Chromatographic method for quantifying nonionic surfactant in a composition comprising nonionic surfactant and a polypeptide

Abstract

The present invention relates to a chromatographic method for quantifying non-ionic surfactant in a composition comprising non-ionic surfactant and a polypeptide. In particular, the present invention provides a method for quantifying non-ionic surfactant in a composition comprising a polypeptide and non-ionic surfactant, wherein the quantification exhibits reduced interference between the non-ionic surfactant and the polypeptide. Also provided is a method wherein the composition further comprises N-acetyl tryptophan, and the quantification exhibits reduced interference between the non-ionic surfactant, the polypeptide and N-acetyl tryptophan.

Description

[0001] This application is a divisional application of PCT application PCT / US2017 / 046725, filed on August 14, 2017, entitled "Chromatographic method for quantifying nonionic surfactants in compositions containing nonionic surfactants and peptides". The PCT application entered the Chinese national phase on March 20, 2019, and the application number is 201780057815.5.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 375,373, filed August 15, 2016, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] This invention provides a method for analyzing the presence of polysorbate in polypeptide formulations. Background Technology

[0005] Polysorbate 20 (PS20) is a surfactant commonly used in peptide 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 the control system of each product. PS20 can be quantified by spectrophotometry, fluorescence micelle assay, or high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) assay (see, for example, Kim, J. and Qiu, J., Analytica chimica acta 806:144-151, 2014; Hewitt et al., Journal of Chromatography A, 1215(1):156-160, 2008).

[0006] Evaporative light scattering detector (ELSD) assays are preferred as a control system because they do not require a long settling time compared to fluorescence micelle assays. The ELSD method also avoids the need to prepare the same polysorbate batches as the standard curves used in production. Furthermore, fluorescence micelle assays are susceptible to nonspecific protein interference, particularly for hydrophobic proteins and antibody-drug conjugates (ADCs). The vcMMAE linker of ADCs introduces additional hydrophobicity into proteins, which can lead to increased protein interference when quantifying PS20. In some cases, this nonspecific protein interference can be mitigated by using HPLC-ELSD assays.

[0007] Although HPLC-ELSD assays can reduce the degree of protein interference, this interference is not completely eliminated. Protein interference becomes particularly problematic at oligosorbate concentrations and at higher hydrophobic and / or concentrated protein concentrations. Furthermore, the impact of protein interference is highly dependent on the batch of column resin used. Strategies to mitigate these problems include: a) adding PS20 to dilute protein interference without reducing PS20 reactivity, and b) removing proteins from the sample via protein precipitation.

[0008] The doping method requires diluting the sample with a PS20 stock solution at the target concentration of the formulation. This sample preparation dilutes the protein concentration while keeping the PS20 concentration approximately constant. The amount of PS20 added to the sample is then subtracted during data analysis. Because the relationship between the ELSD response and the quality of analysis in the detector follows a power law, doping in PS20 disproportionately reduces the protein's contribution to the ELSD signal. In some cases, the doping method has been shown to improve the accuracy of PS20 quantification, but in situations where this is not a feasible solution, protein precipitation must be used. While it effectively removes 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 uses the same HPLC-ELSD conditions but without the significant sample preparation procedures. What is needed is a more robust solution to eliminate protein interference and produce consistent PS20 quantification under all chromatographic conditions.

[0009] All references cited in this article, including patent applications and publications, are incorporated herein by reference in their entirety. Summary of the Invention

[0010] In some aspects, the present invention provides a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein interference between the nonionic surfactant and the polypeptide is reduced during quantification, wherein the method comprises the steps of: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid, wherein the polypeptide binds specifically and nonspecifically to the chromatography material; 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 nonionic surfactant and nonspecifically 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 compared to step c); d) quantifying the nonionic surfactant, wherein interference between the nonionic surfactant and the polypeptide is reduced during quantification. In some embodiments, the ratio of mobile phase B to mobile phase A in step a) is approximately 10:90. In some embodiments, in step b), the ratio of mobile phase B to mobile phase A is increased to approximately 40:60. In some embodiments, in step c), the ratio of mobile phase B to mobile phase A is increased to approximately 100:0. In some embodiments, mobile phase A comprises approximately 2% of an aqueous solution of an acid. In some embodiments, mobile phase B comprises approximately 2% of a methanol solution of an acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid.

[0011] In some embodiments, the flow rate of the chromatography is approximately 1.25 mL / min. In some embodiments, step b) begins approximately 1 minute after the start of chromatography and ends approximately 3.4 minutes after the start of chromatography.

[0012] In some embodiments, step c) begins approximately 3.5 minutes after the start of chromatography and ends approximately 4.6 minutes after the start of chromatography. In some embodiments, the nonionic surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is from 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioning agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase 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... MAX chromatography materials. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0013] In some aspects, the present invention provides a method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant 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 added to the chromatography material in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises a solution of ammonium hydroxide in an organic solvent; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with the 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 from the chromatography material with the 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) quantifying the nonionic surfactant. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the ratio of mobile phase B to mobile phase A in step a) is about 10:90. In some embodiments, the ratio of mobile phase B to mobile phase A in step b) is increased to about 45:55. In some embodiments, in step c), the ratio of mobile phase B to mobile phase A is increased to approximately 100:0. In some embodiments, mobile phase A comprises approximately 2% aqueous ammonium hydroxide solution. In some embodiments, mobile phase B comprises approximately 2% methanolic ammonium hydroxide solution. In some embodiments, the flow rate for chromatography is approximately 1.4 mL / min. In some embodiments, step b) begins approximately 1 minute after the start of chromatography and ends approximately 4.4 minutes after the start of chromatography. In some embodiments, step c) begins approximately 4.5 minutes after the start of chromatography and ends approximately 7.6 minutes after the start of chromatography. In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate in the composition is approximately 0.001% to 1.0% (w / v). In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the ratio of mobile phase B to mobile phase A in step a) is about 10:90. In some embodiments, step b) increases the ratio of mobile phase B to mobile phase A to about 40:60. In some embodiments, the ratio of mobile phase B to mobile phase A in step c) is increased to 100:0. In some embodiments, mobile phase A comprises about 2% aqueous ammonium hydroxide solution or 43% methanol solution. In some embodiments, mobile phase B comprises about 2% ammonium hydroxide solution in acetonitrile. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of poloxamer in the composition is about 0.001% to 1.0% (w / v). 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. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM. In some embodiments, the concentration of the peptide in the composition ranges from about 1 mg / mL to about 250 mg / mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.5. In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tension agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, or antibody-drug conjugate THIOMAB. TM THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography materials. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). Attached Figure Description

[0014] Figure 1 It is a multi-step gradient of a PS20-free ADC and a 0.6 mg / ml PS20 standard using an increase of 5% methanol. The elution solvent contains 2% formic acid.

[0015] Figure 2 The experiment compares the methanol multi-step gradient experiment and the isopropanol stepwise gradient experiment (both containing 2% formic acid) on different columns. For each stack, there is an A1 ADC without PS20 on the first column (trace 1), an A1 ADC without PS20 on the second column (trace 2), and a PS20 standard (trace 3).

[0016] Figure 3 The results of the optimized experimental design are shown. The solid lines represent the directionality of the statistical model fitted to the data using JMP10 software. The dashed lines that define each solid line represent the error associated with the fit. The slope of the lines represents the influence of each factor on the PS20 peak area and PS20 peak width.

[0017] Figure 4 The comparison of PS20 method chromatograms optimized from the DoE method is shown. For these experiments, the flow rate was 1.25 mL / min, the sample loading was 12 μg PS20, and the washing duration was 3 min.

[0018] Figure 5 The mobile phase showed a 10 mg / ml A10 ADC without PS20 and a 0.6 mg / ml PS20 standard using 0.2% trifluoroacetic acid.

[0019] Figure 6 The mobile phase showed a 20 mg / ml A1 ADC without PS20 and a 0.6 mg / ml PS20 standard using 2% formic acid.

[0020] Figure 7 The mobile phase showed a 20 mg / ml A1 ADC without PS20 and a 0.7 mg / ml PS20 standard using 2% acetic acid.

[0021] Figure 8 The comparison between mobile phases containing formic acid and acetic acid using the following samples is shown: water (trace 1), A1 ADC without PS20, 20 mg / mL (trace 2), and 0.2 mg / mL PS20 incorporated into A1 ADC, 20 mg / mL (trace 3).

[0022] Figure 9 The use of methanol / acetic acid as elution in different... Various spectra of PS20 standards running on a MAX column. Typical spectra (trace 1), peaks with tails (trace 2), peaks showing splitting (trace 3), and peaks with slight tails (trace 4). This variability in spectra does not affect the quantification of standards, controls, or protein samples.

[0023] Figure 10 The MeOH / acetic acid method is shown. Typical 20 μL of water for injection (trace 1), PS20-free A1 ADC formulation buffer (trace 2), PS20-free A1 ADC (trace 3), and minimum PS20 standard at 0.1 mg / mL (trace 4).

[0024] Figure 11A and 11B The method 1 of Example 1 is shown to evaluate A16 / A17 without PS20. Figure 11A Display ELSD, Figure 11BDisplayed UV (280nm). Water (trace 1), PS20-free A16 / A17 formulation buffer (trace 2), and PS20-free A16 / A17 protein (trace 3) were evaluated using Method 1 of Example 1.

