on. Tandem MS has been successfully performed using a wide variety of analyzer combinations. The analyzers that are combined for a particular application are determined by many different factors such as sensitivity, selectivity, and speed, as well as size, cost, and availability. The two main categories of tandem MS methods are spatial tandem and temporal tandem, although hybrids also exist in which a temporal tandem analyzer is coupled with a spatial or spatial tandem analyzer.

[0102] Exemplary embodiments Embodiments disclosed herein provide compositions, methods, and systems for the rapid characterization of proteins in a sample.

[0103] As used herein, the terms "include", "includes", and "including" are meant to be open-ended and are understood to mean "comprise", "comprises", and "comprising", respectively.

[0104] The disclosure provides, at least in part, a method for identifying proteins in a sample, the method comprising contacting a sample containing proteins with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin with a mobile phase to obtain an eluent containing proteins, and identifying the proteins in the eluent using an electrospray ionization mass spectrometer.

[0105] The disclosure provides, at least in part, a method for quantifying proteins in a sample, comprising contacting a sample comprising proteins with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin with a mobile phase to obtain an eluent comprising proteins, and quantifying the proteins in the eluent using an electrospray ionization mass spectrometer.

[0106] The disclosure provides, at least in part, a method for quantifying the relative abundance of proteins in a sample, comprising contacting a sample comprising proteins with a chromatography system having a Protein A chromatography resin, washing the Protein A chromatography resin with a mobile phase to obtain an eluent comprising proteins, and quantifying the proteins in the eluent using an electrospray ionization mass spectrometer.

[0107] In some exemplary embodiments, the protein may be a monoclonal antibody.

[0108] In some exemplary embodiments, the protein may be a therapeutic antibody.

[0109] In some exemplary embodiments, the protein may be an immunoglobulin protein.

[0110] In some exemplary embodiments, the protein may be a bispecific antibody.

[0111] In some exemplary embodiments, the protein may be an antibody fragment formed upon digestion of an antibody.

[0112] In one embodiment, the protein may be a post-translationally modified protein.

[0113] In one embodiment, the protein may be an antibody variant.

[0114] In yet another exemplary embodiment, the protein may be an impurity found in a biopharmaceutical.

[0115] In another exemplary embodiment, the protein may be an impurity found during the manufacture of a biopharmaceutical.

[0116] In some exemplary embodiments, the protein may be a digestion product of an antibody. The digestion product may be formed by a hydrolysis agent. The hydrolysis agent may include an agent that performs digestion using enzymatic digestion or non-enzymatic digestion. The hydrolysis agent may be an agent that can perform digestion using enzymatic digestion, which may include trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T (OmpT), immunoglobulin degrading enzyme (IdeS) of Streptococcus pyogenes, chymotrypsin, pepsin, thermolysin, papain, pronase, and protease from Aspergillus Saitoi. The hydrolysis agent may be an agent that can perform digestion using non-enzymatic digestion, which may include the use of high temperature, microwave, ultrasound, high pressure, infrared, and solvents. The digestion product may be an impurity associated with the product.

[0117] In some exemplary embodiments, the proteins can include Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0118] In some exemplary embodiments, the protein may have a pI ranging from about 4.5 to about 9.0.

[0119] In an exemplary embodiment, the protein may have a pI of about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

[0120] In an exemplary embodiment, the number of proteins in a sample may be at least two.

[0121] In some exemplary embodiments, the amount of protein in a sample loaded onto a chromatography system can range from about 10 μg to about 100 μg. In an exemplary embodiment, the amount of protein in a sample loaded onto a chromatography system can be about 10 μg, about 12.5 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg.

[0122] In some exemplary embodiments, the mobile phase used to elute the proteins may be any mobile phase that may be compatible with a mass spectrometer.

[0123] In some particular exemplary embodiments, the mobile phase used to elute the protein may be a mobile phase that may include a volatile salt.

