EA042991B1 - METHOD AND SYSTEM FOR PROTEIN IDENTIFICATION AND QUANTITATION - Google Patents

Description

Technical field

The present invention generally relates to a method and system for the identification and/or quantification of a protein.

State of the art

Protein-based biopharmaceuticals must meet very high standards of purity. Biopharmaceuticals contain several process impurities and related impurities. These impurities do not have properties comparable to those of the desired product in terms of potency, efficacy and safety. One example is post-translational modification (PTM) of a protein, which greatly affects the properties of the protein relevant to their therapeutic use. Another such example includes homodimeric contaminants that may be present during the production of bispecific antibodies, which should ideally be removed by subsequent purification. These impurities may exhibit a different mechanism of action and potential toxicity or immunogenicity compared to the product. In addition, they may have lower stability than the product, which poses a higher risk of aggregation and immunogenicity. Despite recent advances, the problem of developing purity assay methods to quantify such impurities remains relevant. In addition, a key issue in developing assay methods for bispecific antibodies may be that the method must accurately and reproducibly detect impurities present at or below 2% of the major desired species. Therefore, it is important to monitor and characterize such impurities at different stages of drug development and manufacture. Despite the importance of impurities for biological function, their large-scale study is difficult due to the lack of suitable methods.

Purity analytical methods must demonstrate sufficient accuracy and resolution to detect and quantify the desired product and its impurities. The evaluation of impurities such as PTMs in antibodies and homodimers in bispecific antibodies can be difficult due to the similarity between the structural and physicochemical properties of such impurities and the desired product. Direct analysis of such impurities requires isolation of the desired product in a sufficiently large amount for analysis, which is undesirable and only possible in some cases.

Thus, there has long been a need in the art for a method and/or system for identifying and quantifying protein, impurities, and/or a desired product in protein-based biopharmaceuticals.

The essence of the invention

The pace of development, manufacturing scale and growth in sales of protein-based biopharmaceutical products has led to an increase in demand for a method and/or system for identifying and quantifying impurities in products.

The embodiments of the present invention described herein satisfy the aforementioned requirements by providing methods and systems for the rapid characterization of proteins.

The present invention at least partially provides a method for quantifying an impurity in a sample.

In one exemplary embodiment, the method may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, washing the mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing impurities, and quantifying the amount of impurities in the eluent using a mass spectrometer.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include contacting said sample with a chromatography system having a mixed mode size exclusion chromatography resin with a hydrophobic interaction function.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include contacting said sample with a chromatography system having a mixed mode size exclusion chromatography resin with a charge-charge interaction function.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include contacting about 10 μg to about 100 μg of a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include washing a mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing the impurity.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase compatible with the mass spectrometer.

- 1 042991

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase, wherein the mobile phase may be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase containing up to 600 mM total salt concentration.

In one aspect of this embodiment, a method for quantifying an impurity in a sample may include washing a mixed mode size exclusion chromatography resin using a mobile phase with a flow rate of 0.2 to 0.4 ml/min.

In one aspect of this embodiment, the method for quantifying the impurity may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a related impurity.

In one aspect of this embodiment, the method for quantifying the impurity may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a process impurity.

In one aspect of this embodiment, the method for quantifying the impurity may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a protein degradation product.

In one aspect of this embodiment, the method for quantifying the impurity may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a protein cleavage product.

In one aspect of this embodiment, a method for quantifying an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be homodimeric species of a multispecific antibody product.

In one aspect of this embodiment, a method for quantifying an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a post-translational modification of a protein.

In one aspect of this embodiment, a method for quantifying an impurity may include quantifying the amount of an impurity in said eluent using a mass spectrometer, wherein the mass spectrometer may be a tandem mass spectrometer.

In one aspect of this embodiment, a method for quantifying an impurity may include quantifying the amount of an impurity in said eluent using a mass spectrometer, wherein the mass spectrometer may be a native mass spectrometer.

The present invention at least partially provides a method for detecting an impurity in a sample.

In one exemplary embodiment, the method may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, washing the mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing impurities, and quantifying the amount of impurities in the eluent using a mass spectrometer.

In one aspect of this embodiment, a method for detecting an impurity in a sample may include contacting said sample with a chromatography system having a mixed mode size exclusion chromatography resin with a hydrophobic interaction function.

In one aspect of this embodiment, a method for detecting an impurity in a sample may include contacting said sample with a chromatography system having a mixed mode size exclusion chromatography resin with a charge-charge interaction function.

In one aspect of this embodiment, a method for detecting an impurity in a sample may include contacting about 10 μg to about 100 μg of a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality.

In one aspect of this embodiment, a method for detecting an impurity in a sample may include washing a mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing the impurity.

In one aspect of this embodiment, a method for detecting an impurity in a sample may include washing a mixed mode size exclusion chromatography resin using

- 2 042991 mobile phase compatible with the mass spectrometer.

In some specific exemplary embodiments, a method for detecting an impurity in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase, wherein the mobile phase may be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof.

In some specific exemplary embodiments, a method for detecting an impurity in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase containing a total salt concentration of up to 600 mM.

In one aspect of this embodiment, a method for detecting an impurity in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase with a flow rate of 0.2 to 0.4 ml/min.

In one aspect of this embodiment, a method for detecting an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be an impurity associated with a drug product.

In one aspect of this embodiment, a method for detecting an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a process impurity.

In one aspect of this embodiment, a method for detecting an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a protein degradation product.

In one aspect of this embodiment, a method for detecting an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a protein cleavage product.

In one aspect of this embodiment, a method for detecting an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be homodimeric species of a multispecific antibody product.

In one aspect of this embodiment, a method for detecting an impurity may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the impurity may be a post-translational modification of a protein.

In one aspect of this embodiment, a method for detecting an impurity may include detecting the amount of an impurity in an eluent using a mass spectrometer, wherein the mass spectrometer may be a tandem mass spectrometer.

In one aspect of this embodiment, a method for detecting an impurity may include detecting the amount of an impurity in said eluent using a mass spectrometer, wherein the mass spectrometer may be a native mass spectrometer.

The present invention at least partially provides a method for detecting and/or quantifying a target protein in a sample.

In one exemplary embodiment, the method may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, washing the mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent including the target protein, and detecting and/or quantifying the amount of target protein in the eluent using a mass spectrometer.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein in a sample may include contacting said sample with a chromatography system having a mixed mode size exclusion chromatography resin with a hydrophobic interaction function.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein in a sample may include contacting said sample with a chromatography system having a mixed mode size exclusion chromatography resin with a charge-charge interaction function.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein in a sample may include contacting about 10 μg to about 100 μg of the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality.

In one aspect of this embodiment, a method for detecting and/or quantifying a protein of interest in a sample may include washing a mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing the contaminant.

In one aspect of this embodiment, a method for detecting and/or quantifying a protein of interest in a sample may include washing the mixed mode size exclusion chromatography resin using a mass spectrometer compatible mobile phase. In a specific aspect, a method for detecting and/or quantifying a protein of interest in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase, wherein the mobile phase may be selected from ammonium acetate, ammonium bicarbonate, or ammonium formate, or a combination thereof. In another specific aspect, a method for detecting and/or quantifying a target protein in a sample may include washing the mixed mode size exclusion chromatography resin using a mobile phase containing a total salt concentration of up to 600 mM.

In one aspect of this embodiment, a method for detecting and/or quantifying a protein of interest in a sample may include washing a mixed mode size exclusion chromatography resin using a mobile phase with a flow rate of 0.2 to 0.4 ml/min.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be an antibody.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be a bispecific antibody.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be a therapeutic protein.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be an impurity.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be a process admixture of a biopharmaceutical protein manufacturing process.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be a related impurity of a biopharmaceutical protein manufacturing process.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting a sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be a degradation product of the protein.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be a protein cleavage product.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include contacting the sample with a chromatography system having a mixed mode size exclusion chromatography resin with additional functionality, wherein the target protein may be homodimeric species of a multispecific antibody product.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include quantifying the amount of target protein in said eluent using a mass spectrometer, wherein the mass spectrometer may be a tandem mass spectrometer.

In one aspect of this embodiment, a method for detecting and/or quantifying a target protein may include quantifying the amount of target protein in said eluent using a mass spectrometer, wherein the mass spectrometer may be a native mass spectrometer.

In one exemplary embodiment, the present invention at least partially provides a mixed mode chromatography system with a chromatography column having a mixed mode size exclusion chromatography resin with additional functionality and a mass spectrometer.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mixed mode size exclusion chromatography resin with a hydrophobic interaction function.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mixed mode size exclusion chromatography resin with a charge-charge interaction function.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mixed mode size exclusion chromatography resin with additional functionality that can be used to elute about 10 µg to about 100 µg of sample.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mixed mode size exclusion chromatography resin capable of accepting a mobile phase.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mixed mode size exclusion chromatography resin further capable of receiving a sample containing the target protein.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mixed mode size exclusion chromatography resin that can be washed with a mobile phase.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a mass spectrometer coupled to a chromatography column having a mixed mode size exclusion chromatography resin with additional functionality.

In one aspect of this embodiment, the mixed mode chromatographic system may comprise a tandem mass spectrometer.

In one aspect of this embodiment, the mixed mode chromatographic system may comprise a native mass spectrometer.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a chromatography column having a mixed mode size exclusion chromatography resin with additional functionality, wherein the mixed mode size exclusion chromatography resin can be compatible with a mobile phase selected from ammonium acetate, bicarbonate ammonium or ammonium formate, or combinations thereof.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a chromatography column having a mixed mode size exclusion chromatography resin with additional functionality, wherein the mixed mode size exclusion chromatography resin can be washed using a mobile phase containing up to 600 mM total concentration salt.

In one aspect of this embodiment, the mixed mode chromatography system may comprise a chromatography column having a mixed mode size exclusion chromatography resin with additional functionality, wherein the chromatography column can be washed with a mobile phase at a flow rate of 0.2 to 0.4 ml/min. .

Brief description of graphic materials

Fig. 1 shows an example of a system used to quantify and/or detect a protein using size exclusion chromatography or ion exchange chromatography.

Fig. 2 shows an approach to purifying a bispecific antibody from homodimeric species using an exemplary embodiment.

Fig. 3 shows a Hofmeister series showing the effect of anions and cations on protein precipitation (or promoting hydrophobic interaction).

Fig. 4 shows a mixed mode size exclusion chromatography and mass spectrometry system according to an exemplary embodiment.

Fig. 5 shows retention time plots of eight mAbs in a mixture of samples on a BEN200 SEC column obtained with mobile phase concentrations ranging from 30 to 300 mM, with two insets representing base peak chromatograms from MM-analysis. SEC-MS of eight mAbs at appropriate concentrations according to a typical embodiment.

Fig. 6 shows a chromatographic profile of a bispecific antibody sample obtained using a typical mixed mode size exclusion chromatography and mobile phase mass spectrometry system: total salt concentration 150 mM and total concentration

- 5 042991 salts 450 mM.

Fig. 7 shows an extracted ion chromatogram (XIC) and native mass spectra of a bispecific antibody, homodimer 1 and homodimer 2, separated and analyzed by a mixed mode size exclusion chromatography/mass spectrometry system according to an exemplary embodiment.

Fig. 8 shows a comparison of total ion chromatogram (TIC) and native MS spectra for RP LC-MS on Lumos, SEC-MS on EMR, and MM-SEC-MS on EMR antibody-detected spectrometry according to a typical embodiment.

Fig. 9 shows extraction ion chromatograms (XIC) and native mass spectra of serial MM-SEC-MS antibody detection cycles using Zenix SEC-300, 3 μm, 300 A, 7.8x300 mm according to a typical embodiment.

Fig. 10 shows extraction ion chromatograms (XIC) obtained by performing detection of homodimeric species in a bispecific antibody product according to an exemplary embodiment.