[0025] Figure 12A and 12B ELSD chromatograms of different A16 / A17 buffer components using the method of Example 1 are shown. Figure 12A The following buffers with NAT are shown: 20 mM histidine-HCl, 1 mM NAT, 5 mM methionine, 240 mM sucrose (trace 1); 20 mM histidine-HCl, 1 mM NAT, 240 mM sucrose (trace 2); 20 mM histidine-HCl, 5 mM NAT (trace 3). Figure 12B Show the NAT-free buffer: 20 mM histidine-HCl, 5 mM methionine, 240 mM sucrose (trace 1); 20 mM histidine-HCl, 25 mM methionine (trace 2); 20 mM histidine-HCl (trace 3). Inject 50 μL of buffer.

[0026] Figure 13A and 13B ELSD chromatograms of different buffer components using an improved method (MCX column and ammonium hydroxide in the mobile phase) are shown. Figure 13A The buffers containing NAT are shown as follows: 20 mM histidine-HCl, 1 mM NAT, 5 mM methionine, 240 mM sucrose (trace 1); 20 mM histidine-HCl, 1 mM NAT, 240 mM sucrose (trace 2); 20 mM histidine-HCl, 5 mM NAT (trace 3); and a PS20-free protein containing NAT (trace 4). Figure 13B Show the NAT-free buffer: 20 mM histidine-HCl, 5 mM methionine, 240 mM sucrose (trace 1); 20 mM histidine-HCl, 25 mM methionine (trace 2); 20 mM histidine-HCl (trace 3). 50 μL injection.

[0027] Figure 14A and 14B The use of 0.15–1.50% ammonium hydroxide additive in the mobile phase to evaluate PS20-free A16 / A17 proteins was demonstrated. Figure 14A Display the ELSD tomographic diagram. Figure 14BUV (280 nm) chromatography is shown. 50 μL of PS20-free A16 / A17 protein was injected into the mobile phase containing 0.15, 0.29, 0.73, and 1.5% ammonium hydroxide (traces 1, 2, 3, and 4, respectively), and PS20-free A16 / A17 formulation buffer containing 1.5% ammonium hydroxide (traces 5).

[0028] Figure 15A and 15B This shows the evaluation of the 20-60% mobile phase B washing step. Figure 15A Display the ELSD tomographic diagram. Figure 15B UV (280 nm) chromatography is shown. 15 μL of PS20-free A16 / A17 protein was injected. 20, 30, 40, 50, and 60% mobile phase B (washing step) are shown as traces 1, 2, 3, 4, and 5, respectively.

[0029] Figure 16 The washing time and injection volume for evaluating PS20-free A16 / A17 protein by ELSD chromatography are shown. 25 μL PS20-free A16 / A17 sample injection: 3.4 min wash (trace 1). 50 μL PS20-free A16 / A17 sample injection: 3.4 min wash (trace 2) and 2.4 min wash (trace 3).

[0030] Figure 17A and 17B The evaluation of different flow rates for A18 / A19 without PS20 is shown. Figure 17A shows the ELSD chromatogram. Figure 17B UV (280 nm) chromatography is shown. 25 μL injections of PS20-free A18 / A19 (150 mg / mL) were performed at 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.

[0031] Figure 18 The different elution times of PS20 in water were evaluated by ELSD chromatography. 50 μL of 0.1 mg / mL PS20 aqueous solution was injected, followed by a 3.1-minute elution step (trace 1) or a 1.1-minute elution step (trace 2).

[0032] Figure 19 The evaluation of finalization method 2 by ELSD chromatography is shown. 25 μL of water for injection (trace 1), 150 mg / mL of PS20-free A18 / A19 (trace 2), 0.2 mg / mL of PS20 mixed with water (trace 3), and 0.2 mg / mL of PS20 mixed with A18 / A19 (trace 4) were prepared using the finalization parameters of method 2.

[0033] Figure 20A-20F The evaluation shows the specificity of the three different products. Figure 20A (A18 / A19), 20C (A16 / A17) and 20E (A14 / A20) show ELSD tomography. Figure 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 an aqueous solution of 0.1 mg / mL PS20 (trace 3).

[0034] Figure 21 The variability of columns was assessed by ELSD chromatography using either PS20 incorporated into water or A18 / A19 without PS20. Column 2, incorporated with 0.1 mg / mL PS20 in A18 / A19 without PS20 (trace 1); Column 6, incorporated with 0.1 mg / mL PS20 in A18 / A19 without PS20 (trace 2); Column 2, incorporated with 0.2 mg / mL PS20 in water (trace 3); and Column 6, incorporated with 0.2 mg / mL PS20 in water (trace 4).

[0035] Figure 22 The variability of columns was evaluated by ELSD chromatography using either PS20 incorporated into water or A18 / A19 without PS20. Column 4, incorporated with 0.2 mg / mL PS20 in A18 / A19 without PS20 (trace 1); Column 6, incorporated with 0.2 mg / mL PS20 in A18 / A19 without PS20 (trace 2); Column 4, incorporated with 0.2 mg / mL PS20 in water (trace 3); and Column 6, incorporated with 0.2 mg / mL PS20 in water (trace 4).

[0036] Figure 23 The ELSD chromatogram of the control samples used in a sequence comprising 100 A18 / A19 injections on a single column is shown. A stack of 11 control samples injected throughout the sequence is also shown.

[0037] Figure 24 The image shows an ELSD chromatogram of an A18 / A19 sample intended for inclusion in a sequence of 100 A18 / A19 injections on a single column. The 100 A18 / A19 sample injections are stacked throughout the sequence.

[0038] Figure 25A and 25B Chromatographic images of the 1st, 50th, and 100th protein injections from a sequence comprising 100 A18 / A19 injections on a single column are shown.

[0039] Figure 26A and 26B This shows the quantitative results of 100 injections of A18 / A19 samples (nominal 0.2 mg / mL PS20) using column 4. Figure 26A Displays the area of ​​PS20 and the number of injections. Figure 26B The display shows the relationship between PS20 concentration and the number of injections.

[0040] Figure 27A and 27B This shows the quantitative results of 100 injections using column 5 with A18 / A19 formulation buffer (nominal 0.2 mg / mL PS20). Figure 27A The figure shows the area of ​​PS20 versus the number of injections. Figure 27B shows the PS20 concentration versus the number of injections.

[0041] Figure 28A and 28B The results show the quantitative results of 100 injections using column 3 with 0.2 mg / mL PS20 added to water. Figure 28A Displays the area of ​​PS20 relative to the number of injections. Figure 28B The display shows the relationship between PS20 concentration and the number of injections.

[0042] Figures 29A-29F The methods 1 and 2 are shown to evaluate three low pI products by ELSD chromatography. Figure 29A Figures 29C and 29E show tomographic diagrams of Method 1, using A21, A14 / A15, and A14, respectively. Figure 29B Figures 29D and 29F show tomographic diagrams of method 2, using A21, A14 / A15, and A14, respectively.

[0043] Detailed description of the invention

[0044] This invention provides a method for quantifying nonionic surfactants in compositions comprising peptides and nonionic surfactants, wherein the quantification demonstrates reduced interference between the nonionic surfactant and the peptide. A method is also provided in which the composition further comprises N-acetyltryptophan, and the quantification demonstrates reduced interference between the nonionic surfactant, the peptide, and N-acetyltryptophan.

[0045] I. Definition

[0046] The terms “peptide” or “protein” are used interchangeably herein and refer to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers, whether naturally occurring or modified through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component or toxin. The definition also includes, for example, peptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art. The terms “peptide” and “protein” as used herein specifically include antibodies.

[0047] "Purified" peptides (e.g., antibodies or immunoadhesins) refer to peptides with increased purity, such that they exist in a purer form than when they are in their natural environment and / or initially synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0048] The term "antagonist," used in the broadest sense, includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a natural polypeptide. Similarly, the term "agonist," used in the broadest sense, includes any molecule that mimics the biological activity of a natural polypeptide. Suitable agonist or antagonist molecules particularly include agonist or antagonist antibodies or antibody fragments, fragments of natural polypeptides, or amino acid sequence variants, etc. Methods for identifying agonists or antagonists of a polypeptide may include contacting the polypeptide with a candidate agonist or antagonist molecule and measuring detectable changes in one or more biological activities typically associated with the polypeptide.

[0049] "Binding" a target antigen, such as a tumor-associated peptide antigen, refers to a peptide that binds to the antigen with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting cells or tissues expressing that antigen, and without significant cross-reactivity with other peptides. In such embodiments, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA), the degree of binding of the peptide to "non-target" peptides will be less than approximately 10% of the binding of the peptide to its specific target peptide.

[0050] Regarding the binding of peptides to target molecules, the terms "specific binding," "exclusive binding," or "specific to" a particular peptide or epitope on a specific peptide target refer to a binding that is measurably different from nonspecific interactions. For example, specific binding can be measured by comparing the binding of a molecule to that of a control molecule, typically a molecule with a similar structure that does not have binding activity. Specific binding can also be determined by competition with a control molecule similar to the target, such as an excess of an unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of the unlabeled target, it indicates specific binding.

[0051] The term “antibody” is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, provided they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” in this article.

[0052] Antibodies are naturally occurring immunoglobulin molecules with varying structures, all based on immunoglobulin folding. For example, IgG antibodies have two "heavy" chains and two "light" chains that bind together via disulfide bonds to form a functional antibody. Each heavy and light chain contains "constant" (C) and "variable" (V) regions. The V region determines the antibody's antigen-binding specificity, while the C region provides structural support and plays a role in non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody, or an antigen-binding fragment of an antibody, is the antibody's ability to specifically bind to a particular antigen.

