Methods of analyzing protein therapeutics under native conditions

Abstract

This application pertains to methods of characterizing therapeutic proteins involving the use of size exclusion chromatography, ion exchange chromatography, and native mass spectrometry analysis, as well as methods of determining the stability of therapeutic proteins.

Description

43913-02708 / WO (REGE-040 / 001WO)METHODS OF ANALYZING PROTEIN THERAPEUTICS UNDER NATIVE CONDITIONSRELATED APPLICATION

[0001] This application claims the benefit of priority to US Provisional Application No. 63 / 760,791 filed on February 20, 2025, the contents of which are hereby incorporated by reference in their entirety.BACKGROUND

[0002] Conventional methods of characterizing protein therapeutics with mass spectrometry (MS) can be problematic. For example, MS analysis of protein therapeutics commonly involves denaturing conditions, which can make detection of the protein therapeutic challenging. MS analysis is also frequently combined with an immunoprecipitation step, which can introduce analytical bias and / or rely on reagents which can be costly and methods which can be time intensive to develop. In contrast, native MS analysis can yield improved signal selectivity for protein therapeutics. Accordingly, there exists a need for analytical methods that use MS methods compatible with native conditions to analyze protein therapeutics.SUMMARY

[0003] The disclosure provides methods of characterizing protein therapeutics and determining the stability of protein therapeutics with native MS.

[0004] In some aspects, the present disclosure provides a method of characterizing a therapeutic protein, wherein the method comprises:(i) subjecting a sample comprising the therapeutic protein to size exclusion chromatography under conditions whereby the therapeutic protein is eluted;(ii) contacting the therapeutic protein eluted at step (i) with an ion exchange chromatography matrix under conditions sufficient for the therapeutic protein to be bound to the ion exchange chromatography matrix and eluted therefrom, thereby producing an eluate comprising the therapeutic protein; and(iii) subjecting the eluate from step (ii) to native mass spectrometry analysis to characterize the therapeutic protein.

[0005] In some embodiments, the therapeutic protein is an antibody, fusion protein, antibodydrug conjugate, or protein pharmaceutical product.43913-02708 / WO (REGE-040 / 001WO)

[0006] In some embodiments, the fusion protein comprises a receptor fusion protein (a Trap protein).

[0007] In some embodiments, the antibody is a bispecific antibody or monoclonal antibody.

[0008] In some embodiments, the ion exchange chromatography is cation exchange chromatography.

[0009] In some embodiments, the cation exchange chromatography is strong cation exchange chromatography.

[0010] In some embodiments, the mass spectrometry analysis is performed by an electrospray ionization mass spectrometer or nano-electrospray ionization mass spectrometer.

[0011] In some embodiments, the mass spectrometry analysis is high-resolution mass spectrometry analysis.

[0012] In some embodiments, the sample comprises about 0.1 mg / mL to about 1 mg / mL of the therapeutic protein.

[0013] In some embodiments, the sample comprises about 0.1 mg / mL to about 0.3 mg / mL of the therapeutic protein.

[0014] In some embodiments, the method characterizes one or more derivatives of the therapeutic protein.

[0015] In some embodiments, the one or more derivatives of the therapeutic protein comprise a glycoform of the therapeutic protein.

[0016] In some embodiments, at least one derivative of the therapeutic protein is a deamidated form of the therapeutic protein.

[0017] In some embodiments, the therapeutic protein is administered to a patient.

[0018] In some embodiments, the therapeutic protein is generated in vivo in a patient.

[0019] In some embodiments, the sample is prepared from blood collected from a patient.

[0020] In some embodiments, the sample is prepared from blood serum of a patient.

[0021] In some embodiments, the sample is prepared from blood plasma of a patient.

[0022] In some embodiments, the patient is a mammal.

[0023] In some embodiments, the patient is a human.

[0024] In some embodiments, the method does not comprise a step to isolate the therapeutic protein from the sample prior to subjecting the sample to size exclusion chromatography.

[0025] In some embodiments, the method does not comprise an immunoprecipitation step.

[0026] In some embodiments, one or more isoforms of the therapeutic protein are quantified.

[0027] In some embodiments, the one or more isoforms of the therapeutic protein are quantified from mass spectrometry data.43913-02708 / WO (REGE-040 / 001WO)

[0028] In some aspects, the present disclosure provides a method of determining stability of a therapeutic protein, comprising:(i) providing at least a first and a second sample comprising the therapeutic protein, wherein the first sample is collected at an initial time point and the second sample is collected after a period of time;(ii) subjecting the at least first and second samples to a method of characterizing a therapeutic protein described herein, thereby generating at least one mass spectrogram of the therapeutic protein for each sample; and(iii) comparing the at least one mass spectrogram for each sample,thereby determining the stability of the therapeutic protein.

[0029] In some embodiments, the at least first and second samples are generated in vitro.

[0030] In some embodiments, the at least first and second samples are collected from a subject, thereby determining the stability of the therapeutic protein in vivo.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows a proof-of-concept test of the present MS method. 1 mg / mL of a therapeutic monoclonal antibody (mAb) is spiked into mouse plasma and then analyzed without using an immunoprecipitation (IP) step. The mouse plasma is subjected first to sizebased isolation by size exclusion chromatography, followed by charge-based isolation by strong cation exchange chromatography, and finally mass-to-charge ratio (m / z) isolation using high-resolution native MS.

[0032] FIGS. 2 A and 2B show size exclusion chromatography (SEC) and strong cation exchange chromatography (SCX) traces from an exemplary method of the disclosure. The insets show the mass spectrograms (m / z on the x-axis, abundance on the y-axis) of the indicated region (FIG. 2A) or peak (FIG. 2B).

[0033] FIG. 2C shows a m / z-filtered version of the SCX chromatogram of FIG. 2B. XIC: extracted ion chromatogram.

[0034] FIGS. 3A-3E show mass spectra of the Al, A2, Main, Bl, and B2 peaks from FIG. 2C.

[0035] FIGS. 4A and 4B show the quantitative performance of an exemplary 2D-SEC-SCX-MS method of the disclosure. A mAb was spiked into mouse plasma at concentrations from 2.5 pg / mL to 100 pg / mL. 5 pL of plasma was analyzed by 2D-SEC-SCX-MS in “dilute-and-shof ’ mode. The top panel (FIG. 4A) plots the concentration versus intensity (counts) of the43913-02708 / WO (REGE-040 / 001WQ)GOF glycoform of the therapeutic antibody. The bottom panel (FIG. 4B) plots concentration versus intensity (counts) of all glycoforms.

[0036] FIGS. 5A-5F show deconvoluted MS spectra of the Main peak of the samples analyzed in FIGS. 4 A and 4B.

[0037] FIG. 6A shows a general procedure to characterize mAb generated in vivo with the present IP-free SEC-SCX- MS method. AAV comprising a transgene encoding the mAb were injected into mice, and mouse serum was collected, followed by IP-free SEC-SCX- MS.

[0038] FIGS. 6B and 6C show exemplary mass spectra of the mAb generated in vivo (FIG.6B) or for mAb expressed by CHO cells, and then purified and spiked into mouse plasma (FIG. 6C).

[0039] FIG. 7A shows mass spectra of the Al, A2, and Main peaks of FIG. 6B and the Main peak of FIG. 6C.

[0040] FIG. 7B shows quantitation of glycoforms from the samples analyzed in FIGS. 6B and 6C.

[0041] FIGS. 8A-8E show m / z-filtered SCX chromatograms (2D-SEC-SCX-XICs) of samples of mAb spiked in mouse plasma at 1 mg / mL, which was then incubated at 37 °C for 0, 1, 3, 5, or 7 days prior to analysis.

[0042] FIG. 9A shows complementarity determining region (CDR) deamidation over time from the samples analyzed in FIGS. 8A-8E. Time in days is plotted on the x-axis, and relative abundance on the y-axis.