Fig. 11 shows a comparison of extraction ion chromatograms (XIC) obtained when detecting homodimeric species in a bispecific antibody product using Zenix SEC-300, 3 μm, 300 A, 7.8x300 mm at a flow rate of 0.4 ml/min and Zenix SEC-300. 300, 3 µm, 300 A, 4.6x300 mm at a flow rate of 0.3 ml/min according to a typical embodiment.

Fig. 12 shows extraction ion chromatograms (XIC) obtained by performing MM-SEC-MS analysis of a deglycosylated mixture of bispecific antibody, homodimer 1 and homodimer 2 using a mobile phase with different salt concentration according to an exemplary embodiment.

Fig. 13 shows a graph of protein retention time (in minutes) versus total salt concentration in the mobile phase for a deglycosylated mixture of bispecific antibody, homodimer 1, and homodimer 2 in an MM-SEC-MS analysis according to an exemplary embodiment.

Fig. 14 is a graph showing the trend of retention time based on change in total salt concentration when performing an MM-SEC-MS analysis according to an exemplary embodiment.

Fig. 15 is a graph showing the trend of retention time with total salt concentration when performing an MM-SEC-MS analysis according to an exemplary embodiment.

Fig. 16 shows extraction ion chromatograms (XIC) obtained by performing MM-SEC-MS analysis of a deglycosylated mixture of bispecific antibody, homodimer 1 and homodimer 2 on a Waters BEN SEC column according to an exemplary embodiment.

Fig. 17 shows a plot of protein retention time (in minutes) versus total salt concentration in the mobile phase for a deglycosylated mixture of bispecific antibody, homodimer 1, and homodimer 2 in an MM-SEC-MS analysis on a Waters BEN SEC column according to an exemplary embodiment.

Fig. 18 shows extraction ion chromatograms (XIC) obtained by performing MM-SEC-MS analysis of an antibody and its oxidized variant on a Waters BEN SEC column according to an exemplary embodiment.

Fig. 19 shows a graph of protein retention time (in minutes) versus total salt concentration in the mobile phase for an antibody and its oxidized variant in an MM-SEC-MS analysis on a Waters BEN SEC column according to an exemplary embodiment.

Fig. 20 shows a method for preparing a sample mixture containing a bispecific antibody and homodimeric species thereof, according to an exemplary embodiment.

Fig. 21 shows intact level MM-SEC-MS analysis of a mixture of a bispecific antibody and its homodimeric species using a 300 mM salt mobile phase according to an exemplary embodiment.

Fig. 22 shows subunit level MM-SEC-MS analysis of a mixture of a bispecific antibody and its homodimeric species using a 70 mM salt mobile phase according to an exemplary embodiment.

Fig. 23 shows the results of homodimer quantitation from intact level MM-SEC-MS analysis of a bispecific antibody and homodimer species thereof, according to an exemplary embodiment.

Fig. 24 shows the results of homodimer quantitation from MM-SEC-MS analysis at the subunit level of a bispecific antibody and its homodimer species according to an exemplary embodiment.

Fig. 25 shows the analysis of mixtures of bispecific antibodies [four different mixtures of bsAb/H2L2 homodimer/H ⁺ 2L2 homodimer] by MM-SEC-MS either on a BEN column (left) or

- 6 042991 on a Zenix column (right), carried out according to exemplary embodiments, where each curve

HRV is a separate assay using the indicated mobile phase salt concentration.

Fig. 26 shows MM-SEC-MS detection limit study for homodimeric impurities in bsAb2 (left panel) and bsAb4 (right panel) using added standards of 0.01% and 0.1%, respectively, for analysis performed according to exemplary embodiments, where native MS spectra (a, b, c, d, e, and f) are averaged over the respective TIC regions, and the two most common charge states are shown.

Fig. 27 shows a quantitative assay performed according to exemplary embodiments using homodimers added to bsAb2 at serially diluted known ratios (gray line and marked values in left panel) and shows measured ratios of H2L2 homodimer and H*2L2 homodimer in bsAb2 based on intensity XIC of the four most common charge states in the raw mass spectrum (0.1% relative abundance of each homodimer as an example shown in the right panel).

Detailed description

Impurities in biopharmaceuticals can cause changes that could potentially affect the efficacy, clearance, safety, and immunogenicity of the desired product. For example, oxidation of methionine and tryptophan side chains can affect antibody binding to Fc receptors and antigens (Bertolotti-Ciarlet et al., Mol. Immunol. (2009), 46:1878-1882; Pan et al., Protein Sci. (2009 ), 18:424-433; Wei et al., Anal. Chem. (2007), 79:2797-2805; and Wang et al., Mol. Immunol. (2011), 48:860-866).

Traditional separation-based antibody purity assays, such as electrophoresis and high performance liquid chromatography (HPLC) methods, do not have the resolution necessary to distinguish these impurities from the desired product. Peptide mapping using reverse phase liquid chromatography (RPLC) in combination with mass spectrometry used for RTM monitoring has some limitations because sample preparation for RP-LC-MS is lengthy and in some cases, chromatographic conditions such as high temperature, organic solvents, and acidic pH can cause oxidation artifacts.

In addition, some size exclusion chromatography or ion exchange chromatography techniques can also be used to separate impurities from the desired product. The separated impurities and the desired product can be further analyzed with a mass spectrometer. However, the mobile phase from a size exclusion chromatography or ion exchange chromatography column cannot be directly injected into the mass spectrometer and requires additional steps including replacement of the mobile phase (see FIG. 1).

Taking into account the limitations of existing methods, an efficient and effective method for the identification and quantification of impurities was developed using a new system of mixed mode size exclusion chromatography and mass spectrometry, as described in this document. The Mixed Mode Size Exclusion Chromatography and Mass Spectrometry system improves sensitivity and the ability to quantify impurities present at very low levels through efficient mixed mode separations and sensitive online MS detection that cannot be achieved with other generic assays.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by a person of ordinary skill in the field to which this invention belongs. While any methods and materials similar or equivalent to those described herein may be practiced or tested, specific methods and materials are described herein. All publications mentioned are incorporated herein by reference.

When used in the singular, the term is to be understood as meaning at least one; and the terms about and approximately should be understood as allowing for standard variation understood by those skilled in the art; and where ranges are given, endpoints are also included.

target protein.

Biopharmaceutical products must exhibit a high level of potency, purity, and a low level of structural heterogeneity. Structural heterogeneity often affects the bioactivity and efficacy of a drug. Therefore, the characterization and quantification of a therapeutic protein and/or impurities is important in the development of pharmaceuticals. Structural heterogeneity in a protein can result from post-translational modifications, as well as their inherent chemical modifications during manufacture and under certain storage conditions. For proteins produced in the biotechnology industry, additional separation methods may be required both to purify the target protein and to obtain an accurate picture of the quality of the final product. The complexity of the product excludes the use of simple one-dimensional strategies

- 7 042991 separation. Therefore, an accurate and efficient method for detecting and/or quantifying therapeutic protein and/or impurities is needed.

In some exemplary embodiments, the present invention provides a method for quantifying and/or detecting a protein and/or contaminant in a sample.

In this context, the term protein includes any amino acid polymer having covalently linked amide bonds. Proteins contain one or more polymeric chains of amino acids, generally known in the art as polypeptides. A polypeptide refers to a polymer composed of amino acid residues related to naturally occurring structural variants and its synthetic non-natural counterparts linked by peptide bonds, related to naturally occurring structural variants and their synthetic non-natural counterparts. 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. Those skilled in the art are aware of various methods for solid phase peptide synthesis. A protein may contain one or multiple polypeptides to form a single functioning biomolecule. The protein may include any of biotherapeutic proteins, recombinant proteins used in research or therapy, decoy proteins and other chimeric Fc receptor fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another exemplary aspect, a 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 insect baculovirus system, yeast systems (eg Pichia sp.), mammalian systems (eg CHO cells and CHO derivatives such as CHO-K1 cells). For a recent 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. (2012 ), 147-75). In some embodiments, proteins contain modifications, adducts, and other covalently linked moieties. These modifications, adducts, and fragments 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, protein, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST), myc-epitope, fluorescent labels , as well as 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 derived proteins such as primary derived proteins and secondary derived proteins.

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

In this context, the term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains: CH1, CH2, and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains only one domain (C _L 1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the anti-big-ET-1 FR antibodies (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs. As used herein, the term antibody 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 engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be generated, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic cleavage or recombinant DNA and genetic engineering techniques involving the manipulation and expression of DNA encoding the 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. DNA can be sequenced and processed chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a usable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. .

In this context, an antibody fragment includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragment, Fab' fragment, F(ab') ₂ fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment, and complementarity determining region (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. In some exemplary embodiments, an antibody fragment contains sufficient amino acid sequence of the parent antibody of which it is a fragment that binds to the same antigen as the parent antibody; in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and/or competes with the parent antibody for binding to the antigen. The antibody fragment can be obtained by any method. For example, an antibody fragment can be obtained enzymatically or chemically by fragmentation of an intact antibody and/or an antibody fragment can be obtained recombinantly from a gene encoding a partial antibody sequence. In an alternative or additional embodiment, the antibody fragment may be wholly or partially obtained synthetically. An antibody fragment may optionally include a single chain antibody fragment. In an alternative or additional embodiment, an antibody fragment may include multiple chains that are linked together, for example, by disulfide bonds. An antibody fragment may optionally include a multimolecular complex. A functional antibody fragment typically contains at least about 50 amino acids, and more typically contains at least about 200 amino acids.

The phrase bespecifically antibody includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies typically contain two different heavy chains, with each heavy chain specifically binding a different epitope to either two different molecules (eg, antigens) or the same molecule (eg, the same antigen). If the bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will typically be at least one, two, three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. Epitopes recognized by a bispecific antibody may be on the same or different targets (eg, on the same or different protein). Bispecific antibodies can be obtained, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses a light chain. immunoglobulin. A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain, which either does not confer antigen-binding specificity but can be associated with each heavy chain. chain, or which may be associated with each heavy chain and which may bind one or more epitopes linked by antigen-binding regions of the heavy chain, or which may be associated with each heavy chain and which may bind one or both heavy chains to one or both epitopes. BsAbs can be divided into two main classes: those containing an Fc region (IgG-like) and those lacking an Fc region, the latter typically being smaller than IgG and IgG-like Fc-containing bispecific molecules. IgG-like bsAbs can come in a variety of formats, such as, but not limited to, triomab, IgG ridge-in-gap (kih IgG), crossMab, orth-Fab IgG, Ig double variable domains (DVD-Ig), two-in-one, or dual Fab (DAF), IgG-single-chain Fv (IgG-scFv) or κλ-body actions. He-IgG-like different formats include tandem scFv, diabody format, single chain diabodies, tandem diabodies (TandAb), dual affinity reorienting molecule (DART), DART-Fc, nanobodies or dock-and-lock antibodies. lock - DNL) method. Fan et al. and Kontermann and Brinkmann provide a detailed overview of bispecific antibodies (Fan et al., Bispecific antibodies and their applications, J. Hematol. Oncol. (2015), 8:130; Kontermann and Brinkmann, Bispecific

- 9 042991 antibodies, Drug Discov., Today (2015), 20:838-847). Methods for producing BsAb are not limited to quadroma technology based on somatic fusion of two different hybridoma cell lines, chemical conjugation that includes chemical crosslinkers, and genetic approaches using recombinant DNA technology. Examples of bsAbs include those described in the following patent applications incorporated herein by reference in their entirety: US Patent Application No. 8,586,713, filed June 25, 2010; US Patent Publication Application No. 2013/0045492, filed June 5, 2012; U.S. Patent Application No. 9,657,102, filed September 19, 2013; U.S. Patent Publication Application No. 2016/0024147, filed July 24, 2015; U.S. Patent Publication Application No. 2018/0112001, filed September 22, 2017; U.S. Patent Publication Application No. 2018/0104357, filed September 22, 2017; U.S. Patent Publication Application No. 2017/0174779, filed December 21, 2016; U.S. Patent Publication Application No. 2017/0174781, filed December 21, 2016; U.S. Patent Application No. 10179819, filed July 29, 2016; and U.S. Publication Application No. 2018/0134794, filed November 15, 2017. Low levels of homodimeric impurities may be present at several steps in the manufacture of bispecific antibodies. Detection of such homodimeric impurities can be difficult when performing mass analysis of the starting material due to the low content of homodimer impurities and the coeluting of these impurities with the main species when performing a conventional liquid chromatography method (as demonstrated in Fig. 2).