[0053] The antigen-binding specificity of an antibody is determined by the structural features of the V region. Variableness is unevenly distributed across the 110-amino acid span of the variable domain. Conversely, the V region consists of relatively constant segments of 15–30 amino acids called framework regions (FRs), separated by extremely variable shorter regions called “hypervariable regions” (HVRs), each 9–12 amino acids long. The variable domains of the natural heavy and light chains each contain four FRs, predominantly in a β-sheet configuration, linked by three hypervariable regions that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are tightly held together by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as antibody participation in antibody-dependent cytotoxicity (ADCC).

[0054] Each V region typically contains three HVRs, for example, three complementarity-determining regions (“CDRs”, each containing a “hypervariant loop”) and four framework regions. Therefore, the antibody binding site, the smallest structural unit required to bind a specific desired antigen with substantial affinity, will typically include three CDRs, with at least three, preferably four, framework regions dispersed therebetween to maintain and present the CDRs in a suitable conformation. Classical tetrachain antibodies have an antigen-binding site consisting of V... H and V L Domain cooperation is defined. Some antibodies, such as camel and shark antibodies, lack a light chain and rely solely on binding sites formed by heavy chains. Single-domain engineered immunoglobulins can be prepared where the binding site is formed only by either the heavy or light chain, in V... H and V L There is no collaboration between them.

[0055] The term "variable" refers to the fact that certain portions of the variable domain differ significantly in sequence between antibodies and are responsible for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domains of an antibody. It is concentrated in three segments called hypervariable regions within the variable domains of the light and heavy chains. The more conserved portions of the variable domains are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, predominantly in a β-sheet configuration, connected by three hypervariable regions that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held together tightly by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit a variety of effector functions, such as antibody participation in antibody-dependent cytotoxicity (ADCC).

[0056] When used in this document, the term "hypervariant region" (HVR) refers to the amino acid residues in an antibody responsible for antigen binding. The hypervariant region may contain amino acid residues from the "complementarity-determining region" or "CDR" (e.g., in V...). L The middle portion consists of residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), and in V HThe middle portion consists of approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or residues from the “hypervariate ring” (e.g., V). L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the group, and V H 26-32(H1), 52A-55(H2) and 96-101(H3) (Chothia and Lesk J.Mol.Biol.196:901-917(1987)).

[0057] “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues defined in this paper.

[0058] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; tandem biantibodies (taDb); linear antibodies (e.g., 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; microantibodies; single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (bi, tri)-scFv); and bispecific T-cell adaptors (BiTE)).

[0059] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link the antigen.

[0060] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. This region consists of a dimer of a tightly bound, non-covalently linked heavy chain and a light chain variable domain. In this configuration, the three hypervariable regions of each variable domain interact to achieve V H -V LThe surface of the dimer defines the antigen-binding site. The six hypervariable regions collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or only half of the Fv of the three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the intact binding site.

[0061] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the name for Fab' in this paper, where the cysteine ​​residues of the constant domain have at least one free thiol group. The F(ab')2 antibody fragments were originally generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0062] Based on the amino acid sequence of their constant structural domains, the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be designated as one of two distinct types, called κ and λ.

[0063] Antibodies can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0064] The "single-chain Fv" or "scFv" antibody fragment contains the antibody's V. H and V L Domains, wherein these domains are present within a single polypeptide chain. In some embodiments, the Fv polypeptide also contains V. H and V L The polypeptide linkers between the domains enable scFv to form the desired structures 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, pp. 269-315 (1994).

[0065] The term "dual antibody" refers to a small antibody fragment with two antigen-binding sites, which includes a light chain variable domain (V) in the same polypeptide chain. L ) connected heavy chain variable structural domain (V H ) (V H -V L By using a linker that is too short to allow pairing between two domains on the same strand, the domain is forced to pair with a complementary domain on another strand, creating two antigen-binding sites. Biantibodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0066] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies that exhibit multi-epitope specificity. Such multispecific antibodies include, but are not limited to, those containing a heavy chain variable domain (V... H ) and light chain variable structural domain (V L Antibodies, of which V H V L The unit exhibits multi-epitope specificity; it has two or more V... L and V H Antibodies with structural domains, each V H V L Multi-epitope specificity refers to the ability to specifically bind two or more different epitopes on the same or different targets. Monospecificity refers to the ability to bind only one epitope. According to one embodiment, a multi-specific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0067] The terms "single-domain antibody" (sdAbs) or "single-variable-domain (SVD) antibody" typically refer to antibodies containing a single variable domain (V). H or V LA single domain can confer antigen binding to an antibody. In other words, a single variable domain does not need to interact with another variable domain to recognize a target antigen. Examples of single-domain antibodies include those from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks) and those 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; WO00 / 29004; WO 02 / 051870).

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies contained in the population are identical and / or bind to the same epitopes, and these variants are typically present in small amounts, except for possible variants that may arise during the production of the monoclonal antibody. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of not being contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the methods provided herein can be prepared by the hybridoma method first described in Kohler et al., Nature 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J.Mol.Biol.222:581-597 (1991).

[0069] The monoclonal antibodies described herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primate-derived” antibodies, which comprise a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, such as baboons, rhesus monkeys, or cynomolgus monkeys) and a human constant region sequence (US Patent No. 5,693,780).

[0070] "Humanized" non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues from the receptor hypervariable region are replaced by residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capacity for hypervariable regions. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. Typically, humanized antibodies will contain at least one, usually both, variable domains, where all or substantially all hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all FRs are those FRs of the human immunoglobulin sequence, except for FR substitutions as described above. Optionally, humanized antibodies will also contain at least a portion of the immunoglobulin constant region, typically the constant region of the human immunoglobulin. For further details, see 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).

[0071] For the purposes of this document, an "intact antibody" is an antibody that comprises heavy and light variable domains as well as an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. Preferably, an intact antibody has one or more effector functions.

[0072] "Natural antibodies" are typically 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 the heavy chain by a covalent disulfide bond, the number of which varies among heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H Following this are numerous constant structural domains. Each light chain has a variable structural domain (V0) at one end. L The light chain has a constant domain at one end and a variable domain at the other. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the variable domains of the light and heavy chains.

[0073] "Naked antibody" is an antibody that is not conjugated to a foreign molecule, such as a cytotoxic moiety or a radiolabeled substance (as defined herein).

[0074] In some implementations, antibody "effective functions" refer to those biological activities attributable to the antibody's Fc region (native sequence Fc region or amino acid sequence variable Fc region) and that vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors.

[0075] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to cell-mediated reactions in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize antibodies bound to target cells, subsequently causing lysis of the target cells. Primary NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of target molecules, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be evaluated in vivo, for example in animal models, such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0076] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred.

[0077] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave its target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (e.g., a peptide, such as an antibody) that is complexed with a homologous 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).

[0078] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of antibodies. In some embodiments, the FcR is a naturally occurring human FcR. Furthermore, preferred FcRs are FcRs that bind IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activating motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain (see [link to relevant documentation]). Annu.Rev.Immunol.15:203-234(1997)). FcRs have been reviewed in 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). The term “FcR” used in this paper covers other FcRs, including those to be identified in the future. The term also includes neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J.Immunol.117:587(1976) and Kim et al., J.Immunol. 24:249(1994)).

[0079] "Impurity" refers to a substance that is different from the desired polypeptide product. In some embodiments of the invention, impurities include charge variants of the polypeptide. In some embodiments of the invention, impurities include charge variants of antibodies or antibody fragments. In other embodiments of the invention, impurities include, but are not limited to: host cell material, such as CHOP; leached protein A; nucleic acids; variants, fragments, aggregates, or derivatives of the desired polypeptide; another polypeptide; endotoxins; viral contaminants; cell culture medium components, etc.

[0080] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence having the desired binding specificity and an immunoglobulin constant domain sequence, wherein the amino acid sequence having the desired binding specificity differs from the antigen recognition and binding site of an antibody (i.e., is "heterologous"). The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that contains at least a receptor or ligand binding site. The immunoglobulin constant domain sequence in an immunoadhesin can be derived 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.

[0081] As used in this article, "surfactant" refers to a surfactant, preferably a nonionic surfactant. Examples of surfactants in this article include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamer (e.g., poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; dodecyl-, myristoyl-, linoleyl-, or stearyl-sulfobetaine; dodecyl-, myristoyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristoyl-, or hexadecyl-betaine; lauroylaminopropyl-, cocamidopropyl-, linoleamide-, myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamide-propyl); myristamidopropyl-, palmitamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl, or disodium methyloleyl-taurate; and MONAQUAT TM Series (Mona Industries, Inc., Paterson, NJ); copolymers of polyethylene glycol, polypropylene glycol, ethylene glycol, and propylene glycol (such as Pluronics, PF68, etc.); in one embodiment, the surfactant herein is polysorbate 20. In another embodiment, the surfactant herein is poloxamer 188.

[0082] As used in this paper with respect to chromatography, the term "sequence" refers to having a first chromatography followed by a second chromatography. Additional steps may be included between the first and second chromatography.

[0083] As used in this paper regarding chromatography, the term "continuous" refers to a first and second chromatographic material that are directly connected, or some other mechanism that allows continuous flow between two chromatographic materials.

[0084] "Spray density" refers to the amount (e.g., grams) of composition that comes into contact with a given volume of chromatographic material (e.g., liters). In some instances, spray density is expressed in g / L.

[0085] As used herein, the term “interference” in relation to the quantification of a class (e.g., nonionic surfactant) refers to the contribution of a component other than the stated class (e.g., peptide) to the quantification. For example, the ELSD signal of a chromatographic fraction containing polysorbate 20 and a peptide will have contributions from both polysorbate 20 and the peptide, and the quantification of polysorbate 20 in the fraction will have interference from the peptide.