[0043] FIG. 9B shows unconverted N-terminal glutamine (glutamine that has not converted to pyroglutamic acid) over time from the samples analyzed in FIGS. 8A-8E. Time in days is plotted on the x-axis, and relative abundance on the y-axis.DETAILED DESCRIPTION

[0044] The disclosure provides methods for analyzing protein therapeutics under native conditions, a sample of a therapeutic protein in plasma (e.g., obtained in vitro or in vivo) is first subjected to size exclusion chromatography (see FIG. 2 A for a representative chromatogram). Eluate comprising the therapeutic protein is then subjected to ion exchange chromatography (e.g., SCX; see FIG. 2B for a representative chromatogram). Finally, eluate from the ion exchange chromatography that comprises the therapeutic protein is analyzed by native MS (see FIG. 2C for a representative m / z-filtered SCX chromatogram). This method may be used to test the stability of a therapeutic protein over time (e.g., under in vitro or in43913-02708 / WO (REGE-040 / 001WO)vivo conditions; see FIGS. 9A-9B for an exemplary in vitro biotransformation study of a mAb).

[0045] The disclosure provides methods of analyzing therapeutic proteins. In some embodiments, the therapeutic protein can be a drug substance, a formulated drug substance or a drug product (z.e., a “protein pharmaceutical product”).Definitions

[0046] As used herein, the term “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or <1% of the stated reference value.

[0047] As used herein “antibody” refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and antigen binding portions thereof. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CHI, CH2 and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term “antibody” includes antibody molecules prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. The term antibody also includes bispecific antibody, which includes a heterotetrameric immunoglobulin that can bind to more than one different epitope. Bispecific antibodies are generally described in U.S. Pat. No. 8,586,713, which is incorporated by reference into this application.

[0048] The term "antigen-binding portion" of an antibody (or "antibody fragment"), refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments43913-02708 / WO (REGE-040 / 001WO)linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 241 :544-546), which consists of a VH domain, (vi) an isolated CDR, and (vii) an scFv, which consists of the two domains of the Fv fragment, VL and VH, joined by a synthetic linker to form a single protein chain in which the VL and VH regions pair to form monovalent molecules. Other forms of single chain antibodies, such as diabodies are also encompassed under the term "antibody" (see e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1 121 -1 123).

[0049] Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol.Immunol. 31: 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as via papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques commonly known in the art.

[0050] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.

[0051] The term “humanized antibody”, as used herein, includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, or otherwise modified to increase their similarity to antibody variants produced naturally in humans.

[0052] Exemplary antibodies include antibodies selected from the group consisting of an anti -Programmed Cell Death 1 antibody (e.g., an anti-PDl antibody as described in U.S. Pat. Appln. Pub. No. US2015 / 0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g., an anti-PD-Ll antibody as described in in U.S. Pat. Appln. Pub. No. US2015 / 0203580A1), an anti-D114 antibody, an anti-Angiopoetin-2 antibody (e.g., an anti-ANG2 antibody as43913-02708 / WO (REGE-040 / 001WO)described in U.S. Pat. No. 9,402,898), an anti-Angiopoetin-Like 3 antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Pat. No. 9,018,356), an anti-platelet derived growth factor receptor antibody (e.g., an anti-PDGFR antibody as described in U.S. Pat. No.9,265,827), an anti-Erb3 antibody, an anti-Prolactin Receptor antibody (e.g., anti-PRLR antibody as described in U.S. Pat. No. 9,302,015), an anti-Complement 5 antibody (e.g., an anti-CS antibody as described in U.S. Pat. Appln. Pub. No US2015 / 0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g., an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an anti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No. US2015 / 0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g., an anti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. No. 9,540,449), an Anti-Growth and Differentiation Factor-8 antibody (e.g. an anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871,209 or 9,260,515), an anti-Glucagon Receptor (e.g. anti-GCGR antibody as described in U.S. Pat. Appln. Pub. Nos. US2015 / 0337045A1 or US2016 / 0075778A1), an anti-VEGF antibody, an anti-ILlR antibody, an interleukin 4 receptor antibody (e.g., an anti-IL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014 / 0271681A1 or U.S. Pat. Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880), an anti-ILl antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti -interleukin 33 (e.g., anti-IL33 antibody as described in U.S. Pat. Nos. 9,453,072 or 9,637,535), an anti-Respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Pat. Appln. Pub. No. 9,447,173), an anti-Cluster of differentiation 3 (e.g., an anti-CD3 antibody, as described in U.S. Pat. Nos. 9,447, 173and 9,447,173, and in U.S. Application No. 62 / 222,605), an anti-Cluster of differentiation 20 (e.g., an anti-CD20 antibody as described in U.S. Pat. No. 9,657,102 and US20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody, an anti-CD28 antibody, an anti-Cluster of Differentiation-48 (e.g. anti-CD48 antibody as described in U.S. Pat. No.9,228,014), an anti-Fel dl antibody (e.g. as described in U.S. Pat. No. 9,079,948), an antiMiddle East Respiratory Syndrome virus (e.g. an anti-MERS antibody as described in U.S. Pat. Appln. Pub. No. US2015 / 0337029A1), an anti-Ebola virus antibody (e.g., as described in U.S. Pat. Appln. Pub. No. US2016 / 0215040), an anti-Zika virus antibody, an antiLymphocyte Activation Gene 3 antibody (e.g., an anti-LAG3 antibody, or an anti-CD223 antibody), an anti-Nerve Growth Factor antibody (e.g., an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016 / 0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176) and43913-02708 / WO (REGE-040 / 001WO)an anti-Protein Y antibody. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos. US2014 / 0088295A1 and US20150266966A1), an anti-CD3 x antiMucin 16 bispecific antibody (e.g., an anti-CD3 x anti-Mucl6 bispecific antibody), and an anti-CD3 x anti-Prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody).

[0053] Further exemplary antibodies are selected from the group consisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansinealirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, and vedolizumab.

[0054] As used herein, the term "therapeutic protein" may be used interchangeably with "protein therapeutic." Therapeutic proteins can be created or isolated by any means known in the art. These include recombinant means, such as proteins (e.g., antibodies) expressed using a recombinant expression vector transfected into a host cell. Antibodies can be isolated from a recombinant, combinatorial human antibody library, isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.43913-02708 / WO (REGE-040 / 001WO)

[0055] In some embodiments, the therapeutic protein comprises a fragment crystallizable (Fc) domain. For example, the therapeutic protein can be a receptor-Fc-fusion protein or a soluble TCR-Fc fusion protein. In some embodiments, the receptor-Fc-fusion protein is a trap protein.

[0056] Fusion proteins comprise two or more parts of the protein which are not otherwise found together in nature. For example, an “Fc fusion protein” can comprise an Fc portion of an immunoglobulin molecule, which is fused to another heterologous domain, such as a receptor ligand binding domain. Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535, 1991; Bym et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11 , 1992. "Receptor Fc fusion proteins" comprise one or more extracellular domain(s) of a receptor coupled to an Fc moiety, which in some embodiments comprises a hinge region followed by a CH2 and CH3 domain of an immunoglobulin. In some embodiments, the Fc-fusion protein contains two or more distinct receptor chains that bind to a one or more ligand(s). For example, an Fc-fusion protein is a trap, such as for example an interleukin 1 (IL-1) trap (e.g., rilonacept, which contains the IL-1 RAcP ligand binding region fused to the IL-1 R1 extracellular region fused to Fc of hlgGl; see U.S. Pat. No. 6,927,004), or a vascular endothelial growth factor A (VEGF) trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl fused to Fc of hlgGl; see U.S. Pat. Nos. 7,087,411 and 7,279,159).

[0057] In some embodiments, the therapeutic protein is a fusion protein, such as a receptor fusion protein. Receptor fusion proteins can include, inter alia, trap proteins and mini trap proteins.

[0058] The term “fusion protein” refers to a molecule comprising two or more proteins or fragments thereof linked by a covalent bond via their individual peptide backbones, optionally generated through genetic expression of a polynucleotide molecule encoding the fusion protein.