Therapeutic bispecific antibodies (bsAbs) can simultaneously bind to two different targets and promise increased therapeutic efficacy, offering dual functionality or novel mechanisms of action (Marie Godar et al., Therapeutic bispecific antibody formats: a patent applications review (1994-2017), 28 , Expert opinion on therapeutic patents, 251-276 (2018)). To date, more than 60 bispecific molecules have been developed and evaluated for the treatment of various diseases, many of which have an IgG-like structure due to known advantages (stability, serum half-life, etc.) in therapeutic applications (Christoph Spiess, Qianting Zhai & Paul J. Carter, Alternative molecular formats and therapeutic applications for bispecific antibodies, 67, Molecular Immunology, 95-106 (2015) M.X. Sliwkowski & I. Mellman, Antibody therapeutics in cancer., 341, Science, 1192-1198 (2013) Paul J. Carter, Potent antibody therapeutics by design, 6, Nature Reviews Immunology, 343-357 (2006)). Bispecific antibodies are often produced in the same cell by co-expression of different light and heavy chains. Subsequently, assembly of the bsAb construct requires proper pairing of cognate light and heavy chains, as well as heterodimerization of the two different hemimolecules. Unfortunately, this process can also lead to misassembled molecular constructs such as monospecific molecules (eg, homodimeric species). Unlike other contaminants, the removal of homodimeric species through subsequent purification can be challenging as they often exhibit very similar physicochemical properties to the expected bsAb products. In order to improve the fidelity of polypeptide chain pairing and therefore promote bsAb formation, various strategies have been developed in recent years (Shixue Chen et al., Immunoglobulin Gamma-Like Therapeutic Bispecific Antibody Formats for Tumor Therapy, 2019, Journal of immunology research, 1- 13 (2019)). For example, to avoid mismatching between light and heavy chains, it has been particularly successful to use an identical light chain for each antigen-binding arm of a bsAb (A. Margaret Merchant et al., An efficient route to human bispecific IgG, 16, Nature biotechnology, 677-681 ( 1998)). In addition, heterodimerization of various heavy chains can be greatly facilitated by the use of ridge-to-cavity technology (John B.B., Ridgway, Leonard G. Presta & Paul Carter, 'Knobsinto-holes' engineering of antibody CH3 domains for heavy chain heterodimerization, 9, Protein Engineering, Design and Selection, 617-621 (1996)), in which specific mutations are engineered in the Fc portion of the antibody to promote heterodimer formation. Alternatively, by modulating protein A binding affinity through amino acid substitutions in the Fc portion, bsAb can also be efficiently isolated from homodimeric contaminants during the protein A purification step (Adam Zwolak et al., Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding, 1, Scientific Reports, 15521 (2017)). This strategy has already been successfully implemented for the mass production of bsAbs for clinical trials (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)).

Advances in new bsAb formats through protein engineering and process development have made it possible to manufacture high purity therapeutic bsAbs on a large scale. However, the presence of low levels of homodimeric impurities in bsAb drugs is still possible and should be routinely monitored during development and release testing. Homodimeric antibodies, considered as impurities associated with the drug product, can be very similar to the desired bsAb in many ways, making their detection and quantification a unique and challenging task for current analytical methods. Because homodimeric species typically exhibit distinctive molecular weights compared to the corresponding bsAbs, mass measurement at the intact protein level using LC-MS based technologies has been the method of choice for their characterization.

- 10 042991 (R. Jeremy Woods et al., LC-MS characterization and purity assessment of a prototype bispecific antibody, 5 MABS 711-722 (2013);

Wolfgang Schaefer et al., Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry, 8 MABS 49-55 (2015); FrankD. Macchi et al., Absolute Quantitation of Intact Recombinant Antibody Product Variants Using Mass Spectrometry, 87 ANALYTICAL CHEMISTRY 10475-10482 (2015);

Luis Schachner et al., Characterization of Chain Pairing Variants ofBispecific IgG Expressed in a Single Host Cell by High-Resolution Native and Denaturing Mass Spectrometry, 88 Analytical Chemistry 12122-12127 (2016); Yiyuan Yin et al., Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry, 8 MABS 1467-1476 (2016); Chunlei Wang et al., A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture, 10 MABS 1226-1235 (2018); Markus Haberger et al., Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry, 8 MABS 331-339 (2015); Francois Debaene et al., Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and “Bispecific ” Monoclonal Antibody Formation, 85 Analytical Chemistry 9785-9792 (2013)).

For example, several laboratories have reported using reversed phase chromatography (RPLC) in combination with high-resolution accurate-mass (HRAM) mass spectrometry to quantify homodimeric impurities in samples. bsAb (Woods et al., 2013, supra; Schachner et al., 2016, supra; Yin et al., 2016, supra). In most of these studies, homodimeric impurities could be detected and quantified without chromatographic separation from the main bsAb species. Indeed, given the large size (~150 kDa) as well as the similarity in physicochemical properties, it can often be challenging to achieve sufficient separation between homodimeric antibodies and bsAbs using the RPLC method. As a result, RPLC-MS-based methods are often not sensitive in detecting homodimeric species present at low levels (lowest reported LLOQ is ~1% (Schachner et al., 2016, supra; Yin et al., 2016, supra), mainly due to suppression of ions from co-eluting and much more numerous bsAb species.Moreover, without chromatographic separation, detection of homodimeric species with molecular weights close to bsAb can be particularly problematic because variant forms of bsAb from PTM (e.g., +128 Da for C-terminal Lys and +162 for glycation) or adduction formation could potentially interfere with the analysis Finally, in cases where chromatographic separation between homodimer and bsAb is achieved by RPLC, MS-based quantitation can still be difficult due to inconsistencies in performance ionization of antibodies eluted with different retention times in different solvent compositions, which requires the construction of an external calibration curve using added standards when performing quantitative analysis (Risto Kostiainen & Tima J. Kauppila, Effect of eluent on the ionization process in liquid chromatography-mass spectrometry, 1216, Journal of chromatography A, 685-699 (2009)). Alternatively, native mass spectrometry is another valuable method for analyzing intact proteins and is integrated into many routine analytical workflows for assessing monoclonal antibody (mAb) heterogeneity (Haberger et al, 2016, supra; Sara Rosati et al., Qualitative and Semi quantitative Analysis of Composite Mixtures of Antibodies by Native Mass Spectrometry, 84 ANALYTICAL CHEMISTRY 7227-7232 (2012); Anthony Ehkirch et al., Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products, 1086 JOURNAL OF CHROMATOGRAPHY B 176-183 (2018); ; Oscar Hernandez-Alb a et al., Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies, 90 Analytical Chemistry 8865-8872 (2018)).

Due to the more concentrated signal generated by fewer charge states, native MS may have improved sensitivity compared to RPLC-MS. For example, Rosati et al. (above) reported the use of native MS to study a binary mixture of two co-expressed IgG1 antibodies. However, without effective chromatographic separation, detection and quantification of homodimeric species present at low concentrations is still a challenge for the same reasons discussed above.

- 11 042991

To date, there are limited reports of analytical methods based on chromatographic separation for the detection and quantification of homodimeric species in bsAb samples. For example, through hydrophobic interaction chromatography (HIC - English: hydrophobic interaction chromatography) (Wang et al., 2018, supra) or ion exchange chromatography (IEX - English: ion exchange chromatography) (A.F. Labrijn et al., Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange, 110, Proceedings of the national academy of Sciences, 5145-5150 (2013) Michael J. Gramer et al., Production of stable bispecific IgG1 by controlled Fab-arm exchange, 5 mAbs, 962 -973 (2013)) it was demonstrated that bsAb is separated from its homodimers if they have sufficiently different hydrophobicity or isoelectric point (pI) values, respectively. In addition, recent advances in online IEX-native MS technologies (Yuetian Yan et al., Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry, 90, Analytical Chemistry, 13013-13020 (2018) Aaron O. Bailey et al., Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis, 10 mAbs, 1214-1225 (2018)) provide an efficient approach for the sensitive detection of low-level homodimeric species in bsAb. However, due to the high resolution, IEX typically generates a complex charge profile for each of all antibodies based on their charge heterogeneity, which is likely to overlap with each other. Moreover, MS-based quantification using this approach can be difficult, as all of the individual charge variant forms of each molecule must be summed for calculation. In addition, similar to the RPLC-based approach, the IEX method uses gradient elution, which is likely to compromise MS-based quantitation due to the different ionization efficiency of antibodies eluted under different solvent conditions (eg, pH or salt concentration). Recently, mixed-mode SEC-based chromatography (MM-SEC) has been applied to study antibody heterogeneity, which separates analytes by both hydrodynamic volume and hydrophobic interactions with the column matrix (Xiaoyu Yang et al., Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography, 484, Analytical Biochemistry, 173-179 (2015) Cintyu Wong, Camille Strachan-Mills & Sudhir Burman, Facile method of quantification for oxidized tryptophan degradants of monoclonal antibody by mixed mode ultra liquid performance chromatography, 1270, Journal of Chromatography A, 153-161 (2012); Jorge Alexander Pavon et al., Analysis of monoclonal antibody oxidation by simple mixed mode chromatography, 1431, Journal of chromatography A, 154-165 (2016)). In combination with UV or fluorescent detection, the MM-SEC method has been successfully applied to the relative quantification of homodimeric species in a bsAb sample (Yang et al., 2015, supra). However, it is clear that the usefulness of this method may be limited to cases where the homodimeric species and bsAb differ substantially in hydrophobicity such that an initial separation can be achieved for UV or fluorescence based quantitation. Moreover, since the identification of homodimeric species was based solely on comparison of retention times with standards, there was always a risk of overestimating the relative abundance if they were co-eluted with oligomeric or truncated forms of the bsAb molecule.

The concept of mixed mode chromatography using a SEC column originates from undesirable secondary interactions between the analyzed proteins and the column matrix. Ideally, SEC should separate protein analytes based solely on their hydrodynamic volume. In practice, electrostatic, hydrophobic, and hydrogen interactions can contribute to protein retention and separation to varying degrees depending on column matrix, buffer conditions, and protein characteristics (Alexandre Goyon et al., Unraveling the mysteries of modern size exclusion chromatography - the way to achieve character confidentization of therapeutic proteins, 1092, Journal of chromatography B, 368-378 (2018); Tsutomu Arakawa et al., The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals, 99, Journal of pharmaceutical sciences, 1674- 1692 (2010)). The use of these secondary interactions during SEC separation, by appropriately optimizing chromatographic conditions, provides opportunities to improve the separation of antibodies with similar hydrodynamic volume but different surface characteristics (eg, charge and hydrophobicity). When using MS-compatible online mobile phases, the combination of mixed-mode SEC with native MS detection (MM-SEC-MS) can have many advantages over UV-based methods, including unambiguous identification of homodimeric species through accurate mass measurements, minimal co-influence eluting particles and less stringent chromatographic resolution requirements (Terral et al., 2016, supra; Goyon et al., 2017, supra).

In this context, the term multispecific antibody or Mab refers to an antibody with binding specificity for at least two different antigens. Although such molecules usually bind only two antigens (i.e., bispecific antibodies, BsAbs), antibodies with additional specificity, such as trispecific antibodies and trispecific KIHs, can also be

- 12 042991 discussed in the system and method described in this document.

In this context, the term monoclonal antibody is not limited to antibodies obtained using hybridoma technology. A monoclonal antibody 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 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.