[0086] As used in this article, "substantially the same" means that the value or parameter has not been significantly altered by the effect. For example, if the ionic strength does not change significantly, the ionic strength of the chromatographic mobile phase at the column outlet is substantially the same as the initial ionic strength of the mobile phase. For example, the ionic strength at the column outlet within 10%, 5%, or 1% of the initial ionic strength is substantially the same as the initial ionic strength.

[0087] References to “about” values ​​or parameters in this document include (and describe) variations of that value or parameter itself. For example, a description involving “about X” includes a description of “X”.

[0088] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural references unless the context clearly indicates otherwise. It should be understood that aspects and variations of the invention described herein include those “consisting of” and / or “substantially consisting of” aspects and variations.

[0089] II. Chromatography Methods

[0090] In some aspects, the present invention provides a method for analyzing compositions comprising peptides and nonionic surfactants (e.g., polysorbate 20 or PS20), comprising binding the peptides and nonionic surfactants to a mixed-mode ion-exchange chromatography material using a loading buffer, and eluting the peptides and nonionic surfactants from the chromatography material using a buffer, such that the peptides and nonionic surfactants are eluted from the chromatography material in different fractions. In some embodiments, the chromatography method is suitable for compositions comprising multiple peptides (e.g., peptide products), including compositions of peptides with different pIs. For example, the method can be used to analyze compositions comprising nonionic surfactants and many different antibody products (e.g., antibody products with pIs ranging from 6.0 to 9.5). In other embodiments, the chromatography method includes using optimal conditions (e.g., chromatography material, buffer, gradient, step duration, flow rate, sample loading) identified by the methods described herein.

[0091] In some embodiments of any of the methods described herein, the chromatographic material is a mixed-mode material comprising functional groups capable of performing one or more of the following functions: anion exchange, cation exchange, hydrogen bonding, and hydrophobic interactions. In some embodiments, the mixed-mode material is a mixed-mode anion exchange chromatographic material. In some embodiments, the mixed-mode anion exchange chromatographic material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatographic material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatographic material is... MAX chromatography materials. 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 reversed-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... MCX chromatography materials. In some embodiments, the mixed-mode material comprises a solid support. In some embodiments, the mixed-mode material is contained within a column or column body. In some of the embodiments described above, the mixed-mode material is a mixed-mode chromatography column or column body, such as a mixed-mode anion exchange chromatography column or column body, or a mixed-mode cation exchange chromatography column or column body. In some embodiments, the mixed-mode material is a high-performance liquid chromatography (HPLC) material.

[0092] In some embodiments of any of the methods described herein, the ion exchange material may be a conventional chromatographic material or a convection chromatographic material. Conventional chromatographic materials include, for example, perfusion materials (e.g., poly(styrene-divinylbenzene) resin) and diffusion materials (e.g., cross-linked agarose resin). In some embodiments, the poly(styrene-divinylbenzene) resin may be... Resin. In some embodiments, the cross-linked agarose resin may be sulfopropyl- Fast Flow (“SPSFF”) resin. The convection chromatography material can be a membrane (e.g., polyethersulfone) or a bulk material (e.g., a crosslinked polymer). The polyethersulfone membrane can be Mustang. The crosslinked polymer bulk material can be crosslinked poly(glycidyl methacrylate-copolymer-ethylenedimethacrylate).

[0093] In some embodiments of any method of the present invention, the chromatographic material is in a chromatographic column or column body; for example, a mixed-mode cation exchange chromatographic column or column body or a mixed-mode anion exchange chromatographic column or column body. In some embodiments, the chromatographic column or column body is used for liquid chromatography. In some embodiments, the chromatographic column or column body is used for high-performance liquid chromatography (HPLC). In some embodiments, the chromatographic column or column body is an HPLC chromatographic column or column body; for example, a mixed-mode cation exchange HPLC column or column body or a mixed-mode anion exchange HPLC column or column body.

[0094] For example, in some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the peptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the peptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the peptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less) of the total peptides 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of a methanol solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid.

[0095] In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values).

[0096] In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., at least about 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). In some embodiments, the nonionic surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TMDrug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0097] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the steps of: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the peptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c), wherein the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%). Interference from the polypeptide is 0.1%, 0.05%, 0.01%, or less. In some embodiments, the polypeptide binds specifically and non-specifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 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 (e.g., any one of about 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 (e.g., any one of about 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 implementations, the third ratio is between about 80:20 and about 100:0 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of a methanol solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., any one of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). In some embodiments, the nonionic surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, the therapeutic peptide is a fusion protein, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography materials. In some implementations, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0098] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the peptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c), wherein the eluent contains less than about 10% (e.g., less than any one of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less) of the total peptide 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of acid. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid.In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., at least about 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). In some embodiments, the nonionic surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 stabilizers, buffers, and tensioning agents.In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of interference from peptides.

[0099] In some embodiments, a method is provided for quantifying the nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) eluting the peptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the peptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the peptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less) of the total peptides 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of acetic acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). In some embodiments, the nonionic surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0100] In some embodiments, a method is provided for quantifying the nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the steps of: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material comprising a solution of mobile phase B and mobile phase A in a first ratio of about 5:95 to about 15:85, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the peptide from the mixed-mode anion exchange chromatography material with a solution comprising mobile phase B and mobile phase A in a second ratio of about 35:65 to about 45:55; c) eluting the nonionic surfactant from the chromatography material with a solution comprising mobile phase B and mobile phase A in a third ratio of about 90:10 to about 100:0; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 40:60. In some embodiments, the third ratio is about 100:0. In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 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 about 0.5% to about 5% (v / v) of an aqueous solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of a methanol solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some implementations, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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).In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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) after the start of step a) and lasts for at least about 1 minute (e.g., at least about 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). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). In some embodiments, the nonionic surfactant is poloxamer (P188) or polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the nonionic surfactant in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide.In some implementations, the therapeutic peptide is a fusion protein, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0101] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and a polypeptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the polypeptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less) of the total peptides 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of an acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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) after the start of step a) and lasts for at least about 1 minute (e.g., at least about 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). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., any one of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0102] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising 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 added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) washing the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A. The steps are: c) removing the polypeptide, wherein the second proportion is greater than the first proportion; c) eluting polysorbate from the chromatographic material with a solution containing a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c), wherein the quantification of the nonionic surfactant includes less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from the polypeptide. In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91%, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of a methanol solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, the therapeutic peptide is a fusion protein, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography materials. In some implementations, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0103] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the polypeptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c), wherein the eluent contains less than about 10% (e.g., less than any one of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less). 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of acid. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of acid. In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid.In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., any one of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioning agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject.In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0104] In some embodiments, a method for quantifying polysorbate in a composition comprising polysorbate and a polypeptide is provided, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) eluting the polypeptide from the mixed-mode anion exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of acetic acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., any one of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0105] In some embodiments, a method for quantifying polysorbate in a composition comprising polysorbate and a polypeptide is provided, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution of mobile phase B and mobile phase A in a first ratio between about 5:95 and about 15:85, wherein mobile phase A comprises an aqueous solution of an acid and mobile phase B comprises a methanol solution of an acid; b) eluting the polypeptide from the mixed-mode anion exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between about 35:65 and about 45:55; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between about 90:10 and about 100:0; d) quantifying the polysorbate in the eluent of step c). In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 40:60. In some embodiments, the third ratio is approximately 100:0. In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 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 about 0.5% to about 5% (v / v) of an aqueous solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of a methanol solution of an acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the acid is formic acid. In some embodiments, the acid is acetic acid. In some implementations, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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).In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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) after the start of step a) and lasts for at least about 1 minute (e.g., at least about 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). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., any one of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TMOr THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0106] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and a polypeptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution of mobile phase B and mobile phase A in a first ratio between about 5:95 and about 15:85, wherein mobile phase A comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) eluting the polypeptide from the mixed-mode anion exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between about 35:65 and about 45:55; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between about 90:10 and about 100:0; d) quantifying the polysorbate in the eluent of step c), wherein the quantification of the nonionic surfactant comprises less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%). Interference from the polypeptide (any one or less, 1%, 0.5%, 0.1%, 0.05%, 0.01%). In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 40:60. In some embodiments, the third ratio is about 100:0. In some embodiments, the polypeptide binds specifically and non-specifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 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 contains about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of acetic acid. In some embodiments, mobile phase B contains about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanolic solution of acetic acid. In some implementations, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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).In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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) after the start of step a) and lasts for at least about 1 minute (e.g., at least about 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). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., any one of 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB.TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography materials. In some implementations, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0107] In some embodiments, a method for quantifying polysorbate in a composition comprising polysorbate and a polypeptide is provided, wherein the method comprises the following steps: a) applying the composition to a mixed-mode anion exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising mobile phase B and mobile phase A in a first ratio between about 5:95 and about 15:85, wherein mobile phase A comprises an aqueous solution of acetic acid and mobile phase B comprises a methanol solution of acetic acid; b) eluting the polypeptide from the mixed-mode anion exchange chromatography material with a solution comprising mobile phase B and mobile phase A in a second ratio between about 35:65 and about 45:55; c) eluting the polysorbate from the chromatography material with a solution comprising mobile phase B and mobile phase A in a third ratio between about 90:10 and about 100:0; d) quantifying the polysorbate in the eluent of step c) wherein the polypeptide from step c) The eluent contains less than about 10% (e.g., less than any one of about 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 about 10:90. In some embodiments, the second ratio is about 40:60. In some embodiments, the third ratio is about 100:0. In some embodiments, mobile phase A contains about 0.5% to about 5% (v / v) of an aqueous solution of acetic acid (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of acetic acid. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL.In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., at least about 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TMDrug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of interference from peptides.

