EA046855B1 - ONLINE CHROMATOGRAPHY AND ELECTROSPRAY IONIZATION MASS SPECTROMETER - Google Patents

Description

Technical field

This invention generally relates to a method for characterizing protein-containing biopharmaceuticals using online chromatography and electrospray ionization mass spectrometry.

State of the art

Protein-containing biopharmaceuticals have become important therapeutics for the treatment of cancer, autoimmune diseases, infections, and cardiometabolic disorders, and represent one of the fastest growing segments of the pharmaceutical industry.

Biopharmaceuticals containing protein must meet very high standards of purity. Thus, it may be important to monitor and characterize protein-containing biopharmaceuticals at different stages of drug development and manufacturing. The analytical method for characterizing such protein-containing biopharmaceuticals must provide sufficient precision and resolution to detect and quantify the desired product. Evaluation can be difficult due to the similarities between the structural and physicochemical properties of biopharmaceuticals containing the protein compared to its mutated, modified, or cleaved form. Direct analysis may require isolation of the product in sufficiently large quantities for analysis, which is undesirable and has only been possible in isolated cases.

There has been a long-standing need in the art for a method and/or system for characterizing protein-containing biopharmaceuticals.

The essence of the invention

The growth in the development, production, and marketing of protein-containing biopharmaceuticals has led to an increased demand for characterization of a protein-containing biopharmaceutical, along with possible impurities, binding stoichiometry, and its overall composition.

The exemplary embodiments disclosed herein meet the above requirements by providing methods for characterizing, identifying, and/or quantifying a biopharmaceutical containing a protein, along with its possible impurities, binding, and overall composition.

The present invention at least in part provides a method for characterizing a protein. In one exemplary embodiment, said method of characterizing a protein includes contacting a sample containing the protein with a chromatography system containing a chromatography resin, washing said resin using a mobile phase to obtain an eluate containing the protein, and characterizing the protein in said eluate using mass spectrometer with electrospray ionization.

In one aspect of this embodiment, the protein characterization method may include a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the method for characterizing a protein may include coupling an electrospray ionization mass spectrometer to a chromatography system containing a chromatography resin.

In one aspect of this embodiment, the method for characterizing a protein may include coupling an electrospray ionization mass spectrometer to a chromatography system containing a size exclusion chromatography resin.

In one aspect, the method for characterizing a protein may include an electrospray ionization mass spectrometer operating under native conditions.

In one aspect of this embodiment, the protein characterization method may include a nanoelectrospray ionization mass spectrometer.

In one aspect of this embodiment, the protein characterization method may comprise a nanoelectrospray ionization mass spectrometer operating under native conditions.

In one aspect of this embodiment, the protein characterization method may include at least one three-pass flow splitter for connecting the electrospray ionization mass spectrometer to a chromatography system containing a resin.

In one aspect of this embodiment, the protein characterization method may include at least one three-way flow splitter for connecting an ultraviolet detector to a chromatography system containing a resin.

In one aspect of this embodiment, the protein characterization method may include at least one three-pass flow splitter for connecting an ultraviolet detector and electrospray ionization mass spectrometer to a chromatography system containing a resin.

In one aspect of this embodiment, the protein characterization method may include at least one three-way flow splitter for connecting the mass spectrometer to

- 1 046855 electrospray ionization with a chromatography system containing size exclusion chromatography resin.

In one aspect of this embodiment, the protein characterization method may include at least one three-way flow splitter for coupling an ultraviolet detector to a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the protein characterization method may include at least one three-way flow splitter for coupling an ultraviolet detector and electrospray ionization mass spectrometer to a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the protein characterization method may include washing the resin using a mobile phase to obtain an eluate containing the protein, wherein the eluate is introduced into an ultraviolet detector through at least one three-way flow splitter at a flow rate of from about 0.2 to about 0.4 ml/min.

In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase containing a volatile salt.

In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase containing ammonium acetate.

In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase with a total concentration of less than about 100 mM ammonium acetate.

In one aspect of this embodiment, the protein characterization method may include washing the resin with a mobile phase at a flow rate of about 0.2 to about 0.4 mL/min.

In one aspect of this embodiment, the method for characterizing a protein may include a mobile phase with a pH of about 6.8.

In one aspect of this embodiment, the method for characterizing a protein may include a sample containing protein in an amount of from about 10 to about 100 μg of protein.

In one aspect of this embodiment, the method for characterizing a protein may include a protein that is an antibody.

In one aspect of this embodiment, the method for characterizing a protein may include a protein that is an antigen-antibody complex.

In one aspect of this embodiment, the method for characterizing a protein may include a protein that is an antibody-drug conjugate.

In one aspect of this embodiment, the protein characterization method may comprise an electrospray ionization mass spectrometer at a flow rate of from about 10 nL/min to about 50 nL/min.

In one aspect of this embodiment, the protein characterization method may comprise an electrospray ionization mass spectrometer with an electrospray voltage of about 0.8 kV to about 1.5 kV.

In one aspect of this embodiment, the method for characterization may include identifying the protein.

In one aspect of this embodiment, the method for characterizing a protein may include quantifying the protein.

In one aspect of this embodiment, the method of characterizing a protein may include quantifying the relative abundance of the protein.

In one aspect of this embodiment, the method for characterizing a protein may include a sample containing at least two proteins.

The present invention at least in part provides a method for characterizing an antibody-drug conjugate. In one exemplary embodiment, the method for determining the characteristics of an antibody-drug conjugate, comprising contacting a sample, including the antibody-drug conjugate, with a chromatography system containing a chromatography resin, washing said resin using a mobile phase to obtain an eluate containing the antibody-drug conjugate, and characterizing the antibody-drug conjugate in said eluate using an electrospray ionization mass spectrometer.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include coupling an electrospray ionization mass spectrometer to a chromatography system containing a chromatography resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include coupling to an electrospray ionization mass spectrometer.

- 2 046855 dusting with a chromatography system containing resin for size exclusion chromatography.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an electrospray ionization mass spectrometer under native conditions.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a nanoelectrospray ionization mass spectrometer.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may comprise a nanoelectrospray ionization mass spectrometer operating under native conditions.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way flow splitter for connecting the electrospray ionization mass spectrometer to a chromatography system containing a resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way flow splitter for connecting an ultraviolet detector to a chromatography system containing a resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way flow splitter for connecting an ultraviolet detector and electrospray ionization mass spectrometer to a chromatography system containing a resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way flow splitter for connecting the electrospray ionization mass spectrometer to a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way flow splitter for connecting an ultraviolet detector to a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include at least one three-way flow splitter for connecting an ultraviolet detector and electrospray ionization mass spectrometer to a chromatography system containing a size exclusion chromatography resin.

In one aspect of this embodiment, the method of characterizing an antibody-drug conjugate may include washing the resin using a mobile phase to obtain an eluate containing the antibody-drug conjugate, wherein the eluate is introduced into an ultraviolet detector through at least one three-way flow splitter at flow rates of about 0.2 to about 0.4 ml/min.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a mobile phase containing a volatile salt.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a mobile phase containing ammonium acetate.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a mobile phase with a total concentration of less than about 100 mM ammonium acetate.

In one aspect of this embodiment, the method of characterizing an antibody-drug conjugate may include washing the resin with a mobile phase at a flow rate of from about 0.2 ml/min to about 0.4 ml/min.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a mobile phase with a pH of about 6.8.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include a sample containing protein in an amount of from about 10 to about 100 μg of the antibody-drug conjugate.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include washing the resin using a mobile phase to obtain an eluate that is introduced into an electrospray ionization mass spectrometer at a flow rate of less than about 50 μL/min.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an electrospray ionization mass spectrometer at a flow rate of from about 10 to about 50 nL/min.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an electrospray ionization mass spectrometer with

- 3 046855 spray voltage in electrospray from about 0.8 to about 1.5 kV.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is a site-specific antibody-drug conjugate.