Impurities can form during many steps in the manufacture of biopharmaceuticals. Impurities produced during a biotechnological process can be very difficult to characterize and quantify because they are often present in very low amounts and can be very complex species or mixtures of species. It can also be very difficult to obtain a true reference of the peaks of such impurities. In this case, the complete characterization of trace amounts of protein impurities becomes a laborious, lengthy, and often very expensive process. Often, an admixture may include variants, isoforms, degradation products, related impurities, process impurities, minor post-translational modifications, aggregates, or truncated fragments of an intact recombinant protein. There is an almost infinite number of possible impurities, most but not all of which may be known.

As used herein, the term target protein may include the desired product or contaminant, or both.

In this context, the term desired product refers to a protein that has the desired structure, function, or performance profile.

In this context, the term impurity can include any undesirable protein present in a biopharmaceutical product. Impurities may include process impurities 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 fall into three main categories: those originating from the cell substrate, those originating from the cell culture, and those arising from subsequent steps in the process. Impurities derived from the cell substrate include, but are not limited to, proteins derived from the host organism and nucleic acid (genomic, vector or total DNA of the host cell). Impurities originating from cell cultures include, but are not limited to, inducers, antibiotics, serum, and other media components. Impurities generated during subsequent process steps include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers , ligands (eg, monoclonal antibodies) and other leachable agents. Related impurities (eg, precursors, certain degradation products) may be molecular variants that occur during manufacture and/or storage that do not have properties comparable to those of the desired product in terms of potency, efficacy, and safety. Such variants may require considerable effort in isolation and characterization to identify the type of modification(s). Related 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, mismatched S-S-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 amounts 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 during (co-translational modification) or after (post-translational modification) their ribosome synthesis. RTMs are usually administered by specific enzymes or enzyme pathways. Many of them occur at the site of a certain characteristic protein sequence (signature sequence) within the protein scaffold. Several hundred RTMs have been documented, 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 attachment of prosthetic groups, acetylation (adding an acetyl group , usually at the N-terminus of a protein), alkylation (addition of an alkyl group (e.g., methyl, ethyl, propyl) usually on lysine or arginine residues), methylation, adenylation, ADP-ribosylation, covalent cross-links within or between polypeptide chains, sulfonation, preniliro

- 13 042991 vitamin C-dependent modifications (hydroxylation of proline and lysine and carboxy-terminal amidation), vitamin K-dependent modification, in which vitamin K is a cofactor in the carboxylation of glutamic acid residues, leading to the formation of γ-carboxyglutamate (glucose residue), glutamylation (covalent attachment of glutamic acid residues), glycylation (covalent attachment 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 (attachment of a lipoate functionality), phosphopantetheynylation (adding a 4'-phosphopantetheynyl moiety from coenzyme A as in fatty acid, polyketide, nonribosomal peptide and leucine biosynthesis), phosphorylation (adding 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 nature of amino acids include, but are not limited to, citrullination (the conversion of arginine to citrulline by deelimination) and deamidation (the conversion of glutamine to glutamic acid or asparagine to aspartic acid). Post-translational modifications that include structural changes include, but are not limited to, the formation of disulfide bridges (covalent bonding 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 (covalent binding to the ISG15 protein (interferon stimulated gene)), SUMOylation (covalent binding to the SUMO (Small Ubiquitin-Like Modifier) protein), and ubiquitination (covalent binding to ubiquitin protein). See http://www.uniprot.org/docs/ptmlist for a more detailed controlled RTM vocabulary recommended by UniProt.

Mixed mode size exclusion chromatography.

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

As used herein, the term mixed mode chromatography (MMC) or multimodal chromatography includes a chromatographic technique in which solutes interact with a stationary phase through more than one interaction mode or mechanism. MMS can be used as an alternative or addition to traditional reversed-phase (RP - English: reversed-phased), ion-exchange (IEX - English: ion exchange) and normal-phase chromatography (NP - English: normal phase). Unlike RP, NP, and IEX 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. A mixed mode chromatography medium can provide unique selectivity that cannot be replicated with single mode chromatography. Mixed mode chromatography can also offer potential cost savings and operational flexibility over affinity based methods.

The phrase size exclusion chromatography or SEC or gel filtration includes a liquid column chromatography technique that allows the molecules in solution to be sorted by size.

As used herein, the terms SEC resin or SEC medium are used interchangeably herein and can include any kind of solid phase used in SEC that separates impurities from the desired product (e.g., a homodimeric contaminant for a bispecific antibody product). The volume of resin, the length and diameter of the column used, and the dynamic performance and flow rate may depend on several parameters such as the volume of fluid being treated, the concentration of protein in the fluid to be subjected to the process of the present invention, and so on. Determination of these parameters for each step is within the skill of the person skilled in the art.

As used herein, the term mixed mode size exclusion chromatography or MM-SEC can include any chromatographic technique that separates proteins through an additional interaction other than size separation. The additional or secondary interaction may use one or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, charge-charge interaction, hydrogen bond, pi-pi bond, and metal affinity. Mixed mode size exclusion chromatography resin can be any type of solid phase used for MM-SEC separation. Non-limiting examples are Sepax Zenix SEC-300, Waters BEN 300 or Agilent Bio SEC-3.

In this context, the term hydrophobic functionality refers to the hydrophobic interaction of a protein with a SEC chromatographic resin as a secondary interaction. The hydrophobic functionality can also significantly change the shape of the peak, which can have a noticeable effect.

- 14 042991 for process resolution. Hydrophobic interactions are strongest at high ionic strength of the mobile phase. To select a mobile phase to incorporate hydrophobic functional groups into the resin, different ions can be arranged in the so-called soluphobic row depending on whether they promote hydrophobic interactions (salting out effect) or disrupt the structure of water (chaotropic effect) and lead to a weakening of the hydrophobic interaction. (as shown in Fig. 3). The cations are ranked in ascending order of the salting out effect in the following order: Ba~; Ca ⁺⁺ ; Mg ⁺⁺ ; Li ⁺ ; Cs ⁺ ; Na ⁺ ; K ⁺ ; Rb ⁺ ; NH4 ⁺ , while anions can be ranked by increasing chaotropic effect in the following order: RO ^- ; SO4 ^- ; CH3CO2; ^Cl- ; ^Br- ; NO3 ^- ; ClC4 ^- ; I ^- ; ^SCN- . In general, Na, K, or NH4 sulfates are effective in promoting ligand-protein interactions in HIC. Salts can be formulated to influence the strength of the interaction, defined by the following relationship:

(NH ₄ ) ₂ SO ₄ >Na ₂ SO ₄ >NaCl>NH ₄ Cl>NaBr>NaSCN

Mass spectrometry.

In this context, the term mass spectrometer includes a device capable of detecting certain 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 can include 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 an analyte can be transferred to the gas phase and ionized simultaneously (as in electrospray ionization). The choice of ion source depends largely on the application.

In this context, the term mass analyzer includes a device that can separate particles, i.e. atoms, molecules or clusters, according to their mass. Non-limiting examples of mass analyzers that can be used for fast protein sequencing are time-of-flight (TOF), magnetic/electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT). : quadrupole ion trap), orbital trap, Fourier transform ion cyclotron resonance (FTICR) and accelerator mass spectrometry (AMS).

In this context, the term tandem mass spectrometry includes a method in which structural information about the molecules of the sample is obtained using several stages of selection and mass separation. The prerequisite is that the sample molecules can be gas-phased and ionized unchanged and that they can be induced to decay in some predictable and controlled manner after the first mass selection step. Multi-step MS/MS or MS ⁿ can be performed by selecting and isolating the precursor ion (MS ² ), fragmenting it, isolating the primary fragment ion (MS ³ ), fragmenting it, isolating the secondary fragment (MS ⁴ ), etc. as long as meaningful information or a detectable fragment ion signal can be obtained. Tandem MS has been successfully performed with a wide range of analyzer combinations. The choice of 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 hybrids where tandem-in-time analyzers are coupled in space or to tandem-in-space analyzers.

In some exemplary embodiments, mass spectrometry can be performed in vivo.

As used herein, the term native conditions, native MS, or native ESI-MS may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For detailed information on 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 Science, 1176-1192 (2015). Some of the differences between native ESI and conventional ESI are shown in Table 1. 1 (Nao Zhang et al., Native mass spectrometry ofphotosynthetic pigment-protein complexes, 587, FEBS Letters, 1012-1020 (2013)).

- 15 042991

Table 1

Native ESI Regular ESI Sample solution Aqueous solution water, ammonium acetate Partial organic solution water, formic acid, acetonitrile/methanol (pH 1-2) Conditions 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 treatment Desalination out of line Online/offline desalting with RP-HPLC Concentration squirrel 1-10 µM (complex) <1 µM (subunit) Imprint Molecular mass protein complex and subunits Non-covalent interactions Stoichiometry Structure Molecular weight of one subunits

Exemplary Embodiments.

The embodiments described herein are compositions, methods, and systems for rapidly characterizing proteins in a sample.

As used herein, the terms include, includes, and including are not to be understood as limiting, and mean contain, contains, and containing, respectively.

The present invention provides methods for detecting or quantifying an impurity in a sample, comprising contacting the sample with a chromatography system containing a mixed mode chromatography resin, washing the mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing impurities, and detection of impurities in the eluent using a mass spectrometer.

The present invention provides methods for detecting or quantifying a target protein in a sample, comprising contacting the sample with a chromatography system having a mixed mode chromatography resin, washing the mixed mode chromatography resin using a mobile phase to obtain an eluent comprising the target protein, and detecting or quantifying the target protein in the eluent using a mass spectrometer.

In certain specific exemplary embodiments, the chromatography system may comprise a size exclusion chromatography resin with additional interaction.

In certain specific exemplary embodiments, the chromatography system may comprise a hydrophobic interaction size exclusion chromatography resin.

In certain specific exemplary embodiments, the chromatography system may comprise a size exclusion chromatography resin with a charge-charge interaction function.

In some exemplary embodiments, a method for detecting or quantifying an impurity in a sample may include an impurity that may include at least one unwanted protein. The impurity(s) may have a known structure, be partially characterized(s) or unidentified(s).

In some exemplary embodiments, the impurity may be a related impurity. A related impurity may be molecular variants, precursors, degradation products, a fragmented protein, a cleavage product, aggregates, a form of post-translational modification, or combinations thereof.

In some exemplary embodiments, the impurity may be a process impurity. Technological impurities may include impurities obtained during the manufacturing process, ie. host cell nucleic acids and proteins, antibiotics, serum, other media components, enzymes, chemical and biochemical processing reagents, inorganic salts, solvents, carriers, ligands, and other leachables used in the manufacturing process.

- 16042991

In some exemplary embodiments, the impurity may be a protein with a pI ranging from about 4.5 to about 9.0. In one aspect, the impurity 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 some exemplary embodiments, the impurity may be a homodimeric species. In one aspect, the impurity may be a homodimeric species that may be formed during the production of the bispecific antibody. In another aspect, the amount of impurities in the sample may be at least two.

In some exemplary embodiments, the amount of sample loaded into the chromatographic system may range from about 10 µg to about 100 µg. In one exemplary embodiment, the amount of sample loaded into the chromatography system may 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 micrograms, about 50 micrograms, about 55 micrograms, about 60 micrograms, about 65 micrograms, about 70 micrograms, about 75 micrograms, about 80 micrograms, about 85 micrograms, about 90 micrograms, about 95 micrograms, or about 100 micrograms.

In some exemplary embodiments, the mobile phase used to elute the impurity may be a mobile phase that may be compatible with the mass spectrometer.

In certain specific exemplary embodiments, the mobile phase may be ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof. In one aspect, the total mobile phase concentration may be up to about 600 mM. In a particular aspect, the total mobile phase concentration may be 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, about 130 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.

In some exemplary embodiments, the mobile phase may have a flow rate from about 0.1 ml/min to about 0.4 ml/min. In one exemplary embodiment, the flow rate of the mobile phase 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.

In some exemplary embodiments, a method for detecting or quantifying an impurity may include detecting or quantifying an impurity in an eluent using a mass spectrometer. In one aspect, the mass spectrometer may be a tandem mass spectrometer. In another aspect, the mass spectrometer may include a nanosprayer.