[0059] The therapeutic protein can be a trap protein or a mini trap protein. In some embodiments, trap proteins are engineered therapeutic proteins capable of acting as decoy receptors to bind to and antagonize or modulate the activity of a target protein. An exemplary trap protein comprises one or more receptor components that mimic the binding domain of the receptor for its target protein (e.g., the VEGF receptor Ig domain 2 of Fit- 1 and the Ig43913-02708 / WO (REGE-040 / 001WO)domain 3) fused to a human IgG constant region, optionally including additional domains such as linkers, dimerization or multimerization domains, and cleavage sites. In some embodiments, the trap protein is truncated or of reduced size (a mini trap), for example through protein cleavage, which can aid in tissue penetration of the mini trap. Non-limiting examples of trap proteins include an IL-1 trap (e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the IL-1R1 extracellular region which in turn is fused to the Fc of hlgGl) (e.g., SEQ ID NO: 1) (see U.S. Patent No. 6,927,004), or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl which in turn is fused to Fc of hlgGl. See, e.g., U.S. Patent Nos.7,087,411, 7,279,159; see also U.S. Patent No. 5,610,279 for etanercept (TNF trap), the contents of each of which are incorporated by reference in their entirety herein.

[0060] As used herein, the term “glycan” sometimes used interchangeably with “polysaccharide” and “oligosaccharide” refers to a compound comprising or consisting of glycosidically linked monosaccharides. The term glycan can also be used to refer to a carbohydrate linked to a glycoprotein or glycolipid, even if the carbohydrate isa monosaccharide. Glycans may comprise O-glycosidic linkages of monosaccharides.Glycans can be homo- or heteropolymers of monosaccharides, and can be linear or branched. Exemplary glycans can comprise monomers of mannose, N-Acetylglucosamine (GlcNAc), N-Glycolylneuraminic acid (Neu5Gc), galactose, sialic acid, and fucose, among others. Glycans can be linked to a therapeutic via either N-linkages or O-linkages, and a therapeutic protein can comprise N-linked glycans, O-linked glycans or a combination of N-linked and O-linked glycans.

[0061] Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (for example, high performance liquid chromatography, or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Liquid chromatography separates mixtures with multiple components, while mass spectrometry provides structural identity and levels of the individual components with high molecular specificity and detection sensitivity.

[0062] As used herein, the term “chromatography” refers to a process in which a chemical mixture comprising a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around, over, and / or through a stationary liquid or solid phase. “Liquid chromatography” or “LC” refers to a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways.43913-02708 / WO (REGE-040 / 001WO)The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).

[0063] “Retention time” refers to length of time for which a particular analyte, such as an ampholyte composition component, is retained by a liquid chromatography substrate prior to elution.

[0064] “ Size exclusion chromatography” or “SEC” refers to a chromatographic method in which molecules in solution are separated by their shape and / or size.

[0065] Cation exchange chromatography (CEX) refers to a type of ion exchange chromatography and is used to separate molecules in solution based on charge. In cation exchange chromatography, the stationary bed has an ionically negative (-) charged surface while the sample ions are of positive (+) charge.

[0066] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass to charge ratio (m z). MS technology generally includes ionizing the compounds to form charged species (e.g., ions) and detecting the exact mass of the ions divided by their charge, known as m / z. The compounds may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”). See, e.g., U.S. Patent Nos. 6,204,500; 6,107,623; 6,268,144; and 6,124,137.

[0067] “Native mass spectrometry” refers to the analysis of molecules, usually intact biomolecules such as proteins and protein complexes, under conditions in which the native structural features are retained. Native MS is described, for example in Tamara et al., Chem. Rev. 2022, 122, 8, 7269-7326, the contents of which are incorporated by reference in their entirety herein.

[0068] Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.43913-02708 / WO (REGE-040 / 001WO)

[0069] The specific examples and descriptions herein are exemplary in nature and embodiments may be developed by those skilled in the art based on the material taught herein without departing from the scope of the present invention.

[0070] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; inventive embodiments may be practiced otherwise than as specifically described and claimed. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.Methods of Characterization

[0071] In some aspects, the present disclosure provides a method of characterizing a therapeutic protein, wherein the method comprises:(i) subjecting a sample comprising the therapeutic protein to size exclusion chromatography under conditions whereby the therapeutic protein is eluted;(ii) contacting the therapeutic protein eluted at step (i) with an ion exchange chromatography matrix under conditions sufficient for the therapeutic protein to be bound to the ion exchange chromatography matrix and eluted therefrom, thereby producing an eluate comprising the therapeutic protein; and(iii) subjecting the eluate from step (ii) to native mass spectrometry analysis to characterize the therapeutic protein.

[0072] In some embodiments, step (i) is performed under native conditions.

[0073] In some embodiments, step (ii) is performed under native conditions.

[0074] In some embodiments, both steps (i) and (ii) are performed under native conditions.

[0075] In some embodiments, the therapeutic protein is intact under native conditions.

[0076] In some embodiments, non-covalent complexes (e.g., protein-protein complexes) comprising the therapeutic protein are intact under native conditions.43913-02708 / WO (REGE-040 / 001WO)

[0077] In some embodiments, the native conditions do not cause denaturation of the therapeutic protein.

[0078] In some embodiments, the native conditions are in neutral pH water.

[0079] In some embodiments, the native conditions are in physiological ionic strength.

[0080] In some embodiments, the native conditions are between 25 °C and 40 °C.

[0081] In some embodiments, the size exclusion chromatography column is in sequence with the ion exchange chromatography column ( / .< ., the method involves two-dimensional chromatography).

[0082] In some embodiments, the size exclusion chromatography, ion exchange chromatography, and / or native mass spectrometry is described in Anal Chem. 2022 Apr 26;94(16):6355-6362 (doi: 10.1021 / acs.analchem.2c00707); Pharm Res. 2022 Sep 7 (doi: 10.1007 / sl 1095-022-03381-0); Anal Chem. 2024 Jul 10 (doi:10.1021 / acs.analchem.4c00660; JPharm Biomed Anal. 2023 Aug 1;235:115622 (doi:10.1016 / j .j pba.2023.115622); J Pharm Biomed Anal . 2023 May 10;228 : 115337 (doi :10.1016 / j jpba.2023.115337); JBiotechnol. 1998 Dec 11;66(2-3): 125-36; Anal Chem. 2018 Nov 6;90(21): 13013-13020; Anal Chem. 2019 Sep 3;91(17):11417-11424; J Pharm Biomed Anal. 2024 Oct 16;253:116524 (doi: 10.1016 / j .jpba.2024.116524); or J Pharm Biomed Anal 2020 Apr 20; 186: 113313 (doi: 10 1016 / j jpba 2020 113313); the contents of each of which are incorporated by reference in their entirety herein.

[0083] In some embodiments, the therapeutic protein is quantified from mass spectrometry data (e.g., from a calibration curve correlating protein concentration vs. MS signal).

[0084] In some embodiments, the method is run in “dilute-and-shoof ’ mode, wherein the sample is diluted and then directly analyzed.

[0085] In some embodiments, the method does not comprise a step to isolate the therapeutic protein from the sample prior to subjecting the sample to size exclusion chromatography.

[0086] In some embodiments, the method does comprise a step to isolate the therapeutic protein from the sample prior to subjecting the sample to size exclusion chromatography.

[0087] In some embodiments, the method does not comprise an immunoprecipitation step.

[0088] In some embodiments, the method does comprise an immunoprecipitation step.

[0089] In some embodiments, one or more isoforms of the therapeutic protein are quantified.

[0090] In some embodiments, the one or more isoforms of the therapeutic protein are quantified from mass spectrometry data.

[0091] In some embodiments, the method characterizes one or more derivatives of the therapeutic protein.43913-02708 / WO (REGE-040 / 001WO)

[0092] In some embodiments, the at least one derivative of the therapeutic protein is a glycoform of the therapeutic protein.

[0093] In some embodiments, the at least one derivative of the therapeutic protein is a deamidated form of the therapeutic protein.Therapeutic Proteins and Samples Thereof

[0094] In some embodiments, the therapeutic protein is an antibody, fusion protein, antibodydrug conjugate, or protein pharmaceutical product.

[0095] In some embodiments, the therapeutic protein is an antibody.

[0096] In some embodiments, the antibody is a bispecific antibody or monoclonal antibody.

