METHOD FOR QUANTIFYING A NON-IONIC SURFACTANT IN A COMPOSITION COMPRISING THE NON-IONIC SURFACTANT AND A POLYPEPTIDE

Abstract

A method for quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide comprising the steps of: a) applying the composition to a mixed-mode cation-exchange or anion-exchange chromatography material by loading the composition onto the chromatography material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase A comprises ammonium hydroxide or acid in water respectively and mobile phase B comprises ammonium hydroxide in an organic solvent or acid in methanol respectively; b) eluting the polypeptide from the mixed-mode cation-exchange or anion-exchange chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step a);c) eluting the nonionic surfactant, or the nonionic surfactant and the polypeptide not specifically bound from the chromatography material with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase B to mobile phase A is increased compared to step c); and d) quantifying the nonionic surfactant, wherein the interference between the nonionic surfactant and the polypeptide is reduced during the quantification.

Description

[0002] The present invention provides methods for testing polypeptide formulations for the presence of polysorbates. BACKGROUND OF THE INVENTION

[0003] Polysorbate 20 (PS20) is a surfactant commonly used in polypeptide formulations to protect the product from physical damage during processing and storage (Kerwin, B., 2007, J. Pharm. Sci., 97(8): 2924-2935). Due to its importance to product stability, PS20 must be accurately quantified in each product control system. PS20 can be quantified by spectrophotometric assay, fluorescent micelle assay, or high performance liquid chromatography-evaporative light scatter detector (HPLC-ELSD) assay (see, for example, Kim, J. and Qiu, J., Analítica chimica acta 806:144-151, 2014; Hewitt et al., Journal of Chromatography A, 1215(1):156-160, 2008).

[0004] The evaporative light scattering detector (ELSD) assay is preferred as a control system assay because, relative to the fluorescent micelle assay, it does not require long conditioning times. The ELSD method can also obviate the requirement to use the same batch of polysorbate. for preparation of the standard curve when used in production. Additionally, the fluorescent micelle assay is susceptible to interference from non-specific proteins, particularly for hydrophobic proteins and antibody-drug conjugates (ADCs). The vcMMAE linker-drug of ADCs introduces additional hydrophobicity into the protein that can lead to greater protein interference when PS20 is quantified. In some cases, this non-specific protein interference can be mitigated using the HPLC-ELSD assay.

[0005] Although the HPLC-ELSD assay can reduce the degree of protein interference, this interference is not completely eliminated. The issue of protein interference becomes particularly problematic at low polysorbate concentrations and with more hydrophobic and / or concentrated proteins. Additionally, the effect of protein interference is highly dependent on the The strategies for lot of resin in used cartridge. mitigating these issues include a) addition of analyte from PS20 to dilute the interfering proteins without reducing the PS20 response and b) removal of the protein from the sample by precipitation of the proteins.

[0006] The analyte addition approach involves diluting a sample with a stock solution of PS20 to the target concentration of the formulation. This sample preparation dilutes the protein concentration while maintaining a concentration of PS20 approximately unchanged. The amount of PS20 added in the sample is then subtracted during data analysis. Since the analyzed mass ratio between the ELSD response and that at the detector follows a power law, addition in PS20 disproportionately reduces the protein's contribution to the ELSD signal. The analyte addition approach was shown to improve the accuracy of PS20 quantification in some cases, but in cases where this is not a viable solution, protein precipitation should be used. While effective in removing protein interference, the HPLC-ELSD precipitation method is not ideal due to overnight sample preparation time, large sample volumes, and variability in sample preparation. In contrast, protein removal employs the same conditions as HPLC-ELSD, but without significant sample preparation procedures. What is needed is a more robust solution to eliminate protein interference and obtain consistent PS20 quantification across all chromatographic conditions.

[0007] All references mentioned herein, including patent applications and publications, are incorporated herein in their entirety by reference. BRIEF SUMMARY

[0008] In some aspects, the invention provides a method for quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide, wherein interference between the nonionic surfactant and the polypeptide during quantification is reduced , wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the material Yo chromatography in a solution comprising a mobile phase A and a mobile phase B, wherein the mobile phase A comprises acid in water and the mobile phase wherein the polypeptide chromatography B comprises acid in methanol, where it binds with the specific material non-specifically; b) elution of the polypeptide specifically from mixed-mode chromatography linked anion exchange with a solution comprising mobile phase A and mobile phase B, wherein the material ratio of mobile phase B to mobile phase A is increased in comparison with step a); c) elution of the non-ionic surfactant and the non-specifically bound polypeptide from the chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased in comparison with stage c); d) quantification of the nonionic surfactant, wherein the interference between the nonionic surfactant and the polypeptide during quantification is reduced. In some embodiments, the ratio of mobile phase B to mobile phase A in step a) is approximately 10:90. In some embodiments, the ratio of mobile phase B to mobile phase A is increased to approximately 40:60 in step b). In some embodiments, the ratio of mobile phase B to mobile phase A is increased to approximately 100:0 in step c). In some embodiments, mobile phase A comprises approximately 2% acid in water. In some embodiments, mobile phase B comprises approximately 2% acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the L Chromatography flow rate is approximately 1.25 mL / minute. In some embodiments, step b) begins at about 1 min after the start of the chromatography and ends at about 3.4 min after the start of the chromatography. In some embodiments, step c) begins at about 3.5 min after the start of the chromatography and ends at about 4.6 min after the start of the chromatography. In some embodiments, the nonionic surfactant is poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant concentration in the composition is in the range of about 0.001% to 1.0% (w / v). In some embodiments, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL. In some embodiments, the formulation has a pH of from about 4.5 to about 7.5. In some embodiments, the composition also includes excipients selected from the stabilizer, a one or more consisting of a buffer and a tonicity agent. In composition it is some forms of embodiment, the appropriate pharmaceutical formulation to administer to a subject. In some embodiments, the therapeutic polypeptide. In some forms it is an embodiment polypeptide, humanized polyclonal therapeutic antibody polypeptide antibody, is a fusion protein, monoclonal antibody, human antibody, chimeric antibody, glycoengineered multispecific antibody, antibody fragment , an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0009] In some aspects, the invention provides a method for quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a chromatography material mixed mode cation exchange, wherein the composition is loaded onto the chromatography material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the chromatography material of mixed mode cation exchange with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step a); c) elution of the nonionic surfactant from the chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step c); d) quantification of the nonionic surfactant. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the ratio of mobile phase B to mobile phase A in step a) is approximately 10:90. In some embodiments, the ratio of mobile phase B to mobile phase A is increased to approximately 45:55 in step b). In some embodiments, the ratio of mobile phase B to mobile phase A is increased to approximately 100:0 in step c). In some embodiments, mobile phase A comprises approximately 2% ammonium hydroxide in water. In some embodiments, mobile phase B comprises approximately 2% ammonium hydroxide in methanol. In some embodiments, the chromatography flow rate is approximately 1.4 mL / minute. In some embodiments, step b) begins at about 1 min after the start of the chromatography and ends at about 4.4 min after the start of the chromatography. In some embodiments, step c) begins approximately 4.5 min after the start of chromatography and ends approximately 7.6 min after the start of the chromatography. In some embodiments, the nonionic surfactant is a polysorbate. In some In embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v). In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some embodiments, the ratio of mobile phase B to mobile phase A in step a) is approximately 10:90. In some embodiments, the ratio of mobile phase B to mobile phase A is increased to approximately 40:60 in step b). In some embodiments, the ratio of mobile phase B to mobile phase A is increased to 100:0 in step c). In some embodiments, mobile phase A comprises approximately 2% ammonium hydroxide in water or 43% methanol. In some embodiments, mobile phase B comprises approximately 2% ammonium hydroxide in acetonitrile. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of poloxamer in the composition is in the range of about 0.001% to 1.0% (w / v). In some embodiments, and / or embodiments comprise N-acetyltryptophan, the composition methionine. In concentration also some of acetyltryptophan in the composition varies from about 0.1 mM to approximately 10 mM In some forms of methionine in the embodiment, the composition concentration varies from about 0.1 mM to about 100 mM. In some embodiments, the composition concentration is polypeptide at approximately 1 mg / mL the Yo approximately 250mg / mL. In some embodiments, the formulation has a pH of approximately 4.5 to about 7.5. In some embodiments, the composition also includes excipients selected from the one or more consisting of a stabilizer, a buffer, and a tonicity group. In some agent embodiments, the composition is a formulation for administration to a subject. Appropriate Pharmaceutical In some embodiments, the therapeutic polypeptide. antibody polypeptide In some forms it is an embodiment polypeptide, polyclonal, humanized, therapeutic antibody conjugated antibody, is a fusion protein, monoclonal antibody, a human antibody, a multispecific antibody, antibody, chimeric, glycoengineered, antibody fragment -drug, a THIOMAB™, a drug a THIOMAB™. In some embodiments, the strong cation exchange mixed mode cation exchange chromatography material comprises a reverse phase polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG 1 is an overlay of Al ADC without PS20 and a 0.6 mg / ml PS20 standard using the multi-step gradient of 5% methanol build-up. The elution solvent contains 2% formic acid.

[0011] FIG 2 shows the comparison of a methanol multi-step gradient experiment and an isopropanol one-step gradient experiment (both with 2% formic acid) on different cartridges. For each overlay, there is PS20 free Al ADC in a first cartridge (trace 1), PS20 free Al ADC in a second cartridge (trace 2), and PS20 standard (trace 3).

[0012] FIG 3 shows results of the optimization design of the experiment method. The solid lines show the directionality according to the statistical model that the JMP10 software fits to the data. The dashed lines bounding each solid line indicate error associated with the fit. The slopes of the lines indicate the effect each factor had on the PS20 peak area and the PS20 peak width.

[0013] FIG 4 shows a comparison of chromatograms of the PS2 0 method of the DoE method optimization with a 40% MeOH wash and a 50% MeOH wash. For these experiments, the flow rate was 1.25 mL / min, 12 pg of PS20 was loaded, and the wash duration was 3 minutes.

[0014] FIG 5 shows 10 mg / ml PS20-free A10 ADC and 0.6 mg / ml PS2 0 standard with 0.2% trifluoroacetic acid in the mobile phase.

[0015] FIG 6 shows 20 mg / ml Al ADC free PS20 and or, 6 mg / ml PS20 standard with 2% formic acid in the mobile phase.

[0016] FIG 7 shows 20 mg / ml PS20-free Al ADC and 0.7 mg / ml PS20 standard with 2% acetic acid in the mobile phase.

[0017] FIG 8 shows a comparison between acid formic and acetic acid containing mobile phases using the following samples: water (trace 1), To the ADC free of PS2 0, 2 0 mg / mL (trace 2) and 0.2 mg / mL of PS20 added in Al ADC, 20 mg / mL (trace 3).

[0018] FIG 9 shows various profiles of the PS20 standard run on different cartridges OASIS'® MAX using methanol / acetic acid elution. The typical profile (trace 1), tailed beak (trace 2), beak showing splitting (trace 3) and beak with slight tail (trace 4). This variability in the profile does not affect the quantification of standards, controls, or protein samples.

[0019] FIG 10 shows the MeOH / acetic acid method. Normal injections of 20 pL of water (trace 1), formulation buffer of PS20-free Al ADC (trace 2), PS20-free Al ADC (trace 3), and the minimum PS20 standard at 0.1 mg / mL (trace 4).

[0020] FIGS HA and 11B show evaluation of PS20-free A16 / A17 using method 1 of Example 1. FIG 1A shows ELSD and FIG IB shows 'UV (2 80 nm). Water (trace 1), formulation buffer of PS20-free A16 / A17 (trace 2) and PS20-free A16 / A17 protein (trace 3) using method 1 of example 1.

[0021] FIGS 12A and 12B show the ELSD chromatograms of different components of the A16 / A17 buffer using the method of Example 1. FIG 12A shows buffers with NAT: 20 mM histidine HC1, 1 mM NAT, 5 mM histidine methionine, 240 mM sucrose (trace 1); 20 mM histidine HC1, 1 mM NAT, 240 mM sucrose (trace 2); 20 mM histidine HC1, 5 mM NAT (trace 3). FIG 12B shows buffers without NAT: 20 mM Histidine HC1, 5 mM methionine, 240 mM sucrose (trace i); 20 mM histidine HC1, 25 mM methionine (trace 2); 20 mM histidine HC1 i (trace 3). 50 pL of buffer injections.