In some exemplary embodiments, the eluent may contain the target protein in addition to the contaminant. In one aspect, the target protein may include an antibody, a bispecific antibody, an antibody fragment, or a multispecific antibody. In a specific aspect, the target protein may be a monoclonal antibody. In a specific aspect, the target protein may be a therapeutic antibody. In a specific aspect, the target protein may be an immunoglobulin protein. In another specific aspect, the immunoglobulin protein may be IgG1. In yet another specific aspect, the immunoglobulin protein may be IgG4. In one aspect, the target protein may be a bispecific antibody. In a specific aspect, the bispecific antibody may be an anti-CD20/CD3 monoclonal antibody. In one aspect, the target protein may be an antibody generated using the MG87 mouse fibroblast cell line. In one aspect, the target protein may be an antibody fragment resulting from cleavage of an antibody.

In one aspect, the target protein may be a post-translationally modified protein. In a specific aspect, a post-translationally modified protein can be formed by cleavage, N-terminal extensions, protein degradation, N-terminal acylation, biotinylation, C-terminal amidation, oxidation, glycosylation, iodination, covalent attachment, prosthetic groups, acetylation, alkylation, methylation, adenylation, ADP -ribosylation, covalent crosslinks within or between polypeptide chains, sulfonation, prenylation, vitamin C dependent modifications, vitamin K dependent modifications, glutamylation, glycylation, glycosylation, deglycosylation, isoprenylation, lipoylation, phosphopantetheynylation, phosphorylation, sulfation, citrullination , deamidation, formation of disulfide bridges, proteolytic cleavage, ISG-ylation, SUMO-ylation or ubiquitination (covalent binding to the ubiquitin protein).

In another aspect, the target protein may be a protein degradation product.

In yet another aspect, the target protein may be an impurity found in a biopharmaceutical product. In a specific aspect, the target protein may be an impurity found during the manufacture of the biopharmaceutical product.

In one aspect, the target protein may be a protein with a pI in the range of about 4.5 to about 9.0.

In one aspect, the target protein may be a related impurity. A related impurity may be molecular variants, precursors, degradation products, a fragmented protein, a cleavage product, aggregates, a form of post-translational modification, or combinations thereof.

In one aspect, the target protein may be a process impurity. Technological impurities may include impurities obtained during the manufacturing process, ie. host cell nucleic acids and proteins, antibiotics, serum, other media components, enzymes, chemical and biochemical processing reagents, inorganic salts, solvents, carriers, ligands, and other leachables used in the manufacturing process.

In one aspect, the amount of impurities in the sample may be at least two.

In one aspect, a post-translationally modified protein may be formed upon protein oxidation.

In another aspect, the target protein may include a degradation product.

In another aspect, the degradation product may include a post-translational modification of the therapeutic protein

In some exemplary embodiments, washing the mixed mode chromatography resin using a mobile phase requires a time of less than about 30 minutes. In one aspect, the time required to wash the mixed mode chromatography resin using a mobile phase may be about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 26 minutes, about 27 minutes, about 28 minutes , about 29 min or about 30 min.

In some exemplary embodiments, the chromatographic system may be used for at least about 3 sample runs without purification. In one aspect, the chromatography system can be used for at least about 3 sample runs, at least about 4 sample runs, at least about 5 sample runs, at least about 6 sample runs, at least about 7 sample runs. samples or at least about 8 sample runs without cleaning.

It is understood that the methods are not limited to any of the aforementioned proteins, contaminants, columns, and that the detection or quantification methods may be performed by any suitable means.

In some exemplary embodiments, the present invention provides a mixed mode chromatographic system comprising a chromatographic column 110, which can be washed using a mobile phase to provide an eluent comprising the target protein, and a mass spectrometer 120 coupled to the chromatographic column (as shown in FIG. 4). In one aspect, the chromatographic column 110 may be contacted with a sample using a sample loading device 100.

In some exemplary embodiments, the amount of sample that can be loaded into the chromatography column 110 may be in the range of about 10 μg to about 100 μg. In one aspect, the amount of sample loaded into the chromatography column 110 may 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 mcg, about 50 mcg, about 55 mcg, about 60 mcg, about 65 mcg, about 70 mcg, about 75 mcg, about 80 mcg, about 85 mcg, about 90 mcg, about 95 mcg, or about 100 mcg.

In some exemplary embodiments, the chromatographic column 110 may be washed with a mobile phase. In one aspect, the mobile phase may be ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof. In one aspect, the total mobile phase concentration that can be used with the chromatography column 110 can be up to about 600 mM. In a particular aspect, the total mobile phase concentration that can be used with the chromatography column 110 may be 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, about 130 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. In another aspect, the mobile phase that can be used with the chromatography column 110 may have a flow rate of 0.1 ml/min to 0.4 ml/min. In a specific aspect, the mobile phase flow rate 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. In another aspect, a mobile phase that can be used with the chromatography column 110 can be used to elute an impurity.

In some exemplary embodiments, the chromatographic column 110 may be coupled to a mass spectrometer 120. In one aspect, the mass spectrometer 120 may comprise a nanospray.

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

In some exemplary embodiments, the mass spectrometer 120 may be a native mass spectrometer.

In some exemplary embodiments, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 a target protein (see FIG. 4). In one aspect, a mixed mode chromatographic system can be used to detect 140 and/or quantify 130 a single target protein.

In some exemplary embodiments, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 a monoclonal antibody.

In some exemplary embodiments, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 a therapeutic antibody.

In some exemplary embodiments, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 an immunoglobulin protein. In one aspect, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 an IgG1 protein. In another aspect, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 an IgG4 protein. In another aspect, a mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 a bispecific antibody. In yet another aspect, a mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 an anti-CD20/CD3 monoclonal antibody.

In some exemplary embodiments, a mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 an antibody fragment resulting from antibody cleavage. In one aspect, a mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 a target protein, which may be a post-translationally modified protein. In another aspect, a mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 a protein of interest, which may be a protein degradation product. In yet another aspect, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 a protein of interest, which may be an impurity found in a biopharmaceutical product. In another aspect, a mixed mode chromatographic system may be capable of detecting 140 and/or quantifying 130 a protein of interest, which may be an impurity found in a biopharmaceutical product.

In some exemplary embodiments, the mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 a target protein, which may be a protein with a pI ranging from about 4.5 to about 9.0.

In some exemplary embodiments, a mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 a protein of interest, which may be a drug-associated impurity. A related impurity may be molecular variants, precursors, degradation products, a fragmented protein, a cleavage product, aggregates, a form of post-translational modification, or combinations thereof.

In some exemplary embodiments, the mixed mode chromatography system may be capable of detecting 140 and/or quantifying 130 a protein of interest, which may be a process impurity. Technological impurities may include impurities obtained during the manufacturing process, ie. host cell nucleic acids and proteins, antibiotics, serum, other media components, enzymes, chemical and biochemical processing reagents, inorganic salts, solvents, carriers, ligands, and other leachables used in the manufacturing process. In one aspect, the amount of impurities in the sample may be at least two.

In some exemplary embodiments, the implementation of the chromatographic column 110, which can be used for at least about 3 runs of samples without purification. In one aspect, the chromatography column 110 may be used for at least about 3 sample runs, at least about 4 sample runs, at least about 5 sample runs, at least about 6 sample runs, at least about 7 sample runs or at least about 8 sample runs without cleaning.

- 19 042991

It is clear that the system is not limited to any of the above elements: proteins, impurities, mobile phase or chromatographic column.

The sequential marking of the steps of the methods proposed herein with numbers and/or letters is not meant to limit the method or any of its embodiments to the specific order specified.

Various publications are cited in the description text, including patents, patent applications, published patent applications, access numbers, technical articles, and scientific articles. Each of these cited references is incorporated herein by reference in its entirety and for all purposes.

The present invention will be better 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 taken as limiting the scope of the invention.

Examples

Materials.

Deionized water was supplied by a Milli-Q inline water purification system installed with a MilliPak Express 20 filter (MilliporeSigma, Burlington, MA). Ammonium acetate (LC/MS grade), acetic acid, and ammonium bicarbonate (LC/MS grade) were purchased from Sigma-Aldrich (St. Louis, MO). The peptide N-glycosidase F (NGase F) was purchased from New England Biolabs Inc (Ipswich, MA). Invitrogen™ UltraPure™ 1 M Tris-HCl buffer, pH 7.5 was obtained from Thermo Fisher Scientific (Waltham, MA). All monoclonal antibodies and bispecific drugs, including the IgG1 and IgG4 subclasses, were manufactured by Regeneron.

Sample preparation.

In order to reduce mass heterogeneity due to the presence of two N-linked glycans in the Fc portion, each antibody or antibody mixture sample was treated with PNGas F (1IUB milliunit per 10 μg of protein) at 45°C in 100 mM Tris-HCl (pH 7, 5) within 1 hour. To prepare injectable bsAb mixture standards, the bsAb drug substance was first further purified by analytical strong cation exchange chromatography (SCX) to remove any residual homodimeric impurities resulting from the large scale manufacturing process. Detailed SCX fractionation conditions are given below. After fractionation, both the purified BsAb buffer and the corresponding homodimer standards were buffer exchanged with 50 mM Tris-HCl buffer (pH 7.5) and adjusted to 6 μg/μl each based on concentrations determined by Nanodrop (Thermo Fisher Scientific, Bremen, Germany). The bsAb and the two corresponding homodimers were then mixed in a 1:1:1 ratio. Finally, serial dilutions were made using a 2 μg/μl bsAb solution to prepare a series of additional injection standards with homodimer levels ranging from 0.1 to 10%.

Purification of bsAb drug substance bsAb using SCX.

For further bsAb purification, analytical strong cation exchange chromatography (SCX) was performed on each bsAb sample on a Waters I-Class UPLC system equipped with a photodiode array detector (PDA) (Waters, Milford, Massachusetts, USA). Prior to sample injection, the column compartment temperature was set to 45°C and a strong cation exchange YMC-BioPro SP-F (100x4.6 mm, 5 µm) column (YMC Co., LTD., Kyoto, Japan) was used, preconditioned with mobile phase A (20 mM ammonium acetate, pH adjusted to 5.6 with 20 mM acetic acid) at a flow rate of 0.4 ml/min. After injecting an aliquot (200 μg) of protein samples, the gradient was maintained at 100% mobile phase A for 2 min followed by a linear increase to 100% mobile phase B (140 mM ammonium acetate, 10 mM ammonium bicarbonate, pH 7.4) over 16 min . The gradient was maintained at 100% mobile phase B for 4 minutes and then returned to 100% mobile phase A to rebuild the column for 7 minutes before the next injection. After fractionation, the BsAb buffer was changed to the same buffer as for the mismatched homodimer samples before mixing to prepare injectable standards.

MM-SEC-MS method.

Mixed mode size exclusion chromatography was performed on a Waters I-Class UPLC system equipped with a photodiode array (PDA) detector (Waters, Milford, MA, USA). For mass measurements, a Thermo Exactive Plus EMR mass spectrometer equipped with a Nanospray Flex™ ion source (Thermo Fisher Scientific, Bremen, Germany) was used. Prior to sample injection, the column (Waters BEN200 SEC 4.6x300 mm, 200 A, 1.7 µm or Sepax Zenix SEC-300 4.6x300 mm, 300 A, 3 µm) was pre-equilibrated at a flow rate of 0.2 ml/min using a mobile phase based on ammonium acetate and ammonium bicarbonate of various concentrations (from 30 to 450 mm). This was achieved by a fixed percentage cycle of mobile phase B using a dual solvent system (mobile phase A: water; mobile phase B: 420 mM ammonium acetate and 30 mM ammonium bicarbonate). After the introduction of the antibody sample (2-10 µg) within 24 min was performed isocratic elution. To provide simultaneous UV and MS detection

- 20 042991 A post-column splitter (~200:1 ratio) was applied after SEC separation to reduce the flow to ~1 µl/min for nano-ESI-MS analysis while diverting the remaining high flow to the PDA detector for UV monitoring at 280 nm. To achieve nano-ESI, a disposable PicoTip emitter (uncoated, tip: 10±1 µm) (New Objective, Inc., Woburn, MA, USA) was used. For mass spectrometric analysis, resolution was set to 17,500, capillary sputter voltage to 1.5 kV, source fragmentation energy to 100, collision energy to 10, capillary temperature to 350°C, S-lens RF level to by 200, and HCD entrained gas pressure by 3. Mass spectra were obtained with a m/z range window from 2000 to 15000.