[0097] In some embodiments, the therapeutic protein is a fusion protein.

[0098] In some embodiments, the fusion protein comprises a receptor fusion protein (a Trap protein).

[0099] In some embodiments, the fusion protein is described in Huang, C. Current Opinion in Biotechnology, 2009, 20 (6), pgs. 692-699, orHolash, J. et al. PNAS, 2002, 99(17), pgs.11393-11398, the contents of each of which are incorporated by reference in their entirety herein.

[0100] In some embodiments, the therapeutic protein is an antibody-drug conjugate.

[0101] In some embodiments, the therapeutic protein is a protein pharmaceutical product.

[0102] In some embodiments, the therapeutic protein has a molecular weight of <25 kDa, <50 kDa, <100 kDa, <150 kDa <200 kDa, <250 kDa, <300 kDa, <350 kDa, <400 kDa, <450 kDa, <500 kDa, <550 kDa, <600 kDa, <650 kDa, <700 kDa, <750 kDa, <800 kDa, <850 kDa, <900 kDa, <950 kDa, or <1000 kDa.

[0103] In some embodiments, the therapeutic protein has a molecular weight of >25 kDa, >50 kDa, >100 kDa, >150 kDa >200 kDa, >250 kDa, >300 kDa, >350 kDa, >400 kDa, >450 kDa, >500 kDa, >550 kDa, >600 kDa, >650 kDa, >700 kDa, >750 kDa, >800 kDa, >850 kDa, >900 kDa, >950 kDa, or >1000 kDa.

[0104] In some embodiments, the therapeutic protein has a molecular weight of about 25 kDa, about 50 kDa, about 100 kDa, about 150 kDa about 200 kDa, about 250 kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa, about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, about 950 kDa, or about 1000 kDa.43913-02708 / WO (REGE-040 / 001WO)

[0105] In some embodiments, the therapeutic protein has a molecular weight of <30 kDa, <40 kDa, <50 kDa, <60 kDa, <70 kDa, <80 kDa, <90 kDa, <100 kDa, <110 kDa, <120 kDa, <130 kDa, <140 kDa, <150 kDa, <160 kDa, <170 kDa, <180 kDa, <190 kDa, or <200 kDa.

[0106] In some embodiments, the therapeutic protein has a molecular weight of >30 kDa, >40 kDa, >50 kDa, >60 kDa, >70 kDa, >80 kDa, >90 kDa, >100 kDa, >110 kDa, >120 kDa, >130 kDa, >140 kDa, >150 kDa, >160 kDa, >170 kDa, >180 kDa, >190 kDa, or >200 kDa.

[0107] In some embodiments, the therapeutic protein has a molecular weight of about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, or about 200 kDa.

[0108] In some embodiments, the therapeutic protein is generated in vivo in a patient.

[0109] When the therapeutic protein is generated in vivo in the patient, a polynucleotide encoding the therapeutic protein can be delivered to the using a vector, such as a viral vector or a non-viral vector. Exemplary viral vectors that can encode the therapeutic protein include adeno-associated virus (AAV) vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors. AAV is widely used due to its ability to infect both dividing and non-dividing cells, its mostly episomal persistence, low immunogenicity and range of tissue trophisms.Engineered AAV capsids have enabled targeted delivery to muscle, eye, central nervous system, and liver, although the relatively small packaging capacity of about 4.7 kilobases pose challenges. Lentiviral vectors are derived from HIV-1, integrate stably into the host genome and can accommodate larger transgenes (around 8-10 kb). Adenoviral vectors remain in the nucleus without integrating, allowing them to deliver large genetic payloads (up to ~36 kb in gutted vector forms) with high transduction efficiency. Alternatively, a polynucleotide encoding the therapeutic protein can be delivered to a subject using a non-viral vector such as lipid nanoparticle, liposome or micelle. As a further alternative, polynucleotide encoding the therapeutic protein can be delivered to a subject using a viruslike particle. Suitable viral and non-viral vectors for the delivery of therapeutic proteins will be known to persons of ordinary skill in the art.

[0110] In some embodiments, the therapeutic protein is administered to a patient.[OHl] In some embodiments, the sample is prepared from cells collected from a patient.

[0112] In some embodiments, the sample is cell lysate prepared from cells collected from a patient.

[0113] In some embodiments, the sample is prepared from tissues collected from a patient.

[0114] In some embodiments, the sample is prepared from fluid collected from a patient.43913-02708 / WO (REGE-040 / 001WO)

[0115] In some embodiments, the sample is prepared from saliva collected from a patient.

[0116] In some embodiments, the sample is prepared from urine collected from a patient.

[0117] In some embodiments, the sample is prepared from cerebrospinal fluid collected from a patient.

[0118] In some embodiments, the sample is prepared from bile collected from a patient.

[0119] In some embodiments, the sample is prepared from blood collected from a patient.

[0120] In some embodiments, the sample is prepared from blood serum of a patient.

[0121] In some embodiments, the sample is prepared from blood plasma of a patient.

[0122] In some embodiments, the sample is prepared from blood buffy coat of a patient.

[0123] In some embodiments, the patient is a mammal.

[0124] In some embodiments, the patient is a mouse.

[0125] In some embodiments, the patient is a human.

[0126] In some embodiments, the sample comprises about 2.5 pg / mL to about 100 pg / mL of the therapeutic protein.

[0127] In some embodiments, the sample comprises <2.5 pg / mL, <5 pg / mL, <10 pg / mL, <20 pg / mL, <30 pg / mL, <40 pg / mL, <50 pg / mL, <60 pg / mL, <70 pg / mL, <80 pg / mL, <90 pg / mL, or <100 pg / mL of the therapeutic protein.

[0128] In some embodiments, the sample comprises >2.5 pg / mL, >5 pg / mL, >10 pg / mL, >20 pg / mL, >30 pg / mL, >40 pg / mL, >50 pg / mL, >60 pg / mL, >70 pg / mL, >80 pg / mL, >90 pg / mL, or >100 pg / mL of the therapeutic protein.

[0129] In some embodiments, the sample comprises about 2.5 pg / mL, about 5 pg / mL, about 10 pg / mL, about 20 pg / mL, about 30 pg / mL, about 40 pg / mL, about 50 pg / mL, about 60 pg / mL, about 70 pg / mL, about 80 pg / mL, about 90 pg / mL, or about 100 pg / mL of the therapeutic protein.

[0130] In some embodiments, the sample comprises about 0.1 mg / mL to about 1 mg / mL of the therapeutic protein.

[0131] In some embodiments, the sample comprises <0.1 mg / mL, <0.2 mg / mL, <0.3 mg / mL, <0.4 mg / mL, <0.5 mg / mL, <0.6 mg / mL, <0.7 mg / mL, <0.8 mg / mL, <0.9 mg / mL, or <1 mg / mL of the therapeutic protein.

[0132] In some embodiments, the sample comprises >0.1 mg / mL, >0.2 mg / mL, >0.3 mg / mL, >0.4 mg / mL, >0.5 mg / mL, >0.6 mg / mL, >0.7 mg / mL, >0.8 mg / mL, >0.9 mg / mL, or >1 mg / mL of the therapeutic protein.43913-02708 / WO (REGE-040 / 001WO)

[0133] In some embodiments, the sample comprises about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL of the therapeutic protein.

[0134] In some embodiments, the sample comprises about 0.1 mg / mL to about 0.3 mg / mL of the therapeutic protein.

[0135] In some embodiments, the sample comprises <0.1 mg / mL, <0.2 mg / mL, or <0.3 mg / mL of the therapeutic protein.

[0136] In some embodiments, the sample comprises >0.1 mg / mL, >0.2 mg / mL, or >0.3 mg / mL of the therapeutic protein.

[0137] In some embodiments, the sample comprises about 0.1 mg / mL, about 0.2 mg / mL, or about 0.3 mg / mL of the therapeutic protein.Size Exclusion Chromatography

[0138] In some embodiments, the size exclusion chromatography is performed under native conditions (e.g., in neutral pH water).