[0022] FIGS 13A and 13B show ELSD chromatograms of different buffer components using a modified method (MCX cartridge and ammonium hydroxide in mobile phase). FIG 13A shows NAT buffers: 20 mM histidine HC1, 1 mM NAT, 5 mM methionine, 240 mM sucrose (trace 1); 20 mM histidine HC1, 1 mM NAT, 240 mM sucrose (trace 2); 20 mM histidine HC1, 5 mM NAT (trace 3); and PS20 free protein with NAT (trace 4). FIG 13B shows buffers without NAT: 20 mM histidine HC1, 5 mM methionine, 240 mM sucrose (trace 1); 20 mM histidine HC1, 25 mM methionine (trace 2); 20 mM histidine HC1 (trace 3). 50 pL injections.

[0023] FIGS 14A and 14B show the evaluation of PS20-free A16 / A17 protein with 0.15-1.50% ammonium hydroxide additive in the mobile phase. FIG 14A shows ELSD chromatograms. FIG 14B shows UV (280 nm) chromatograms. Injection of 50 pL of PS20-free A16 / A17 protein with 0.15, 0.29, 0.73, and 1.5% ammonium hydroxide in the mobile phase (traces 1, 2, 3, and 4, respectively). and formulation buffer Α16 / Α17 free of PS20 with 1.5% ammonium hydroxide (trace 5).

[0024] FIGS 15A and 15B show the 2060% evaluation of the mobile phase wash step B. FIG 15A shows ELSD chromatograms. FIG 15B shows UV (280 nm) chromatograms. Injection of 15 pL of PS20-free A16 / A17 protein. 20, 30, 40, 50 and 60% mobile phase B (wash step), shown as traces 1, 2, 3, 4 and 5, respectively.

[0025] FIG 16 shows evaluation of wash times and injection volumes for PS20-free A16 / A17 protein by ELSD chromatography. PS20-free A16 / A17 25 pL sample injection: 3.4 minute wash step (trace 1). PS20-free A16 / A17 50 pL sample injection: 3.4 minute wash step (trace 2) and 2.4 minute wash step (trace 3).

[0026] FIGS 17A and 17B show evaluation of different flow rates for PS20-free A18 / A19. FIG 17A shows ELSD chromatograms. FIG 17B shows UV (280 nm) chromatograms. Injection of 25 pL of PS20-free A18 / A19 (150 mg / mL) with different flow rates: 1.6, 1.4, 1.25, 1.0, and 0.8 mL / min, corresponding to traces 1, 2, 3, 4 and 5, respectively.

[0027] FIG 18 shows the evaluation of different elution times for PS20 in water by ELSD chromatography. Injection of 50 pL of 0.1 mg / mL PS20 in water, 3.1 minute elution step (trace 1) or 1.1 minute elution step (trace 2).

[0028] FIG 19 shows the evaluation of the finished method 2 by ELSD chromatography. Injection of 25 pL of water (trace 1), 150 mg / mL of PS20-free A18 / A19 (trace 2), 0.2 mg / mL of PS20 added in water (trace 3), and 0.2 mg / mL PS20 added in A18 / A19 (trace 4) using the finalized parameters for method 2.

[0029] FIGS 20A-20F show the evaluation of specificity for three different products. FIGS 20A (A18 / A19), 20C (A16 / A17) and 20E (A14 / A20) show ELSD chromatograms. FIGS 20B (A18 / A19), 20D (A16 / A17) and 20F (A14 / A20) show UV (280 nm) chromatograms. PS20 free formulation (trace 1), PS20 free protein (trace 2) and 0.1 mg / mL PS20 in water (trace 3).

[0030] FIG 21 shows the evaluation of cartridge-to-cartridge variability using PS20 added in water or PS20-free A18 / A19 by ELSD chromatography. Cartridge 2, 0.1 mg / mL PS20 added in A18 / A19 free of PS20 (trace 1); Cartridge 6, 0.1 mg / mL PS20 spiked in A18 / A19 free of PS20 (trace 2); Cartridge 2, 0.2 mg / mL PS20 added in water (trace 3); and cartridge 6, 0.2 mg / mL PS20 added in water (trace 4).

[0031] FIG 22 shows the assessment of cartridge-to-cartridge variability using PS20 added in water or PS20-free A18 / A19 by ELSD chromatography. Cartridge 4, 0.2 mg / mL PS20 added in A18 / A19 free of PS20 (trace 1); cartridge 6, 0.2 mg / mL PS20 added in A18 / A19 free of PS20 (trace 2); Cartridge 4, 0.2 mg / mL PS20 added in water (trace 3); and cartridge 6, 0.2 mg / mL PS20 added in water (trace 4).

[0032] FIG 23 shows ELSD chromatograms for control samples used in a sequence including 100 A18 / A19 injections in a single cartridge. Overlay of 11 control samples injected throughout the sequence.

[0033] FIG shows chromatograms of ELSD for A18 / A19 samples used in a sequence including 100 injections of A18 / A19 in a single cartridge. Yo Overlapping of 100 A18 / A19 sample injections throughout the sequence.

[0034] FIGS 25A and 25B show chromatograms of the protein injection 1, 50 and 100 of a sequence including 100 injections of A18 / A19 in a single cartridge.

[0035] FIGS 26A and 26B show results of quantification of 100 sample injections of A18 / A19 (0.2 mg / mL nominal PS20) using cartridge 4. FIG 2 6A shows area of ​​PS20 vs. injection number. FIG 26B shows the concentration of PS20 vs. injection number.

[0036] FIGS 27A and 27B show quantification results from 100 injections of A18 / A19 formulation buffer (0.2 mg / mL nominal PS20) using cartridge 5. FIG 27A shows area of ​​PS20 vs. injection number. FIG 27B shows PS20 concentration vs injection number.

[0037] FIGS 28A and 28B show quantification results of 100 injections of 0.2 mg / mL PS20 added in water using cartridge 3. FIG 28A shows PS20 area vs injection number. FIG 28B shows PS20 concentration vs injection number.

[0038] FIGS 29A-29F show the evaluation of three low pl products using method 1 and method 2 by ELSD chromatography. FIGS 29A, 29C and 29E show chromatograms for method 1 with A21, A14 / A15 and A14, respectively. FIGS 29B, 29D and 29F show the chromatograms for method 2 with A21, A14 / A15 and A14, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0039] The invention provides methods for quantifying a nonionic surfactant in a composition comprising a polypeptide and the nonionic surfactant, where the quantification exhibits less interference between the nonionic surfactant and the polypeptide. Methods are also provided where the composition also includes N-acetyltryptophan and the quantitation exhibits less interference between the nonionic surfactant, polypeptide, and N-acetyltryptophan. I. Definitions

[0040] The terms polypeptide or protein are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that is modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or toxin. Also included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically comprise antibodies.

[0041] Purified polypeptide (eg, antibody or immunoadhesin) means that the purity of the polypeptide has been increased, such that it exists in a form that is more pure than that in which it exists in its natural environment and / or when initially synthesized and / or amplified under laboratory conditions. The term purity is a relative term and does not necessarily mean absolute purity.

[0042] The term antagonist is used in its broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide. Similarly, the term agonist is used in its broadest sense and includes any molecule that mimics a biological activity of a native polypeptide. Suitable agonist or antagonist molecules e specifically include antibodies or antibody fragments, polypeptide amino acid sequence fragments or variants, etc., with agonist or antagonist properties. Methods for identifying agonists or antagonists of a polypeptide may comprise contacting a polypeptide with a candidate agonist or antagonist molecule, and measuring a detectable change in one or more normally associated biological activities on the peptide.

[0043] A polypeptide that binds an antigen of interest, eg, a tumor-associated polypeptide target antigen, is one that binds the antigen with sufficient affinity such that the polypeptide is useful as a diagnostic and / or therapeutic agent. in targeting a cell or tissue expressing the antigen and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to a non-target polypeptide will be less than about 10% of the binding of the polypeptide to its polypeptide. particular target as determined by fluorescence activated cell sorting (FACS) or radioimmunoprecipitation (RIA) analysis.

[0044] With respect to binding of a polypeptide to a target molecule, the terms "specific binding" or "binds specifically to" or "is specific with respect to a particular polypeptide or to an epitope on a particular target polypeptide" means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding to a target molecule compared to binding to a control molecule, which is typically a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the blank, eg, an excess of unlabeled blank. In this case, specific binding is indicated if the binding of labeled target to a probe is competitively inhibited by excess unlabeled target.

[0045] The term antibody is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), made up of at least two intact antibodies, and antibody fragments as long as they exhibit the desired biological activity. The term immunoglobulin (Ig) is used interchangeably with antibody herein.

[0046] Antibodies are naturally occurring immunoglobulin molecules that have various structures, all based on the immunoglobulin fold. For example, IgG antibodies they have two heavy and two light chains that are disulfide-linked to form a functional antibody. Each heavy and light chain itself comprises a constant region (C) and a variable region (V). The V regions determine the antigen-binding specificity of the antibody, while the V regions C provide structural support and function in non-antigenic specific interactions with immune effectors. The antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a particular antigen.

[0047] The antigen-binding specificity of an antibody is determined by the structural features of the V region. Variability is not evenly distributed across the 110 amino acid gap of variable domains. Instead, V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter, highly varied regions called hypervariable regions (HVRs) that are 9-12 amino acids in length. The native heavy and light chain variable domains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops that connect, in some cases are part of, the structure of the native heavy and light chains. β sheet. The hypervariable regions on each chain are held together in close proximity by the FRs and, with the hypervariable regions on the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as involvement of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0048] Typically, each V region comprises three HVRs, eg CDRs (complementarity determining regions), each containing a hypervariable loop, and four framework regions. Thus, an antigen-binding site, the minimal structural unit required to bind with substantial affinity to an antigen, typically includes all three CDRs, and at least three, preferably four, framework regions interspersed in a uniform fashion between them. support and present the CDRs in their proper conformation. Classic four-chain antibodies have antigen-binding sites that are defined by the cooperating VH and VL domains. Certain antibodies, such as camel and shark antibodies, lack light chains and rely on binding sites made up of heavy chains only. It is possible to prepare single-domain engineered immunoglobulins in which the binding sites are formed by heavy chains or light chains only, in the absence of cooperation between VH and VL.

[0049] The term variable refers to the fact that certain portions of the variable domains differ greatly in sequence between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across all antibody variable domains. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). The native heavy and light chain variable domains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops that connect, in some cases form part of, the structure of the native heavy and light chains. β sheet. The hypervariable regions on each chain are held together in close proximity by the FRs and, with the hypervariable regions on the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as involvement of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0050] The term hypervariable region (HVR), when used herein, refers to antibody amino acid residues that are responsible for antigen binding. The hypervariable region may comprise amino acid residues from a complementarity determining region or CDR (for example, around approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in Vl, and around of approximately 31-35B (Hl), 50-65 (H2) and 95-102 (H3) in VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues of a hypervariable loop ((for example residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in VL, and 2632 (Hl), 52A-55 (H2) and 96-101 (H3) in Vh (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). .

[0051] Framework or FR residues are those variable domain residues other than hypervariable region residues as defined herein.

[0052] Antibody fragments comprise a portion of an intact antibody, preferably comprising its counterpart. region Examples of antigen-binding antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (eg, U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single arm antibodies, single variable domain antibodies, minibodies, single chain antibody molecules; multispecific antibodies formed from antibody fragments (eg, including, but not limited to, Db-Fc, taDb-Fc, taDbCH3, (scFV)4-Fc, di-scFv, bi-scFv, or (di,tri)- tandem scFv); and bispecific T cell couplers (BITES).

[0053] Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a unique antigen-binding site, and a residual Fe fragment, the name of which reflects its ability to crystallize rapidly. Pepsin treatment produces a F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0054] Fv is the minimal antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a variable domain dimer of one heavy chain and one light chain in tight non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the Vh~Vl dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific hypervariable regions) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

[0055] The Fab fragment also contains the light chain constant domain and the first constant domain. Fab' fragments differ from Fab fragments by a few residues at the carboxy terminus the addition of ones from the domain Heavy chain CH1 including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation, herein, for Fab' wherein the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0056] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, termed kappa (k) and lambda (λ), based on the amino acid sequences of their domains. constants.

[0057] Based on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are designated a, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0058] Single chain FV antibody fragments or scFv comprise the antibody VH and Vl domains, where these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the Vh and Vl domains which allows the scFv to form the desired structure for antigen binding. For a review of scFv see: Plückthun in: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, p. 269-315 (1994).