[0108] In some embodiments, a method is provided for quantifying polysorbate 20 in a composition comprising 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 added to the chromatography material comprising a mobile phase B and a mobile phase A in a ratio of about 10:90, wherein mobile phase A comprises an aqueous solution of about 2% acetic acid and mobile phase B comprises a methanol solution of about 2% acetic acid; b) using a mixture comprising about 40:60... The process involves: c) eluting the polypeptide from a mixed-mode anion exchange chromatography material using a solution of mobile phase B in a ratio of approximately 100:0 to mobile phase A; d) quantifying the polysorbate 20 in the eluent from step c), wherein in some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 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 flow rate of the chromatography is about 0.5 to 2.5 (e.g., about 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 / min. In some embodiments, the flow rate of the chromatography is about 1.25 mL / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, the volume of the composition applied to the chromatographic material is about 20 μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 1 minute (e.g., at least about 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 any one of more) after step a). In some implementations, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 0.5 minutes (e.g., at least about 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3 or more).In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic peptide is a fusion protein, polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode anion exchange chromatography material comprises a reversed-phase strong anion exchange polymer. In some embodiments, the mixed-mode anion exchange chromatography material comprises a quaternary ammonium moiety. In some embodiments, the mixed-mode anion exchange chromatography material comprises a solid-phase support. In some embodiments, the mixed-mode anion exchange chromatography material is contained in a column or column body. 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... MAX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of interference from peptides.

[0109] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less) of the total peptides in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one of more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, or any one of more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 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). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 contains N-acetyltryptophan (also known as N-acetyl-DL-tryptophan) and / or methionine.In some embodiments, the concentration of N-acetyltryptophan in the composition is from about 0.1 mM to about 10 mM (e.g., any one of about 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 concentration of methionine in the composition is from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0110] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the steps of: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the peptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c), wherein the quantification of the nonionic surfactant includes less than about 10% (e.g., less than any one or less of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from the peptide. In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 implementations, the third ratio is between about 80:20 and about 100:0 (e.g., any one of about 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 about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of ammonium hydroxide. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, or any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 further 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 (e.g., any one of about 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 concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography materials. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0111] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the steps of: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the peptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c), wherein the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%). The total polypeptide in the composition is 1%, 0.5%, 0.1%, 0.05%, 0.01% or less. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of ammonium hydroxide. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an organic solvent (e.g., methanol or acetonitrile) solution of ammonium hydroxide.In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 contains 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 (e.g., any one of about 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 concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0112] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises a methanol solution of ammonium hydroxide; b) eluting the peptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the peptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the peptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total peptides 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of ammonium hydroxide. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 contains 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 (e.g., any one of about 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 concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or any lower) of peptide interference.

[0113] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a peptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) eluting the peptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the peptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the peptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total peptides 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 contains 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 (e.g., any one of about 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 concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than any one or lower of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of peptide interference.

[0114] In some embodiments, a method is provided for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant 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 added to the chromatography material comprising a solution of mobile phase B and mobile phase A in a first ratio between approximately 5:95 and approximately 15:85, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between approximately 40:60 and approximately 50:50; c) eluting the nonionic surfactant from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between approximately 90:10 and approximately 100:0; and d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 implementations, the third ratio is between about 80:20 and about 100:0 (e.g., any one of about 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 about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of ammonium hydroxide. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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, any one or more) after the start of step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 further 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 (e.g., any one of about 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 concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of nonionic surfactants includes less than about 10% (e.g., less than any one or lower of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of peptide interference.

[0115] When products containing N-acetyltryptophan (NAT) in formulations were tested using HPLC-ELSD conditions, significant interference was observed in the PS20 region. Therefore, in some cases, alternative conditions are needed to eliminate NAT and protein-related interference.

[0116] In some embodiments, a method is provided for quantifying the nonionic surfactant in a composition comprising a nonionic surfactant, a peptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the peptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the nonionic surfactant from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the nonionic surfactant in the eluent of step c). In some embodiments, the peptide binds specifically and nonspecifically to the chromatography material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the peptide is eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total peptides in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than any one or less of about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 implementations, the third ratio is between about 80:20 and about 100:0 (e.g., any one of about 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 about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of ammonium hydroxide. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after the start of step a) and continues for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, or any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the nonionic surfactant is poloxamer. In some embodiments, the poloxamer is poloxamer P188. In some embodiments, the concentration of the nonionic surfactant (e.g., polysorbate or poloxamer) in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the nonionic surface detergent is quantified to contain less than about 10% (e.g., less than any one or lower of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of peptide interference.

[0117] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate and a polypeptide, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total polypeptide in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate of chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than any one or lower of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of peptide interference.

[0118] In some embodiments, a method is provided for quantifying 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 added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c), wherein the quantification of the polysorbate comprises less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%). Interference from peptides and NATs (any one or less) of 0.1%, 0.05%, 0.01%. In some embodiments, the peptides bind specifically and non-specifically to the chromatographic material, and at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the peptides are eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of the total peptides in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than any one or less of about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 implementations, the second ratio is between about 35:65 and about 55:45 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography materials. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0119] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, a polypeptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c), wherein the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%). The total polypeptide in the composition is 0.1%, 0.05%, 0.01% or less, and the total NAT in the composition is less than about 5% (e.g., less than about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less). In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of 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 (e.g., any one of about 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 (e.g., any one of about 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 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 an organic solvent (e.g., methanol or acetonitrile). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL. In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine.In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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).

[0120] In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tension agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from peptides and NAT.

[0121] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, a polypeptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises a methanol solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 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 (e.g., any one of about 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 (e.g., any one of about 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 implementations, the third ratio is between about 80:20 and about 100:0 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of a methanol solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after the start of step a) and continues for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, or any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine.In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from peptides and NAT.

[0122] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, a polypeptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising a first proportion of mobile phase B and mobile phase A, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising a second proportion of mobile phase B and mobile phase A, wherein the second proportion is greater than the first proportion; c) eluting the polysorbate from the chromatography material with a solution comprising a third proportion of mobile phase B and mobile phase A, wherein the third proportion is greater than the second proportion; d) quantifying the polysorbate in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 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 (e.g., any one of about 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 (e.g., any one of about 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 implementations, the third ratio is between about 80:20 and about 100:0 (e.g., any one of about 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 about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of an acetonitrile solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after the start of step a) and continues for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, or any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of polysorbate is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine.In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from peptides and NAT.

[0123] In some embodiments, a method is provided for quantifying 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 added to the chromatography material in a solution of mobile phase B and mobile phase A in a first ratio between approximately 5:95 and approximately 15:85, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between approximately 40:60 and approximately 50:50; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between approximately 90:10 and approximately 100:0; and d) quantifying the polysorbate in the eluent of step c). In some embodiments, the polypeptide binds specifically and nonspecifically to the chromatographic material, and at least about 90% (e.g., at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of the polypeptide is eluted in step b). In some embodiments, the eluent from step c) contains nonspecifically bound polypeptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total polypeptide in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% or less) of the total NAT in the composition. In some embodiments, the organic solvent of mobile phase B is methanol. In some embodiments, the organic solvent of mobile phase B is acetonitrile. In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 45:55. In some embodiments, the third ratio is about 100:0. In some embodiments, mobile phase A comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an aqueous solution of ammonium hydroxide. In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of an organic solvent (e.g., methanol or acetonitrile) for ammonium hydroxide.In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values).

[0124] In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tension agents. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from peptides and NAT.

[0125] In some embodiments, a method is provided for quantifying 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 added to the chromatography material in a solution of mobile phase B and mobile phase A in a first ratio between approximately 5:95 and approximately 15:85, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises a methanol solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between approximately 40:60 and approximately 50:50; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between approximately 90:10 and approximately 100:0; d) quantifying the polysorbate in the eluent of step c), wherein the quantification of the polysorbate comprises less than about 10% (e.g., less than about 9%, 8%, 7%). Interference from peptides and NATs (any one or less) of 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% is present. In some embodiments, the peptides bind specifically and non-specifically to the chromatographic material, and at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the peptides are eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% or less) of the total peptides in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than any one or less of about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 45:55. In some embodiments, the third ratio is about 100:0. In some embodiments, mobile phase A contains about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B contains about 0.5% to about 5% (v / v) of a methanol solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography materials. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0126] In some embodiments, a method is provided for quantifying polysorbate in a composition comprising polysorbate, a polypeptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material comprising a solution of mobile phase B and mobile phase A in a first ratio between approximately 5:95 and approximately 15:85, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises a methanol solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between approximately 40:60 and approximately 50:50; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between approximately 90:10 and approximately 100:0; d) quantifying the polysorbate in the eluent of step c), wherein the eluent from step c) comprises less than about 10% (e.g., less than about 9%, 8%). The composition contains 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less of total polypeptides and less than about 5% (e.g., less than about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less of total NAT) in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 45:55. In some embodiments, the third ratio is about 100:0. In some embodiments, mobile phase A comprises about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values) of a methanol solution of ammonium hydroxide. In some embodiments, the flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL.In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more) after step a). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 stabilizers, buffers, and tensioning agents.In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, the nonionic detergent is quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of interference from the peptide.