[0139] Size exclusion chromatography is described, for example, in Hong, P. et al. Journal of Liquid Chromatography & Related Technologies, 2012, 35(20), 2923-2950, the contents of which are incorporated by reference in their entirety herein.

[0140] In some embodiments, the size exclusion chromatography is gel filtration chromatography (GFC).

[0141] In some embodiments, the size exclusion chromatography is gel permeation chromatography (GPC).

[0142] In some embodiments, the size exclusion chromatography comprises a stationary phase and a mobile phase.

[0143] In some embodiments, the stationary phase comprises porous beads (e.g., hydrophilic porous beads) packed within a column.

[0144] In some embodiments, the porous beads are agarose beads.

[0145] In some embodiments, the porous beads are plain agarose beads.

[0146] In some embodiments, the porous beads are cross-linked agarose beads.

[0147] In some embodiments, the agarose beads (e.g., plain agarose beads or cross-linked agarose beads) comprise <2%, 2%, 4%, 6%, 8%, 10%, or greater than 10% of agarose.

[0148] In some embodiments, the porous beads are polyacrylamide beads.

[0149] In some embodiments, the porous beads are silica-based polymers.

[0150] In some embodiments, the porous beads are microparticles.43913-02708 / WO (REGE-040 / 001WO)

[0151] In some embodiments, the porous beads are <5 pm, <10 pm, <20 pm, <30 pm, <40 pm, <50 pm, <60 pm, <70 pm, <80 pm, <90 pm, <100 pm, <110 pm, <120 pm, <130 pm, <140 pm, <150 pm, <160 pm, <170 pm, <180 pm, <190 pm, <200 pm, <210 pm, <220 pm, <230 pm, <240 pm, or <250 pm in diameter, on average.

[0152] In some embodiments, the porous beads are >5 pm, >10 pm, >20 pm, >30 pm, >40 pm, >50 pm, >60 pm, >70 pm, >80 pm, >90 pm, >100 pm, >110 pm, >120 pm, >130 pm, >140 pm, >150 pm, >160 pm, >170 pm, >180 pm, >190 pm, >200 pm, >210 pm, >220 pm, >230 pm, >240 pm, or >250 pm in diameter, on average.

[0153] In some embodiments, the porous beads are about 5 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, or about 250 pm in diameter, on average.

[0154] In some embodiments, the porous beads have a mean size distribution of <0.1 pm, <0.2pm, <0.3pm, <0.4pm, <0.5pm, <0.6pm, <0.7pm, <0.8pm, <0.9pm, <lpm, <2pm, <3pm, <4pm, <5pm, <6pm, <7pm, <8pm, <9pm, or <10 pm,

[0155] In some embodiments, the porous beads have a mean size distribution of >0.1 pm, >0.2pm, >0.3pm, >0.4pm, >0.5pm, >0.6pm, >0.7pm, >0.8pm, >0.9pm, >lpm, >2pm, >3pm, >4pm, >5pm, >6pm, >7pm, >8pm, >9pm, or >10 pm,

[0156] In some embodiments, the porous beads have a mean size distribution of about 0.1pm, about 0.2pm, about 0.3pm, about 0.4pm, about 0.5pm, about 0.6pm, about 0.7pm, about 0.8pm, about 0.9pm, about 1pm, about 2pm, about 3pm, about 4pm, about 5pm, about 6pm, about 7pm, about 8pm, about 9pm, or about 10 pm,

[0157] In some embodiments, the stationary phase has a fractionation range suitable for isolating the therapeutic protein.

[0158] In some embodiments, the stationary phase has a fractionation range of Mr comprising about 25 kDa, about 50 kDa, about 100 kDa, about 150 kDa about 200 kDa, about 250 kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa, about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, about 950 kDa, or about 1000 kDa.

[0159] In some embodiments, the stationary phase has a fractionation range of Mr comprising about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa,43913-02708 / WO (REGE-040 / 001WO)about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, or about 200 kDa.

[0160] In some embodiments, the size exclusion chromatography is performed with a column inlet pressure less than 2,000 psi; less than 3,000 psi; less than 4,000 psi; less than 5,000 psi; less than 6,000 psi; less than 7,000 psi; less than 8,000 psi; less than 9,000 psi; less than 10,000 psi; less than 15,000 psi; or less than 20,000 psi.

[0161] In some embodiments, the size exclusion chromatography is performed with a column inlet pressure greater than 2,000 psi; greater than 3,000 psi; greater than 4,000 psi; greater than 5,000 psi; greater than 6,000 psi; greater than 7,000 psi; greater than 8,000 psi; greater than 9,000 psi; greater than 10,000 psi; greater than 15,000 psi; or greater than 20,000 psi.

[0162] In some embodiments, the size exclusion chromatography is performed with a column inlet pressure about 2,000 psi; about 3,000 psi; about 4,000 psi; about 5,000 psi; about 6,000 psi; about 7,000 psi; about 8,000 psi; about 9,000 psi; about 10,000 psi; about 15,000 psi; or about 20,000 psi.

[0163] In some embodiments, the size exclusion chromatography is monitored with a detector.

[0164] In some embodiments, the detector is a refractive index detector, UV detector, lightscattering detector, or mass spectrometer.

[0165] In some embodiments, the detector is a UV detector (e.g., detecting 280 nm).

[0166] In some embodiments, the mobile phase is water.

[0167] In some embodiments, the mobile phase is neutral pH water ( / .< ., pH of 7).

[0168] In some embodiments, the mobile phase is a mixture of water and one or more polar organic solvent (e.g., acetonitrile, methanol, ethanol, or isopropanol).

[0169] In some embodiments, the mobile phase is <60%, <65%, <70%, <75%, <80%, <85%, <90%, or <95% water by volume.

[0170] In some embodiments, the mobile phase is >60%, >65%, >70%, >75%, >80%, >85%, >90%, or >95% water by volume.

[0171] In some embodiments, the mobile phase is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% water by volume.

[0172] In some embodiments, the mobile phase is <95%, <96%, <97%, <98%, or <99% water by volume.

[0173] In some embodiments, the mobile phase is >95%, >96%, >97%, >98%, or >99% water by volume.43913-02708 / WO (REGE-040 / 001WO)

[0174] In some embodiments, the mobile phase is about 95%, about 96%, about 97%, about 98%, or about 99% water by volume.

[0175] In some embodiments, the mobile phase is buffered.

[0176] In some embodiments, the mobile phase is buffered with a volatile salt (e.g., ammonium salt).

[0177] In some embodiments, the ammonium salt is ammonium acetate.

[0178] In some embodiments, the ammonium salt is ammonium bicarbonate.

[0179] In some embodiments, the buffer concentration in the mobile phase is <10 mM, <20 mM, <30 mM, <40 mM, <50 mM, <60 mM, <70 mM, <80 mM, <90 mM, <100mM, <110 mM, <120 mM, <130 mM, <140 mM, <150 mM, <160 mM, <170 mM, <180 mM, <190 mM, <200 mM, <210 mM, <220 mM, <230 mM, <240 mM, or <250 mM.

[0180] In some embodiments, the buffer concentration in the mobile phase is >10 mM, >20 mM, >30 mM, >40 mM, >50 mM, >60 mM, >70 mM, >80 mM, >90 mM, >100mM, >110 mM, >120 mM, >130 mM, >140 mM, >150 mM, >160 mM, >170 mM, >180 mM, >190 mM, >200 mM, >210 mM, >220 mM, >230 mM, >240 mM, or >250 mM.

[0181] In some embodiments, the buffer concentration in the mobile phase is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about lOOmM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM.

[0182] In some embodiments, the mobile phase is not buffered.

[0183] In some embodiments, the mobile phase is buffered to a pH of <6.0, <6.1, <6.2, <6.3, <6.4, <6.5, <6.6, <6.7, <6.8, <6.9, <7.0, <7.1, <7.2, <7.3, <7.4,. 7.5, <7.6, <7.7, <7.8, <7.9, or <8.0.

[0184] In some embodiments, the mobile phase is buffered to a pH of >6.0, >6.1, >6.2, >6.3, >6.4, >6.5, >6.6, >6.7, >6.8, >6.9, >7.0, >7.1, >7.2, >7.3, >7.4,. 7.5, >7.6, >7.7, >7.8, >7.9, or >8.0.