In one aspect of this embodiment, the method of characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is not a site-specific antibody-drug conjugate.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is a cysteine-constructed antibody-drug conjugate.

In one aspect of this embodiment, the method for characterizing an antibody-drug conjugate may include an antibody-drug conjugate that is an antibody-drug conjugate constructed from a non-specific cysteine.

In one aspect of this embodiment, the method of characterizing an antibody-drug conjugate may include determining the ratio of drug to antibody.

In one aspect of this embodiment, the method of characterizing an antibody-drug conjugate may include identifying the antibody.

In one aspect of this embodiment, the method of characterizing an antibody-drug conjugate may include quantifying the antibody.

The present invention at least in part provides a system comprising a chromatography column containing a chromatography resin. In another exemplary embodiment, the system comprises a chromatography column containing a chromatography resin, the chromatography column is capable of receiving a mobile phase and a sample containing a protein, and an electrospray ionization mass spectrometer.

In one aspect of this embodiment, the system may comprise a chromatography column containing a size exclusion chromatography resin.

In one aspect of this embodiment, the system may comprise an electrospray ionization mass spectrometer that can be coupled to the chromatography column.

In one aspect of this embodiment, the system may comprise an electrospray ionization mass spectrometer that can operate under native conditions.

In one aspect of this embodiment, the system may comprise a nanoelectrospray ionization mass spectrometer.

In one aspect of this embodiment, the system may comprise a chromatography column that can be coupled to a mass spectrometer using a three-way flow splitter.

In one aspect of this embodiment, the system may comprise a chromatography column that may be coupled to an ultraviolet detector using a three-way flow splitter.

In one aspect of this embodiment, the system may comprise a chromatography column that can be coupled to an ultraviolet detector and a mass spectrometer using a three-way flow splitter.

In one aspect of this embodiment, the system may be capable of characterizing the ratio of drug to antibody in an antibody-drug conjugate.

In one aspect of this embodiment, the system may be capable of characterizing a protein.

In one aspect of this embodiment, the system may be capable of characterizing an antigen-antibody complex.

Brief description of graphic materials

In fig. Figure 1 shows spectra obtained using conventional and native electrospray ionization mass spectrometry.

In fig. 2 shows an exemplary implementation of a system capable of characterizing protein-containing biopharmaceuticals.

In fig. 3 shows an exemplary implementation of a system capable of characterizing protein-containing biopharmaceuticals.

In fig. 4 illustrates a setup for a system capable of characterizing protein-containing biopharmaceuticals according to one exemplary embodiment.

In fig. 5A and 5B demonstrate an antigen-antibody complex assay with a system capable of characterizing protein-containing biopharmaceuticals according to one exemplary embodiment.

- 4 046855

In fig. 6 shows the results of an antigen-antibody titration between Bet v 1 and Fab-1, characterized in accordance with an exemplary embodiment.

In fig. 7A illustrates the mass spectrometer signal resulting from simultaneous dual-wavelength ultraviolet detection and electrospray ionization mass spectrometer antigen-antibody interaction under native conditions between Bet v 1 and Fab-1 under native conditions according to an exemplary embodiment.

In fig. 7B shows the ultraviolet spectrum resulting from simultaneous dual-wavelength ultraviolet detection and electrospray ionization mass spectrometer antigen-antibody interaction under native conditions between Bet v 1 and Fab-1 under native conditions according to an exemplary embodiment.

In fig. 8A shows the raw spectrum of a drug-to-antibody ratio analysis of the parent mAb-1 for site-specific conjugated cysteine ADC-1 using online SEC-nano-ESI-MS tools according to an exemplary embodiment.

In fig. 8B shows the raw spectrum of an ADC-1 site-specific conjugated cysteine ADC-1 drug-to-antibody ratio analysis using online SEC-nano-ESI-MS tools according to an exemplary embodiment.

In fig. 8C shows a convolution of the drug-to-antibody ratio analysis spectrum of the parent mAb-1 for site-specific conjugated cysteine ADC-1 using online SEC-nano-ESI-MS tools according to an exemplary embodiment.

In fig. 8D shows a convolution spectrum analysis of the drug-to-antibody ratio of ADC-1 site-specific conjugated cysteine ADC-1 using online SEC-nano-ESI-MS tools according to an exemplary embodiment.

In fig. 9 demonstrates drug-to-antibody ratio analysis of site-specific cysteine conjugated mAb-1 and ADC-1 using FabRICATOR digestion and online SEC-nano-ESI-MS according to an exemplary embodiment.

In fig. 10A shows raw drug-to-antibody ratio analysis spectra of the parent mAb-2 for the site-specific cysteine conjugated ADC-2 using online SEC-nano-ESI-MS tools according to an exemplary embodiment, where N indicates that the antibody does not have glycan, S means that glycosylation occurs on one chain of the antibody, and D means that glycosylation occurs on both chains of the antibody.

In fig. 10B shows a convolution of the parent mAb-2 drug-to-antibody ratio analysis spectrum for the site-specific cysteine conjugated ADC-2 using online SEC-nano-ESI-MS tools according to an exemplary embodiment, where N indicates that the antibody does not have glycan, S means that glycosylation occurs on one chain of the antibody, and D means that glycosylation occurs on both chains of the antibody.

In fig. 10C shows raw drug-to-antibody ratio analysis spectra of ADC-2 site-specific conjugated cysteine ADC-2 using SEC-nano-ESI-MS online tools according to an exemplary embodiment, where N indicates that the antibody does not have a glycan , S means that glycosylation occurs on one chain of the antibody, and D means that glycosylation occurs on both chains of the antibody.

In fig. 10D shows a convolution of the drug-to-antibody ratio analysis spectrum of the parent ADC-2 site-specific conjugated cysteine ADC-2 using online SEC-nano-ESI-MS tools according to an exemplary embodiment, where N indicates that the antibody does not have a glycan. S means that glycosylation occurs on one chain of the antibody, and D means that glycosylation occurs on both chains of the antibody.

Detailed description of the invention

Identification and quantification of proteins in protein-containing biopharmaceuticals can be very important during drug manufacturing and development. The presence of impurities and the mode of binding of a biopharmaceutical can be critical to the development of a safe and effective drug. Therefore, a reliable method and/or workflow for characterizing a protein-containing biopharmaceutical, its binding mode, and characterizing any associated impurities may be useful.

One method involves the use of size exclusion chromatography (SEC) to characterize biomolecular aggregation and fragmentation in the biotechnology industry (Hong Paule et al., Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates, 35 JOURNAL OF LIQUID CHROMATOGRAPHY AND RELATED TECHNOLOGY 2923- 2950 (2012)). Molecular separation by SEC is based on the differential interaction of molecules with a controlled porous structure on the stationary phase. Because SEC uses buffer conditions that preserve the native structure of proteins in solution, it allows the characterization of biomolecules without disturbing their native conformation. Among the various detection modes that can be associated with SEC, mass spectrometry (MS) allows for the accurate and reliable identification of individual components in complex samples. The combination of SEC and MS has been previously reported, including collection of SEC peaks with

- 5 046855 subsequent MS by direct infusion (Basak Kukrer et al., Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography, 27 Pharmaceutical Research 2197-2204 (2010); Francois Debaene et al., Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment, 86 Analytical Chemistry 10674-10683 (2014)) or online SEC-MC (Khaja Muneeruddin et al., Characterization of Small Protein Aggregates and Oligomers Using Size Exclusion Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry, 86 Analytical Chemistry 10692-10699 (2014); S. F. McDonagh et al., Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment, 19 PROTEIN ENGINEERING Design and Selection 299-307 (2006). However, direct ionization of the high flux generated by SEC separations requires stringent ionization conditions that are often incompatible with native MS analysis, thereby limiting the usefulness of combining these technologies for the analysis of non-covalent interactions. In addition, the sensitivity of the mass spectrometer can be compromised by the high salt concentrations used in SEC buffers.