Example 1 Secondary interactions during SEC using an MS compatible buffer.

The protein surface is highly heterogeneous and consists of many different functional groups that can promote hydrogen bonding (hydroxyl, amine and amide groups), electrostatic (charged groups) and hydrophobic (hydrophobic groups) interactions with the SEC column matrix based on silica or polysaccharides . These interactions typically have different binding strengths and are highly dependent on the pH, temperature, and mobile phase composition used in the SEC. For example, when performing an assay at near neutral pH, the silanol groups from the silica SEC column can be negatively charged and therefore promote electrostatic interaction with basic proteins. In the past, mobile phases with moderate ionic strength (Goyon et al., 2018, see above) or low pH (less than 5) (Pavon et al., 2016, see above) were often required to suppress such interactions in the past.

More recently, through derivatization of the silica surface (e.g., short alkyl chains or functional group bonds), it has become possible to effectively screen residual silanol groups from a modern SEC column, which therefore drastically reduces the presence of electrostatic interactions. However, these newly introduced chemical groups may also lead to other enhanced secondary interactions (e.g. hydrophobic interaction) with the analyzed protein, as reported in recent studies (Yang et al., 2015, supra; Yan He et al., Online coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product, 1262, Journal of Chromatography A, 122-129 (2012)). According to Arakawa et al. electrostatic interactions predominate at low salt concentrations, while hydrophobic interactions are preferred for mobile phases with high ionic strength, especially when using salts of higher rank in the Hofmeister series (see Fig. 3). Although the choice of salts compatible with online SEC-MS is usually limited (e.g., ammonium formate, ammonium acetate, and ammonium bicarbonate), various types of secondary interactions between the analyzed proteins and the column matrix can still be modulated by varying the salt concentrations and investigated for separation purposes. proteins.

To evaluate salt concentration-associated mixed-mode interactions, a mixture of eight antibodies (IgG1 and IgG4 subclasses) with different surface characteristics was analyzed on a Waters BEH200 SEC column using ammonium acetate and ammonium bicarbonate mobile phases at varying concentrations ranging from 30 to 300 mm, acceptable for subsequent analysis by native MS. The molar ratio between ammonium acetate and ammonium bicarbonate was kept constant at 14:1 to achieve a pH of about 7.4. The chromatographic behavior of eight mAbs was illustrated by plotting their SEC retention times, as determined by extracted ion chromatograms (XIC), as a function of mobile phase concentration (FIG. 5). In total, eight mAbs were best separated by SEC using a mobile phase salt concentration of 30 mM. As the salt concentration increased, the elution profiles of the eight mAbs became more converged and less separated (Fig. 5 insets). These shifts in retention time can be explained by changes in the secondary interactions between the mAb molecule and the column matrix. On the one hand, when the residual silanol groups from the column matrix became negatively charged at pH 7.4, they could interact with the positively charged protein surface via electrostatic interaction. This interaction was enhanced with the use of lower salt concentrations. On the other hand, the hydrophobic interaction between the mAb molecule and the column matrix can be enhanced by using higher salt concentrations. It is of interest that, based on the pI values, these eight mAbs can be divided into three different groups in which a similar relationship between retention time and salt concentration was observed. The first group includes three acidic mAb molecules, mAb5 (pI=6.3), mAb6 (pI=6.4) and mAb1 (pI=6.7), which are expected to carry fewer positive charges on the protein surface at pH 7.4 and therefore show the least electrostatic interaction with the column matrix. As shown in FIG. 5, these three molecules all showed a tendency for retention time to increase as salt concentration increased, indicating that since weak electrostatic interactions are eliminated at higher ionic strength, hydrophobic interaction plays a dominant role during SEC separation. In contrast, the three main mAb molecules, mAb3 (pI=8.0), mAb4 (pI=8.3) and mAb8 (pI=7.6), are expected to carry more positive charges by

- 21 042991 protein surfaces at pH 7.4 all showed a tendency for retention time to decrease as salt concentration increased. This inverse correlation between salt concentration and retention time is mainly due to the dominant electrostatic interaction, which is enhanced at low salt concentration and suppressed at high salt concentration. Finally, in contrast to acidic or basic mAbs, mAb2 (pI=7.3) and mAb7 (pI=6.9) are a neutral group that likely carries a moderate amount of positive charges on the protein surface and therefore exhibits an average the level of electrostatic interactions with the column matrix. This group of molecules maintained a relatively constant retention time at various salt concentrations (30 to 300 mM). In this case, as the salt concentration was increased, the increase in hydrophobic interaction was likely close to and counteracted by the decrease in electrostatic interaction, resulting in a slight shift in retention time. These results demonstrate that by properly modulating salt concentrations, it is possible to separate or partially separate different antibodies (eg, bsAbs versus homodimers) using mixed mode interactions on a SEC column for subsequent MS analysis.

Although it appears that greater chromatographic resolution has been achieved at low salt concentrations on a Waters BEN column, care should be taken with major mAbs as strong peak broadening can occur under these conditions, which can significantly affect protein recovery (Alexandre Goyon et al., Characterization of 30 therapeutic antibodies and related products by size exclusion chromatography: Feasibility assessment for future mass spectrometry hyphenation, 1065-1066, Journal of Chromatography B, 35-43 (2017)).

Example 2 Detection of O-glycan variant of a bispecific antibody, bispecific Ab by MM-SEC-MS.

2.1. Preparation of bispecific antibody samples.

The anti-CD20xCD3 bispecific antibody (BsAb1) is a hinge-stabilized full-length anti-CD20xCD3 antibody (Ab) based on an IgG4 isotype modified to reduce Fc binding. The specified antibody is designed to bind T cells (via CD3) and cells expressing CD20. Bispecific antibodies were generated following the procedure described by Smith et al. (Sci. Rep. (2015), 5:17943).

2.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Zenix SEC-300 MK (7.8 x 300 nm, 3 µm) column in the system as described above. Elution was controlled by UV radiation at 280 nm.

Two series of experiments were carried out. In the first experiment, the mobile phase contained 140 mM ammonium acetate and 10 mM ammonium bicarbonate, and in the second experiment, the mobile phase contained 420 mM ammonium acetate and 30 mM ammonium bicarbonate. Elution was carried out at a flow rate of 0.4 ml/min. The balancing was carried out using the mobile phase.

For assay runs, loadings administered included 100 µg of total protein. Elution was carried out using an isocratic gradient consisting of ammonium acetate (buffer A) and ammonium bicarbonate (buffer B). Mass spectrometry data was analyzed using Protein Metrics Intact software.

Two cycles with mobile phases of different concentrations demonstrated that a higher salt concentration can enhance the hydrophobic interaction during SEC separation, as observed by the elution time of the bispecific antibody in different mobile phases. This effect resulted in increased separation between the bispecific antibody and its O-glycan variant (see FIG. 6).

Example 3 Detection of Homodimeric Species with Zenix SEC-300, 3 µm, 300 A, 7.8 x 300 mm.

3.1. Preparation of sample standards of a mixture of bispecific antibodies and homodimers.

Two homodimeric impurities are formed during the production of the bispecific antibody (BsAb1) (Fc*/Fc): homodimer 1 (Fc*-Fc*) and homodimer 2 (Fc/Fc) (see FIG. 2).

3.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Zenix SEC-300 MK column (7.8 x 300 nm, 3 µm) in the system as described above. Elution was controlled by UV radiation at 280 nm.

Two series of experiments were carried out. In the first experiment, the mobile phase contained 140 mM ammonium acetate and 10 mM ammonium bicarbonate, and in the second experiment, the mobile phase contained 420 mM ammonium acetate and 30 mM ammonium bicarbonate. Elution was carried out at a flow rate of 0.4 ml/min.

For assay runs, loadings administered included 50 µg of total protein. Elution was carried out using an isocratic gradient consisting of ammonium acetate (buffer A) and ammonium bicarbonate (buffer B). Similar to the results obtained in 2.2, two runs with different concentrations of mobile phases demonstrated that higher salt concentration can enhance the hydrophobic interaction during SEC separation, as observed by the elution times of the bispecific antibody and homodimers in different mobile phases. The mobile phase with a total salt concentration of 450 mm improved the separation of homodimer 1 and homodimer 2 from bispecific antibodies (see Fig. 7).

Example 4 Comparison of detection of homodimeric impurities in a bispecific antibody by MM-SEC-MS, SEC-MS and RP LC-MS.

4.1. Preparation of sample standards of a mixture of bispecific antibodies and homodimers.

The sample was prepared according to the procedure illustrated in example 3.

4.2. RPLC-MS.

The sample was diluted to 0.5 mg/ml. This solution was administered in an amount of 0.5 μg for LC-MS analysis. The LC-MS experiment was performed on a ThermoFisher Fusion Lumos Tribrid mass spectrometer. A Waters BioResolve™ mAb Polyphenyl, 450 A, 2.7 µm, 2.1x50 mm column (O.N. 186008944) was used for reverse phase separation. The sample temperature was set to 5°C and the column temperature to 80°C. Mobile phase A is 0.1% FA in water, mobile phase B is 0.1% FA in acetonitrile. The mass spectrometric experiment was carried out in the positive mode. MS ion source conditions were set as follows: spray voltage 3.8 kV, ion transfer tube temperature 325°C, evaporator temperature 250°C, shield gas 40 (Arb), auxiliary gas 10 (Arb), purge gas 2 (Arb), lens RF (%) 60 and source fragmentation energy 40 V. MS data were acquired with orbital trap in high mass range mode with m/z range from 1500 to 4000. Resolution was set to 15000 at m/z 200 with 10 microscans, AGC target was 105, the maximum injection time was 50 ms. Mass spectrometry data was analyzed using Xaclibur software.

4.3. SEC-MS.

Size exclusion chromatography (SEC) was performed on an ACQUITY UPLC Protein BEN SEC column (200 A, 1.7 μm, 4.6 x 300 mm) using a mobile phase containing 140 mM ammonium acetate and 10 mM ammonium bicarbonate. SEC experiments were performed on a Waters Acquity UPLC Class I system at room temperature with wavelength detection at 280 nm, a flow rate of 0.2 ml/min, and a protein loading of 50 μg.

4.4. MM-SEC-MS.

An analytical run on the MM-SEC-MS system was carried out isocratically using a mobile phase containing 420 mM ammonium acetate and 30 mM ammonium bicarbonate according to the procedure illustrated in Example 3.2.

4.5. Results.

Comparison of total ion chromatogram (TIC) and native MS spectra for RP LC-MS on Lumos (see FIG. 8), SEC-MS on EMR, and MM-SEC-MS on EMR demonstrates significant separation and detection of homodimers from the bispecific antibody. The original mass spectrogram, obtained using RP LC-MS, did not allow differentiation of homodimers and bispecific antibody. The raw mass spectrum from SEC-MS allowed separation and detection of homodimer 1 and bispecific antibody, but separation was not sufficient to separate and detect homodimer 2 and bispecific antibody. Only raw mass spectra from MM-SEC-MS showed sufficient separation and detection of homodimer 1, homodimer 2 and bispecific antibody. This comparison validates the concept of the superiority of MM-SEC-MS over SEC-MS and RP LC-MS for the detection of impurities in biopharmaceuticals.

Example 5 Sequential MM-SEC-MS detection cycles with Zenix SEC-300, 3 µm, 300 A, 7.8x300 mm.

To evaluate the quality of detection data in consecutive runs, three analytical runs were performed on a sample containing bispecific Ab, homodimer 1, and homodimer 2 (prepared as described in section 4.1) using the MM-SEC-MS system. Analytical runs were performed according to the methodology illustrated in section 4.2 and with a mobile phase containing 280 mM ammonium acetate and 20 mM ammonium bicarbonate.