[0185] In some embodiments, the mobile phase is buffered to a pH of 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,. 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0186] In some embodiments, the size exclusion chromatography is performed at <10 °C, <15 °C, <20 °C, <25 °C, <30 °C, <35 °C, <40 °C, or <45 °C.43913-02708 / WO (REGE-040 / 001WO)

[0187] In some embodiments, the size exclusion chromatography is performed at >10 °C, >15 °C, >20 °C, >25 °C, >30 °C, >35 °C, >40 °C, or >45 °C.

[0188] In some embodiments, the size exclusion chromatography is performed at about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, or about 45 °C.Ion Exchange Chromatography

[0189] In some embodiments, the ion exchange chromatography is performed under native conditions (e.g., in neutral pH water).

[0190] In some embodiments, the ion exchange chromatography is cation exchange chromatography.

[0191] In some embodiments, the cation exchange chromatography is strong cation exchange chromatography.

[0192] Ion exchange chromatography is described, for example, in Zhai, Z. et al. Journal of Separation Science, 2025, 48(9), e70268, the contents of which are incorporated by reference in their entirety herein.

[0193] In some embodiments, the ion exchange chromatography comprises a stationary phase and a mobile phase.

[0194] In some embodiments, the stationary phase comprises porous beads (e.g., acidic porous beads or anionic porous beads) packed within a column.

[0195] In some embodiments, the porous beads are agarose beads.

[0196] In some embodiments, the porous beads are plain agarose beads.

[0197] In some embodiments, the porous beads are cross-linked agarose beads.

[0198] In some embodiments, the agarose beads (e.g., plain agarose beads or cross-linked agarose beads) comprise <2%, 2%, 4%, 6%, 8%, 10%, or greater than 10% of agarose.

[0199] In some embodiments, the porous beads are polyacrylamide beads.

[0200] In some embodiments, the porous beads are silica-based polymers.

[0201] In some embodiments, the porous beads are polystyrene beads.

[0202] In some embodiments, the porous beads are sulfonated polystyrene.

[0203] In some embodiments, the porous beads are microparticles.

[0204] In some embodiments, the porous beads are anionic porous beads (e.g., comprising sulfonate or carboxylate groups).

[0205] In some embodiments, the porous beads comprise sulfonate groups (i.e., for strong cation exchange chromatography).43913-02708 / WO (REGE-040 / 001WO)

[0206] In some embodiments, the porous beads comprise carboxylate groups ( / .< ., for weak cation exchange chromatography).

[0207] In some embodiments, the porous beads are <5 pm, <10 pm, <20 pm, <30 pm, <40 pm, <50 pm, <60 pm, <70 pm, <80 pm, <90 pm, <100 pm, <110 pm, <120 pm, <130 pm, <140 pm, <150 pm, <160 pm, <170 pm, <180 pm, <190 pm, <200 pm, <210 pm, <220 pm, <230 pm, <240 pm, or <250 pm in diameter, on average.

[0208] In some embodiments, the porous beads are >5 pm, >10 pm, >20 pm, >30 pm, >40 pm, >50 pm, >60 pm, >70 pm, >80 pm, >90 pm, >100 pm, >110 pm, >120 pm, >130 pm, >140 pm, >150 pm, >160 pm, >170 pm, >180 pm, >190 pm, >200 pm, >210 pm, >220 pm, >230 pm, >240 pm, or >250 pm in diameter, on average.

[0209] In some embodiments, the porous beads are about 5 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, or about 250 pm in diameter, on average.

[0210] In some embodiments, the porous beads have a mean size distribution of <0.1 pm, <0.2pm, <0.3pm, <0.4pm, <0.5pm, <0.6pm, <0.7pm, <0.8pm, <0.9pm, <lpm, <2pm, <3pm, <4pm, <5pm, <6pm, <7pm, <8pm, <9pm, or <10 pm,

[0211] In some embodiments, the porous beads have a mean size distribution of >0.1 pm, >0.2pm, >0.3pm, >0.4pm, >0.5pm, >0.6pm, >0.7pm, >0.8pm, >0.9pm, >lpm, >2pm, >3pm, >4pm, >5pm, >6pm, >7pm, >8pm, >9pm, or >10 pm,

[0212] In some embodiments, the porous beads have a mean size distribution of about 0.1pm, about 0.2pm, about 0.3pm, about 0.4pm, about 0.5pm, about 0.6pm, about 0.7pm, about 0.8pm, about 0.9pm, about 1pm, about 2pm, about 3pm, about 4pm, about 5pm, about 6pm, about 7pm, about 8pm, about 9pm, or about 10 pm,

[0213] In some embodiments, the ion exchange chromatography is performed with a column inlet pressure less than 2,000 psi; less than 3,000 psi; less than 4,000 psi; less than 5,000 psi; less than 6,000 psi; less than 7,000 psi; less than 8,000 psi; less than 9,000 psi; less than 10,000 psi; less than 15,000 psi; or less than 20,000 psi.

[0214] In some embodiments, the ion exchange chromatography is performed with a column inlet pressure greater than 2,000 psi; greater than 3,000 psi; greater than 4,000 psi; greater than 5,000 psi; greater than 6,000 psi; greater than 7,000 psi; greater than 8,000 psi; greater than 9,000 psi; greater than 10,000 psi; greater than 15,000 psi; or greater than 20,000 psi.43913-02708 / WO (REGE-040 / 001WO)

[0215] In some embodiments, the ion exchange chromatography is performed with a column inlet pressure about 2,000 psi; about 3,000 psi; about 4,000 psi; about 5,000 psi; about 6,000 psi; about 7,000 psi; about 8,000 psi; about 9,000 psi; about 10,000 psi; about 15,000 psi; or about 20,000 psi.

[0216] In some embodiments, the ion exchange chromatography is monitored with a detector.

[0217] In some embodiments, the detector is a refractive index detector, UV detector, lightscattering detector, or mass spectrometer.

[0218] In some embodiments, the detector is a UV detector (e.g., detecting 280 nm).

[0219] In some embodiments, the mobile phase is water.

[0220] In some embodiments, the mobile phase is neutral pH water ( / .< ., pH of 7).

[0221] In some embodiments, the mobile phase is a mixture of water and one or more polar organic solvent (e.g., acetonitrile, methanol, ethanol, or isopropanol).

[0222] In some embodiments, the mobile phase is <60%, <65%, <70%, <75%, <80%, <85%, <90%, or <95% water by volume.

[0223] In some embodiments, the mobile phase is >60%, >65%, >70%, >75%, >80%, >85%, >90%, or >95% water by volume.

[0224] In some embodiments, the mobile phase is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% water by volume.

[0225] In some embodiments, the mobile phase is <95%, <96%, <97%, <98%, or <99% water by volume.

[0226] In some embodiments, the mobile phase is >95%, >96%, >97%, >98%, or >99% water by volume.

[0227] In some embodiments, the mobile phase is about 95%, about 96%, about 97%, about 98%, or about 99% water by volume.

[0228] In some embodiments, the mobile phase is buffered.

[0229] In some embodiments, the mobile phase is buffered with a volatile salt e.g., ammonium salt).

[0230] In some embodiments, the ammonium salt is ammonium acetate.

[0231] In some embodiments, the ammonium salt is ammonium bicarbonate.

[0232] In some embodiments, the buffer concentration in the mobile phase is <10 mM, <20 mM, <30 mM, <40 mM, <50 mM, <60 mM, <70 mM, <80 mM, <90 mM, <100mM, <110 mM, <120 mM, <130 mM, <140 mM, <150 mM, <160 mM, <170 mM, <180 mM, <190 mM, <200 mM, <210 mM, <220 mM, <230 mM, <240 mM, or <250 mM.43913-02708 / WO (REGE-040 / 001WO)

[0233] In some embodiments, the buffer concentration in the mobile phase is >10 mM, >20 mM, >30 mM, >40 mM, >50 mM, >60 mM, >70 mM, >80 mM, >90 mM, >100mM, >110 mM, >120 mM, >130 mM, >140 mM, >150 mM, >160 mM, >170 mM, >180 mM, >190 mM, >200 mM, >210 mM, >220 mM, >230 mM, >240 mM, or >250 mM.