Because noncovalent protein interactions mediate such a wide range of biological functions, there is growing interest in developing methods that facilitate the study of their structure, stoichiometry, and dynamics. Such methods can help probe noncovalent protein interactions that occur widely in nature and coordinate the interactions of protein biopharmaceuticals with a variety of molecules, including other proteins and peptides, nucleic acids, lipids, and small inorganic and organic molecules required. Size exclusion chromatography (SEC) allows the isolation of biomolecules from heterogeneous mixtures of molecular components, and since buffer conditions maintain proteins in their native conformation, SEC is an ideal method for preserving non-covalent biomolecular complexes during their isolation. Among the various detection methods that can be combined with SEC analysis, mass spectrometry (MS) allows the confident identification and characterization of individual components of complex mixtures. However, the high-flow non-volatile salts used in SEC are often incompatible with downstream MS analysis.

Considering the limitations of existing methods, an efficient and effective method for the analysis of protein-containing biopharmaceuticals was developed using online chromatography with an electrospray ionization MS platform.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. The invention includes any equivalent methods and materials disclosed in the specification that may be used in practice or testing. The invention is further described in terms of specific methods and materials. All publications cited in this document are incorporated by reference in their entirety.

Singular forms should be understood to mean at least one; and the terms about and about are to be understood as being subject to standard variations as will be understood by those skilled in the art; and if ranges are specified, endpoints are included.

In some exemplary embodiments, the present invention provides a method for characterizing, identifying, and/or quantifying a biopharmaceutical containing a protein.

As used herein, the term protein-containing biopharmaceutical includes an active substance that is wholly or partly biological in nature. In some exemplary embodiments, the protein-containing biopharmaceutical may comprise a protein, vaccine, allergen, nucleic acid, virus, antibody-drug conjugate, cell, gene, tissue, or combinations thereof. In some other exemplary embodiments, the protein-containing biopharmaceutical may contain a recombinant, engineered, modified, mutated or truncated version of a protein, vaccine, allergen, nucleic acids, viruses, antibody-drug conjugates, cells, genes, tissues, or combinations thereof .

The term protein, as used herein, includes any polymer of an amino acid having covalently linked amide bonds. Proteins contain one or more polymer chains of amino acids, commonly known in the art as polypeptides. A polypeptide refers to a polymer consisting of amino acid residues, related naturally occurring structural variants and their synthetic non-natural analogues linked by peptide bonds, related naturally occurring structural variants and their synthetic non-natural analogues. Synthetic peptides or polypeptides refer to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automatic polypeptide synthesizer. Various methods of solid-phase peptide synthesis are known. A protein may contain one or more polypeptides to form a single functioning biomolecule. The protein may include any of biotherapeutic proteins, recombinant proteins,

- 6 046855 in research or therapy, decoy proteins and other chimeric receptor fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies and bispecific antibodies. In another exemplary aspect, the protein may 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 the baculovirus system in insect cells, yeast systems (eg, Pichia sp.), mammalian systems (eg, CHO cells and CHO derivatives such as CHO-Κι cells). For a review discussing 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. 147-175 (2012)). In some illustrative embodiments, the proteins contain modifications, adducts, and other covalently linked 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 protein, maltose binding protein (MBP) ), chitin binding protein (CBP), glutathione^-transferase (GST), myc epitope, fluorescent tags and other dyes, etc. Proteins can be classified based on composition and solubility and thus can include simple proteins such as globular proteins and fibrous proteins; conjugated proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins and lipoproteins; and protein derivatives, such as primary protein derivatives and secondary protein derivatives.

In some illustrative embodiments, the protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a host cell protein, or combinations thereof.

The term antibody, as used herein, includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as multimers thereof (eg, IgM). Each heavy chain includes a heavy chain variable region (abbreviated HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region includes three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (C _L1 ). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various exemplary embodiments, the anti-big-ET-1 antibody FR (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. A consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs. The term antibody, as used herein, also includes antigen-binding fragments of full-length antibody molecules. The terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be prepared, for example, from full-length antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or using molecular biology techniques, for example, to organize one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or remove amino acids, and so on.

The term antibody fragment, as used herein, includes a portion of an intact antibody, such as, for example, an antigen binding or antibody variable region. Examples of antibody fragments include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, Fc fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment and highlighted region complementarity determining factor (CDR), as well as tribodies, tetrabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Fv fragments are a combination of immunoglobulin heavy and light chain variable regions, and ScFv proteins are recombinant single-chain polypeptide molecules in which the immunoglobulin light and heavy chain variable regions are connected by a peptide linker. The antibody fragment can be produced in various ways. For example, frag

- 7 046855 ment of an antibody can be produced enzymatically or chemically by fragmentation of an intact antibody and/or it can be produced recombinantly from a gene encoding a partial sequence of the antibody. Alternatively or additionally, the antibody fragment may be produced in whole or in part synthetically. The antibody fragment may optionally comprise a single chain antibody fragment. Alternatively or additionally, an antibody fragment may contain multiple chains that are linked together, for example, by disulfide bonds. The antibody fragment may optionally contain a multimolecular complex.

The term monoclonal antibody, as used herein, is not limited to antibodies produced using hybridoma technology. A monoclonal antibody can be obtained from a single clone, including any eukaryotic, prokaryotic or phage clone, by any methods available or known in the art. Monoclonal antibodies useful in accordance with the present description can be obtained using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies, or combinations thereof.

As used herein, the term antibody-drug conjugate or ADC can refer to an antibody attached to a biologically active drug(s) by labile linker(s). An ADC may contain multiple molecules of biologically active drug (or payload) that can be covalently linked to the side chains of amino acid residues of the antibody (Siler Panowski et al., Site-specific antibody drug conjugates for cancer therapy, 6 MABS 34-45 (2013 )). The antibody used for the ADC may be able to bind with sufficient affinity for selective accumulation and long-term retention at the target site. Most ADCs can have Kd values in the nanomolar range. The payload may have potency in the nanomolar/picomolar range and may reach intracellular concentrations achieved after distribution of the ADC into the target tissue. Finally, the linker that forms the link between the payload and the antibody may be stable enough in circulation to take advantage of the pharmacokinetic properties of the antibody fragment (i.e., long half-life) and allow the payload to remain attached to the antibody as it is distributed in tissues, but must provide effective release of the biologically active drug d after the ADC is taken up by target cells.

Linkers can be those that are not cleaved during cellular processing and those that are cleaved when the ADC reaches the target site. In the case of non-cleavable linkers, the biologically active drug released during the call includes the payload and all linker elements still attached to the amino acid residue of the antibody, typically a lysine or cysteine residue, after complete proteolytic degradation of the ADC within the lysosome. Cleavable linkers are linkers whose structure includes a cleavage site between the payload and the amino acid attachment site on the antibody. Cleavage mechanisms may include hydrolysis of acid-labile bonds in acidic intracellular compartments, enzymatic cleavage of amide or ester bonds by an intracellular protease or esterase, and reductive cleavage of disulfide bonds by the reducing environment within cells.

In certain exemplary embodiments, the invention also provides a method for determining a drug-to-antibody ratio (DAR) for an antibody-drug conjugate.