[0124] In some exemplary embodiments, the mobile phase may be ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0125] In an exemplary embodiment, the total concentration of the mobile phase can range up to about 600 mM. In an exemplary embodiment, the total concentration of the mobile phase can range up to about 5 mM, about 6 mM, 7 mM, about 8 mM, 9 mM, about 10 mM, 12.5 mM, about 15 mM, 17.5 mM, about 20 mM, 25 mM, about 30 mM, 35 mM, about 40 mM, 45 mM, about 50 mM, 55 mM, about 60 mM, 65 mM, about 70 mM, 75 mM, about 80 mM, 75 mM, about 95 mM, 100 mM, about 1100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 400 mM, 450 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 75 mM, 95 mM, 100 mM, 1100 mM, 120 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 1 mM, about 150 mM, 140 mM, about 150 mM, 160 mM, about 170 mM, 180 mM, about 190 mM, 200 mM, about 225 mM, 250 mM, about 275 mM, 300 mM, about 325 mM, 350 mM, about 375 mM, 400 mM, about 425 mM, 450 mM, about 475 mM, 500 mM, about 525 mM, 550 mM, about 575 mM, or about 600 mM.

[0126] In some exemplary embodiments, the mobile phase can have a flow rate of about 0.1 ml / min to about 0.4 ml / min in the chromatography system. In an exemplary embodiment, the flow rate of the mobile phase can be about 0.1 ml / min, about 0.15 ml / min, about 0.20 ml / min, about 0.25 ml / min, about 0.30 ml / min, about 0.35 ml / min, or about 0.4 ml / min.

[0127] In some exemplary embodiments, the flow rate in the electrospray ionization mass spectrometer can be from about 10 nL / min to about 50 nL / min.

[0128] In some exemplary embodiments, the electrospray ionization mass spectrometer can have a spray voltage of about 0.8 kV to about 1.5 kV.

[0129] In some exemplary embodiments, identification may include protein sequencing, protein de novo sequencing, identification of post-translational modifications, or compatibility analysis, or a combination thereof.

[0130] In an exemplary embodiment, the electrospray ionization mass spectrometer may be a tandem mass spectrometer.

[0131] In another exemplary embodiment, the electrospray ionization mass spectrometer may be a nano-electrospray mass spectrometer.

[0132] It is understood that the methods are not limited to any of the proteins, fragments, impurities, and columns described above, and that the methods for identifying or quantitating may be performed by any suitable means.

[0133] In some exemplary embodiments, the present disclosure provides a system including a chromatography column 110 that can be washed with a mobile phase to obtain an eluent, and a mass spectrometer 120 coupled to the chromatography column 110 (see FIG. 2 ).

[0134] In some exemplary embodiments, the chromatography column 110 may include a Protein A chromatography resin 130 .

[0135] FIG. 2 shows a chromatography column containing a Protein A chromatography resin 150 that is fluidly connected or coupled to an electrospray ionization mass spectrometer 120 that can be operated under non-denaturing conditions.

[0136] In one exemplary embodiment, the chromatography column 110 can be made contactable with a sample that includes proteins.

[0137] In some exemplary embodiments, the amount of sample that can be loaded onto the chromatography column 110 can range from about 10 μg to about 100 μg. In one exemplary embodiment, the amount of sample that can be loaded onto the chromatography column 110 can be about 10 μg, about 12.5 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg.

[0138] In some exemplary embodiments, the chromatography column 110 can be washable with a mobile phase.

[0139] In some exemplary embodiments, the chromatography column 110 can be made washable with a mobile phase that includes a volatile salt.

[0140] In one exemplary embodiment, the mobile phase may be ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof.

[0141] In one exemplary embodiment, the total concentration of the mobile phase that can be used to wash the chromatography column 110 can range up to about 600 mM.