[0059] The term diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable domain chain heavy (VH) linked to a variable light (VL) domain on the same polypeptide chain (VH VL). By using a linker that is too short to allow pairing between the two domains on the same strand, the domains are forced to pair with complementary domains on another strand and create two antigen binding sites. Diabodies are described in greater detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. nati. Acad. Sel. USA, 90:6444-6448 (1993).

[0060] The term "multispecific antibody" is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain (Vh) variable domain and a light chain (VL) variable domain, wherein the VHVL unit has polyepitopic specificity, antibodies having two or more domains VL and Vh with each VhVl unit binding a different epitope, antibodies having two or more unique variable domains with each unique variable domain binding a different epitope, full-length antibodies, antibody fragments such as Fab, Fv , dsFv, scFv, diabodies, diabodies, triabodies, functional antibodies, and antibody fragments that have been covalently or non-covalently ligated. Polyepitopic specificity refers to the ability to specifically bind two or more different epitopes on the same or different targets. Monospecific refers to the ability to bind to only one epitope. According to one embodiment a specific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μΜ to 0.001 pM, 3 μΜ to 0.001 pM, 1 μΜ to 0.001 pM, 0.5 μΜ to 0.001 pM, or 0.1 μΜ to 0.001 pM.

[0061] The term single domain antibodies (sdAbs) or single variable domain antibodies (SVD) generally refers to antibodies in which a single variable domain (Vh or Vl) can confer antigen binding. In other words, the unique variable domain need not interact with another variable domain in order to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (eg nurse sharks) and those derived from recombinant methods of human and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870).

[0062] The term "monoclonal antibody" as used herein refers to one obtained from an antibody population, that is, from individual, substantially homogeneous antibodies that comprise and / or bind to the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Contrary to polyclonal antibody preparations which typically include the population are identical different antibodies directed against different monoclonal determinants (epitopes), each antigen-determinant antibody is directed against a unique one. In addition to their specificity, monoclonal antibodies are advantageous because they are not bound by other immunoglobulins. The monoclonal contaminated modifier indicates that the character of the antibody has been obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular procedure. For example, monoclonal antibodies to used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al. , Nature 256:495 (1975) or can be prepared by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581597 (1991), for example.

[0063] Monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequences in the antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Nati. Acad. Sel. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (eg, Old World monkey, such as baboon, rhesus, or cynomolgus monkey) and human constant region sequences ( US Patent No. 5,693,780).

[0064] Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain non-human immunoglobulin-derived minimal sequence, humanized antibodies For the most part, they are human immunoglobulins in which residues of a recipient are replaced by (recipient antibody) hypervariable region residues of a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or primate. non-human having the desired specificity, affinity, and ability. In some cases, the Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. In general humanized, the antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs. are those of The humanized antibody is a portion of a human immunoglobulin, optionally comprising at least constant region (Fe from English immunoglobulin, typically from human. For further details, see Constant Region) of an immunoglobulin Jones et al., Nature. 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0065] For purposes herein, an intact antibody is one that comprises heavy and light chain variable domains as well as an Fe region. Constant domains can be native sequence constant domains (eg, human native sequence constant domains ) or their variant amino acid sequences. It is preferable that the intact antibody has one or more effector functions.

[0066] Native antibodies are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of different immunoglobulin subtypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has at one end a variable (VH) domain followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. Particular amino acid residues are believed to form an interface between the light chain and the variable domain of the heavy chains.

[0067] A naked antibody is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0068] In some embodiments, the effector functions of an antibody refer to the biological activities attributable to the Fe region (a native Fe sequence region or a variant Fe amino acid sequence region) of an antibody, and vary with the isotype of the antibody. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity; Fe receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors.

[0069] Antibody-dependent cell-mediated cytotoxicity or ADCC refers to a cell-mediated reaction wherein non-specific cytotoxic cells expressing Fe receptors (FcRs) (eg, Natural Killer (NK) cells, neutrophils, and macrophages) recognize an antibody binds to a target cell and subsequently causes lysis of the target cell. The primary cells to mediate ADCC, NK cells, express only FciRIII, whereas monocytes express FciRI, FciRII, and FciRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:45792 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337. Effector cells useful for these assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, by For example, in an animal model as disclosed in Clines et al., Proc. nati. Acad. Seth (United States) 95:652-656 (1998).

[0070] Human effector cells are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcyRIII and carry out the effector function of ADCC. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with preference being given to PBMCs and NK cells.

[0071] Complement-dependent cytotoxicity or CDC refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (Clq) to a molecule (eg, polypeptide (eg, an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

[0072] The terms Fe receptor or FcR are used to describe a receptor that binds to the Fe region of an antibody. In some embodiments, the FcR is human native sequence FcR. Furthermore, a preferred FcR is one that binds to an IgG antibody (a receptor gamma) and includes receptors from the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and, alternatively, cleaved forms of those receptors. FcyRII receptors include FcyRIIA (an activating receptor) and FcyRIIB (an inhibiting receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibition receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain {see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed on Ravetch and Kinet, Annu. Rev Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al • , J. Lab· clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term FcR herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guier et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)).

[0073] Impurities refer to materials that are different from the desired polypeptide product. In some embodiments of the invention, the impurities include charge variants of the polypeptide. In some embodiments of the invention, impurities include charge variants of an antibody or antibody fragment. In other embodiments of the invention, the impurity includes, without limitation: host cell materials, such as CHOP; protein To leachate; nucleic acid; a variant, fragment, aggregate, or derivative of the desired polypeptide; another polypeptide; endotoxin; viral contaminant; component of cell culture medium, etc.

[0074] As used herein, the term immunoadhesin refers to molecules similar to antibodies that combine the binding specificity of a heterologous polypeptide (an adhesin) with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity, which is other than that of an antibody's antigen recognition and binding site (i.e., heterologous), and a constant domain sequence of the immunoadhesins. immunoglobulin. The adhesin part of an immunoadhesin molecule is usually a contiguous amino acid sequence comprising at least one receptor or ligand binding site. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE , IgD or IgM.

[0075] As used herein, a surfactant refers to a surfactant, preferably a nonionic surfactant. Examples of surfactants herein include polysorbate (eg, polysorbate 20 and polysorbate 80); poloxamer (eg, poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl-betaine (eg lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl-dimethylamine; sodium or disodium methylcocoyl- or methyloleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.); polyethylene glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (eg, Pluronics, PF68, etc.); etc. In one embodiment, the surfactant herein is polysorbate 20. In yet another embodiment, the surfactant herein is poloxamer 188.

[0076] The term sequential as used herein with respect to chromatography refers to a first chromatography followed by a second chromatography. Additional steps may be included between the first chromatography and the second chromatography.

[0077] The term "continuous" as used herein with respect to chromatography refers to a first chromatography material and a second chromatography material directly connected to each other or connected to each other by some other mechanism that allows for continuous flow. continuous between the two chromatography materials.

[0078] The term "charge density" refers to the amount, eg in grams, of composition brought into contact with a volume of chromatography material, eg in liters. In some examples, the charge density is expressed in g / L.

[0079] The term interference as used herein with respect to the quantification of a species (eg, nonionic surfactant) refers to the contribution of some component other than the species (eg, polypeptide) to the quantification. For example, an ELSD signal for a chromatographic fraction containing both polysorbate 20 and polypeptide will have contributions from both polysorbate 20 and polypeptide and quantification of the polysorbate 20 in the fraction will have interference from the polypeptide.

[0080] As used herein, essentially the same indicates that a value or parameter was not altered by a significant effect. For example, an ionic strength of a chromatography mobile phase at the outlet of the column is essentially the same as the initial ionic strength of the mobile phase if the ionic strength was not changed significantly. For example, an ionic strength at the outlet of the column that is within 10%, 5%, or 1% of the initial ionic strength is essentially the same as the initial ionic strength.

[0081] Reference to about a value or parameter herein includes (and describes) variations that are directed at that value or parameter itself. For example, a description that refers to about X includes the description of X.

[0082] As used herein and in the appended claims, the singular forms a, an, or, and the include plural referents unless the context clearly indicates otherwise. It is understood that certain aspects and variations of the invention described herein include consisting or consisting essentially of aspects and variations. II. Chromatography Methods

[0083] In some aspects, the invention provides methods of analyzing compositions comprising a polypeptide and a nonionic surfactant (eg, polysorbate 20 or PS20), comprising binding the polypeptide and nonionic surfactant to a material Mixed mode ion exchange chromatography using a loading buffer and elution of the polypeptide and nonionic surfactant from the chromatography material using buffers such that the polypeptide and nonionic surfactant elute from the chromatography material in separate fractions. In some embodiments, chromatography methods are appropriate for compositions comprising multiple polypeptides (eg, polypeptide products), including variant pls polypeptides. For example, the methods can be used to test for compositions comprising a nonionic surfactant and a number of different antibody products, such as antibody products with pls ranging from 6.0 to 9.5. In other embodiments, chromatography methods include the use of optimal conditions (eg, chromatography material, buffers, gradients, step duration, flow rate, sample loading) identified by the methods described herein. .

[0084] In some embodiments of any of the methods described herein, the chromatography material is a mixed-mode material comprising functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, binding of hydrogen and hydrophobic interactions. In some embodiments, the mixed mode material is a mixed mode anion exchange chromatography material. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mode anion exchange chromatography material mixed is an Oasis® MAX chromatography material. In some embodiments, the mixed mode material is a mixed mode cation exchange chromatography material. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the mixed mode material comprises a solid support. In some embodiments, the mixed mode material is contained in a column or cartridge. In some embodiments of the foregoing, the mixed-mode material is a mixed-mode chromatography column or cartridge, such as a mixed-mode anion exchange chromatography column or cartridge or a mixed mode. In some embodiments, the mixed mode material is a high performance liquid chromatography (HPLC) material.

[0085] In some embodiments of any of the methods described herein, the ion exchange material may use a conventional chromatography material or a convective chromatography material. Conventional chromatography materials include, for example, perfusive materials (eg, poly(styrenedivinylbenzene) resin) and diffusive materials (eg, crosslinked agarose resin). In some forms of embodiment, the poly(styrene-divinylbenzene) resin may be Poros® resin. In some embodiments, the crosslinked agarose resin may be Sulfopropyl-Sepharose® Fast resin. The convective chromatography material can be a membrane material (eg, polyethersulfone) or a monolithic material (eg, crosslinked polymer). The polyethersulfone membrane can be Mustang. The crosslinked polymer monolithic material may be crosslinked poly(glycidyl methacrylate-co-ethylene dimethacrylate).

[0086] In some embodiments of any of the methods of the invention, the chromatography material is in a chromatography column or cartridge; for example, a mixed mode cation exchange chromatography column or cartridge a mixed mode anion exchange chromatography column or cartridge. In some embodiments, column or cartridge chromatography is used for liquid chromatography. In some embodiments, column or cartridge chromatography is used for high performance liquid chromatography (HPLC). In some embodiments, the chromatography column or cartridge is an HPLC chromatography column or cartridge; for example, a mixed mode cation exchange HPLC column or cartridge or a mixed mode anion exchange HPLC column or cartridge.