Raw mass spectra and extracted ion chromatogram (XIC) for three cycles showed a decrease in data quality and signal-to-noise ratio (see Fig. 9). This effect can be observed due to the use of a large column (7.8x300 mm) which requires a large amount of protein sample (~50 µg) to provide MS intensity, which can lead to protein precipitation at high salt concentration, and therefore requires more frequent cleaning of the flow path (run a maximum of 3 samples before cleaning).

In addition, the large column and the relatively low flow rate at which the nanoseparator can operate (max ~0.4 ml/min) resulted in a large peak width (~1.5 min), which in some cases affected MS intensity and resolution. . The late elution time also slowed down the total analysis time (30 min for each sample).

- 23 042991

Example 6 Detection of homodimeric species with Zenix SEC-300, 3 µm, 300 A, 4.6 x 300 mm.

6.1. Preparation of sample standards of a mixture of bispecific antibodies and homodimers.

Bispecific antibody and homodimer mixture standards can be prepared according to the methods described in section 3.1.

6.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Zenix SEC-300 MK (4.6 x 300 nm, 3 µm) column in the system as described above.

Elution was controlled by UV radiation at 280 nm.

Four series of experiments were carried out. In the first experiment, the mobile phase contained 140 mm ammonium acetate and 10 mm ammonium bicarbonate, in the second experiment, the mobile phase contained 280 mm ammonium acetate and 20 mm ammonium bicarbonate, in the third experiment, the mobile phase contained 420 mm ammonium acetate and 30 mm of ammonium bicarbonate and in the second In the experiment, the mobile phase contained 560 mM ammonium acetate and 40 mM ammonium bicarbonate. Elution was carried out at a flow rate of 0.3 ml/min.

For assay runs, loadings administered included 10 µg of protein. Elution was carried out using an isocratic gradient consisting of ammonium acetate (buffer A) and ammonium bicarbonate (buffer B). The use of concentrations greater than 150 mm total salt concentration showed improved separation and detection of homodimers and bispecific antibodies (see Fig. 10). The total analysis time using the smaller column (4.6 x 300 nm) was reduced to about 18 min and the peak width was reduced to less than 1 min compared to the total analysis time and peak width using the larger column (7.8 x 300 nm). The differences are shown in Fig. eleven

Example 7 MM-SEC-MS analysis of a deglycosylated mixture of bispecific antibodies, homodimer 1 and homodimer 2 on a Zenix-SEC column.

7.1. Preparation of a deglycosylated mixture of bispecific antibodies, homodimer 1 and homodimer 2.

Each protein was treated with N-glycosidase F peptide (PNGase F; 1 IUB milliu per 10 μg of protein) at 45° C. for 1 hour to completely remove glycan chains from each heavy chain constant region.

7.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Zenix SEC-300 MK (4.6 x 300 nm, 3 µm) column in the system as described above. Elution was controlled by UV radiation at 280 nm.

Six series of experiments were carried out. In the first experiment the mobile phase contained 9.3 mM ammonium acetate and 0.7 mM ammonium bicarbonate, in the second experiment the mobile phase contained 46.7 mM ammonium acetate and 3.3 mM ammonium bicarbonate, in the third experiment the mobile phase contained 93.3 mM ammonium acetate and 6.7 mM ammonium bicarbonate, in the fourth experiment the mobile phase contained 186.7 mM ammonium acetate and 13.3 mM ammonium bicarbonate, in the fifth experiment the mobile phase contained 280 mM ammonium acetate and 20 mM ammonium bicarbonate and in the sixth experiment the mobile phase the phase contained 420 mM ammonium acetate and 30 mM ammonium bicarbonate. Elution was carried out at a flow rate of 0.3 ml/min.

For assay runs, loadings administered included 10 µg of protein.

The use of a total salt concentration of 10 mM showed a significant separation of homodimer 1, bispecific antibody and homodimer 2. At a salt concentration of 10 mM, homodimer 2 had a later retention time than bispecific antibody, which showed a later retention time than homodimer 1. However, at concentrations greater than The 10 mM homodimer 1 had a later retention time than the bispecific antibody, which showed a lower retention time than homodimer 2 (see FIGS. 12 and 13). This effect may be due to a different type of interaction: charge, shape, or hydrophobicity of the three proteins with the used size exclusion chromatography resin. The charge of a protein at a given salt concentration depends on their pI values (Table 2). Significant separations were obtained either when using a mobile phase with a low salt concentration of 10 mm, or when using a mobile phase with a high salt concentration of more than 100 mm. At lower salt concentrations, retention can be caused by charge-charge interaction. For example, basic molecules can be separated using a mobile phase with a lower salt concentration in the MM-SEC-MS system. At higher salt concentrations, retention occurs through hydrophobic interaction. For example, acidic or hydrophobic molecules can be separated using a mobile phase with a higher salt concentration in the MM-SEC-MS system (see Fig. 14 and 15).

The ideal SEC separation should be based only on the hydrodynamic volume of the protein and there should be no other interaction between the protein and the stationary phase. Because the silica column matrix can exhibit negative charges due to silanol groups (ion exchange characteristics), derivatization of the silica particles helps to reduce

- 24 042991 effect of silanol, but at the same time may introduce a new interaction mechanism (hydrophobicity). In this way, it is possible to create SEC resins with functionality, as seen with Zenix SEC columns. This explains the difference in the elution order of proteins at different concentrations when using a Zenix-SEC column.

table 2

mAb pi Molecular mass Bispecific antibody 7.66 145 337 Homodimer 1 (bispecific antibody HC* homodimer) 8.03 144 677 Homodimer 2 (bispecific antibody HC homodimer) 7.28 145 998

Example 8 MM-SEC-MS analysis of a deglycosylated mixture of bispecific antibodies, homodimer 1 and homodimer 2 on a Waters BEN SEC column.

8.1. Preparation of a deglycosylated mixture of bispecific antibodies, homodimer 1 and homodimer 2.

The deglycosylated mixture was prepared according to the same procedure as in section 7.1.

8.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Waters BEN SEC column in the system as described above. Elution was controlled by UV radiation at 280 nm.

Six series of experiments were carried out. In the first experiment the mobile phase contained 14 mM ammonium acetate and 1 mM ammonium bicarbonate, in the second experiment the mobile phase contained 18.7 mM ammonium acetate and 1.3 mM ammonium bicarbonate, in the third experiment the mobile phase contained 28 mM ammonium acetate and 2 mM bicarbonate ammonium acetate, in the fourth experiment the mobile phase contained 70 mM ammonium acetate and 5 mM ammonium bicarbonate, in the fifth experiment the mobile phase contained 93.3 mM ammonium acetate and 6.7 mM ammonium bicarbonate and in the sixth experiment the mobile phase contained 280 mM ammonium acetate and 20 mM ammonium bicarbonate. Elution was carried out at a flow rate of 0.2 ml/min.

For assay runs, loadings administered included 10 µg of protein.

The use of a total salt concentration of 15 mM showed a significant separation of homodimer 1, bispecific antibody and homodimer 2. At a salt concentration of 15 mM, homodimer 2 had an earlier retention time than the bispecific antibody, which showed an earlier retention time than homodimer 1. With increasing concentration of mobile phase differences in retention times decreased. In addition, at a mobile phase salt concentration of 300 mM, homodimer 1 had an earlier retention time than the bispecific antibody, which showed an earlier retention time than homodimer 2 (see FIGS. 16 and 17). As shown in FIG. 14 and 15, this effect is associated with the development of an additional interaction on the SEC column. Additional interaction depends on the concentration of salt in the mobile phase. At lower concentrations, charge-charge interactions dominate the column and determine the retention of proteins on the column.

Example 9 MM-SEC-MS analysis of the IgG1 molecule and its oxidized variant on a Waters BEN SEC column.

9.1. Preparation of the oxidized version of the IgG1 molecule - Ab1.

Ab1 was treated with N-glycosidase F peptide (PNGase F; 1 IUB milliu per 10 μg of protein) at 45° C. for 1 hour to completely remove glycan chains from each heavy chain constant region.

9.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Waters BEN SEC column in the system as described above. Elution was controlled by UV radiation at 280 nm.

Three series of experiments were carried out. In the first experiment, the mobile phase contained 93.3 mM ammonium acetate and 6.7 mM ammonium bicarbonate, in the second experiment, the mobile phase contained 140 mM ammonium acetate and 10 mM ammonium bicarbonate, and in the third experiment, the sixth experiment, the mobile phase contained 280 mM ammonium acetate and 20 mM ammonium bicarbonate. Elution was carried out at a flow rate of 0.2 ml/min.

For assay runs, loadings administered included 10 µg of protein.

Significant separation of the Ab1 antibody and its oxidized variant was noted in the MM-SEC-MS system using mobile phase salt concentrations of 100, 150 and 300 mM (see FIG. 18, top panel). The pI value of the IgG1 Ab1 antibody was 8.65. For IgG1 molecules with higher PI, charge interaction played a more dominant role compared to IgG4 molecules, which have low PI values.

- 25 042991

Example 10 MM-SEC-MS analysis of the IgGl molecule on a Waters BEN SEC column.

10.1. Preparation of the IgG1 - Ab2 molecule.

Ab2 was treated with N-glycosidase F peptide (PNGase F; 1 IUB milliu per 10 μg of protein) at 45° C. for 1 hour to completely remove glycan chains from each heavy chain constant region.

10.2. MM-SEC-MS.

MM-SEC-MS analysis was performed isocratically using a Waters BEN SEC column in the system as described in Example 8.2. When comparing the retention times of two IgG1 molecules: Ab1 and Ab2, it was observed that the Ab2 molecule had a lower retention time (see Fig. 18, bottom panel).

This can be explained by the difference in hydrophobicity between Ab1 and Ab2. For more hydrophobic molecules, the salting out effect begins to appear at a lower salt concentration compared to less hydrophobic molecules. This point is also called the transition point (see Fig. 19).

Example 11 Quantification of homodimeric impurities in bispecific antibodies by MM-SEC-MS.

Standards were generated according to the methodology shown in FIG. 20.

MM-SEC-MS analysis was performed isocratically using a Zenix SEC-300 MK (4.6 x 300 nm, 3 µm) column in the system as described above. Mobile phases with a salt concentration of 300 mM and a salt concentration of 70 mM were used to elute proteins. For concentrations at both intact and subunit levels, higher detection by MM-SEC-MS was observed in samples with more homodimers (see FIGS. 21 and 22). At a salt concentration of 70 mM for the mobile phase, an additional Fc contaminant of the bispecific antibody was also detected.

At an intact level, a plot of the theoretical homodimer 1/bispecific antibody ratio versus the detected homodimer 1/bispecific antibody ratio and the theoretical homodimer 2/bispecific antibody ratio versus the detected homodimer 2/bispecific antibody ratio showed good linearity to quantify the homodimers present from 0, 1 to 50% (see Fig. 23).

At the subunit level, a plot of the theoretical homodimer 1/bispecific antibody ratio versus the detected homodimer 1/bispecific antibody ratio and a plot of the theoretical homodimer 2/bispecific antibody ratio versus the detected homodimer 2/bispecific antibody ratio showed good linearity to quantify the homodimers present from 0 ,1 to 50% (see Fig. 24). Compared to the intact level, better accuracy was obtained at the subunit level.

Example 12 Mixed Mode SEC Separation of Bispecific and Homodimeric Antibodies to Detect Native MS.

Four bsAb molecules with different pIs and hydrophobicities (Table 3) were mixed with the corresponding homodimeric antibodies and used as test standards. Each bsAb molecule (HH*L2) contained two identical light chains (LC) and two different heavy chains (HC and HC*), while each homodimeric antibody (H2L2 or H*2L2) contained two identical light chains and two identical heavy chains (NS or NS*).