ADCs can be generated by conjugation to endogenous amino acid residues of an antibody, carefully controlling the average degree of modification to obtain an optimal drug/antibody ratio (DAR). This ratio can be chosen based on (a) minimizing the amount of unconjugated antibody and (b) excluding species in the mixture with very high DAR, which can be problematic in production and formulation due to higher hydrophobicity and lower solubility, and can lead to deterioration of pharmacokinetic properties. The addition of too few bioactive drug molecules will result in decreased efficacy, whereas too many may render the ADC unstable with altered pharmacokinetic properties, increased plasma clearance, decreased half-life, and increased systemic toxicity. The optimal DAR is often undefined and highly dependent on other ADC variables; however, most often ADCs aim to achieve a DAR close to 4. A non-limiting example of conjugating a biologically active drug to an antibody may involve conjugating a biologically active drug to lysine or cysteine residues on the antibody. Lysine conjugation can result in the formation of 0-8 conjugated biologically active drug molecules per antibody and can occur on both the heavy and light chains at different lysine residues. Another non-limiting example of conjugation of a biologically active drug to an antibody may involve cysteine conjugation, which occurs after reduction of four interchain disulfide bonds, and thus the conjugation is limited to eight exposed sulfides

- 8 046855 ryl groups and, therefore, the bound biologically active drug molecules per antibody can range from 0 to 8. The heterogeneity of an ADC mixture is twofold, since these types of ADCs differ in drug loading and conjugation site. Therefore, each species may have different properties, which may result in a wide range of PK properties in vivo. Additionally, ensuring batch-to-batch consistency in ADC production can be challenging and may require the necessary manufacturing capacity.

Site-specific antibody-drug conjugation, in which a known number of biologically active drug molecules are continuously conjugated to specific sites, is one way to overcome these problems. Heterogeneity is minimized and ADC properties are more predictable, with consistent conjugate production from batch to batch. The drug/antibody ratio (DAR) is precisely controlled and can be tailored to various related bioactive drugs producing either 2- or 4-DAR site-specific ADCs. Non-limiting examples of site-specific conjugation include the binding of a biologically active drug molecule to an antibody via an engineered cysteine residue, a glutamine residue, unnatural amino acids (e.g., pacetylphenylalanine, N-((2-azidoethoxy)carbonyl)-E-lysine, p-azidomethyl-b -phenylalanine, selenocysteine), glycans or short peptide tags, as indicated by Qun Zhou in the review of Site-Specific Antibody Conjugation for ADC and Beyond which is incorporated by reference (Qun Zhou, Site-Specific Antibody Conjugation for ADC and Beyond, 5 BIOMEDICINES 64 ( 2017)).

In some exemplary embodiments, the present invention provides a method for characterizing, identifying and/or quantifying at least one impurity in a biopharmaceutical containing a protein.

The term impurity, as used herein, can include any undesirable protein present in a biopharmaceutical product containing the protein. Impurities may include process and related impurities. In addition, the impurity may have a known structure, partially characterized or unidentified. Process impurities can be generated during the manufacturing process and can include three main categories: cell substrate-derived, cell culture-derived, and product isolation and purification. Impurities derived from the cellular substrate include, but are not limited to, host-derived proteins and nucleic acid (host cell genomic, vector, or total DNA). Impurities derived from cell cultures include, but are not limited to, inducers, antibiotics, serum and other media components. Impurities produced downstream include, but are not limited to, enzymes, chemical and biochemical processing agents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ions), solvents, carriers , ligands (eg monoclonal antibodies) and other substances that can be removed by leaching. Product-associated impurities (e.g., precursors, certain degradation products) may be molecular variants arising during manufacturing and/or storage that do not have properties comparable to those of the desired product with respect to potency, efficacy, and safety. Such variants may require significant extraction and characterization efforts to identify the type of modification(s). Product-associated impurities may include truncated forms, modified forms, and aggregates. Truncated forms are formed by hydrolytic enzymes or chemicals that catalyze the cleavage of peptide bonds. Modified forms include, but are not limited to, deamidated, isomerized, non-SS-linked, oxidized, or altered conjugated forms (eg, glycosylation, phosphorylation). Modified forms may also include any form of post-translational modification. Aggregates include dimers and higher order formations of the desired product. (Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, ICH August 1999, U.S. Dept. of Health and Humans Services).

As used herein, the general term post-translational modifications or PTMs 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 of them arise at a site of a specific characteristic protein sequence (signature sequence) within the protein backbone. Several hundred PTMs have been reported, and these modifications invariably affect some aspect 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, cleavage, N-terminal extension, protein degradation, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent addition of prosthetic groups, acetylation (adding acetyl group, usually at the N-terminus of the protein), alkylation (adding an alkyl group

- 9 046855 (e.g. methyl, ethyl, propyl) usually at lysine or arginine residues), methylation, adenylation, ADP-ribosylation, covalent cross-links within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (hydroxylation of proline and lysine and amidation of the carboxyl terminus), vitamin K-dependent modification, where vitamin K is a cofactor for the carboxylation of glutamic acid residues leading to the formation of γ-carboxyglutamate (glu residue), glutamylation (covalent binding of glutamic acid residues), glycation (covalent binding of glycine residues ), glycosylation (adding a glycosyl group to asparagine, hydroxylysine, serine or threonine, resulting in a glycoprotein), isoprenylation (adding an isoprenoid group such as farnesol and geranylgeraniol), lipoylation (adding lipoate functionality), phosphopantetheinylation (adding a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis), phosphorylation (adding a phosphate group, usually at a serine, tyrosine, threonine or histidine) and sulfation (adding a sulfate group, usually at a tyrosine residue). Post-translational modifications that change the chemical nature 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, disulfide bridge formation (the covalent linking of two cysteine amino acids) and proteolytic cleavage (cleavage of a protein at a peptide bond). Some post-translational modifications include the addition of other proteins or peptides, such as ISGylation (covalently linked to the ISG15 (interferon-stimulated gene) protein), SUMOylation (covalently linked to the SUMO (Small Ubiquitin-Like Modifier) protein), and ubiquitination (covalently linked to the ubiquitin protein ). 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 Jan 15 , 2019) for a more detailed controlled vocabulary of PTMs curated by UniProt.

As used herein, the term desired product refers to a biopharmaceutical containing a protein that has a desired structure, function, or efficacy profile.

In some exemplary embodiments, the present invention also provides a method for characterizing the binding of a biopharmaceutical containing a protein. For example, antibodies can bind antigens through highly specific, high-affinity noncovalent interactions, a property that has allowed the development of therapeutic antibodies to target disease-specific antigens in the treatment of various diseases (Andrew C. Chan & Paul J. Carter, Therapeutic antibodies for autoimmunity and inflammation, 10 NATURE REVIEWS IMMUNOLOGY 301-316 (2010)). To develop effective antibody therapeutics, understanding how antibody binding affects the function of the target protein may be critical.

In some exemplary embodiments, the present invention also provides a method for determining the binding ratio of an antibody to an antigen from an antigen-antibody complex.

In certain exemplary embodiments, the invention also provides a method for identifying an antigen to which an antibody binds. In some other exemplary embodiments, the method may include: determining whether cleaved, modified, or mutated versions of an antigen and/or antibody are responsible for an antigen-antibody complex.

In some exemplary embodiments, the invention also provides a method for quantifying the relative abundance of individual proteins in a solution.

In some exemplary embodiments, a method for characterizing, identifying and/or quantifying a protein-containing biopharmaceutical, its possible impurities, binding, or composition may include contacting a sample, protein-containing biopharmaceutical, with a chromatography system containing a chromatography resin.

As used herein, the term chromatography refers to the method in which a chemical mixture carried by a liquid or gas can be separated into components as a result of the differential distribution of chemical entities as they are carried around or over a stationary liquid or solid. Non-limiting examples of chromatography include conventional reverse phase (RP), ion exchange (IO), mixed mode, and normal phase (NP) chromatography.

As used herein, the term mixed mode chromatography (MCC) or multimodal chromatography includes a chromatographic technique in which solutes interact with a stationary phase through more than one interaction mode or mechanism. CCP can be used as an alternative or complement to traditional reverse phase (RP), ion exchange (IE), and normal phase (NP) chromatography. Unlike OF,

- 10 046855

NF and IO chromatography, in which hydrophobic interaction, hydrophilic interaction and ionic interaction, respectively, are the dominant interaction modes, mixed mode chromatography can use a combination of two or more of these interaction modes. Mixed-mode chromatography media can provide unique selectivity that cannot be achieved with single-mode chromatography. Mixed mode chromatography can also provide potential cost savings and operational flexibility compared to affinity-based methods.