[0087] For example, in some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase A, wherein mobile phase A mobile phase B a comprises acid in water and mobile phase B elution of anion exchange polypeptide comprising acid in methanol; of the mixed mode chromatography material with a b) solution comprising a second mobile phase ratio A, where the second is the first ratio; ionic material c) elution of the mobile phase B to the is greater than that of the surfactant chromatography with a solution not comprising a third ratio of the mobile phase B to the mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the ionic surfactant in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material nonspecifically and about 90% nonspecifically and at least (such as at least about any of the 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in some embodiments, the eluate of step b). The phase In c) the polypeptide, not specifically some embodiments, comprises the ligation eluate. The phase In c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the embodiment, the first composition. In some forms the ratio of mobile phase B to mobile phase A is approximately 20:80 between approximately 0:100 as approximately (such any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 18:82, 14:86, 16:84 and these relations). In any range between including some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1 .3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, less 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of the stage a) and continues for at least about 1 (such as at least about any of 1,2, 1,4, 1.6, 1.8, 2, 2,2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after the end of the stage b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) the surfactant min. In some embodiments, the non-ionic is poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the composition concentration is from about protein in the mg / mL to about 250 mg / mL (such as approximately any of 2 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation appropriate for administering the subject's embodiment, a therapeutic polypeptide. polypeptide antibody antibody antibody antibody in some ways In some forms, it is a polypeptide embodiment, a polyclonal, humanized therapeutic, a fusion protein, a monoclonal antibody, a human antibody, a multispecific antibody, a chimeric antibody, a glycoengineer, an antibody fragment, a conjugate of antibody-drug, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. in some ways As an embodiment, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0088] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode anion exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises acid in water and the mobile phase B comprises acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant in the eluate of step c), wherein the quantification of the nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase is between about 0:100 and approximately 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, these relations). In including any range between some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3 .5%, 4% and 4.5%, including any range between these values) of acid in water. In some embodiments, the phase Mobile B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1.3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of the stage a) and continues for at least about 1 (such as at least about any of 1,2, 1,4, 1.6, 1.8, 2, 2,2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after the end of the stage b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the surfactant polysorbate. In nonionic is poloxamer (P188) or embodiment, some forms a polysorbate is some forms non-polysorbate 20 or embodiment, polysorbate 80 surfactant. the ionic concentration in the composition is from about 0.001% to 1.0% in the In the range approximately any of 0.005, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and (w / v) 0.01, 0.9%, (such 0.05, as 0.1, including any range between these values). in some ways of embodiment, the concentration of ] proteins in i composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, , 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, this values). In including any range among some embodiments, the composition also comprises the group that one or more excipients selected from consists of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the therapeutic polypeptide. In some forms it is an embodiment polypeptide, polyclonal therapeutic antibody polypeptide, is a fusion protein, monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific, glycoengineered antibody, an antibody fragment, a an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0089] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material of mixed mode anion exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises acid in water and the mobile phase B comprises acid in methanol; b) elution of the polypeptide from the anion exchange chromatography material of mixed mode with a solution comprising a second ratio of mobile phase B to mobile phase A, where the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantifying the nonionic surfactant in the eluate from step c), wherein the eluate comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1.3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least approximately any of 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, completed stage b) and 1.5, 2 or more) min after it continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1 ,2, 1,3, 1,4, 1.5, 2, 3 or more) min. In some embodiments, the nonionic surfactant is poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) ( such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 %, including any range between these values). In some embodiments, the protein concentration in the composition is about 1 mg / mL at approximately 250 mg / mL (such as approximately any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values ). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In In some embodiments, the composition also comprises one or more excipients selected from the group that composition agent is one to administer, consisting of a stabilizer, a buffer and a In some embodiments, the formulation tonicity. the a subject, therapeutic appropriate pharmaceutical polypeptide. polypeptide antibody antibody antibody In some polyclonal, humanized, chimeric therapeutics, a is a forms In some forms it is an embodiment polypeptide, a fusion protein, a monoclonal antibody, a human antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0090] In some embodiments, * provides a method for quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition chromatography in a solution ratio of a mobile phase B to is loaded in which comprises a mobile phase material of a first A, wherein mobile phase A comprises acetic acid in water and mobile phase B comprises acetic acid in methanol; b) elution of the polypeptide from the mixed-mode anion exchange chromatography material with a solution comprising a second mobile phase ratio A, wherein the second mobile phase ratio B a is greater than the first ratio; ionic material c) elution of the surfactant chromatography with a solution not comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant la in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least 91, 92, 93, about any 97, 98 or 99%) of the polypeptide is eluted in some embodiments, the eluate of 94, 95, 96, step b). The phase In c) the polypeptide, not specifically some embodiments, comprises the ligation eluate. The phase In c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the embodiment, the composition. In some forms of first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such and like roughly any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and these relations). In 18:82, any range between including some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3 .5%, 4% and 4.5%, including any range between these values) of acetic acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including acetic acid any range between these in methanol. In values) of embodiments, the flow rate of some chromatography is between approximately 0.5 2.5 (such as approximately any of 0.7, 0.9, 1.1, 1.2, 1.25, 1.3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least approximately any of 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, less than about 0.05 step c) begins at (such as at least about any 0.1, 0.2, 0.3, 0.4, 0.5, 1, completed stage b) and 0.06, 0.07, 0.08, 0.09, 1.5, 2 or more) min after it continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1 ,2, 1,3, 1,4, 1.5, 2, 3 or more) the surfactant min. In some nonionic forms it is poloxamer (P188) or, in embodiment, a polysorbate. In polysorbate it is some forms polysorbate 20 or embodiment, polysorbate 80. the In some embodiments, the concentration of nonionic surfactant in the composition is (w / v) from about 0.001% to about 1.0% any of 0.005, 0.01, in the (such 0.05, range as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range of realization, between these values). the concentration of In some forms protein in composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administering the embodiment, a subject. the polypeptide In some forms it is a therapeutic polypeptide. polypeptide In some embodiments, the antibody is a polyclonal, humanized, chimeric, therapeutic antibody, a fusion protein, a monoclonal antibody, a human antibody, a multispecific, glycoengineered antibody, an antibody fragment, a conjugate of antibody-drug, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a detector. aerosol charged (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0091] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode anion exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein mobile phase A comprises acid in water and mobile phase B comprises acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 35:65 and about 45:55; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between about 90:10 and about 100:0; and d) quantification of the nonionic surfactant in the eluate of step c). In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 40:60. In some embodiments, the third ratio is approximately 100:0. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide elutes in stage b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1 .3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40 and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least about any of 1.2 , 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the nonionic surfactant is poloxamer (P188) or a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) ( such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 %, including any range between these values). In some embodiments, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as approximately any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the formulation. composition is a pharmaceutical suitable for administering to a subject. In some embodiments, the therapeutic polypeptide. In some forms it is an embodiment polypeptide, polyclonal, humanized, polyclonal, humanized, is a polypeptide antibody, is a fusion protein, is a monoclonal antibody, is a human antibody, is a multispecific antibody, is a chimeric, is a glycoengineered, is a fragment of antibody. , an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is a Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0092] In some embodiments, a method of quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material in mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises acid in water and mobile phase B comprises acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide embodiments, in the composition. In first relation of some the mobile phase B to the mobile phase is between approximately 0:100 and approximately 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, these relations). In including any range between some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3 .5%, 4% and 4.5%, including any range between these values) of acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1.3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as about any of 0.6, 0.7, 0.8, at least 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of the stage a) and continues for at least about 1 (such as at least about any of 1,2, 1,4, 1.6, 1.8, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as < approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is about 1 mg / mL at approximately 250 mg / mL (such as approximately any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values ). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the formulation. composition is a pharmaceutical appropriate to administer the subject, therapeutic polypeptide, polyclonal antibody polypeptide antibody antibody antibody polyclonal polypeptide, in it's ways In some forms, it is an embodiment, a humanized polypeptide, a fusion protein, a monoclonal antibody, a human antibody, a multispecific antibody, a chimeric antibody, a glycoengineer, an antibody fragment, an antibody-drug conjugate, a THIOMAB™ or ΤΗΙΟΜΆΒ™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the chromatography material mixed mode anion exchange comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0093] In some embodiments, a method of quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material in mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises acid in water and mobile phase B comprises acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of step c), wherein the quantification of the nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%). %, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1.3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least approximately any of 1,2, 1,4, 1.6, 1.8, 2, 2,2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after the end of the stage b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1,1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is from about mg / mL to about 250 mg / mL (such as approximately any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about to about 7.5 (such as any 4.6, 4.5 as 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, including any range between 4.8, 5.0, 5.2, 6.8, 7.0, 7.2, 5,4, 7.4, these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent, composition is one to manage performance, the In some embodiments, the therapeutic formulation. polypeptide antibody antibody antibody antibody In appropriate pharmaceutical forms In some forms, it is a polypeptide embodiment, it is a fusion protein, a monoclonal antibody, a human antibody, a multispecific antibody, a subject. polyclonal, humanized, chimeric, a glycoengineer, an antibody fragment, an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate polypeptide some therapeutic. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0094] In some embodiments, a method is provided for quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode anion exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises acid in water and mobile phase B comprises acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantifying the polysorbate in the eluate from step c), wherein the eluate comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4% , 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some forms of realization, the third relationship is between approximately approximately 80:20 and about 100:0 (such as any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase approximately A comprises between 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, range between these values) 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any of the acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 9O f 2.5%, 3%, 3.5%, 4% 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1,3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the stage b) starts at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 , 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least approximately any of 1,2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is about 1 mg / mL at about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values ). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is an appropriate pharmaceutical formulation to administer realization, the to a subject, polypeptide In some forms of it is a therapeutic polypeptide, polypeptide antibody antibody antibody antibody antibody In some embodiments, the chimeric, humanized, polyclonal therapeutic, a fusion protein, a monoclonal antibody, a human antibody, a glycoengineered, multispecific antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0095] In some embodiments, a method is provided for quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises acetic acid in water and mobile phase B comprises acetic acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 30:70 and about 50:50 (such as about any of 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54 and 48:52, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of acetic acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of acetic acid in methanol. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1 .3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some forms of embodiment, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least approximately any of 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these realization, the values). In some forms composition has a pH of about about 4.5 5.6, 5.8, 6.0, to about 7.5 (such as any 4.6, 4.8, 5.0, 5.2, like 5,4, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also consists of one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the therapeutic polypeptide. In some forms it is an embodiment polypeptide, polyclonal therapeutic antibody polypeptide, is a fusion protein, monoclonal antibody, chimeric, humanized antibody, human antibody, multispecific antibody, glycoengineered antibody, antibody fragment , an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0096] In some embodiments, a method of quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material in mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein the mobile phase A comprises acid in water and mobile phase B comprises acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 35:65 and about 45:55; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between approximately 90:10 and approximately 100:0; and d) quantification of the polysorbate in the eluate of step c). In some In embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 40:60. In some embodiments, the third ratio is approximately 100:0. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acid in methanol. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1 .3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least about any of 1.2 , 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and an agent. In some embodiments, tonicity. composition is one to administer pharmaceutical formulation appropriate performance, the a subject, polypeptide In some forms it is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a fusion protein, a polyclonal, humanized, chimeric antibody, a monoclonal antibody, a human antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, a conjugate antibody-drug, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the material Mixed Mode Anion Exchange Chromatography is a High Performance Liquid Chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0097] In some embodiments, a method of quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material in mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein the mobile phase A comprises acetic acid in water and mobile phase B comprises acetic acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 35:65 and about 45:55; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between approximately 90:10 and approximately 100:0; and d) quantification of the polysorbate in the eluate of step c), wherein the quantification of the nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%). %, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference. In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 40:60. In some embodiments, the third ratio is approximately 100:0. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or, *7 9· Όj / o r or less) of the total polypeptide in the composition. In some mobile phase A comprises between about 5% (v / v) (such as about any of the 0.75% embodiments, the about 0.5% and 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acetic acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acetic acid in methanol. In some embodiments, the flow rate of the and 2.5 (such chromatography is between about 0.5 and about any of 0.7, 0.9, 1.1, 1.2, any 1.25, 1.3, 1.5, 1.7, 1.9, 2.1, and 2.3, including range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about and about any about 50 (such as from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the stage b) starts at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) step a) and continues for minus about 1 min after starting (such as at least about any of 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some forms it starts at least approximately as stage c) 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, after completion of stage minus approximately 0.5 completion, any one of 0.5, 1, 1.5, b) and continues (such as 0.05 (such 0.06, 0.07, or more) min for at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 80. In some polysorbate 20 embodiments, the concentration of polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). in some ways embodiment, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a formulation for administering the embodiment to a subject. therapeutic polypeptide. polypeptide antibody antibody antibody antibody some therapeutic polyclonal, humanized, In is an appropriate dosage form In some forms is an embodiment polypeptide, a fusion protein, a monoclonal antibody, a human antibody, a multispecific antibody, a chimeric antibody, a glycoengineer, an antibody fragment, an antibody conjugate -drug, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mode anion exchange chromatography material mixed comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0098] In some embodiments, a method of quantifying a polysorbate in a composition comprising the polysorbate and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material in mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein the mobile phase A comprises acetic acid in water and mobile phase B comprises acetic acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 35:65 and about 45:55; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between approximately 90:10 and approximately 100:0; and d) quantification of the polysorbate in the eluate of the step c), wherein the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 40:60. In some embodiments, the third ratio is approximately 100:0. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of acetic acid in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of acetic acid in methanol. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1 .3, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least approximately any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 0.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 1 (such as at least about any of 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the polysorbate is polysorbate 20, or polysorbate 80. concentration. In that of polysorbate in composition it is in the range of about the 0.001% to 1.0% (w / v) (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises group that composition agent is a to administer performance, one or several excipients selected from consisting of a stabilizer, a buffer and a In some embodiments, the tonicity formulation. the one subject, therapeutic polypeptide. polypeptide antibody antibody antibody antibody In some therapeutic polyclonal, humanized, appropriate dosage forms In some forms, the embodiment is a polypeptide, a fusion protein, a monoclonal antibody, a human antibody, a multispecific antibody, a chimeric antibody, a glycoengineer, an antibody fragment, an antibody-drug conjugate, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some forms of embodiment, the quantification of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0099] In some embodiments, a method for quantifying polysorbate 20 in a composition comprising polysorbate 20 and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to an anion exchange chromatography material mixed mode, wherein the composition is loaded onto the chromatography material in a solution comprising about a 10:90 ratio of mobile phase B to mobile phase A, wherein mobile phase A comprises about 2% acid acetic acid in water and mobile phase B comprises approximately 2% acetic acid in methanol; b) elution of the polypeptide from the mixed mode anion exchange chromatography material with a solution comprising a ratio of approximately 40:60 of mobile phase B to mobile phase A; c) elution of the polysorbate 20 from the chromatography material with a solution comprising a ratio of approximately 100:0 of ​​mobile phase B to mobile phase A; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.2, 1.25, 1.3, 1.5, 1.7, 1.9, 2.1 and any range between these values) 2.3, including mL / minute. In some embodiments, the chromatography flow rate is approximately 1.25 mL / min. In some embodiments, the volume of composition applied between about i and about any to about 50 chromatography material (such is as of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the volume of the composition applied to the chromatography material is approximately 20 pL. In some embodiments, it starts at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1,1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of the stage a) and continues for at least about 1 (such as at least about any of 1,2, 1,4, 1.6, 1.8, 2, 2,2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, more) min. In some embodiments, step c) begins at least about 0.05 about any (such as at least from 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1,1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more) min. In some embodiments, the concentration of polysorbate 20 in the composition is (w / v) in the range of about 0.001% to 1.0% (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In In some embodiments, the protein concentration in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140 , 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administering the embodiment to a subject. therapeutic polypeptide. polypeptide antibody antibody antibody antibody in some ways In some forms of a polypeptide embodiment, it is polyclonal, humanized therapeutic, a fusion protein, monoclonal antibody, a human antibody, a multispecific, glycoengineered antibody, an antibody fragment, chimeric, a conjugate of antibody-drug, a THIOMAB™ or THIOMAB™ drug conjugate. In some embodiments, the mixed mode anion exchange chromatography material comprises a reverse phase strong anion exchange polymer. In some embodiments, the mixed mode anion exchange chromatography material comprises a quaternary amine moiety. In some embodiments, the mixed mode anion exchange chromatography material comprises a solid support. In some embodiments, the mixed mode anion exchange chromatography material is contained in a column or cartridge. In some embodiments, the mixed mode anion exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode anion exchange chromatography material is an Oasis® MAX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0100] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant in the eluate. of stage o). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84 and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about 52:48 and 54:46, including any range 48:52, 50:50, between these relationships). In some embodiments, the third relationship is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 % and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (eg, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of the nonionic surfactant (eg, polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, acetyltryptophan and / or tryptophan) embodiment, composition composition also comprises N(also referred to as N-acetyl-DLla methionine. In some forms the concentration of N-acetyltryptophan at about 0.1 mM varies from about 10 mM (such as approximately any of 0.2, 0.5, one, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the methionine concentration in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 or 90 mM, including any range between performance, these values). concentration In composition it is approximately 250 some polypeptide forms in about i mg / mL (such as about any 2 mg / mL, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these performance, values). In some forms the composition has a PH of approx. 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises the group that one or more excipients selected from consists of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some forms © For embodiment, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0101] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant in the eluate of step c), wherein the quantification of the nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of the 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (for example, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1 and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) starts at j least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 , 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2, 2, 4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of the nonionic surfactant (eg, polysorbate or is in the poloxamer range) in the composition about 0.001% to 1.0% about any of 0.005 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 z any range these values). Come in 0.01, 0.05, 0.1, (w / v) (such as 0.9%, including In some embodiments, the composition also comprises N-acetyltryptophan and / or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 or 9 mM, 100 including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as any 0.2, 0.5, 1, 2, 3, 4, such as about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the composition concentration is approximately any 2, 250 polypeptide at approximately i mg / mL (such as 5, 10, twenty, 40, 60, 80, the mg / mL approximately 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some compositions it has a pH of approximately approximately 4.5 5.6, 5.8, 6.0, to about 7.5 (such as any 4.6, 6.2, 6.4, 6.6, like 4.8, 5.0, 5.2, 5,4, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the therapeutic polypeptide. In some forms it is an embodiment polypeptide, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate , a THIOMAB™, a THIOMAB™ drug conjugate. In some embodiments, the chromatography material 101 mixed mode cation exchange comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0102] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second 102 ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantifying the nonionic surfactant in the eluate from step c), wherein the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) 103 (such as approximately any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values ) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (eg, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min . In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such 104 as at least approximately any of 2,2, 2,4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of the nonionic surfactant (eg, polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range of realization, between In some forms composition also comprises and / or these values). Nmethionine. In some forms of acetyltryptophan embodiment, the concentration of N-acetyltryptophan in composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, one, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values), some embodiments, the concentration The composition of methionine varies from about mM to about 100 mM (such as any of 0.2, 0.5, 1, 2, 3, 4, 0.1 as approximately 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the approximately mg / mL composition is approximately 250 mg / mL (such as any of 2, 5, 10, 20, 40, 60, 160, 180, 200, 220, and 240 mg / mL, like , 80, approximately 100, 120, 140, including any 105 range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material 106 high performance liquid (HPLC). In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0103] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in methanol; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant in the eluate of 107 stage c). In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically at least about the same. 90% (such as at least approximately any of the 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from the step c) comprises the polypeptide not specifically bound. In some embodiments, the eluate from the step c) comprises less than about 10% (such as less than about any of 9%, 89-7 Q Z Q. o i o f O Ό f R 9- Λ 9□ or ¡ 4: Ό 3%, 9- 19Z o f -Lo f 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2.5%, O 9J Ό¡ 3.5%, 4% and 4.5%, including any 108 range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in methanol. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the surfactant does not 109 ionic is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. ionic (for In some embodiments, the concentration of the non-example surfactant, polysorbate or poloxamer) in about 0.001% composition to about any of the 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, any range between realization, the and / or is in the range of 1.0% (w / v) (such as 0.005, 0.8 and these values). 0.01, 0.05, 0.1, 0.9%, including In some composition forms it also comprises methionine. In some forms of acetyltryptophan embodiment, the concentration of N-acetyltryptophan in composition varies from approximately 0.1 mM N of about 10 mM (such as about any of 0.2, 0.5, one, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the methionine concentration in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is from about mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140 , 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as 110 approximately any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material 111 Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0104] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in acetonitrile; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least 112 about 90% (such as about any of 91, 92, 93, at least 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate comprises the non-specifically bound polypeptide. c) In some embodiments, the eluate comprises less than about 10% of the step c) (such as less than about any of 9%, C 9- / 19- 9 9- 99- 19J Ό f ΤΌ / O Ό f Z Ό / X Ό f or less) of the total polypeptide in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase approximately A comprises between 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 % and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, the mobile phase B comprises between about 0.5% and about 113 5% (v / v) (such as approximately any of the 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in acetonitrile . In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the 114 nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of the nonionic surfactant (eg, polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the composition also comprises N-acetyltryptophan and / or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, including any range between these values). one, 2, 3, 4, 5, 6, 7, 8 or 9 mM, In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is about mg / mL about 250 mg / mL (such as approximately any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In 115 In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a polypeptide; some forms therapeutic protein is a therapeutic antibody. In embodiment, polyclonal, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™, a THIOMAB drug conjugate ™. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a detector charged with 116 aerosol (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0105] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide is provided, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 40:60 and about 50:50; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between about 90:10 and about 100:0; and d) quantification of the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in the 117 stage b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, the mobile phase B comprises between about 0.5% and about 118 5% (v / v) (such as approximately any of the 0.75%, 1%, 1.5%, 9 what / 2.5%, 3.5%, 4% and 4.5%, including any range between these ammonium hydroxide in the organic solvent methanol or acetonitrile). In some embodiments, the flow rate of values) of (for example, forms of chromatography is between about 0.5 2.5 (such as approximately any of 0.7, 0.9, 1,1, 1,3, 1,4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3,2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the step c) starts at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after the end of the stage b) and continues for at least about (such as at least about any of 2.2 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the nonionic surfactant is a polysorbate. In embodiment, the polysorbate is some polysorbate 20 forms of 119 polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of the nonionic surfactant (eg, polysorbate or poloxamer) in the composition from about 0.001% to about any of the 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, any range between performance, acetyltryptophan and / or is in the range of 1.0% (w / v) (such as 0.005, 0.8 and these values). 0.01, 0.05, 0.1, 0.9%, including In some composition forms it also comprises methionine. In some embodiments, the concentration of N-acetyltryptophan in composition ranges from about 0.1 mM N of about 10 mM (such as about any of 0.2, 0.5, one, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the concentration of methionine in the composition ranges from about 100 mM to about 100 mM (such as any of 0.2, 0.5, 1, 2, 3, 4, 0.1 as approximately 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of the composition is approximately 250 of about i mg / mL (such as polypeptide in mg / mL about any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some compositions it has a pH of approximately 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 120 including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by light scattering. 121 evaporative (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic surfactant comprises less than about 10% (such as less than about any of the 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.05%, 0.01% or less) interference from the polypeptide.