[0127] In some embodiments, a method is provided for quantifying 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 added to the chromatography material comprising a solution of mobile phase B and mobile phase A in a first ratio between approximately 5:95 and approximately 15:85, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between approximately 40:60 and approximately 50:50; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between approximately 90:10 and approximately 100:0; d) quantifying the polysorbate in the eluent of step c), wherein the quantification of the polysorbate comprises less than about 10% (e.g., less than about 9%, 8%, 7%). Interference from peptides and NATs (any one or less) of 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% is present. In some embodiments, the peptides bind specifically and non-specifically to the chromatographic material, and at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the peptides are eluted in step b). In some embodiments, the eluent from step c) contains non-specifically bound peptides. In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% or less) of the total peptides in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than any one or less of about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of total NAT in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 45:55. In some embodiments, the third ratio is about 100:0. In some embodiments, mobile phase A contains about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) of an acetonitrile solution of ammonium hydroxide (e.g., any one of about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values).In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., any one of about 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). In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography materials. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

[0128] In some embodiments, a method is provided for quantifying 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 added to the chromatography material comprising a solution of mobile phase B and mobile phase A in a first ratio between approximately 5:95 and approximately 15:85, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an acetonitrile solution of ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution of mobile phase B and mobile phase A in a second ratio between approximately 40:60 and approximately 50:50; c) eluting the polysorbate from the chromatography material with a solution of mobile phase B and mobile phase A in a third ratio between approximately 90:10 and approximately 100:0; d) quantifying the polysorbate in the eluent of step c), wherein the eluent from step c) comprises less than about 10% (e.g., less than about 9%, 8%). The composition contains 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less of total polypeptides and less than about 5% (e.g., less than about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less of total NAT) in the composition. In some embodiments, the first ratio of mobile phase B to mobile phase A is about 10:90. In some embodiments, the second ratio is about 45:55. In some embodiments, the third ratio is about 100:0. In some embodiments, mobile phase A comprises about 0.5% to about 5% (v / v) of an aqueous solution of ammonium hydroxide (e.g., about 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%, including any range between these values). In some embodiments, mobile phase B comprises about 0.5% to about 5% (v / v) (e.g., any one of about 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 flow rate for chromatography is about 0.5 to 2.5 (e.g., any one of about 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 / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values) μL.In some implementations, step b) begins at least about 0.5 minutes (e.g., at least about 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 any one or more) after step a) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5 any one or more). In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, any one or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 2.2, 2.4, 2.6, 2.8, 3, 3.1, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, any one or more). 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) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 the peptide in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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 stabilizers, buffers, and tensioning agents.In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the therapeutic protein is a polyclonal antibody, monoclonal antibody, humanized antibody, human antibody, chimeric antibody, multispecific antibody, glycoengineered antibody, antibody fragment, antibody-drug conjugate, or THIOMAB. TM Or THIOMAB TM Drug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of peptide interference.

[0129] In some embodiments, a method is provided for quantifying polysorbate 20 in a composition comprising polysorbate 20, a polypeptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material comprising a mobile phase B and a mobile phase A in a ratio of about 10:90, wherein mobile phase A comprises about 1.5% aqueous ammonium hydroxide and mobile phase B comprises a methanol solution of about 1.5% ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising mobile phase B and mobile phase A in a ratio of about 45:55; c) eluting polysorbate 20 from the chromatography material with a solution comprising mobile phase B and mobile phase A in a ratio of about 100:0; d) quantifying polysorbate 20 in the eluent of step c). In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total peptide in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than any one or less of about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total NAT in the composition. In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., about any one 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). In some embodiments, the flow rate of the chromatography is about 1.40 mL / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, the volume of the composition applied to the chromatographic material is about 25 μL. In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least any one or more of about 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) after the start of step a) and lasts for at least about 2 minutes (e.g., at least any one or more of about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5).In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 2 minutes (e.g., 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). In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TMDrug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of interference from peptides and NAT.

[0130] In some embodiments, a method is provided for quantifying polysorbate 20 in a composition comprising polysorbate 20, a polypeptide, and N-acetyltryptophan, wherein the method comprises the following steps: a) applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material comprising a mobile phase B and a mobile phase A in a ratio of about 10:90, wherein mobile phase A comprises about 1.5% aqueous ammonium hydroxide and mobile phase B comprises an acetonitrile solution of about 1.5% ammonium hydroxide; b) eluting the polypeptide from the mixed-mode cation exchange chromatography material with a solution comprising mobile phase B and mobile phase A in a ratio of about 45:55; c) eluting polysorbate 20 from the chromatography material with a solution comprising mobile phase B and mobile phase A in a ratio of about 100:0; d) quantifying polysorbate 20 in the eluent of step c). In some embodiments, the eluent from step c) contains less than about 10% (e.g., less than any one or less of about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total peptide in the composition. In some embodiments, the eluent from step c) contains less than about 5% (e.g., less than any one or less of about 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) of the total NAT in the composition. In some embodiments, the flow rate of the chromatography is about 0.5 to 2.5 mL / min (e.g., about any one 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). In some embodiments, the flow rate of the chromatography is about 1.40 mL / min. In some embodiments, the volume of the composition applied to the chromatographic material is about 1 to about 50 μL (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45, including any range between these values). In some embodiments, the volume of the composition applied to the chromatographic material is about 25 μL. In some embodiments, step b) begins at least about 0.5 minutes (e.g., at least any one or more of about 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) after the start of step a) and lasts for at least about 2 minutes (e.g., at least any one or more of about 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5).In some embodiments, step c) begins at least about 0.05 minutes (e.g., at least about 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or more) after step b) and lasts for at least about 2 minutes (e.g., at least about 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). In some embodiments, the concentration of polysorbate 20 in the composition is in the range of about 0.001% to 1.0% (w / v) (e.g., any one of about 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 (e.g., any one of about 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 contains methionine. In some embodiments, the concentration of methionine in the composition ranges from about 0.1 mM to about 100 mM (e.g., any one of about 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 peptide concentration in the composition is from about 1 mg / mL to about 250 mg / mL (e.g., any one of about 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 pH of the composition is from about 4.5 to about 7.5 (e.g., any one of about 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, and 7.4, including any range between these values). In some embodiments, the composition further comprises one or more excipients selected from stabilizers, buffers, and tensioners. In some embodiments, the composition is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the peptide is a therapeutic peptide. In some implementations, 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, or THIOMAB. TM Or THIOMAB TMDrug conjugates. In some embodiments, the mixed-mode cation exchange chromatography material comprises a reversed-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-phase 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... MCX chromatography material. In some embodiments, nonionic detergents are quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD). In some embodiments, the quantification of polysorbate contains less than about 10% (e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or less) of interference from peptides and NAT.

[0131] In some embodiments of any of the methods described above, the nonionic surfactant is quantified in the composition containing the nonionic surfactant before the peptide is added to the composition. In some embodiments, the concentration of the nonionic surfactant in the composition will be higher before the peptide is added (e.g., the nonionic surfactant in the composition is diluted upon addition of the peptide). In some embodiments of any of the methods described above, the nonionic surfactant is quantified in a composition containing the nonionic surfactant but not the peptide. This quantification can be used as a control or comparative for compositions containing both the nonionic surfactant and the peptide.

[0132] In some embodiments of any of the methods described above, a sample of the composition to be analyzed is added to the autosampler of a chromatography instrument (e.g., an HPLC instrument). In some embodiments, the sample in the autosampler is refrigerated (e.g., 5 ± 3 °C). In some embodiments, one or more columns containing the chromatographic material are placed in the column chamber of the chromatography instrument. In some embodiments, temperature control features may be employed to maintain the column chamber temperature within a narrow range (e.g., ± 1 °C) from a set point during analysis. In some embodiments, column effluent is monitored at 280 nm.

[0133] In some embodiments of any of the above methods, the composition sample to be analyzed is diluted with loading buffer to a target peptide concentration of about 0.1 mg / mL to about 75 mg / mL (e.g., any one of about 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).

[0134] In some embodiments of any of the methods described above, the chromatography instrument includes a gradient pump (e.g., a low-pressure quaternary gradient pump), an autosampler (e.g., an autosampler with temperature control), a column chamber (e.g., a thermally controlled column chamber), a UV detector (e.g., a diode array UV detector), and an evaporative light scattering detector (ELSD). In some embodiments, the chromatography instrument also includes pH and conductivity monitors (e.g., PCM-3000) to collect pH and conductivity data in real time. Instrument control, data acquisition, and data analysis are performed using appropriate software (e.g., JMP10).

[0135] In some embodiments of the invention, the ionic strength of the mobile phase, for example, the elution buffer, is measured by the conductivity of the mobile phase. Conductivity refers to the ability of an aqueous solution to conduct current between two electrodes. In a solution, current flows through ion transport. Therefore, as the amount of ions present in an aqueous solution increases, the solution will have a higher conductivity. The basic unit of conductivity is Siemens (or mH0), mH0 (mS / cm), and it can be measured using a conductivity meter, such as various models of Orion conductivity meters. Since electrolytic conductivity is the ability of ions in a solution to carry current, the conductivity of the solution can be changed by altering the concentration of ions therein. For example, the concentration of the buffer and / or the salt (e.g., sodium chloride, sodium acetate, or potassium chloride) in the solution can be changed to obtain the desired conductivity. Preferably, the salt concentration of various buffers is changed to obtain the desired conductivity.

[0136] In some embodiments, the mobile phase for chromatography has a concentration greater than approximately 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, and 16 mS / cm. An initial conductivity of any one of 17.0 mS / cm, 18.0 mS / cm, 19.0 mS / cm, or 20.0 mS / cm. In some embodiments, the conductivity of the mobile phase increases during chromatography, for example, through an ionic strength gradient. In some embodiments, the conductivity of the mobile phase at the completion of elution is greater than approximately 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, and 16 mS / cm. The conductivity is any one of 17.0 mS / cm, 18.0 mS / cm, 19.0 mS / cm, or 20.0 mS / cm. In some embodiments, the conductivity of the mobile phase increases via a linear gradient. In some embodiments, the conductivity of the mobile phase increases via a stepwise gradient comprising one or more steps.