In some exemplary embodiments, the chromatography may be size exclusion chromatography.

As used herein, the terms SEC chromatography resin or SEC chromatography medium are used interchangeably and can include any type of solid phase used in SEC that separates the impurity from the desired product (eg, a homodimeric impurity for a bispecific antibody product). The volume of resin, the length and diameter of the column used, and the dynamic capacity and flow rate can depend on several parameters such as the volume of liquid being processed, the protein concentration of the liquid to be processed.

In some exemplary embodiments, a method for characterizing, identifying, and/or quantifying a biopharmaceutical containing a protein, its possible impurities, binding, or composition may include contacting a sample of the biopharmaceutical containing the protein with a chromatography system comprising a size exclusion chromatography resin. by size, washing said size exclusion chromatography resin using the mobile phase to obtain an eluate containing the protein; and characterizing the protein in said eluate using an electrospray ionization mass spectrometer.

As used herein, the term mass spectrometer includes a device capable of identifying specific molecular species and accurately measuring their masses. The term is intended to include any molecular detector into which a polypeptide or peptide can be eluted for detection and/or characterization. A mass spectrometer may have three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to create ions in the gas phase. Atoms, molecules or clusters of the analyte can be transferred into the gas phase and ionized simultaneously (as in electrospray ionization). The choice of ion source is highly dependent on the application.

As used herein, the term electrospray ionization or ESI refers to a spray ionization process in which cations or anions in solution are transferred into the gas phase through the formation and desolvation at atmospheric pressure of a stream of highly charged droplets that results from the application of a potential difference between the tip of an electrospray needle. containing the solution, and a counter electrode. There are usually three main stages of obtaining ions in the gas phase from electrolyte ions in solution. These are: (a) the formation of charged droplets on the ER infusion tip; (b) compression of charged droplets through solvent evaporation and repeated droplet disintegration, resulting in small, highly charged droplets capable of producing ions in the gas phase; and (c) the mechanism by which ions in the gas phase are formed from very small and highly charged droplets. Steps (a)-(c) typically occur in a region of the device where the pressure is atmospheric pressure.

As used herein, the term electrospray infusion unit refers to an electrospray ionization system that is compatible with a mass spectrometer used for protein mass analysis. In electrospray ionization, the orifice of the electrospray needle is located adjacent to the inlet of the spectrometer. A sample containing the protein of interest can be pumped through a syringe needle. The electrical potential between the syringe needle hole and the hole leading to the mass analyzer creates an aerosol (electrospray) of the solution. Electrospray can be performed at atmospheric pressure and produces highly charged solution droplets. The electrospray infusion apparatus may include an electrospray emitter, a nebulizing gas, and/or an ESI power source. The setup can be further automated to perform aspiration, sample distribution, sample delivery, and/or sample nebulization.

In some exemplary embodiments, the electrospray ionization mass spectrometer may be a nanoelectrospray ionization mass spectrometer.

The term nanoelectrospray or nanospray as used herein refers to electrospray ionization at very low solvent flow rates, typically hundreds of nanoliters per minute or lower of a sample solution, often without the use of external solvent delivery. The electrospray infusion apparatus forming the nanoelectrospray may use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small volumes of sample solution (analyte) over an extended period of time. The dynamic nanoelectrospray emitter uses a capillary column and solvent delivery system to perform chromatographic separations of mixtures prior to analysis by a mass spectrometer.

As used herein, the term mass analyzer includes a device that can

- 11 046855 separate particles, that is, atoms, molecules or clusters, according to their mass. Non-limiting examples of mass analyzers that can be used for rapid protein sequencing include time-of-flight (TOF), magnetic/electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, ion cyclotron resonance conversion Fourier transform (FTICR), as well as with the accelerator mass spectrometry (AMS) method.

In some exemplary embodiments, mass spectrometry can be performed under native conditions.

As used herein, the term native conditions, native MS, or native ESI-MS can include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE1176-1192 (2015). Some differences between native ESI and conventional ESI are shown in the table and Fig. 1 (Hao Zhang et al., Native mass spectrometry of photosynthetic pigment-protein complexes, 587 FEBS Letters 1012-1020 (2013)).

Native IER Conventional ESI Solution sample Water solution water, ammonium acetate Partially organic solution water, formic acid, Acetonitrile/methanol (pH 1-2) State spraying 10-50 nl/min Spray voltage 0.8-1.5 kV Temperature 20-30 °C 10-50 nl/min Spray voltage 0.8-1.5 kV Temperature 20-30 °C Salt processing Offline desalting Online/offline desalting using RP-HPLC Protein concentration 1-10 µM (complex) <1 µM (subunit) Output information Molecular weight of protein complex and subunit Non-covalent interactions Stoichiometry Structure Molecular weight of one subunit

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

As used herein, the term tandem mass spectrometry includes a technique in which structural information about the molecules of a sample is obtained using multiple steps of mass sampling and mass separation. The precondition is that the sample molecules can be brought into the gas phase and ionized unchanged, and that they can be made to decay in some predictable and controllable manner after the first stage of mass selection. Multi-step MS/MS, or MC ⁿ , can be performed by first selecting and isolating the precursor ion ( ^MS2 ), fragmenting it, isolating the primary fragment ion ( ^MS3 ), fragmenting it, isolating the secondary fragment ( ^MS4 ), etc. as long as meaningful information can be obtained or the fragment ion signal can be detected. Tandem MS has been successfully performed with a wide range of analyzer combinations. Which analyzers to combine for a particular application is 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 tandem-in-space and tandem-in-time, but there are also hybrid categories where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m/z separation functions can be designed so that in one section of the instrument, ions are selected, dissociated in an intermediate region, and the product ions are then transferred to another analyzer for m/z separation and data collection. In a time-tandem mass spectrometer, ions produced in the ion source can be captured, isolated, fragmented, and separated by m/z ratio in the same physical device.

Peptides identified by mass spectrometer can be used as surrogate

- 12 046855 representatives of the intact protein and their post-translational modifications. They can be used to characterize proteins by matching experimental and theoretical MS/MS data, the latter generated from candidate peptides in a protein sequence database. Characterization may include, but is not limited to, amino acid sequencing of protein fragments, protein sequencing determination, de novo protein sequencing determination, location of post-translational modifications or identification of post-translational modifications, or comparability analysis, or combinations thereof.

As used herein, the term database refers to bioinformatics tools that provide the ability to search uninterpreted MS/MS spectra against all possible sequences in the database(s). Non-limiting examples of such databases include Mascot (http://www.matrixscience.com), Spectrum Mill (http://www.chem.agilent.com), PLGS (http://www.waters.com), PEAKS ( http://www.bioinformaticssolutions.com), Proteinpilot (http://download.appliedbiosystems.com//proteinpilot), Phenyx (http://www.phenyx-ms.com), Sorcerer (http://www. sagenresearch.com), OMSSA (http://www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem (http://www.thegpm.org/TANDEM/), Protein Prospector (http: //www.http://prospector.ucsf.edu/prospector/mshome.htm), Byonic (https://www.proteinmetrics.com/products/byonic) or Sequest (http://fields.scripps.edu/ sequest).

Example implementation options.

Embodiments disclosed herein provide compositions, methods, and systems for rapidly characterizing proteins in a sample.

This invention provides a method for identifying a protein, comprising contacting a sample, including a protein, with a chromatography system containing a chromatography resin, washing said chromatography resin with Protein A using a mobile phase to obtain an eluate comprising the protein, and characterizing the protein in said eluate using an electrospray ionization mass spectrometer.