Table 3

pi Hydrophobicity (apparent retention factor HIC*; min) bsAb2 bsAb2 (HH*L2) 6.5 5.2 HC homodimer (H2L2) 6.1 4.3 mixture HC* homodimer (H*2L2) 7.2 6.1 bsAb3 bsAb3 (HH*L2) 7.4 4.8 HC homodimer (H2L2) 6.6 4.2 mixture HC* homodimer (H*2L2) 8.3 5.6 bsAb4 bsAb4 (HH*L2) 7.7 7.0 HC homodimer (H2L2) 7.3 8.4 mixture HC* homodimer (H*2L2) 8.0 5.7 bsAb 5 bsAb5 (HH*L2) 8.1 6.3 HC homodimer (H2L2) 7.4 5.2 mixture HC* homodimer (H*2L2) 8.5 7.7

* The apparent retention factor of the HIC was calculated based on the retention time of the protein molecule analyzed by the HIC. Mobile phase A from 3.3 M ammonium buffer and mobile phase B water were applied to a YMC BioPro HIC BF column (4 μm, 100x4.6 mm). The gradient was performed from 100 to 97% A in 18 minutes at a flow rate of 0.4 ml/min. The apparent retention factor HIC was calculated by normalizing the retention time over 18 min on a scale of 10.

The four bsAb mixtures were then analyzed on a Waters BEN column using three different salt concentrations (75, 150 and 300 mM) followed by online native MS detection. The resulting chromatograms of the main peaks (base peak chromatogram - BPC) are shown in the left panel of Fig. 25. Consistent with observations from a previous study, when the salt concentration was increased, mAb molecules with different pI values showed different retention time trends. Given the different retention characteristics of each antibody at different salt concentrations, the authors investigated the possibility of separating homodimers from bsAbs by modulating salt concentrations. For example, in a bsAb2 mixture, when the salt concentration decreased from 300 to 75 mM, two acidic molecules, the H2L2 homodimer (pI=6.1) and HH*L2 bsAb (pI=6.5), eluted earlier, probably due to a decrease in hydrophobic and electrostatic interactions at low salt concentration. In contrast, the neutral molecule, the H*2L2 homodimer (pI=7.2), remained almost unchanged in retention time with varying salt concentration, presumably because the reduced hydrophobic interaction was counteracted by increased electrostatic interaction at low salt concentration. In addition, since the H2L2 homodimer showed a greater reduction in retention time compared to the HH*L2 bsAb (perhaps due to its lower pI value and therefore weaker electrostatic interaction), improved separation between them was also achieved at a lower salt concentration. As a result, a good chromatographic separation between the homodimers and bsAb2 was achieved at a salt concentration of 75 mM.

Similarly, the separation between bsAb3 and its two homodimers also improved significantly when the salt concentration decreased from 300 to 75 mM. This is due to the fact that the retention times of the H2L2 homodimer (pI=6.6), HH*L2 bsAb (pI=7.4) and the H*2L2 homodimer (pI=8.3) decreased, remained unchanged or increased, respectively. Notably, although initial resolution was not achieved for either of the two examples, the identification and quantification of homodimers should not be significantly affected by coeluting species, as would be the case for UV-based quantification due to the high specificity of MS as a detector. .

In the bsAb5 mixture, better separation was again achieved with a salt concentration of 75 mM on a BEN column compared to high salt conditions. This is because the relatively basic molecules, HH*L2 bsAb (pI=8.1) and the H*2L2 homodimer (pI=8.5), both showed later retention times, while the relatively neutral H2L2 homodimer (pI= 7.4) showed no change in retention time with decreasing salt concentration. However, despite a good chromatographic separation, this condition was not ideal for homodimer quantitation, because for the H*2L2 homodimer at low salt concentration, due to its high basic characteristics, peak broadening and a decrease in protein recovery began to occur. Moreover, the bsAb4 mixture demonstrated that improved separation cannot be achieved by lowering the salt concentration from 300 to 75 mM. This is probably due to the fact that all three molecules have close to neutral pI values (Table 3) and thus exhibit similar retention characteristics with corresponding changes in salt concentration. Although lowering the salt concentration further to increase the electrostatic interaction may improve the separation, a strong peak broadening is likely to occur, disturbing the quantitation. Also of interest is the fact that the elution profile of the bsAb4 mixture broadened with increasing salt concentration. At a salt concentration of 300 mM, the elution order of the three molecules was determined by XIC (data not shown) for the H*2L2 homodimer, HH*L2 bsAb homodimer, and the H2L2 homodimer, which corresponded to their hydrophobicity ranking determined using hydrophobic interaction chromatography (Table 3). ). Therefore, further enhancement of the hydrophobic interaction by using even higher salt concentration is likely to improve the separation of the bsAb4 mixture on this column. Unfortunately, in our experience, salt concentrations above 300 mM usually create a desolvation problem and greatly desensitize native MS.

To further explore the separation of bsAb mixtures using salt concentrations suitable for MS analysis, a Sepax Zenix SEC-300 column was evaluated for mixed mode interaction. The 3 µm silica beads in this column are coated with a chemically bonded stable monolayer that likely contributes to the mild hydrophobicity of this column, as reported in previous studies (Yang et al., 2016, supra; Wong et al., supra; Pavon et al. , higher). At salt concentrations of 150 and 300 mm, each of the four bsAb mixtures was separated on this column for subsequent detection by native mass spectrometry; the generated HRVs are shown in the right panels of FIG. 25. As expected, the bsAb4 mixture showed improved separation on the Zenix column compared to the BEN column at the same salt concentrations. The elution order of the three molecules also corresponded to their relative hydrophobicity, with the most hydrophobic

- 27 042991 The H2L2 homodimer eluted last. Note that the chromatographic resolution of the bsAb4 mixture was further improved at a salt concentration of 300 mM compared to that at 150 mM, which is expected since a higher salt concentration promotes hydrophobic interaction. In addition, the bsAb5 mixture was more efficiently separated on the Zenix column compared to the BEN column, presumably due to large differences in hydrophobic interaction with the column matrix between the mixture components. Thus, by modulating the salt concentration on two SEC columns with different properties, we demonstrate that good chromatographic separation can be achieved for all four bsAb mixtures at salt concentrations favorable for subsequent detection by native MS. It is also likely that other SEC columns not tested in this study could further expand the applicability of this method by offering new mixed-mode interactions.

Example 13 Quantification of homodimeric impurities using native MM-SEC-MS.

Relative quantification with MS-based approaches often requires a well-characterized understanding of the MS response (eg, ionization efficiency and ion transfer efficiency) from each analyte. Due to their similar size, bsAbs and homodimers should show the same ion transfer efficiency during native MS analysis. On the other hand, both the composition of the solvent at the time of elution and the presence of co-eluting particles can affect the efficiency of ionization. Since the MM-SEC-MS method uses isocratic elution, the effect on ionization due to different solvent composition, which is usually observed in gradient elution methods (eg, IEX-MS), can be eliminated. To evaluate the performance of the MM-SEC-MS method for relative quantification of homodimeric impurities, a series of spiked bsAb2 samples containing homodimeric impurities ranging from 0.1% to 10% was prepared for analysis. A Waters BEN column with a 75 mM salt mobile phase was used to achieve MM-SEC separation between bsAb2 and the corresponding homodimers. To estimate the relative quantitation of each homodimer present in bsAb2 samples, XICs based on the m/z of the four most common charge states of either the homodimer species or bsAb2 were generated, and the peak areas were integrated and used to quantify the amount of each homodimer. As shown in FIG. 26, this method can easily achieve reliable quantitative determination of homodimeric impurities in the range from 0.1 to 10%. Furthermore, even at the 0.1% level added, high quality native mass spectra of the H2L2 and H*2L2 homodimers can still be obtained (FIG. 27, right panel), resulting in high confidence identification and quantitation.

A new MM-SEC-MS method has been developed and tested for the highly sensitive detection and quantification of homodimeric impurities in bsAb samples. We first investigated the mixed mode interactions between the antibody molecule and the column matrix during SEC separation at various salt concentrations. Using eight different antibodies with different pI values, it was found that under certain pH conditions, basic molecules exhibit stronger electrostatic interactions with the column matrix compared to acidic molecules, and this interaction can be enhanced by lowering the salt concentration.

On the other hand, increasing the salt concentration during SEC separation can reduce electrostatic interaction while promoting hydrophobic interactions between the antibody and column matrix. These mixed mode interactions provide a unique opportunity to separate antibodies with similar hydrodynamic volume but different surface characteristics. Taking advantage of the different properties of the column, the chromatographic separation of the four bsAb mixtures was performed by MM-SEC using either electrostatic interaction or hydrophobic interaction, which was easily achieved by adjusting the salt concentration. We have also demonstrated that the achieved chromatographic separation is critical to improve the detection of low content homodimeric impurities by subsequent native MS analysis. In two examples of bsAbs, homodimeric impurities present at 0.01% (bsAb2) and 0.1% (bsAb4) were successfully detected by this MM-SEC-MS method. To our knowledge, this new development represents the most sensitive method for detecting homodimeric impurities in bsAb samples. Finally, using a series of added standards, the authors demonstrated that the MM-SEC-MS method can provide reliable quantitative analysis of homodimeric impurities present at various levels. Due to the high sensitivity, identification and quantification with a high degree of confidence can be achieved even at levels as low as 0.1%. Thus, this newly developed MM-SEC-MS method provides a highly sensitive approach for the detection and quantification of homodimeric impurities in bsAb samples and thus can be applied to support the development of a therapeutic bsAb. Finally, the application of this method can be extended to other areas, such as the characterization of a mixture of antibodies present in co-formulated therapeutic agents.

Claims (18)

1. A method for quantifying an impurity in a protein sample, comprising: contacting said sample with a chromatography system containing a mixed mode size exclusion chromatography resin with additional functionality that results in a mixed mode separation; washing said mixed-mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing the impurity; and determining the amount of impurities in said eluent using a mass spectrometer. 2. The method of claim 1, wherein the mobile phase used to elute the impurity contains ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof. 3. The method of claim 1 wherein the mobile phase used to elute the impurity has a total concentration of less than about 600 mM ammonium acetate and ammonium bicarbonate. 4. The method of claim 1 wherein the mobile phase used to elute the impurity has a flow rate of about 0.2 ml/min to about 0.4 ml/min. 5. The method of claim 1, wherein the amount of sample loaded onto the mixed mode size exclusion chromatography resin is from about 10 µg to about 100 µg. 6. The method of claim 1 wherein the impurity is an impurity associated with the product. 7. The method of claim 1 wherein the impurity is a homodimer. 8. The method of claim 1, wherein the sample includes an impurity and at least one target protein that are separated upon elution. 9. The method of claim 8, wherein the target protein is an antibody. 10. The method of claim 9 wherein the antibody is a bispecific antibody. 11. The method of claim 8 wherein the target protein is a therapeutic antibody. 12. The method of claim 1 wherein the mass spectrometer is connected to a chromatographic system. 13. The method of claim 1 wherein the additional functionality is a hydrophobic interaction functionality. 14. The method of claim 1, wherein the additional functionality is charge-charge interaction functionality. 15. The method of claim 1 wherein the mass spectrometer is a native mass spectrometer. 16. A method for detecting an impurity in a protein sample, comprising: bringing said sample into contact with a chromatographic system containing a mixed mode size exclusion chromatography resin with additional functionality; washing said mixed-mode size exclusion chromatography resin using a mobile phase to obtain an eluent containing the impurity; and detecting an impurity in said eluent using a mass spectrometer. 17. A method for quantifying a target protein in a sample, comprising contacting said sample with a chromatographic system containing a mixed mode size exclusion chromatography resin with additional functionality; washing the mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent comprising the target protein; and quantifying the target protein in said eluent using a mass spectrometer. 18. A method for detecting a target protein in a sample, comprising contacting said sample with a chromatographic system containing a mixed mode size exclusion chromatography resin with additional functionality; washing the mixed mode size exclusion chromatography resin using a mobile phase to obtain an eluent comprising the target protein; and detection of the target protein in said eluent using a mass spectrometer.