[0142] In an exemplary embodiment, the total concentration of the mobile phase that can be used to wash the chromatography column 110 is about 5 mM, about 6 mM, 7 mM, about 8 mM, 9 mM, about 10 mM, 12.5 mM, about 15 mM, 17.5 mM, about 20 mM, 25 mM, about 30 mM, 35 mM, about 40 mM, 45 mM, about 50 mM, 55 mM, about 60 mM, 65 mM, about 70 mM, 75 mM, about 80 mM, 75 mM, about 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, 550 mM, 550 mM, 560 mM, 570 mM, 580 mM, 590 mM, 600 mM, 610 mM, 620 mM, 630 mM, 640 mM, 650 mM, 660 mM mM, about 1100 mM, 120 mM, about 150 mM, 140 mM, about 150 mM, 160 mM, about 170 mM, 180 mM, about 190 mM, 200 mM, about 225 mM, 250 mM, about 275 mM, 300 mM, about 325 mM, 350 mM, about 375 mM, 400 mM, about 425 mM, 450 mM, about 475 mM, 500 mM, about 525 mM, 550 mM, about 575 mM, or about 600 mM.

[0143] In another exemplary embodiment, the mobile phase that can be used with the chromatography column 110 can have a flow rate between 0.1 ml / min and 0.4 ml / min.

[0144] In an exemplary embodiment, the flow rate of the mobile phase that can be used with the chromatography column 110 may be about 0.1 ml / min, about 0.15 ml / min, about 0.20 ml / min, about 0.25 ml / min, about 0.30 ml / min, about 0.35 ml / min, or about 0.4 ml / min.

[0145] In some exemplary embodiments, the chromatography column 110 can be connectable to a mass spectrometer 120 .

[0146] In one exemplary embodiment, the mass spectrometer 120 may include a nanospray.

[0147] In some exemplary embodiments, the mass spectrometer 120 may be a tandem mass spectrometer.

[0148] In some exemplary embodiments, the mass spectrometer 120 may be an electrospray tandem mass spectrometer.

[0149] In some exemplary embodiments, the mass spectrometer 120 may be a nanoelectrospray tandem mass spectrometer.

[0150] In some exemplary embodiments, the mass spectrometer 120 may be a native electrospray tandem mass spectrometer.

[0151] In some exemplary embodiments, the mass spectrometer 120 may be a native nano-electrospray tandem mass spectrometer.

[0152] In some exemplary embodiments, the system may allow for identification 140 and / or quantification 150 of proteins (as depicted in FIG. 2). Proteins may include antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, antibody variants, post-translationally modified antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0153] In yet another exemplary embodiment, the system may enable identification 140 and / or quantification 150 of proteins that may be impurities found in biopharmaceuticals.

[0154] In another exemplary embodiment, the system may enable identification 140 and / or quantification 150 of proteins that may be impurities found during the manufacture of a biopharmaceutical.

[0155] In some exemplary embodiments, the system may allow for the identification 140 and / or quantification 150 of proteins that may have a pI ranging from about 4.5 to about 9.0.

[0156] In some exemplary embodiments, the system may enable identification 140 and / or quantification 150 of proteins that may be impurities associated with a product.

[0157] In one exemplary embodiment, the system can be used to identify 140 and / or quantify 150 more than one protein. In one exemplary embodiment, the system can be used to identify 140 and / or quantify 150 two proteins.

[0158] Another exemplary embodiment of the system is shown in Figure 3. To enable UV / MS dual detection, a post-column splitter 160 with at least three paths is used. A minor fraction can be directed to the MS 120 while a major fraction is sent to a UV detector 170. The detectors share approximately the same retention time. The fraction from the UV detector can be collected for sample recovery.

[0159] It is understood that the system is not limited to any of the proteins, impurities, mobile phases, mass spectrometers, or chromatography columns described above.

[0160] Sequential number and / or letter labeling of method steps presented herein is not intended to limit the method, or any embodiment thereof, to the particular order presented.

[0161] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and journal articles, are cited throughout the specification. Each of these cited references is incorporated herein by reference in its entirety for all purposes.