This invention provides a method for identifying an antibody-drug conjugate, comprising contacting a sample, including the antibody-drug conjugate, with a chromatography system containing a chromatography resin, washing said chromatography resin with Protein A using a mobile phase to obtain an eluate, containing an antibody-drug conjugate, and characterizing the antibody-drug conjugate in said eluate using an electrospray ionization mass spectrometer.

This invention provides a method for identifying an antigen-antibody complex, comprising contacting a sample, including the antigen-antibody complex, with a chromatography system containing a chromatography resin, washing said chromatography resin with Protein A using a mobile phase to obtain an eluate containing the complex antigen-antibody, and characterizing the antigen-antibody complex in said eluate using an electrospray ionization mass spectrometer.

In some exemplary embodiments, the chromatography system may include conventional reverse phase (RP), ion exchange (IE), or normal phase (NP) chromatography.

In some exemplary embodiments, the chromatography resin may be selected from affinity chromatography, anion exchange chromatography, cation exchange chromatography, affinity chromatography, mixed mode chromatography, hydrophobic interaction chromatography, or size exclusion chromatography resin.

In some exemplary embodiments, the electrospray ionization mass spectrometer may be a nanoelectrospray ionization mass spectrometer.

In some exemplary embodiments, the electrospray ionization mass spectrometer may be coupled to a chromatography system with a chromatography resin.

In some exemplary embodiments, the electrospray ionization mass spectrometer can be operated under native conditions.

In some exemplary embodiments, the chromatography system may be coupled to an electrospray ionization mass spectrometer using a three-pass flow splitter.

In some exemplary embodiments, the chromatography system may be coupled to an ultraviolet detector using a three-way flow splitter.

In some exemplary embodiments, the chromatography system may be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-pass flow splitter.

In some exemplary embodiments, the chromatography system may be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-way flow splitter, wherein the electrospray ionization mass spectrometer

- 13 046855 em is a nanoelectrospray ionization mass spectrometer.

In some exemplary embodiments, the chromatography system may be coupled to a mass spectrometer and to an ultraviolet detector using a three-pass flow splitter, wherein the mass spectrometer is an electrospray ionization mass spectrometer operating under native conditions.

In some exemplary embodiments, the chromatography system may be coupled to an electrospray ionization mass spectrometer and an ultraviolet detector using a three-pass flow splitter, wherein the electrospray ionization mass spectrometer is a nanoelectrospray ionization mass spectrometer operating under native conditions.

In some exemplary embodiments, the eluate containing the protein or antigen-antibody complex or antibody-drug conjugate, after washing the resin, is introduced into the ultraviolet detector through at least one three-way flow splitter at a flow rate of about 0.2 to about 0.4 mL. /min.

In some exemplary embodiments, the mobile phase for washing has a flow rate of from about 0.2 to about 0.4 ml/min.

In some exemplary embodiments, the mobile phase may contain a volatile salt. In some specific embodiments, the mobile phase may contain ammonium acetate, ammonium bicarbonate or ammonium formate, or combinations thereof.

In some exemplary embodiments, the mobile phase used may be compatible with the mass spectrometer.

In some exemplary embodiments, the mobile phase may have a pH of about 6.0-8.0.

In some exemplary embodiments, the sample can be used in an amount of from about 10 to about 100 μg of protein or antigen-antibody complex or antibody-drug conjugate.

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

In some exemplary embodiments, the electrospray ionization mass spectrometer may have a spray voltage of from about 0.8 to 1.5 kV.

In some exemplary embodiments, characterization may include identifying and/or quantifying a protein. In one aspect, characterization may include protein sequencing, de novo protein sequencing, identification of post-translational modifications, or comparability analysis, or combinations thereof. In another aspect, characterization may include quantifying the relative abundance of a protein.

In some exemplary embodiments, characterization may include identifying and/or quantifying an antibody in an antibody-drug conjugate. In one aspect, characterization may include protein sequencing, de novo protein sequencing, identification of post-translational modifications, or comparability analysis, or combinations thereof. In another aspect, characterization may include quantifying the relative abundance of antibody in an antibody-drug conjugate.

In some exemplary embodiments, characterization may include identifying and/or quantifying an antibody and/or antigen in an antigen-antibody complex. In one aspect, characterization may include protein sequencing, de novo protein sequencing, identification of post-translational modifications, or comparability analysis, or combinations thereof, for an antibody or antigen. In another aspect, characterization may include quantifying the relative abundance of antibody and/or antigen in an antigen-antibody complex.

In some exemplary embodiments, the sample may contain at least two proteins.

In some exemplary embodiments, the antibody-drug conjugate may include a site-specific ADC or a non-site-specific ADC. In one aspect, the antibody-drug conjugate may include non-site-specific ADCs linked via cysteine or lysine residues of the antibody. In another aspect, the antibody-drug conjugate may include site-specific ADCs linked through natural amino acids, non-natural amino acids, glycans, a short peptide tag, or combinations thereof.

In some exemplary embodiments, the electrospray ionization mass spectrometer may be a tandem mass spectrometer.

In some exemplary embodiments, the protein may be a therapeutic antibody, an antibody, a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment, a fusion protein, or combinations thereof. In one aspect, the fragment may include a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a dAb fragment, an Fd' fragment, an Fd fragment, and a complementarity determining region (CDR) region. ), tribodies, tetrabodies, linear antibodies, single-chain antibody molecules and multispecies

- 14 046855 digital antibodies formed from antibody fragments.

In some exemplary embodiments, the protein may be a cleavage product of an antibody. The cleavage product may be formed by a hydrolyzing agent. The cleavage product may be an impurity associated with the product.

In some exemplary embodiments, the protein may be a product-related impurity present in a biopharmaceutical.

In some exemplary embodiments, the protein may have a pI ranging from about 4.5 to about 9.0. In one aspect, the protein may be a protein with 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.

In one exemplary embodiment, the sample may contain at least two proteins.

It should be understood that the methods are not limited to any of the above protein, impurity, and column, and that the identification or quantification methods can be carried out using any suitable means.

In some exemplary embodiments, this disclosure provides a system comprising a chromatography column 100 containing a chromatography resin containing Protein A, the chromatography column being capable of receiving a mobile phase and a sample containing the protein, and an electrospray ionization mass spectrometer 110 (see Lig. 2).

In some exemplary embodiments, the chromatography column 100 may comprise a resin selected from hydrophobic interaction chromatography, anion exchange chromatography, anion exchange chromatography, affinity chromatography, size exclusion chromatography, mixed mode chromatography resins, or combinations thereof.

In some exemplary embodiments, electrospray ionization mass spectrometer 110 may be coupled to said chromatography column 100.

In some exemplary embodiments, electrospray ionization mass spectrometer 110 may be operated under native conditions.

In some exemplary embodiments, electrospray ionization mass spectrometer 110 may be a nanoelectrospray ionization mass spectrometer.

In some exemplary embodiments, electrospray ionization mass spectrometer 110 may be a nanoelectrospray ionization mass spectrometer operating under native conditions.

In some exemplary embodiments, the chromatography column 100 may be coupled to an electrospray ionization mass spectrometer 100 using a three-pass flow splitter 120.

In some exemplary embodiments, chromatography column 100 may be coupled to ultraviolet detector 130 using a three-way splitter 120.

In some exemplary embodiments, the chromatography column 100 may be coupled to an ultraviolet detector 130 and an electrospray ionization mass spectrometer 110 using a three-way splitter 120.

In some exemplary embodiments, the three-pass flow splitter 120 may be capable of disproportionately splitting the flow to provide flow from the chromatography column 100 to the ultraviolet detector 130 and the electrospray ionization mass spectrometer 110.

In some exemplary embodiments, the system may be capable of characterizing the drug-antibody ratio of an antibody-drug conjugate.

In some exemplary embodiments, the system may be capable of characterizing a protein.

In some exemplary embodiments, the system may be capable of characterizing an antigen-antibody complex.