[0234] In some embodiments, the buffer concentration in the mobile phase is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about lOOmM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM.

[0235] In some embodiments, the mobile phase is not buffered.

[0236] In some embodiments, the mobile phase is buffered to a pH of <6.0, <6.1, <6.2, <6.3, <6.4, <6.5, <6.6, <6.7, <6.8, <6.9, <7.0, <7.1, <7.2, <7.3, <7.4,. 7.5, <7.6, <7.7, <7.8, <7.9, or <8.0.

[0237] In some embodiments, the mobile phase is buffered to a pH of >6.0, >6.1, >6.2, >6.3, >6.4, >6.5, >6.6, >6.7, >6.8, >6.9, >7.0, >7.1, >7.2, >7.3, >7.4,. 7.5, >7.6, >7.7, >7.8, >7.9, or >8.0.

[0238] In some embodiments, the mobile phase is buffered to a pH of 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,. 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0239] In some embodiments, the ion exchange chromatography is performed at <10 °C, <15 °C, <20 °C, <25 °C, <30 °C, <35 °C, <40 °C, or <45 °C.

[0240] In some embodiments, the ion exchange chromatography is performed at >10 °C, >15 °C, >20 °C, >25 °C, >30 °C, >35 °C, >40 °C, or >45 °C.

[0241] In some embodiments, the ion exchange chromatography is performed at about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, or about 45 °C.Native Mass Spectrometry

[0242] Native mass spectrometry is described, for example, in Tamara, S, et al. Chemical Reviews, 2021, 122(8), pgs. 7269-7326, the contents of which are incorporated by reference in their entirety herein.

[0243] In some embodiments, sample desalting is required prior to mass spectrometry analysis.43913-02708 / WO (REGE-040 / 001WO)

[0244] In some embodiments, sample desalting is not required prior to mass spectrometry analysis.

[0245] In some embodiments, buffer exchange of the sample is required prior to mass spectrometry analysis.

[0246] In some embodiments, buffer exchange of the sample is not required prior to mass spectrometry analysis.

[0247] In some embodiments, the mass spectrometry analysis is performed in positive ion mode.

[0248] In some embodiments, the mass spectrometry analysis is performed in negative ion mode.

[0249] In some embodiments, the mass spectrometry analysis is performed by an electrospray ionization mass spectrometer.

[0250] In some embodiments, the mass spectrometry analysis is performed by an nanoelectrospray ionization mass spectrometer.

[0251] In some embodiments, the mass spectrometry analysis is high-resolution mass spectrometry analysis.

[0252] In some embodiments, the mass spectrometry analysis is performed by an electrospray ionization mass spectrometer or nano-electrospray ionization mass spectrometer. Methods of Determining Stability

[0253] In some aspects, the present disclosure provides a method of determining stability of a therapeutic protein, comprising:(i) providing at least a first and a second sample comprising the therapeutic protein, wherein the first sample is collected at an initial time point and the second sample is collected after a period of time;(ii) subjecting the at least first and second samples to a method of characterizing a therapeutic protein described herein, thereby generating at least one mass spectrogram of the therapeutic protein for each sample; and(iii) comparing the at least one mass spectrogram for each sample,thereby determining the stability of the therapeutic protein.

[0254] In some embodiments, step (iii) comprises comparing the MS signal intensity for one or more m / z value for each sample.

[0255] In some embodiments, the method comprises preparing a calibration curve that correlates therapeutic protein concentration and the MS signal intensity for one or more m / z value.43913-02708 / WO (REGE-040 / 001WO)

[0256] In some embodiments, the at least first and second samples are generated in vitro.

[0257] In some embodiments, the at least first and second samples are collected from a subject, thereby determining the stability of the therapeutic protein in vivo.

[0258] In some embodiments, the period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more hours.

[0259] In some embodiments, the period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more days.

[0260] In some embodiments, the period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks.

[0261] In some embodiments, the period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more months.EXEMPLARY EMBODIMENTS

[0262] Exemplary Embodiment No. 1. A method of characterizing a therapeutic protein, wherein the method comprises:(i) subjecting a sample comprising the therapeutic protein to size exclusion chromatography under conditions whereby the therapeutic protein is eluted;(ii) contacting the therapeutic protein eluted at step (i) with an ion exchange chromatography matrix under conditions sufficient for the therapeutic protein to be bound to the ion exchange chromatography matrix and eluted therefrom, thereby producing an eluate comprising the therapeutic protein; and(iii) subjecting the eluate from step (ii) to native mass spectrometry analysis to characterize the therapeutic protein.

[0263] Exemplary Embodiment No. 2. The method of Exemplary Embodiment No. 1, wherein the therapeutic protein is an antibody, fusion protein, antibody-drug conjugate, or protein pharmaceutical product.

[0264] Exemplary Embodiment No. 3. The method of Exemplary Embodiment No. 2, wherein the fusion protein comprises a receptor fusion protein (a Trap protein).

[0265] Exemplary Embodiment No. 4. The method of Exemplary Embodiment No. 2, wherein the antibody is a bispecific antibody or monoclonal antibody.

[0266] Exemplary Embodiment No. 5. The method of any one of Exemplary Embodiment Nos. 1-4, wherein the ion exchange chromatography is cation exchange chromatography.

[0267] Exemplary Embodiment No. 6. The method of Exemplary Embodiment No. 5, wherein the cation exchange chromatography is strong cation exchange chromatography.43913-02708 / WO (REGE-040 / 001WO)

[0268] Exemplary Embodiment No. 7. The method of any one of Exemplary Embodiment Nos. 1-6, wherein the mass spectrometry analysis is performed by an electrospray ionization mass spectrometer or nano-electrospray ionization mass spectrometer.

[0269] Exemplary Embodiment No. 8. The method of any one of Exemplary Embodiment Nos. 1-7, wherein the mass spectrometry analysis is high-resolution mass spectrometry analysis.

[0270] Exemplary Embodiment No. 9. The method of any one of Exemplary Embodiment Nos. 1-8, wherein the sample comprises about 0.1 mg / mL to about 1 mg / mL of the therapeutic protein.

[0271] Exemplary Embodiment No. 10. The method of Exemplary Embodiment No. 9, wherein the sample comprises about 0.1 mg / mL to about 0.3 mg / mL of the therapeutic protein.

[0272] Exemplary Embodiment No. 11. The method of any one of Exemplary Embodiment Nos. 1-10, wherein the method characterizes one or more derivatives of the therapeutic protein.

[0273] Exemplary Embodiment No. 12. The method of Exemplary Embodiment No. 11, wherein at least one derivative is a glycoform of the therapeutic protein.

[0274] Exemplary Embodiment No. 13. The method of Exemplary Embodiment No. 11, wherein at least one derivative is a deamidated form of the therapeutic protein.

[0275] Exemplary Embodiment No. 14. The method of any one of Exemplary Embodiment Nos. 1-13, wherein the therapeutic protein is generated in vivo in a patient.

[0276] Exemplary Embodiment No. 15. The method of any one of Exemplary Embodiment Nos. 1-14, wherein the sample is prepared from blood collected from a patient.

[0277] Exemplary Embodiment No. 16. The method of any one of Exemplary Embodiment Nos. 1-15, wherein the sample is prepared from blood serum of a patient.

[0278] Exemplary Embodiment No. 17. The method of any one of Exemplary Embodiment Nos. 1-15, wherein the sample is prepared from blood plasma of a patient.

[0279] Exemplary Embodiment No. 18. The method of any one of Exemplary Embodiment Nos. 14-17, wherein the patient is a mammal.

[0280] Exemplary Embodiment No. 19. The method of any one of Exemplary Embodiment Nos. 14-18, wherein the patient is a human.

[0281] Exemplary Embodiment No. 20. The method of any one of Exemplary Embodiment Nos. 1-19, wherein the method does not comprise a step to isolate the43913-02708 / WO (REGE-040 / 001WO)therapeutic protein from the sample prior to subjecting the sample to size exclusion chromatography.