[0106] When a product containing N-acetyltryptophan (NAT) in the formulation is tested using conditions of HPLC-ELSD, there is significant interference observed in the region of the PS20. Thus, under certain circumstances, alternative conditions are needed to eliminate both NAT and protein related interference.

[0107] In some embodiments, a method of quantifying a nonionic surfactant in a composition comprising the nonionic surfactant, a polypeptide, and N-acetyltryptophan is provided, wherein the method comprises the steps of a) applying the composition to a material of mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the nonionic surfactant from the chromatography material with a solution comprising a third ratio of the mobile phase B to the 122 mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the nonionic surfactant in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from the step c) comprises the polypeptide not specifically bound. In some embodiments, the eluate from the step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 59- / 19- Q Q. 0 9- 19o f o r □ Ό f Z Ό f X ό , 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any 4%, Or g. ηg. i g O or j or ¡ X or r 0.5%, 0.1%, 0.05%, 0.01% or less) of the Full NAT in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for the mobile phase is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase is between about 0:100 and about 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48 and 54:46, including any range 123 between these relationships). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 9Z or f 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (eg, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at 124 least about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the nonionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is a poloxamer. In some embodiments, the poloxamer is P188 poloxamer. In some embodiments, the concentration of the nonionic surfactant (eg, polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 125 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, polypeptide in the composition is mg / mL at approximately the concentration of 250 of approximately mg / mL (such as approximately any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some forms composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between some embodiments, the group comprising these values). In composition also one or several excipients selected from consists of a stabilizer, a buffer and a In some embodiments, tonicity. composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the therapeutic polypeptide. In some forms it is an embodiment polypeptide, the therapeutic protein is a polyclonal antibody, monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, antibody, glycoengineered conjugate, antibody-drug fragment , a THIOMAB™, a conjugate of embodiments, the drug THIOMAB™. In some mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. in some ways 126 For embodiment, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the amount of nonionic detergent comprises less than about 10% (such as less than about 1 any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0108] In some embodiments, sef provides a method for quantifying a polysorbate in a composition comprising the polysorbate and a* polypeptide, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second 127 ratio of mobile phase B to mobile phase A, where the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some forms of 128 embodiment, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 % and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (eg, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such like at least 129 about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 ,3, 0.4, 0.5, 1, 1.5, 2 or more) min after the end of the stage b) and continues for at least about 2 (such as at least about any of 2,2, 2,4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the composition also comprises N-acetyltryptophan and / or methionine. In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the methionine concentration in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the polypeptide in the composition is the concentration of from about mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 130 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the exchange chromatography material 131 Mixed-mode cationic is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about (such as less than about any of 9%, 8%, 7%, 6%, 5%, 2%, Ί91 or r 0.5%, 0.1%, 0.05%, 0.01% or less) interference from the polypeptide.