An exemplary implementation of the system is shown in Fig. 3. Three-way post-column flow splitter is used for dual UV/MS detection. The smaller volume fraction can be sent to the MS, while the larger volume fraction is sent to the UV detector. The retention time is almost the same. Fractions after the UV detector can be collected for sample recovery.

Fig. 4 installation reflects a specific embodiment of the invention.

It is understood that the system is not limited to any of the above protein, chromatography column, mass spectrometer, antibody-drug conjugate, antigen-antibody complex.

The sequential labeling of process steps as set forth herein by numbers and/or letters is not intended to limit the process or any variations thereof to the particular order stated.

Various references, including patents, patent applications, published patent applications, accession numbers, technical articles and scientific articles are cited throughout the document. Every source

- 15 046855 is incorporated herein by reference in its entirety and for all purposes.

The invention will be more fully understood with reference to the following examples, which are provided to describe the invention in more detail. They are intended to be illustrative and should not be construed as limiting the scope of the invention.

Examples

Materials and reagents. Water was purchased from Honeywell (Muskegon, MI). Ammonium acetate was purchased from Sigma-Aldrich (St. Louis, MO). 1 M Tris-HCl, pH 7.5, was purchased from Teknova (Hollister, CA). Fused silica tubing (inner diameter (ID) 150 μm, outer diameter (OD) 360 μm), 3-way connector, and sleeve were purchased from IDEX (Oak Harbor, WA). Silica PicoTip EMITTER (FS360-20-10-D-20-7CT) was purchased from New Objective (Woburn, MA). ACQUITY UPLC Protein VEN SEC, 200A, 1.7 μm, 4.6 x 300 mm column was purchased from Waters (Milford, MA). Hot pocket column heater was purchased from Thermo-Fisher (Waltham, MA). All reagents were used without additional purification.

Online SEC-nano-ESI-MS analysis. A Class I ACQUITY HPLC system (Waters, Milford, MA) was coupled to a Q Exactive HF hybrid quadrupole orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) for all online SEC-nano-ESI-MS analyses. The temperature of an ACQUITY UPLC Protein BEH SEC column (200A, 1.7 µm, 4.6 x 300 mm) was set to 30°C and used to separate mAbs and ADCs. The mobile phase was 100 mM ammonium acetate at pH 6.8. Each separation lasted 30 min at a flow rate of 0.3 mL/min and the injection amount was 40 μg. A three-way flow splitter (T-splitter) was connected after the SEC column. A fused silica tube (L: 140 cm, ID: 150 μm) and a silica tip (SilicaTip) (L: 5 cm, ID: 10 μm) were connected to the T-splitter. The larger volume fraction was applied to the UV detector via a fused silica tube, while the smaller volume fraction was applied to the MS via a SilicaTip. The following MS parameters were used for online SEC-nano-IERMS data acquisition. Each data collection lasted 25 min, starting from the moment of sample injection. The samples were ionized in positive mode at a spray voltage of 3 kV, capillary temperature of 200 °C and RF level of 70 S lenses. The CID at the source was set to 75 eV. Full MS scans were acquired at a resolution of 15 K with a mass range of m/z 2000–8000. A maximum injection time of 100 ms, an AGC target of 3e6, and 10 microscans were used for a full MS scan.

Data analysis. Protein Metrics Intact Mass software was used to deconvolve the raw data. The Thermo Xcalibur Qual browser was used to analyze the extracted ion chromatogram. Microsoft Excel was used to calculate DAR ADC.

Example 1: Investigation of antigen-antibody interactions using online SEC-nano-ESI-MS.

To develop effective antibody therapeutics, understanding how antibody binding affects the function of the target protein may be critical.

1.1. Online measuring instruments SEC-nano-ESI-MS.

SEC and MS technologies are commonly used to characterize protein samples. SEC allows you to isolate and characterize proteins under conditions that minimize changes in protein structure, while MS allows you to identify individual components in complex samples. Integrating the individual capabilities of SEC and MS into a single platform would be highly desirable but has proven challenging because the high flow rates and nonvolatile salts used for SEC assays are incompatible with native MS. To overcome this limitation, reduction of solvent and salt input into the MS was accomplished by splitting the eluate stream from the SEC using a post-column T-splitter (see Figure 3). An image of the installation is shown in Fig. 4. The Splitter was then connected to the MS via a SilicaTip and in parallel to the UV detector via a fused silica tube. This arrangement allowed for simultaneous dual UV/MS detection of SEC eluates. By varying the length and diameter of the fused silica tube, the flow rate to the MS through the SilicaTip can be adjusted (e.g., longer/narrower tubes can create higher resistance, causing increased flow into the SilicaTip and MS). Protein samples were separated using a 4.6 mm SEC column at a flow rate of 0.3 mL/min. The fused silica tube connecting the T-splitter and UV detector, 140 cm long and 150 μm internal diameter, provided the desired flow rate of ∼1 μL/min to the SilicaTip. The length and diameter of the fused silica tube also allowed near-simultaneous detection of molecules in UV and MS.

1.2. Antigen-antibody complex.

A complex formed between a previously described antibody (Qian Zhang et al., Epitope Mapping by HDX-MS Elucidates the Surface Coverage of Antigens Associated with High Blocking Efficiency of Antibodies to Birch Pollen Allergen, 90 ANALYTICAL CHEMISTRY 11315-11323 (2018)) and a recombinant Bet v 1 and Bet v 1 antigen under native MS conditions.

In addition to the naked form, two major glycosylated forms were observed for Bet v 1, including G2S1F and G2S2F, as presented in FIG. 5. After incubation with equal molar amounts of an

- 16 046855 of the Fab-1 antibody, all three forms of Bet v 1 (naked, G2S1F and G2S2F) bound Fab-1 as a single antigen to a single Fab complex. in fig. 5A, the deconvoluted spectrum of the native MS antigen Bet v 1 only revealed three different species of Bet v 1: naked Bet v 1 and two major glycosylated forms, Bet v 1 G2S1F and G2S2F. Incubation of Bet v 1 antigen with equal molar amounts of Bet v 1 Fab-1 antibody demonstrated that all three forms of Bet v 1 (naked, G2S1F, and G2S2F) complexed with Fab-1 in a ratio of one antigen to one Fab (Fig. 5B). It appears that glycosylation does not affect the formation of the antibody/antigen complex, since the relative amount of MS signal for all forms of Bet v 1 was the same under unbound and bound Fab-1 conditions.

To further characterize the antigen–antibody interaction, a titration experiment was performed on an MS and UV dual detection platform. Several different ratios of Bet v 1 and Fab-1 were examined by UV, as shown in FIG. 6. Only Bet v 1 (black) elutes at 8.5 min, while only Fab-1 (turquoise) and Bet v 1: Fab-1 compodex elute at 9.8 min and 7.9 min, respectively. The stoichiometry of the Bet v 1:Fab-1 complex revealed a 1:1 binding ratio. No additional stoichiometries were observed for excess amounts of antigen or antibody. Additionally, mixing Bet v1 antigen with Fab-1 in a precise 1:1 molar ratio minimized the amount of free antigen and antibody (blue).

While UV peaks can reflect the relative abundance of individual proteins in a solution, MS allows the identification of individual components in complex samples. With dual UV and MS detection, all species co-eluting within the same UV peak or across different UV peaks can be identified. As shown in FIG. 7, MS analysis of the UV peak that elutes at 7.9 min shows three different complexes formed by Fab-1 bound to each of three different Bet v 1 species (naked, G2S1F, and G2S2F). We found that the Fab-1:naked complex eluted after Fab1 in complex with glycosylated Bet v 1 particles, which may be due to the large hydrodynamic radius provided by the glycans. Minimizing the detection delay between UV and MS allowed collection of fractions after the UV detector for sample recovery. This method is particularly useful for determining whether cleaved, modified or mutated versions of an antigen and/or antibody retain binding without the need to purify specific forms of the proteins of interest.