[0137] In some embodiments of any of the methods described herein, a composition comprising a polypeptide and a nonionic surfactant is added to the chromatographic material in an amount of polypeptide greater than any one 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 μg. In some embodiments, the composition is added to the chromatographic 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 being added to the chromatographic material; for example, at a dilution of 1:1, 1:2, 1:5, 1:10, or greater 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 a loading buffer.

[0138] In some embodiments of the methods described herein, the chromatographic material is contained in a column or column body. In some embodiments, the column is an HPLC column or column body. The column or column body may have any size compatible with the chromatographic instrument. For example, in some embodiments, the column or column body has any of the following dimensions: 2.1 x 20 mm, 4 × 50 mm, 4 × 100 mm, 4 × 150 mm, 4 × 200 mm, 4 × 250 mm, or 2 × 250 mm.

[0139] III. Polypeptides

[0140] The peptides are provided for use in any ion exchange chromatography method, wherein the separation conditions are optimized as described herein. In some embodiments of the invention, compositions of peptides are analyzed by ion exchange chromatography. Such methods can be used to identify charge variants of the peptides in the composition. In some embodiments, the peptide is an antibody or a fragment thereof. In some embodiments, the peptide has a pI of about 6.0 to about 9.5. In some embodiments, the peptide is an antibody with a pI range of about 6.0 to about 9.5. In some embodiments, the inflection point (IP) of the charge-to-pH profile of the peptide is provided by the method of the invention. In some embodiments, the change of IP as a function of temperature (dIP / dT) is provided by the method of the invention.

[0141] In some embodiments, the peptide is a therapeutic peptide. In some embodiments, the peptide is an antibody. In some embodiments, the peptide is an immunoadhesive.

[0142] In some embodiments, the polypeptide has a molecular weight greater than any one of about 5,000 Daltons, 10,000 Daltons, 15,000 Daltons, 25,000 Daltons, 50,000 Daltons, 75,000 Daltons, 100,000 Daltons, 125,000 Daltons, or 150,000 Daltons. The polypeptide may have a molecular weight between about 50,000 Daltons and 200,000 Daltons or between 100,000 Daltons and 200,000 Daltons. Alternatively, the polypeptide used herein may have a molecular weight of about 120,000 Daltons or about 25,000 Daltons.

[0143] pI is the isoelectric point and the pH at which a particular molecule or surface does not carry a net charge. In some embodiments, the methods of the present invention can be used in various compositions comprising peptides, wherein the peptides, such as antibodies, in the composition have a pI range of about 6.0 to about 9.5. In some embodiments, the pI of the peptide is greater than about 9.5; for example, about 9.5 to about 12. In some embodiments of any of the methods described herein, the pI of the peptide, such as an antibody, may be less than about 7; for example, about 4 to about 7.

[0144] In embodiments of any of the methods described herein, one or more contaminants in a composition comprising a peptide and one or more contaminants are peptide charge variants. In some embodiments, the peptide charge variant is a peptide that has been modified from its native state, thereby altering the peptide's charge. In some embodiments, the charge variant is more acidic than the parent peptide; i.e., has a lower pI than the parent peptide. In other embodiments, the charge variant is more basic than the parent peptide; i.e., has a higher pI than the parent peptide. In some embodiments, the peptide charge variant is engineered. In some embodiments, the peptide charge variant is the result of a natural process; for example, oxidation, deamidation, C-terminal processing of lysine residues, N-terminal pyroglutamate formation, and glycosylation. In some embodiments, the peptide charge variant is a glycoprotein in which a protein-linked glycan is modified such that the glycoprotein's charge is altered compared to the parent glycoprotein; for example, by adding sialic acid or a derivative thereof. In some embodiments, the peptide charge variant is an antibody charge variant.

[0145] Peptides analyzed using the methods described herein are typically generated using recombinant techniques. Methods for generating recombinant proteins are described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, which are expressly incorporated herein by reference. In some embodiments, the target protein is generated in CHO cells (see, for example, WO 94 / 11026). In some embodiments, the target peptide is generated in *E. coli* cells. See, for example, U.S. Patent Nos. 5,648,237; 5,789,199 and 5,840,523, which describe translation initiation regions (TIRs) and signaling sequences for optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*. When using recombinant techniques, peptides can be generated intracellularly, in the periplasmic space, or secreted directly into the culture medium.

[0146] Peptides can be recovered from culture medium or host cell lysates. Cells used to express peptides can be disrupted by various physical or chemical methods, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis agents. If the peptide is produced intracellularly, as a first step, particulate debris from the host cell or lysate is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for isolating peptides secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and benzyl sulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. In the case of peptide secretion into culture medium, the supernatant from such expression systems is usually concentrated first using a commercially available peptide concentrator, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0147] In some embodiments, the peptide in the composition comprising the peptide and one or more contaminants has been purified or partially purified prior to analysis by the method of the present invention. For example, the peptide obtained by this method is in the eluent from affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, and hydrophobic interaction chromatography. In some embodiments, the peptide is in the eluent from protein A chromatography.

[0148] Examples of peptides that can be analyzed by the methods of the present invention include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fc-containing proteins, immune conjugates, cytokines, and interleukins.

[0149] (A) Antibody

[0150] In some embodiments of any of the methods described herein, the peptide used in any method for analyzing peptides and formulations containing peptides by the methods described herein is an antibody.

[0151] The molecular targets of antibodies include (i) CD proteins and their ligands, such as, but not limited to: CD3, CD4, CD8, CD19, CD11a, CD20, CD22, CD27, CD28, CD34, CD40, CD79α (CD79a), CD79β (CD79b), CD122, and CD137; (ii) cytokines, such as, but not limited to: IL-13, IL-17, IL-22, and IL-33; and (iii) members of the ErbB receptor family, such as the EGF receptor, HER2, HER3 or HER4 receptors; (iv) cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin, including their α or β subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); (v) growth factors such as VEGF; TGFβ, IgE; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptors; mpl receptors; CTLA-4; protein C, BR3, c-met, tissue factor, β7, etc.; (vi) immunomodulatory proteins such as OX40, GITR, ICOS, PD-1, PD-L1, PD-L2, LAG3, TIM-3, and VISTA; (vii) cell surface and transmembrane tumor-associated antigens (TAAs), such as those described in U.S. Patent No. 7,521,541, including but not limited to NaPi2b.

[0152] Other exemplary antibodies include, but are not limited to, those selected from, but not limited to, the following: anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-HER-2 / neu antibodies, anti-EGFR antibodies, anti-TGFβ antibodies, anti-OX40 antibodies, anti-GITR antibodies, anti-ICOS antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-TIM-3 antibodies, anti-VISTA antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and anti-CD5 antibodies. Antibodies, including anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CD11a antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, and anti-CD95 / Fas antibody. Antibodies, including anti-CD99 antibody, anti-CD106 antibody, anti-CD122 antibody, anti-CD137 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-StaphA antibody, anti-FcRH5 antibody, anti-Ly6E antibody, anti-STEAP antibody, anti-FluB antibody, anti-VEGF antibody, anti-Ang2 antibody, anti-FGFR1 antibody, anti-KLB antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-κ light chain antibody, anti-λ light chain antibody, anti-melanosome antibody, anti-prostate-specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, anti-Tn antigen antibody, and MetMab.

[0153] (i) Monoclonal antibodies

[0154] In some implementations, the antibody is a monoclonal antibody. Monoclonal antibodies are derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies contained in the group are identical and / or bind to the same epitopes, except for possible variants generated during the production of the monoclonal antibody, which are typically present in small amounts. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete or polyclonal antibodies.

[0155] For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by the recombinant DNA method (US Patent No. 4,816,567).

[0156] In the hybridoma approach, mice or other suitable host animals, such as hamsters, are immunized as described herein to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to peptides used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0157] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0158] In some embodiments, the myeloma cells are efficiently fused cells that support stable, high-level antibody production via selected antibody-producing cells and are sensitive to culture media such as HAT medium. In some embodiments, the myeloma cell line is a mouse myeloma cell line, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heterologous myeloma cell lines 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)).

[0159] The production of monoclonal antibodies against antigens in the culture medium in which hybridoma cells are grown is measured. In some embodiments, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0160] The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0161] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clone can be subcloned using a limiting dilution procedure and cultured using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 media. Additionally, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0162] Monoclonal antibodies secreted by subclones are purified using conventional immunoglobulin purification methods (e.g., peptide A-). Hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography (HAP) can be used to separate the fluid from the culture medium, ascites, or serum as appropriate.

[0163] DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). In some implementations, hybridoma cells are used as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin peptides, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., *Curr. Opinion in Immunol.* 5:256-262 (1993) and Plückthun, *Immunol. Revs.* 130:151-188 (1992).

[0164] In another implementation, antibodies or antibody fragments can be isolated from an antibody phage library 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. 222:581-597 (1991) describe the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications describe the generation of high-affinity (nM range) human antibodies via chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), as well as combined infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0165] DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (US Patent No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently linking immunoglobulin coding sequences to all or part of the coding sequences of non-immunoglobulin polypeptides.

[0166] Typically, these non-immunoglobulin peptides replace the constant domain of an antibody, or they replace the variable domain of an antigen-binding site of an antibody to produce chimeric bivalent antibodies, which contain an antigen-binding site specific to one antigen and another antigen-binding site specific to a different antigen.