[0162] The present disclosure may be more fully understood by reference to the following examples, which are set forth to more fully illustrate the present disclosure, and are intended for illustrative purposes and should not be construed as limiting the scope of the disclosure. EXAMPLES

[0163] An exemplary implementation can be performed using an Acquity system (Waters, Milford, Mass., USA) coupled to a UV detector and an electrospray ionization mass spectrometer (Thermo Exactive EMR, USA). The mass spectrometer can be operated in positive resolution mode.

[0164] Sample preparation. Allow sample mAb to undergo forced oxidation in the presence of 0.001%-0.02% (v / v) hydrogen peroxide (HO) for 24 h.

[0165] System. On-line Protein A affinity-based separation of monoclonal and bispecific antibody variants can be performed using a Thermo Scientific MAbPac Protein A column (4 x 35 mm). Differentially bound species were eluted using a gradient of ammonium acetate-based mobile phase pH 7.5 to 3.0 at a flow rate of 0.4 mL / min. An analytical flow splitter (approximately 1:400) can be used to reduce the post-column flow rate to the mass spectrometer to approximately 1 μL / min. A Thermo Q-Exactive UHMR mass spectrometer equipped with a Nanospray Flex™ ion source can be used for data acquisition.

[0166] Example 1. The retention times of different mAb variants, as determined by extracted ion chromatograms (XIC), can be utilized to rank the affinity of the mAb variants to Protein A as a result of the different modifications.

[0167] Example 2. This method can also be applied to a mixture of a bispecific antibody (BsAb 1) and two corresponding monospecific mAbs (mAb1 and mAb2) that exhibit successively decreasing Protein A affinity.

[0168] Example 3. Furthermore, mAb samples after different treatments (eg, deglycosylation, forced oxidation, and various stress conditions) can be tested with this novel technology platform.

Claims

1. contacting a sample containing protein with a chromatography system having a Protein A chromatography resin; washing the Protein A chromatography resin with a mobile phase to obtain an acidic eluate comprising the protein; and identifying said proteins in said eluate using an electrospray ionization mass spectrometer operated under non-denaturing conditions.

1. A method for identifying at least one protein comprising: The method, wherein at least one splitter having at least three paths is used to couple the chromatography system to the mass spectrometer and to an ultraviolet detector.

2. 2. The method of claim 1, wherein the electrospray ionization mass spectrometer is coupled online to the chromatography system having the Protein A chromatography resin.

3. The method of claim 1 , wherein the electrospray ionization mass spectrometer is a nano-electrospray ionization mass spectrometer.

4. 2. The method of claim 1, wherein the mobile phase used to wash the Protein A chromatography resin comprises a volatile salt.

5. 2. The method of claim 1, wherein the eluate from washing the Protein A chromatography resin is introduced into the electrospray ionization mass spectrometer, and the flow rate of the electrospray ionization mass spectrometer is from about 10 nL / min to about 50 nL / min.

6. The method of claim 1 , wherein the protein is a monoclonal antibody.

7. The method of claim 1 , wherein the protein is a product-associated impurity.

8. The method of claim 1 , wherein the protein is a bispecific antibody.

9. The method of claim 1 , wherein the protein is an impurity.

10. The method of claim 1 , wherein the protein is a monoclonal antibody variant.

11. The method of claim 1 , wherein the sample comprises at least two proteins.

12. 10. The method of claim 1, wherein the sample is subjected to conditions selected from the group consisting of deglycosylation, oxidation, heat, ultraviolet light, low temperature white light, or combinations thereof.

13. contacting a sample containing protein with a chromatography system having a Protein A chromatography resin; washing the Protein A chromatography resin with a mobile phase to obtain an acidic eluate comprising the protein; and identifying said proteins in said eluate using an electrospray ionization mass spectrometer operated under non-denaturing conditions.

1. A method for characterizing at least one protein, comprising:

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