[0109] In some embodiments, a method of quantifying a polysorbate in a composition comprising the polysorbate is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein The composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) 132 quantification of polysorbate in the eluate of the step c), wherein the quantification of the polysorbate comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% , 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide and NAT interference. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any of 4%, O 9- 99. 19DOf h o f _L o / 0.5%, 0.1%, 0.05%, 0.01% or less) of the Full NAT in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for the mobile phase is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase is between about 0:100 and about 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, including any range between these ratios). In some embodiments, the second ratio is between approximately 35:65 and 133 approximately 55:45 (such as approximately any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 % and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (eg, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least 134 approximately any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 0.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the methionine concentration in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some forms of 135 embodiment, the concentration of polypeptide in the composition is from about 1 mg / mL to about 250 mg / mL (such as about any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises the group that one or more excipients selected from consists of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a support 136 solid. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD) ·

[0110] In some embodiments, a method is provided for quantifying a polysorbate in a composition comprising polysorbate, a polypeptide and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of the step 137 c), wherein the eluate from step c) comprises less than about the 10% (such as less than about any of 9%, 89 7 9ό t l ό f 6%, 5%, 4%, 9- 1 9Z Ό -Lo ¡ 0.5%, 0.1%, 0.05%, less) of the total polypeptide in the composition and less than about the 5% (such as less than about any of 4%, 3%, 2%, 91 or , 0.5%, 0.1%, 0.05%, 0.01% or less) of Full NAT in the composition. In some embodiments, the organic solvent in mobile phase B is methanol. In some embodiments, the organic solvent for the mobile phase is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase is between about 0:100 and about 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48 and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and approximately the 5% (v / v) (such as about any of 0.75%, 1%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in 138 Water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of the 0.75%, 1%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (for example, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is 139 polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values) In some embodiments, the composition also comprises methionine. embodiments, the concentration of In some methionine in composition varies from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the composition concentration is about polypeptide in mg / mL to about 250 mg / mL (such as approximately any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a 140 tonicity agent. composition is a In some embodiments, the appropriate pharmaceutical formulation to administer to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about the 10% (such as less than 141 approximately any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of interference from the polypeptide and the NAT.

[0111] In some embodiments, a method is provided for quantifying a polysorbate in a composition comprising the polysorbate, a polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in methanol; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as 142 less than approximately any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as any 4%, 0.1%, 0.05%, 0.01% or less) of at least 9- 9 9- 1 9J Ό f ¿Oj ± O f of 0.5%, Full NAT in the composition. In some ways of relating the mobile phase B to the embodiment, the first mobile phase A is between about 0:100 and approximately 20:80 (such as approximately any of 2:98, 4:96, 6:94, 8:92, 10:90, 12:88, 14:86, 16:84 and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48, and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 % and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of the 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of 143 ammonium hydroxide in methanol. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). 144 In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 or 90 mM, including any range between performance, these values). concentration In composition it is approximately i mg / mL some forms polypeptide in mg / mL approximately of the 250 (such as any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6 , 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a 145 monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of the polysorbate comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide and NAT interference.

[0112] In some embodiments, a method is provided for quantifying a polysorbate in a composition comprising the polysorbate, a 146 polypeptide and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in acetonitrile; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A, wherein the second ratio is greater than the first ratio; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A, wherein the third ratio is greater than the second ratio; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any of 4%, 3%, 2%, 1%, 0.5%, 147 0.1%, 0.05%, 0.01% or less) of the total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is between about 0:100 and about 20:80 (such as about any of 2:98, 4:96, 6:94, 8: 92, 10:90, 12:88, 14:86, 16:84, and 18:82, including any range between these ratios). In some embodiments, the second ratio is between about 35:65 and about 55:45 (such as about any of 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, 50:50, 52:48, and 54:46, including any range between these ratios). In some embodiments, the third ratio is between about 80:20 and about 100:0 (such as about any of 82:18, 84:16, 86:14, 88:12, 90:10, 92:8, 94:6, 96:4 and 98:2, including any range between these values). In some embodiments, the mobile phase A comprises between approximately 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 % and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of the 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in acetonitrile . In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some forms of 148 embodiment, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the 149 composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the composition concentration is approximately 250 of about ' mg / mL polypeptide in mg / mL (such as about any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some forms, a therapeutic protein is a monoclonal antibody, a polyclonal, humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the chromatography material 150 Mixed mode cation exchange comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about 10% (such as less than about any of 9%, , 8%, 7 g c g c: g / o , Ό Ό , □ Ό , 4%, No. 9 5- 15- «jOf Ζο^ X o f 0.5%, 0.1% , 0.05%, -0.01% or less) interference from polypeptide and NAT.

[0113] In some embodiments, provides a method for quantifying a polysorbate in a composition comprising the polysorbate, a polypeptide and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase 151 A between about 5:95 and about 15:85, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in an organic solvent; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 40:60 and about 50:50; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between approximately 90:10 and approximately 100:0; and d) quantification of the polysorbate in the eluate of step c). In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the Full NAT in the composition. In some embodiments, the organic solvent for mobile phase B is methanol. In some embodiments, the organic solvent for mobile phase B is acetonitrile. In some forms of 152 embodiment, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 45:55. In some embodiments, the third ratio is approximately 100:0. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in the organic solvent (eg, methanol or acetonitrile). In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, and 45, including any range between these values) pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such like at least 153 about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step or) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is about mg / mL about 250 mg / mL (such as approximately any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 154 160, 180, 200, 220, and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about to about 7.5 (such as any 4.6, 4.5 as 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, including any range between some embodiments, the composition also one or more excipients selected from the comprises 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, these values) 7.4, In a group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation appropriate for administering the embodiment, the a subject, polypeptide In some forms of it is a polypeptide, some forms therapeutic protein is a therapeutic antibody. In embodiment, polyclonal, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™, a THIOMAB drug conjugate ™. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the exchange chromatography material 155 Mixed-mode cationic is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the polysorbate quantification comprises less than about 10% (such as less than about any 7 9 Όf / 'ot 6%, 5%, Or 9 99 19. D o f Δ o / 1 o f 0.5%, 0.1%, 0.05%, 0.01% or less) interference from polypeptide and NAT.

[0114] In some embodiments, a method is provided for quantifying a composition comprising a polysorbate polysorbate, in a polypeptide and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to an exchange chromatography material mixed mode cationic, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in methanol; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 40:60 and about 50:50; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase A between approximately 90:10 and 156 about 100:0; and d) quantification of the polysorbate in the eluate of step c), wherein the quantification of the polysorbate comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide and NAT interference. In some embodiments, the polypeptide binds to the chromatography material specifically and non-specifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98 or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate comprises the non-specifically bound polypeptide. c) In some embodiments, the eluate comprises less than about 10% of the step c) (such as less than about any of 9%, 8%, 7%, 6%, R9- 79- 9 9- 19ü o f nt o r 3 o f o r Ιό ¡ 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any 4%, 39- 0 9- 19Ό f Δ O f X Ό f 0.5%, 0.1%, 0.05%, 0.01% or less of Full NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 45:55. In some embodiments, the third ratio is approximately 100:0. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of 157 ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2.5% I O or, or f 3 or f 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in methanol. In some embodiments, the chromatography flow rate is between about 0.5 And 2.5 (such as approximately any of 0.7, 0.9, 1,1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1 and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the step b) starts at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 ( such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5 , 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or 158 polysorbate 80. In some embodiments, the concentration of polysorbate in the composition ranges from about 0.001% (w / v) to 1.0% (such as about any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is to mg / mL of approximately approximately 250 mg / mL (such as approximately any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a 159 In some embodiments, the tonicity agent formulation, composition is a I appropriate pharmaceutical to administer realization, the one subject. therapeutic polypeptide. in some ways In some forms of a polypeptide embodiment, a therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-antibody conjugate. drug, a THIOMAB™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0115] In some embodiments, a method is provided for quantifying a polysorbate in 160 I a composition comprising the polysorbate, a polypeptide and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the material of chromatography in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises hydroxide ammonium in methanol; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 40:60 and about 50:50; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase At between about 90:10 and about 100:0; and d) quantifying the polysorbate in the eluate from step c), wherein the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition and less than about 5% (such as less than approximately any of 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 45:55. In some embodiments, the third ratio is approximately 100:0. In some embodiments, 161 mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3 %, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of ammonium hydroxide in methanol. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the step b) starts at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 , 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2 , 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at 162 least about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the approximately composition range is in (w / v) 0.001% to 1.0% (such as approximately any of the 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the composition concentration is approximately 250 of about mg / mL polypeptide in mg / mL (such as about any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 163 including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is an Oasis® MCX chromatography material. In some embodiments, the nonionic detergent is quantified by light scattering. 164 evaporative (ELSD) or using an aerosol (CAD). In some embodiments, charged detector forms of the polysorbate quantification comprise less than about any 10% or so 30. no. not. Ό f Ό f X Ό / 0.5%, 0.1%, (such as 9%, 0.05%, as less than 8%, 7%, 6%, 5%, 4%, 0.01% or less) interference from the polypeptide.

[0116] In some forms of provides a method for quantifying performance, a polysorbate is in a composition comprising the polysorbate, a polypeptide and N-acetyltryptophan, where the method comprises the steps of a) applying the composition to an exchange chromatography material mixed mode cationic, wherein the composition is loaded onto the chromatography material in a solution comprising a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein the mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in acetonitrile; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 40:60 and about 50:50; c) elution of the polysorbate from the chromatography material with a solution comprising a third ratio of mobile phase B to mobile phase between approximately 90:10 approximately 100:0; and d) quantification of the polysorbate in the eluate of step c), wherein the quantification of the polysorbate comprises less than approximately the 10% (such as less than approximately any 8%, 7%, 6%, 5%, 4%, 39- 9 9 19O , O and i or f 0.5%, 0.1%, 0.01% or less) of 165 polypeptide interference and NAT. In some embodiments, the polypeptide binds to the chromatography material specifically and nonspecifically and at least about 90% (such as at least about any of 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluate from step c) comprises the non-specifically bound polypeptide. In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any 4%, 9- 7 9- 19J O f O / X Ό f 0.5%, 0.1%, 0.05%, 0.01% or less) of the Full NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 45:55. In some embodiments, the third ratio is approximately 100:0. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in 166 of acetonitrile flow. In some embodiments, the chromatography rate is between about 0.5 and 2.5 (such as approximately any of 0.7, 0.9, 1.1, any 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about about and about to about 50 (such as from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the stage b) starts at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) step a) and continues for at least 1, 1.1, 1.2, 1.3, 1.4, min after onset minus about 2 (such as at least about any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the composition is in the range of about 0.001% to 1.0% (w / v) (such as approximately any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about either about 0.2, 5, 6, 7, 8, or 9 mM, including any 0.5, 1, 2, 3, 4, range between these values). In some composition forms it also comprises methionine. realization, the In some embodiments, the concentration of methionine in the composition ranges from about 100 mM to about 0.1 mM to (such as approximately any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In embodiment, the concentration of some polypeptide forms in the composition is approximately 250 of approximately i mg / mL (such as mg / mL of approximately any 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the approximately 4.5 composition has a to approximately approximately any of 4.6, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, pH 4.8, 6.8, 7.5 (such as 5.0, 5.2, 5.4, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer and a In some embodiments, tonicity. composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In therapeutic protein is a polyclonal antibody, some embodiments, the one 168 monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD).

[0117] In some embodiments, a method for quantifying a polysorbate in a composition comprising the polysorbate, a polypeptide, and N-acetyltryptophan is provided, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising 169 a first ratio of a mobile phase B to a mobile phase A between about 5:95 and about 15:85, wherein mobile phase A comprises ammonium hydroxide in water and mobile phase B comprises ammonium hydroxide in acetonitrile; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising a second ratio of mobile phase B to mobile phase A between about 40:60 and about 50:50; c) elution of the polysorbate from the material comprises a chromatography with a solution that third ratio of the mobile phase B to the mobile phase At between approximately 90:10 approximately 100:0; and d) quantifying the polysorbate in the eluate from step c), wherein the eluate from step c) comprises less than about 10% (such as less than about any 9%, 8%, 9- £ 9- R 9 / O f Ό o f □ 'O f 4%, 0.5%, 0.1%, 0.01% or less) of the total polypeptide in the composition and less than about 5% (such as less than about any 4%, either. not. ίο. or f Z ό ¡ ± Ό f 0.5%, 0.1%, 0.05%, 0.01% or less) of the total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is approximately 10:90. In some embodiments, the second ratio is approximately 45:55. In some embodiments, the third ratio is approximately 100:0. In some embodiments, mobile phase A comprises between about 0.5% and about 5% (v / v) (such as about any of 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% and 4.5%, including any range between these values) of ammonium hydroxide in water. In some embodiments, mobile phase B comprises between 170 about 0.5% and about 5% (v / v) any of 0.75%, 1%, (such as about 1.5%, 2.5%, 3.5%, 4% and 4.5%, including any ammonium hydroxide in the range between these values) acetonitrile. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as approximately any of 0.7, 0.9, 1.1, any 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including range between these values) mL / minute. In some embodiments, the volume of the composition applied to the chromatography material is between about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) realization, the pL stage. In some forms of b) begins at least approximately 0.5 (such as at least approximately any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least about any of 2.2 , 2.4, 2.6, 2,8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, one, 1.5, 2 or more) min after completion of step b) less approximately 2 (such continues for at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, or more) min. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the polysorbate in the embodiment, the composition is in 171 the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 5, 6, 7, 8, or 9 mM, including any 0.5, 1, 2, 3, 4, range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2. 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is about mg / mL about 250 mg / mL (such as approximately any of 2 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5,4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is an appropriate pharmaceutical formulation 172 to administer to a subject. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB ™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about the 10% (such as less than approximately any of 9%, 8%, 7%, 173 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of polypeptide interference.