[0167] 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.

[0168] (ii) Humanized antibodies

[0169] In some embodiments, the antibody is a humanized antibody. Methods for humanizing nonhuman antibodies have been described in the art. In some embodiments, the humanized antibody has one or more amino acid residues from a nonhuman source introduced therein. These nonhuman amino acid residues are generally referred to as “input” residues, which are typically derived from the “input” variable domain. Humanization can be performed substantially as described by Winter and colleagues (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)) by replacing the corresponding sequence of the human antibody with a hypervariable region sequence. Thus, such a “humanized” antibody is a chimeric antibody (US Patent No. 4,816,567) in which substantially less than the complete human variable domain has been replaced by a corresponding sequence from a nonhuman species. In practice, humanized antibodies are usually human antibodies, with some hypervariable residues and possibly some FR residues replaced by residues from similar sites in rodent antibodies.

[0170] The selection of human variable domains (light and heavy chains) used to prepare humanized antibodies is crucial for reducing antigenicity. According to the so-called "best-fit" method, sequences of variable domains for rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent is then accepted as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another approach uses specific framework regions of common sequences from all human antibodies derived from specific subgroups of the light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0171] More importantly, the antibody is humanized, retaining its high affinity for the antigen and other favorable biological properties. To achieve this, in some embodiments of this method, humanized antibodies are prepared using three-dimensional models of the parental and humanized sequences via analytical methods of the parental sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs exist that can illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examining these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the receptor and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. Typically, hypervariable region residues are directly and fundamentally involved in influencing antigen binding.

[0172] (iii) Human antibodies

[0173] In some implementations, the antibody is a human antibody. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that, upon immunization, can produce a complete library of human antibodies in the absence of endogenous immunoglobulin production. For example, antibody heavy chain linkers (J) in chimeric and germline mutant mice have been described. HHomozygous deletion of the gene leads to complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice will result in the production of human antibodies after antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and U.S. Patent Nos. 5,591,669; 5,589,369; and 5,545,807.

[0174] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from a gene library of immunoglobulin variable (V) domains from non-immunized donors. According to this technology, antibody V domain genes are cloned frame-compliantly into the major or minor coat polypeptide genes of filamentous phages such as M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on antibody functional characteristics also leads to the selection of genes encoding antibodies that display these characteristics. Thus, phages mimic some characteristics of B cells. Phage display can be performed in various forms; a review can be found, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated multiple antibodies from a small randomized library of the V gene derived from the spleen of immunized mice. Zyrazole antibodies. A V gene library derived from non-immunized human donors can be constructed, and antibodies against a variety of antigens, including autoantigens, can be isolated substantially according to the techniques described in Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0175] Human antibodies can also be produced by activated B cells in vitro (see U.S. Patents 5,567,610 and 5,229,275).

[0176] (iv) Antibody fragments

[0177] In some implementations, the antibody is an antibody fragment. Various techniques have been developed for generating antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be generated directly from recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from *E. coli* and chemically conjugated to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for generating antibody fragments are readily apparent to those skilled in the art. In other implementations, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The antibody fragment may also be a “linear antibody,” for example, as described in U.S. Patent 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0178] In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, scFv, Fv, and biantibodies.

[0179] (v) Bispecific antibodies

[0180] In some embodiments, the antibody is a bispecific antibody. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. An exemplary bispecific antibody may bind to two different epitopes. Alternatively, the binding arm of a bispecific antibody may be combined with an arm that binds to triggering molecules on leukocytes (e.g., T cell receptor molecules (e.g., CD2 or CD3), or Fc receptors (FcγR) of IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)) to focus cellular defense mechanisms on the cell. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody).

[0181] Methods for preparing bispecific antibodies are known in the art. Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). Due to the random sorting of the immunoglobulin heavy and light chains, these hybridomas (tetralog hybridomas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually by affinity chromatography, is quite cumbersome and yields low product yields. Similar methods are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0182] Depending on the method, an antibody variable domain (antibody-antigen binding site) with the desired binding specificity is fused to an immunoglobulin constant domain sequence. In some embodiments, the fusion is with an immunoglobulin heavy chain constant domain, which includes at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides considerable flexibility in adjusting the relative proportions of the three polypeptide fragments in the embodiments when unequal proportions of the three polypeptide chains used in the construction provide optimal yield. However, when expressing at least two polypeptide chains in equal proportions results in high yield or when the proportions are not particularly important, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector.

[0183] In some embodiments of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from the unwanted combination of immunoglobulin chains, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy separation method. This method is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Sureshet al., Methods in Enzymology 121:210 (1986).

[0184] According to another method described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers recovered from recombinant cell cultures. In some embodiments, the interface comprises the C0 of the antibody constant structural domain. H At least a portion of the 3-domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by la...

Claims

1. A method for quantifying a nonionic surfactant in a composition comprising a nonionic surfactant and a polypeptide, wherein the method comprises the following steps: a) Applying the composition to a mixed-mode cation exchange chromatography material, wherein the composition is added to the chromatography material in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution of ammonium hydroxide and mobile phase B comprises an organic solvent solution of ammonium hydroxide; b) Elute the polypeptide from the mixed-mode cation exchange chromatography material with a solution containing 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) Elute the nonionic surfactant from the chromatographic material with a solution containing mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step b); d) Quantify the nonionic surfactant; The composition further comprises N-acetyltryptophan and / or methionine.

2. The method according to claim 1, wherein the organic solvent of the mobile phase B is methanol.

3. The method according to claim 1 or 2, wherein the ratio of mobile phase B to mobile phase A in step a) is 10:

90.

4. The method according to claim 1, wherein in step b), the ratio of mobile phase B to mobile phase A is increased to 45:

55.

5. The method according to claim 1, wherein in step c), the ratio of mobile phase B to mobile phase A is increased to 100:

0.

6. The method of claim 1, wherein the mobile phase A comprises 2% aqueous ammonium hydroxide solution.

7. The method of claim 1, wherein the mobile phase B comprises a 2% methanol solution of ammonium hydroxide.

8. The method according to claim 1, wherein the flow rate of the chromatography is 1.4 mL / min.

9. The method of claim 8, wherein step b) begins 1 minute after the start of chromatography and ends 4.4 minutes after the start of chromatography.

10. The method of claim 8, wherein step c) begins 4.5 minutes after the start of chromatography and ends 7.6 minutes after the start of chromatography.

11. The method according to claim 1, wherein the nonionic surfactant is a polysorbate.

12. The method of claim 11, wherein the polysorbate is polysorbate 20 or polysorbate 80.

13. The method of claim 11, wherein the concentration of polysorbate in the composition is in the range of 0.001% to 1.0% (w / v).

14. The method according to claim 1, wherein the organic solvent of the mobile phase B is acetonitrile.

15. The method according to claim 14, wherein the ratio of mobile phase B to mobile phase A in step a) is 10:

90.

16. The method according to claim 14 or 15, wherein the ratio of mobile phase B to mobile phase A in step b) is increased to 40:

60.

17. The method of claim 14, wherein in step c), the ratio of mobile phase B to mobile phase A is increased to 100:

0.

18. The method of claim 14, wherein the mobile phase A comprises a solution of 2% ammonium hydroxide in water or in 43% methanol.

19. The method of claim 14, wherein the mobile phase B comprises a 2% ammonium hydroxide acetonitrile solution.

20. The method of claim 14, wherein the nonionic surfactant is poloxamer.

21. The method of claim 20, wherein the poloxamer is poloxamer P188.

22. The method of claim 20, wherein the concentration of poloxamer in the composition is in the range of 0.001% to 1.0% (w / v).

23. The method according to claim 1, wherein the concentration of N-acetyltryptophan in the composition ranges from 0.1 mM to 10 mM.

24. The method according to claim 1, wherein the concentration of methionine in the composition ranges from 0.1 mM to 100 mM.

25. The method according to claim 1, wherein the concentration of the polypeptide in the composition is from 1 mg / mL to 250 mg / mL.

26. The method of claim 1, wherein the composition has a pH of 4.5 to 7.

5.

27. The method of claim 1, wherein the composition further comprises one or more excipients selected from stabilizers, buffers and tension agents.

28. The method of claim 1, wherein the composition is a pharmaceutical preparation suitable for administration to a subject.

29. The method of claim 1, wherein the polypeptide is a therapeutic polypeptide.

30. The method of claim 29, wherein the therapeutic polypeptide is a fusion protein, a polyclonal antibody, a monoclonal antibody, a human antibody, a multispecific antibody, a glycoengineered antibody, an antibody fragment, or an antibody-drug conjugate.

31. The method of claim 29, wherein the therapeutic polypeptide is a chimeric antibody.

32. The method of claim 29, wherein the therapeutic polypeptide is a humanized antibody.

33. The method of claim 1, wherein the mixed-mode cation exchange chromatography material comprises an antiphase strong cation exchange polymer.

34. The method of claim 1, wherein the mixed-mode cation exchange chromatography material comprises a sulfonic acid portion.

35. The method of claim 1, wherein the mixed-mode cation exchange chromatography material comprises a solid support.

36. The method of claim 1, wherein the mixed-mode cation exchange chromatography material is contained in a column.

37. The method according to claim 1, wherein the mixed-mode cation exchange chromatography material is a high-performance liquid chromatography (HPLC) material.

38. The method according to claim 1, wherein the mixed-mode cation exchange chromatography material is MCX chromatography material.

39. The method of claim 1, wherein the nonionic surfactant is quantified by evaporative light scattering (ELSD) or by using an electrosol detector (CAD).

Images