[0282] Exemplary Embodiment No. 21. The method of any one of Exemplary Embodiment Nos. 1-20, wherein the method does not comprise an immunoprecipitation step.

[0283] Exemplary Embodiment No. 22. The method of any one of Exemplary Embodiment Nos. 1-21, wherein one or more isoforms of the therapeutic protein are quantified.

[0284] Exemplary Embodiment No. 23. The method of Exemplary Embodiment No. 22, wherein the one or more isoforms of the therapeutic protein are quantified from mass spectrometry data.

[0285] Exemplary Embodiment No. 24. A method of determining stability of a therapeutic protein, comprising:(i) providing at least a first and a second sample comprising the therapeutic protein, wherein the first sample is collected at an initial time point and the second sample is collected after a period of time;(ii) subjecting the at least first and second samples to the method of Exemplary Embodiment No. 1, thereby generating at least one mass spectrogram of the therapeutic protein for each sample; and(iii) comparing the at least one mass spectrogram for each sample,thereby determining the stability of the therapeutic protein.

[0286] Exemplary Embodiment No. 25. The method of Exemplary Embodiment No. 24, wherein the at least first and second samples are generated in vitro.

[0287] Exemplary Embodiment No. 26. The method of Exemplary Embodiment No. 24, wherein the at least first and second samples are collected from a subject, thereby determining the stability of the therapeutic protein in vivo.EXAMPLESExample 1. Proof-of-concept Test of Immunoprecipitation (IP)-free Antibody Analysis

[0288] A monoclonal antibody (mAb) was spiked into mouse plasma to generate a 1 mg mAb / mL solution. The antibody solution was analyzed by IP -free 2D-Native-SEC-SCX-MS, a two-dimensional chromatography, wherein the solution is first subjected to size exclusion chromatography, then subjected to strong cation exchange chromatography, and finally analyzed by high-resolution native mass spectrometry (see, FIGS. 2A-2C and 3A-3E).43913-02708 / WO (REGE-040 / 001WQ)Example 2. Quantitation of IP-Free, 2D-Native-SEC-SCX-MS Method

[0289] A mAb was spiked into mouse plasma at concentrations of 2.5 pg mAb / mL, 5 pg mAb / mL, 10 pg mAb / mL, 25 pg mAb / mL, 50 pg mAb / mL, and 100 pg mAb / mL. 5 pL of the antibody solutions were analyzed by IP-free 2D-SEC-SCX-MS in “dilute-and-shot” mode. FIGS. 4 A and 4B provide representative calibration curves that correlate mAb concentration and the resulting MS signal intensity. Additionally, FIGS. 5A-5F show the deconvoluted MS spectra of the Main peak of the samples analyzed in FIGS. 4 A and 4B.Example 3. Characterization of AAV antibody by IP-Free, 2D-Native-SEC-SCX-MS Method

[0290] As depicted in FIG. 6A, AAV comprising a transgene encoding the mAb were injected into mice, and then mouse serum was collected. The mouse serum was then analyzed by IP-free 2D-Native-SEC-SCX-MS.

[0291] FIG. 6B provides the extracted ion chromatogram from IP-free 2D-Native-SEC-SCX-MS data of mAb expressed in vivo. For reference, FIG. 6C provides the extracted ion chromatogram of mAb expressed in CHO cells, which were purified and spiked in plasma at 0.2 mg / mL for analysis by IP-free 2D-Native-SEC-SCX-MS. FIG. 7A shows mass spectra of the Al, A2, and Main peaks of FIG. 6B and the Main peak of FIG. 6C.FIG. 7B shows quantitation of glycoforms from the samples analyzed in FIGS. 6B and 6C.Example 4. In vitro Antibody Biotransformation Study

[0292] A mAb was spiked in mouse plasma at 1 mg / mL, and then incubated at 37 °C for 0, 1, 3, 5, or 7 days prior to analysis by IP-free 2D-Native-SEC-SCX-MS. FIGS. 8A-8E show m / z-filtered SCX chromatograms of the mAb samples. FIG. 9A shows complementarity determining region (CDR) deamidation over time from the samples analyzed in FIGS. 8A-8E, and FIG. 9B shows unconverted N-terminal glutamine (glutamine that has not converted to pyroglutamic acid) over time from the samples analyzed in FIGS. 8A-8E.

Claims

43913-02708 / WO (REGE-040 / 001WO)CLAIMSWhat is claimed is:

1. A method of characterizing a therapeutic protein, wherein the method comprises:(i) subjecting a sample comprising the therapeutic protein to size exclusion chromatography under conditions whereby the therapeutic protein is eluted;(ii) contacting the therapeutic protein eluted at step (i) with an ion exchange chromatography matrix under conditions sufficient for the therapeutic protein to be bound to the ion exchange chromatography matrix and eluted therefrom, thereby producing an eluate comprising the therapeutic protein; and(iii) subjecting the eluate from step (ii) to native mass spectrometry analysis to characterize the therapeutic protein.

2. The method of claim 1, wherein the therapeutic protein is an antibody, fusion protein, antibody-drug conjugate, or protein pharmaceutical product.

3. The method of claim 2, wherein the fusion protein comprises a receptor fusion protein (a Trap protein).

4. The method of claim 2, wherein the antibody is a bispecific antibody or monoclonal antibody.

5. The method of any one of claims 1-4, wherein the ion exchange chromatography is cation exchange chromatography.

6. The method of claim 5, wherein the cation exchange chromatography is strong cation exchange chromatography.

7. The method of any one of claims 1-6, wherein the mass spectrometry analysis is performed by an electrospray ionization mass spectrometer or nano-electrospray ionization mass spectrometer.

8. The method of any one of claims 1-7, wherein the mass spectrometry analysis is high-resolution mass spectrometry analysis.43913-02708 / WO (REGE-040 / 001WO)9. The method of any one of claims 1-8, wherein the sample comprises 0.1 mg / mL to 1 mg / mL of the therapeutic protein.

10. The method of claim 9, wherein the sample comprises 0.1 mg / mL to 0.3 mg / mL of the therapeutic protein.

11. The method of any one of claims 1-10, wherein the method characterizes one or more derivatives of the therapeutic protein.

12. The method of claim 11, wherein the one or more derivatives of the therapeutic protein comprises a glycoform of the therapeutic protein.

13. The method of claim 11, wherein at least one derivative is a deamidated form of the therapeutic protein.

14. The method of any one of claims 1-13, wherein the therapeutic protein is administered to a patient, or generated in vivo in a patient.

15. The method of any one of claims 1-14, wherein the sample is prepared from blood collected from a patient.

16. The method of any one of claims 1-15, wherein the sample is prepared from blood serum of a patient.

17. The method of any one of claims 1-15, wherein the sample is prepared from blood plasma of a patient.

18. The method of any one of claims 14-17, wherein the patient is a mammal.

19. The method of any one of claims 14-18, wherein the patient is a human.

20. The method of any one of claims 1-19, wherein the method does not comprise a step to isolate the therapeutic protein from the sample prior to subjecting the sample to size exclusion chromatography.43913-02708 / WO (REGE-040 / 001WO)21. The method of any one of claims 1-20, wherein the method does not comprise an immunoprecipitation step.

22. The method of any one of claims 1-21, wherein one or more isoforms of the therapeutic protein are quantified.

23. The method of claim 22, wherein the one or more isoforms of the therapeutic protein are quantified from mass spectrometry data.

24. A method of determining stability of a therapeutic protein, comprising:(i) providing at least a first and a second sample comprising the therapeutic protein, wherein the first sample is collected at an initial time point and the second sample is collected after a period of time;(ii) subjecting the at least first and second samples to the method of claim 1, thereby generating at least one mass spectrogram of the therapeutic protein for each sample; and(iii) comparing the at least one mass spectrogram for each sample,thereby determining the stability of the therapeutic protein.

25. The method of claim 24, wherein the at least first and second samples are generated in vitro.

26. The method of claim 24, wherein the at least first and second samples are collected from a subject, thereby determining the stability of the therapeutic protein in vivo.

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