[0118] In some embodiments, a method is provided for quantitating polysorbate 20 in a composition comprising polysorbate 20, a polypeptide, and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a material mixed mode cation exchange chromatography, wherein the composition is loaded onto the chromatography material in a solution comprising about a 10:90 ratio of mobile phase B to mobile phase A, wherein mobile phase A comprises about 1.5% ammonium hydroxide in water and mobile phase B comprises approximately 1.5% ammonium hydroxide in methanol; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising an approximately 45:55 ratio of mobile phase B to mobile phase A; c) elution of the polysorbate 20 from the chromatography material with a solution comprising a ratio of approximately 100:0 of ​​mobile phase B to mobile phase A; and d) quantification of polysorbate 20 in the eluate of step c). In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, no, 9 9 19O o f Δ o / _L o 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any 4%, 3%, 99- 19 ZΌf 0.5%, 0.1%, 0.05%, 0.01% or less) of the Full NAT in the composition. In some embodiments, the rate of 174 chromatography flux is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1.5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In some embodiments, the chromatography flow rate is approximately 1.40 mL / min. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the volume of the composition applied to the chromatography material is approximately 25 pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0 ,one, 175 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the methionine concentration in the composition ranges from about 0.1 mM to about 100 mM such as about any of 0.2, 0.5, 1, 2, (such 3. 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is from about mg / mL to about 250 mg / mL (such as approximately any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises the group that one or more excipients selected from consists of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the 176 therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, an antibody-drug conjugate, a THIOMAB™, a THIOMAB drug conjugate ™. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about the 10% (such as less than about any of 9%, 9 9Όf / Όf 6%, 5%, 4%, 9- 9 Q. 1 Q. O f 4.O f J-'Of 0.5%, 0.1%, 0.05%, 0.01% or less) interference from polypeptide and NAT.

[0119] In some embodiments, a method is provided to quantitate polysorbate 20 in 177 a composition comprising polysorbate 20, a polypeptide and N-acetyltryptophan, wherein the method comprises the steps of a) applying the composition to a mixed mode cation exchange chromatography material, wherein the composition is loaded onto the material chromatography on a solution comprising about a 10:90 ratio of mobile phase B to mobile phase A, wherein mobile phase A comprises about 1.5% ammonium hydroxide in water and mobile phase B comprises about 1.5% 0.5% ammonium hydroxide in acetonitrile; b) elution of the polypeptide from the mixed mode cation exchange chromatography material with a solution comprising an approximately 45:55 ratio of mobile phase B to mobile phase A; c) elution of the polysorbate 20 from the chromatography material with a solution comprising a ratio of approximately 100:0 of ​​mobile phase B to mobile phase A; and d) quantification of polysorbate 20 in the eluate of step c). In some embodiments, the eluate from step c) comprises less than about 10% (such as less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluate from step c) comprises less than about 5% (such as less than about any 4%, ng. no. i o. O O ¿ O f ± O f 0.5%, 0.1%, 0.05%, 0.01% or less) of the Full NAT in the composition. In some embodiments, the chromatography flow rate is between about 0.5 and 2.5 (such as about any of 0.7, 0.9, 1.1, 1.3, 1.4, 1 .5, 1.7, 1.9, 2.1, and 2.3, including any range between these values) mL / minute. In 178 In some embodiments, the chromatography flow rate is approximately 1.40 mL / min. In some embodiments, the volume of the composition applied to the chromatography material is between about 1 and about 50 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, and 45, including any range between these values) pL. In some embodiments, the volume of the composition applied to the chromatography material is approximately 25 pL. In some embodiments, step b) begins at least about 0.5 (such as at least about any of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more) min after the start of step a) and continues for at least about 2 (such as at least approximately any of 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, step c) begins at least about 0.05 (such as at least about any of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0 0.3, 0.4, 0.5, 1, 1.5, 2 or more) min after completion of step b) and continues for at least about 2 (such as at least about any of 2, 2, 2, 4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 or more) min. In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w / v) (such as about any of 0.005, 0.01, 0.05, 0 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9%, including any range between these values). In some embodiments, the concentration of N-acetyltryptophan in the composition ranges from about 0.1 mM to 179 about 10 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, or 9 mM, including any range between these values). In some embodiments, the composition also comprises methionine. In some embodiments, the methionine concentration in the composition ranges from about 0.1 mM to about 100 mM (such as about any of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90 mM, including any range between these values). In some embodiments, the concentration of polypeptide in the composition is from about 1 mg / mL to about 250 mg / mL (such as approximately any of 2, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220 and 240 mg / mL, including any range between these values). In some embodiments, the composition has a pH of from about 4.5 to about 7.5 (such as about any of 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, including any range between these values). In some embodiments, the composition also comprises one or more excipients selected from the group consisting of a stabilizer, a buffer, and a tonicity agent. In some embodiments, the composition is a formulation to be administered to a subject. appropriate pharmaceutical In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the therapeutic protein is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, a glycoengineered antibody, a fragment of 180 antibody, an antibody-drug conjugate, a THIOMAB™, a THIOMAB™ drug conjugate. In some embodiments, the mixed mode cation exchange chromatography material comprises a reverse phase strong cation exchange polymer. In some embodiments, the mixed mode cation exchange chromatography material comprises a sulfonic acid moiety. In some embodiments, the mixed mode cation exchange chromatography material comprises a solid support. In some embodiments, the mixed mode cation exchange chromatography material is contained in a column. In some embodiments, the mixed mode cation exchange chromatography material is a high performance liquid chromatography (HPLC) material. In some embodiments, the mixed mode cation exchange chromatography material is a chromatography material Oasis® MCX. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or using a charged aerosol detector (CAD). In some embodiments, the quantification of polysorbate comprises less than about the 10% (such as less than about any of 9%, 6%, 5%, 4%, 9- 7 9J O / á, Q i 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) interference from polypeptide and NAT.

[0120] In some embodiments of any of the methods described above, the nonionic surfactant is quantitated in the composition comprising the nonionic surfactant prior to addition of the polypeptide to the composition. In some embodiments, the concentration of surfactant 181 The nonionic surfactant in the composition will be higher before the addition of the polypeptide (eg, the nonionic surfactant in the composition is diluted after the addition of the polypeptide). In some embodiments of any of the methods described above, the nonionic surfactant is quantitated in the composition comprising the nonionic surfactant above, but without the polypeptide in the composition. These quantifications can be used as controls or comparators with the compositions comprising the nonionic surfactant and the polypeptide.

[0121] In some embodiments of any of the methods described above, samples of the compositions to be tested are added to an autosampler of a chromatographic instrument (eg, HPLC instrument). In some embodiments, the samples in the autosampler are refrigerated (eg, 5 ± 3 °C). In some embodiments, one or more columns comprising the chromatography material are placed in a column compartment of the chromatographic instrument. In some embodiments, a temperature control feature may be employed to maintain the column compartment temperature within a narrow range (eg, ±1°C) from the set point during analysis. In some embodiments, the effluent from the column is monitored at 280 nm.

[0122] In some embodiments of any of the methods described above, samples of the compositions to be tested are diluted with loading buffer to a concentration of target polypeptide between about 0.1 mg / mL and about 182 mg / mL (such as approximately any of 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mg / mL, including any range between these values).

[0123] In some embodiments of any of the methods described above, the chromatographic instrument includes a gradient pump (eg, a low-pressure quaternary gradient pump), an autosampler (eg, an autosampler with temperature control), a column compartment (eg, a thermally controlled column compartment), a UV detector (eg, a diode array UV detector), and an evaporative light scattering detector (ELSD). In some embodiments, the chromatographic instrument also comprises a pH and conductivity monitor (eg, PCM-3000) to collect pH and conductivity data in real time. Instrument control, data acquisition and data analysis is carried out using appropriate software (eg JMP10).

[0124] In some embodiments of the invention, the ionic strength of the mobile phase, eg, the elution buffer, by conductivity of the mobile phase. Conductivity refers to the ability of an aqueous solution to conduct an electrical current between two electrodes. In solution, current flows by ionic transport. Consequently, with a greater amount of ions present in the aqueous solution, the solution will have a higher conductivity. The basic unit of measurement for conductivity is Siemens (or mho), mho (mS / cm) and can be measured using a conductivity meter. 183 conductivity, such as various models of Orion conductivity meters. While electrolytic conductivity is the ability of ions in a solution to carry electrical current, the conductivity of a solution can be altered by changing the concentration of ions within. For example, the concentration of a buffering agent and / or the concentration of a salt (eg, sodium chloride, sodium acetate, or potassium chloride) in the solution can be altered in order to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is modified to achieve the desired conductivity.

[0125] In some embodiments, the mobile phase of the chromatography has an initial conductivity of greater than about any of 0.0 mS / cm, 0.5 mS / cm, 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, 3.5 mS / cm, 4.0 mS / cm, 4.5 mS / cm, 5.0 mS / cm, 5.5 mS / cm, 6.0 mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0 mS / cm, 8.5 mS / cm, , 9.0 mS / cm, 9.5 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm , 14 mS / cm, 15 mS / cm , 16 mS / cm, 17.0 mS / cm, 18.0 mS / cm, 19.0 mS / cm, or 20.0 mS / cm. In In some embodiments, the conductivity of the mobile phase is increased in the course of chromatography, for example, by an ionic strength gradient. In some embodiments, the conductivity of the mobile phase at completion of 1. approximately any of mS / cm, 2.5 mS / cm, 3.0 mS / cm, mS / cm, 5.0 mS / cm, 5.5 mS / cm, mS / cm, 7.5 mS / cm, 8.0 mS / cm, uction is more than 1.0 mS / cm, 1.5 mS / cm, 2.0 3.5 mS / cm, 4.0 mS / cm, 4.5 6.0 mS / cm, 6.5 mS / cm, 7.0 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, mS / cm, 16 mS / cm, 17.0 mS / cm, 18.0 mS / cm, 19.0 mS / cm or 184 20.0 mS / cm. In some embodiments, the conductivity of the mobile phase is increased by a linear gradient. In some embodiments, the conductivity of the mobile phase is increased by a gradual gradient that comprises one or more steps. realization of any form of

[0126] In some of the methods described in which the composition comprises a polypeptide and present, a nonionic surfactant is loaded onto the chromatography material in an amount of polypeptide greater than any of about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 , 9000 or 10000 pg. In some embodiments, the composition is loaded onto the chromatography material at a concentration greater than any of about 0.5, 1, 1.5, 2, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg / mL. In some embodiments, the composition is diluted before loading onto the chromatography material; eg, diluted 1:1, 1:2, 1:5, 1:10, or more than 1:10. In some embodiments, the composition is diluted in the mobile phase of the chromatography. In some embodiments, the composition is diluted in loading buffer.