Example 2: Characterization of cysteine ADCs using online SEC-nano-ESI-MS.

Antibody-drug conjugates (ADCs) are highly effective therapeutics that can specifically deliver small molecule drugs to target tissue by conjugating them to antibodies (Francois Debaene et al., Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MSfor Average DAR and DAR Distribution Assessment, 86 ANALYTICAL CHEMISTRY 10674-10683 (2014)). The activity, efficacy, and toxicity of ADCs can be highly dependent on the number of small molecule drugs conjugated to each antibody. Therefore, it may be critical to determine the drug-antibody ratio (DAR) for each ADC. Conjugation via interchain cysteines is one of the most common approaches for conjugating small molecule drugs to antibodies. For cysteine-based interchain ADCs, unpaired interchain cysteine residues can be introduced by engineering primary sequence mutations (site-specific cysteine-based interchain conjugates) or by partial reduction of the antibody (random cysteine-based interchain conjugates). Site-specific conjugation of engineered mAbs provides better control over the DAR ADCs, whereas conjugation performed on partially reconstituted mAbs results in a more variable range of DARs (zero to eight). However, it may still be necessary to identify the DARs of ADCs generated by either method to understand and interpret the biological effects of these drug conjugates. The most common method for detecting cysteine-based DAR ADCs is the HIC-UV method (Laura R. Saunders et al., A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo, 7 SCIENCE TRANSLATIONAL MEDICINE (2015) or RPLC-MS under reducing/denaturing conditions However, an off-line SEC desalting method coupled with native MS and high-resolution LM-MS to measure average DAR was recently reported by Debaene et al. DAR for cysteine-conjugated ADCs without destroying the intact molecule.

1.1. Online measuring instruments SEC-nano-ESI-MS.

We used the devices presented in 1.1.

1.2. Site-specific cysteine-based conjugated ADC.

The DAR of site-specific cysteine-based conjugated ADCs was assessed using an online SEC-nano-ESI-MS system. Interchain cysteine mutations on the mAb heavy chains introduced two unpaired cysteines on the mAb light chains, which were conjugated to the drug to form DAR 2 antibodies. As shown in FIG. 8, raw and deconvoluted spectra of parent mAb-1 and ADC-1 show that only ADC is present in the sample

- 17 046855 form with DAR 2. Deconvoluted spectra in Fig. 8A-C show that different glycans are present on both the parent mAb-1 and the conjugated ADC-1. Along with non-glycosylated mAb-1, partially and fully glycosylated mAb-1 species having various combinations of GOF, GIF and G2F were all conjugated to 2 drugs. These results were confirmed by analysis of manufacturer-digested mAb-1 and ADC-1 as shown in FIG. 9. Several ADCs with similar conjugation chemistry were tested and showed only the DAR 2 form (data not shown).

1.3. Non-specific cysteine-conjugated ADC.

In addition to analyzing site-specific ADCs, drug DARs conjugated to interchain cysteines of partially reduced mAbs were also examined. Raw and deconvoluted spectra of the nonspecific cysteine-conjugated ADC obtained using similar chemistry are shown in FIG. 10. ADC-2 was generated with an unmodified parent antibody with normal glycosylation. The raw and deconvoluted spectra of the original mAb-2 (Fig. 10A,B) and ADC-2 (Fig. 10C-D) show DAR values that range from 2-8 for ADC-2. The deconvoluted spectra also show different glycans present in mAb-2 (Fig. 10B) and ADC-2 (Fig. 10D).

An online platform for SEC-nano-electrospray ionization (nano-ESI)MS with dual ultraviolet (UV) and MS detection was developed. The utility of this platform was validated by studying non-covalent protein interactions, using it to characterize antigen-antibody complexes obtained from titration experiments and determining the drug-antibody ratio (DAR) of cysteine-based antibody-drug conjugates (ADCs). This platform can be easily modified and therefore can be adapted for the analysis of other native MS projects, such as the characterization of charge variants of monoclonal antibodies (mAbs) or large aggregated protein complexes.

A three-way flow splitter was used to disproportionately separate SEC eluates between the MS and UV detector, with the smaller volume fraction sent to the MS and the larger volume fraction to the UV detector. The current platform enables complementary dual detection by UV and native MS with fraction collection capabilities and can be used to characterize antigen-antibody complexes and DAR assays of interchain cysteine-conjugated ADCs. Further modifications to this online SEC-nano-ESI-MS platform, such as changing the column chemistry or using the Q Exactive UHMR instrument, will allow it to be adapted for other applications, such as the analysis of charge variants or very large protein complexes. The method described herein has opened the possibility of combining high-salt separation methods (e.g., HIC, WCX) with mass spectrometry-based detection. In conclusion, the online SEC-nano-ESI-MS platform can be widely applied to analyze protein-containing biopharmaceuticals for various applications.

Claims (17)

1. A method for characterizing an antibody-drug conjugate, comprising: contacting a sample containing the antibody-drug conjugate with a chromatography system containing a size exclusion chromatography resin; washing said size exclusion chromatography resin using a mobile phase to obtain an eluate containing the antibody-drug conjugate; and characterizing the antibody-drug conjugate in said eluate using an electrospray ionization mass spectrometer under native conditions, wherein at least one three-pass is used to couple the electrospray ionization mass spectrometer to a chromatography system containing a size exclusion chromatography resin stream separator. 2. The method of claim 1, wherein the electrospray ionization mass spectrometer is coupled to a chromatography system containing a size exclusion chromatography resin, or the electrospray ionization mass spectrometer is coupled online to a chromatography system containing a size exclusion chromatography resin. size. 3. The method according to claim 1, characterized in that the electrospray ionization mass spectrometer is a nanoelectrospray ionization mass spectrometer. 4. The method of claim 1, wherein at least one three-way flow splitter is used to couple the ultraviolet detector to a chromatography system containing a size exclusion chromatography resin. 5. The method according to claim 4, characterized in that the eluate after washing the resin for size exclusion chromatography is introduced into an ultraviolet detector through at least one three-way flow divider at a flow rate of 0.2 to 0.4 ml/min. 6. The method according to claim 1, characterized in that the mobile phase used for washing the resin for - 18 046855 size exclusion chromatography, contains ammonium acetate. 7. The method according to claim 1, characterized in that the mobile phase used to wash the size exclusion chromatography resin contains a volatile salt. 8. The method of claim 1, wherein the mobile phase used to wash the size exclusion chromatography resin has a total concentration of about 100 mM. 9. The method according to claim 1, characterized in that the mobile phase used for washing the size exclusion chromatography resin has a flow rate of 0.2 to 0.4 ml/min. 10. The method of claim 1, wherein the mobile phase used to wash the size exclusion chromatography resin has a pH of about 6.8. 11. The method according to claim 1, characterized in that the amount of sample, including the antibody-drug conjugate, brought into contact with the chromatographic system is from 10 to 100 μg. 12. The method of claim 1, wherein the eluate obtained from washing the size exclusion chromatography resin is introduced into the electrospray ionization mass spectrometer at a flow rate of less than about 50 μL/min. 13. The method of claim 1, wherein the eluate obtained from washing the size exclusion chromatography resin is introduced into an electrospray ionization mass spectrometer, wherein the electrospray flow rate in the electrospray ionization is from 10 to 50 nL/min. 14. The method according to claim 1, characterized in that the eluate obtained by washing the size exclusion chromatography resin is introduced into an electrospray ionization mass spectrometer, and the electrospray spray voltage is from 0.8 to 3.0 kV. 15. The method of claim 1, wherein the antibody-drug conjugate is an antibody-drug conjugate with genetically engineered cysteines. 16. The method according to claim 1, characterized in that the antibody-drug conjugate is an antibody-drug conjugate with non-specific cysteines. 17. The method of claim 1, wherein characterization of the antibody-drug conjugate includes characterization of the drug-antibody ratio.