[0127] In some embodiments of the methods described herein, the chromatography material is in a column or cartridge. In some embodiments, the column is a column or cartridge of HPLC. The column or cartridge can have any dimension compatible with the chromatographic instrument. For example, in some embodiments, the column or cartridge has any of the following 185 Dimensions: 2.1 x 20mm, 4*50mm, 4 x 100mm, 4 x 150mm, 4 x 200mm, 4 x 250mm or 2 x 250mm. III. Polypeptides

[0128] Polypeptides are provided for use in any of the ion exchange chromatography methods, where separation conditions are optimized as described herein. In some embodiments of the invention, compositions of a polypeptide are analyzed by ion exchange chromatography. These methods are useful in identifying charge variants of the polypeptide within the composition. In some embodiments, the polypeptide is an antibody or its fragment. In some embodiments, the polypeptides have a pl ranging from about 6.0 to about 9.5. In some embodiments, the polypeptide is an antibody having a pl ranging from about 6.0 to about 9.5. In some embodiments, the inflection point (IP) in a load vs. Polypeptide pH is provided by the methods of the invention. In some embodiments, the change in IP with a change in temperature (dIP / dT) is provided by the methods of the invention.

[0129] In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the polypeptide is an immunoadhesin.

[0130] In some embodiments, the polypeptide has a molecular weight of greater than about any of 5,000 Daltons, 10,000 Daltons, 15,000 Daltons, 25,000 Daltons, 50,000 Daltons, 186 75,000 Daltons, 100,000 Daltons, 125,000 Daltons, or 150,000 Daltons. The polypeptide can have a molecular weight of between about any of 50,000 Daltons to 200,000 Daltons or 100,000 Daltons to 200,000 Daltons. Alternatively, the polypeptide for use herein can have a molecular weight of about 120,000 Daltons or about 25,000 Daltons.

[0131] pl is the isoelectric point and is the pH at which a particular molecule or surface carries no electrical lattice charge. In some embodiments, the method of the invention can be used for a plurality of compositions comprising a polypeptide where the pl of the polypeptide in the composition, eg, an antibody, ranges from about 6.0 to about 9.5. In some embodiments, the polypeptide has a pl of greater than about 9.5; eg, from about 9.5 to about 12. In some embodiments of any of the methods described herein, the pl of the polypeptide, eg, an antibody, can be less than about 7; for example, from about 4 to about 7.

[0132] In embodiments of any of the methods described herein, the one or more contaminants in a composition comprising a polypeptide and one or more contaminants are charge variants of polypeptides. In some embodiments, the polypeptide charge variant is a polypeptide that has been modified from its native state in such a way that the charge of the polypeptide is altered. In some embodiments, the charge variants are more acidic than the parent polypeptide; that is, they have a lower pl than the main polypeptide. In other embodiments, 187 cargo variants are more basic than the parent polypeptide; that is, they have a larger pl than the parent polypeptide. In some embodiments, polypeptide charge variants are engineered. In some embodiments, the polypeptide charge variant is the result of natural processes; for example, oxidation, deamidation, C-terminal processing of lysine residues, N-terminal formation of pyroglutamate, and glycation. In some embodiments, the polypeptide charge variant is a glycoprotein in which the glycan attached to the protein is modified such that the charge of the glycoprotein is altered compared to the parent glycoprotein; for example, by addition of sialic acid or its derivatives. In some embodiments, the polypeptide charge variant is an antibody charge variant.

[0133] Polypeptides to be analyzed using the methods described herein are generally produced using recombinant techniques. Methods for producing recombinant proteins are described, for example, in U.S. Pat. Nos. 5,534,615 and 4,816,567, specifically incorporated herein by reference. In some embodiments, the protein of interest is produced in a CHO cell (see, for example, WO 94 / 11026 ). In some embodiments, the polypeptide of interest is produced in an E. coli cell. See, for example, U.S. Patent No. 5,648,237; U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523, which describe translation initiation region (TIR) ​​and signal sequences to optimize expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana 188 Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli. When using recombinant techniques, the polypeptides can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.

[0134] Polypeptides can be recovered from culture medium or from host cell lysates. The cells used in the expression of the peptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. If the polypeptide is produced intracellularly, as a first step, particulate debris, either from host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a procedure for isolating polypeptides that are secreted into the periplasmic space of E. coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 min. Cell debris can be removed by centrifugation. When polypeptide is secreted into the medium, supernatants from such expression systems are generally concentrated first, using a commercially available polypeptide concentrator filter, eg, an Amicon or Millipore Pellicon filter unit. A protease inhibitor such as PMSF may be included in any of the preceding steps to inhibit proteolysis and antibiotics may be included to 189 effects of preventing the development of adventitious contaminants.

[0135] In some embodiments, the polypeptide in the composition comprising the polypeptide and one or more contaminants was purified or partially purified prior to analysis by the methods of the invention. For example, the polypeptide of the methods is in an eluent from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, and hydrophobic interaction chromatography. In some embodiments, the polypeptide is in an eluent from a protein A chromatography.

[0136] Examples of polypeptides that can be analyzed by the methods of the invention include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fe-containing proteins, immunoconjugates, cytokines, and interleukins. (A) Antibodies

[0137] In some embodiments of any of the methods described herein, the polypeptide for use in any of the methods of polypeptide analysis and formulations comprising the polypeptides by means of the methods described herein it is an antibody.

[0138] Molecular targets for antibodies include (i) CD proteins and their ligands such as, but not limited to: CD3, CD4, CD8, CD19, CDlla, CD20, CD22, CD27, CD28, CD34, CD40, CD79a (CD79a) , CD79p (CD79b), CD122 and CD137; (ii) cytokines such as, but not limited to: IL-13, IL-17, IL-22, and IL-33; (iii) members 190 from the ErbB family of receptors such as the EGF, HER2, HER3 or HER4 receptor; (iv) cell adhesion molecules such as LFA-1, Macl, pl50.95, VLA-4, ICAM-1, VCAM, and αν / β3 integrin, including their alpha or beta subunits (for example, anti-CDlla, anti-CD18 antibodies). or anti-CDIIb); (v) growth factors such as VEGF; TGFP, IgE; blood group antigen; flk2 / flt3 receiver; obesity receptor (OB); receiver mpl; CTLA-4; protein C, BR3, c-met, tissue factor, β7, etc.; (vi) immunomodulatory proteins such as 0X40, GITR, ICOS, PD-1, PD-L1, PD-L2, LAG3, TIM-3 and VISTA; and (vii) transmembrane tumor and cell surface associated antigens (TAAs), such as those described in U.S. Patent No. 7,521,541, including, without limitation, NaPy2b.

[0139] Other examples of antibodies include those selected without limitation from anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-OX40 antibody, anti-p53 antibody, anti-EGFR antibody, anti-GITR antibody, anti-HER-2 / neu antibody, anti-PD-II antibody, anti-CTLA-4, antibody 3ηίί-ΤΟΕβ, anti-PD-1 antibody, anti-ICOS antibody, anti-PD-L2 antibody, anti-VISTA antibody, anti-Bcl-2 antibody, anti-TIM-3 antibody, anti-CA19-9 , anti-c-erbB-2 antibody, anti-Pglycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody. anti~CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, 191 anti-CDllc antibody, anti-CD20 antibody, anti-CD34 antibody, anti-CD10 antibody, anti-CD15 antibody, anti-CD27 antibody, anti-CD31, anti-CD40 antibody, anti-CD13 antibody, anti-CD22 antibody, antibody anti-CD35 antibody, anti-CDlla antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD33, anti-CD41 antibody, anti-CD14 antibody, anti-CD2 3 antibody, anti-CD30, anti-CD38 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-CD122 antibody, anti- CD137, anti-ubiquitin antibody, anti-ubiquitin antibody CD71, anti-StafA antibody, anti-FluB antibody, anti-FcRH5 antibody, anti-c-myc antibody, anti-Li6E, anti-STEAP antibody, anti-VEGF, anti-KLB antibody, anti-cytokeratin antibody, anti- FGFRl, anti-Ang2 antibody, anti-vimentin antibody, anti-HPV antibody, protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, antiprostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, anti-Tn-antigen antibody and MetMab. (i) Monoclonal Antibodies

[0140] In some embodiments, the antibodies are monoclonal antibodies. Monoclonal antibodies are derived from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope with the exception of possible variants that occur during 192 production of the monoclonal antibody, such variants being generally present in minor amounts. Therefore, the monoclonal modifier indicates that the character of the antibody is not from a mixture of discrete polyclonal antibodies.

[0141] For example, monoclonal antibodies may be prepared using the method first described by Kohler (1975), or recombinant (U.S. Patent No. 4,816,567, Nature 256:495).

[0142] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described herein to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells whereby a suitable fusion agent, such as polyethylene glycol, is used to form a hybridoma cell (Goding, Monoclonal antibodies: Principles and practice, p. 59-103 (Academic Press, 1986)).

[0143] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium)), all of which substances prevent the growth of HGPRT-deficient cells. 193

[0144] In some embodiments, myeloma cells are those that fuse efficiently, support stable high-level production by antibody-producing cells, and are sensitive to a medium such as HAT medium. Among them, in some embodiments, the myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors provided by Salk Institute Cell Distribution Center, San Diego, California USA. .USA, and SP-2 or X63Ag8-653 cells provided by the American Type Culture Collection, Rockville, Maryland USA. Human myeloma cell lines and mouse and human heteromyeloma cell lines are also available. have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications pp. 51-63 (Marcel Dekker, Inc., New York, 1987 ) ) .

[0145] The culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assays (ELISA). enzyme-linked).

[0146] The binding affinity of the monoclonal antibody can be determined for example by the Scatchard assay of Munson et al., Anal. Biochem. 107:220 (1980). 194

[0147] After hybridoma cells producing antibodies with the desired specificity, affinity and / or activity have been identified, clones can be subcloned by dilution procedures and grown by standard procedures (Goding, Monoclonal antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in an animal.

[0148] The monoclonal antibodies secreted by the subclones are conveniently separated from the culture medium, ascites fluid, or serum, by conventional immunoglobulin A purification procedures such as, for example, polypeptide A-Sepharose, hydroxylapatite chromatography, electrophoresis in gel, dialysis, or affinity chromatography.

[0149] The DNA encoding the monoclonal antibodies is readily isolated by sequencing by conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding murine antibody heavy and light chains). In some embodiments, the hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that would otherwise do not produce immunoglobulin polypeptide, so as to obtain the synthesis of monoclonal antibodies in the cells 195 recombinant host. Reference articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

[0150] In another embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), as well as combinatorial injection and indirect recombination. vivo as a strategy for building very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0151] DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the murine homologous sequences (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nati Acad. Sci. USA 81:6851 (1984)), or by covalently linking to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. 196

[0152] Typically, such non-immunoglobulin polypeptides are substituted in terms of their constant domains of an antibody, or are substituted in the variable domains of an antigen combining site, of an antibody so as to create a bivalent chimeric antibody comprising a site one antigen-combining site having specificity for one antigen and another antigen-combining site having specificity for a different antigen.

[0153] In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody. (ii) Humanized antibodies

[0154] In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically taken from an imported variable domain. Humanization can be accomplished essentially by following the method of Winter et al., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988), substituting the rodent hypervariable region sequences for the corresponding sequences from a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than one human variable domain 197 intact has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

[0155] The choice of human variable domains, both light and heavy, to be used to prepare the humanized antibodies is very important to reduce antigenicity. According to the so-called best-fit method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent is then accepted as the human framework region (FR) for the humanized antibody. Sims et al. , j. Immunol., 151;2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987). Another method uses a particular framework region derived from the consensus sequence of all human antibodies to a particular subset of heavy or light chain variable regions. The same framework can be used for several different humanized antibodies. Carter et al. , Proc. nati. Acad. Sel. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993).

[0156] It is also important that the antibodies be humanized with high affinity retention for the antigen and other favorable biological properties. To achieve this goal, in some embodiments of the methods, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products. 198 using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are commonly obtainable and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selectable candidate immunoglobulin sequences. Inspection of these displays allows analysis of the probable role of the residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences such that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, hypervariable region residues are directly and more substantially involved in influencing antigen binding. (rii) Human antibodies

[0157] In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (eg, mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (Jh) gene in chimer...

Claims

1. A method for quantifying a nonionic surfactant in a composition comprising the nonionic surfactant and a polypeptide, characterized in that it comprises the steps of a) applying the composition to a mixed-mode anion-exchange chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising a mobile phase A and a mobile phase B, wherein mobile phase A comprises acetic acid in water and mobile phase B comprises acetic acid in methanol, wherein the polypeptide binds to the chromatography material in a specific and nonspecific manner; b) eluting the specifically bound polypeptide from the mixed-mode anion-exchange chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step a);c) eluting the non-ionic surfactant and the non-specifically bound polypeptide from the chromatography material with a solution comprising mobile phase A and mobile phase B wherein the ratio of mobile phase B to mobile phase A is increased with respect to step b); d) quantifying the non-ionic surfactant. 22 Claims follow;