High-throughput liquid chromatography-mass spectrometry-based peptide mapping

Abstract

Provided herein are methods of separating peptides of an enzyme digestion which may be useful in analyzing a protein for post-translational modifications. In exemplary embodiments, the method comprises (a) applying a sample comprising enzyme-digested peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first RPLC column in tandem with a second RPLC column, whereby digested peptides of the sample bind to the first and / or second RPLC column and (b) applying a mobile phase to the MCLC system to obtain eluted peptides, wherein the first RPLC column is (i) different from the second RPLC column, (ii) joined to the second RPLC column through a connector, and (iii) in fluid communication with the second RPLC column. Fluids applied flow from the first RPLC column to the second RPLC column through the connector without flowing through any intervening valves or diverters.

Description

HIGH-THROUGHPUT LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY-BASED PEPTIDEMAPPINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 655,412, filed on June 3, 2024; U.S. Provisional Patent Application No. 63 / 755,105, filed on February 6, 2025; and U.S. Provisional Patent Application No. 63 / 812,612, filed on May 27 , 2025, is hereby claimed, each of which is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as: 57,394 byte (XML) file named "10932-W001-SEC_Seqlisting.xml"; created on May 30, 2025.BACKGROUND

[0003] The ability to monitor for chemical liabilities within a protein is of critical importance to the development of therapeutic proteins, including, for instance, antibodies, since those chemical changes have the potential to impact the safety and / or efficacy of the therapeutic protein. While some post translational modifications (PTMs), such as, oxidation or glycation, can be readily detected by their modified mass, other PTMs prove more challenging to detect. For example, the deamidation of asparagine and the isomerization of aspartic acid are considerably more difficult to monitor, because the isotopic envelope of the modified protein overlaps with that of the unmodified protein. In addition, because aspartic acid and isomerized aspartic acid are isobaric, these species are indistinguishable by conventional mass spectrometry (MS) without specialized fragmentation. Chromatographic resolution of the peptides of such modified proteins is a time limiting factor and can significantly slow down the peptide mapping process. Thus, there exists a need for improved methods of analyzing a protein for post-translational modifications.SUMMARY

[0004] Presented herein are data demonstrating the feasibility and advantages of methods of separating peptides (e.g., peptides of an enzyme digestion) using a multi-column liquid chromatography (MCLC) system comprising a first chromatography column in tandem with a second chromatography column. As supported herein, the methods provide high resolution separation of unmodified peptides (peptides without any PTMs) from their post-translationally modified counterpart(s). The presently disclosed methods provide a faster, more efficient way of detecting the presence of a post translationally modified peptide when analyzing a protein for PTMs. Suchmethods may be useful in characterizing a drug substance or drug product from a quality or safety standpoint. The presently disclosed methods are advantageously adept at separating challenging post-translationally modified peptides, such as peptides with a deamidated Asn or an isomerized Asp. Furthermore, the methods of the present disclosure advantageously separate peptides with high resolution, and these results are highly reproducible. The methods can achieve high resolution, e.g., marked by peak-to-peak resolution of about 0.8 to about 10.9. Also, the methods exhibit high repeatability in that retention times between replicate injections are highly reproducible. In exemplary aspects, the methods exhibit a separation wherein the maximum retention time variability is less than 1 sec, e.g., less than 0.75 sec. In exemplary aspects, the methods exhibit a separation wherein the maximum retention time is about 0.6 s (RSD < 0.05%). The methods of the present disclosure also are marked by narrow peaks widths (FWHM ranging from 1.2 - 2.6 s), in various aspects. The present inventive methods are faster, requiring less than 50% of the time required of traditional separation methods.

[0005] Accordingly, the present disclosure provides methods of separating peptides of a protein, e.g., peptides of an enzyme digestion. In exemplary embodiments, the method comprises (a) applying a sample comprising peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first chromatography column in tandem with a second chromatography column, whereby peptides of the sample bind to the first chromatography column and / or second chromatography column; and (b) applying a mobile phase to the MCLC system to elute the bound peptides from the first chromatography column and second chromatography column to obtain eluted peptides. In exemplary aspects, the first chromatography column is different from the second chromatography column. In exemplary instances, the first chromatography column is joined to the second chromatography column through a connector. In various aspects, the first chromatography column is in fluid communication with the second chromatography column and fluids applied to the first chromatography column flow from the first chromatography column to the second chromatography column through the connector. In various instances, there are no intervening valves or diverters between the first chromatography column and second chromatography column. In exemplary instances, the first chromatography column is a reversed- phase liquid chromatography column (RPLC) and the second chromatography column is a RPLC column.

[0006] Also provided herein are methods of analyzing a protein for post-translational modifications. In exemplary aspects, the method comprises (a) applying a sample comprising peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first chromatography column in tandem with a second chromatography column, whereby peptides of thesample bind to the first chromatography column and / or second chromatography column; (b) applying a mobile phase to the MCLC system to elute the bound peptides from the first chromatography column and second chromatography column to obtain eluted peptides; and (c) analyzing the eluted peptides by mass spectrometry for post-translational modifications. In exemplary aspects, the first chromatography column is different from the second chromatography column. In exemplary instances, the first chromatography column is joined to the second chromatography column through a connector. In various aspects, the first chromatography column is in fluid communication with the second chromatography column and fluids applied to the first chromatography column flow from the first chromatography column to the second chromatography column through the connector. In various instances, there are no intervening valves or diverters between the first chromatography column and second chromatography column. In exemplary instances, the first chromatography column is a reversed-phase liquid chromatography column (RPLC) and the second chromatography column is a RPLC column.

[0007] Also provided are MCLC systems as described herein.

[0008] Methods of characterizing a drug substance or drug product, or a process preparing the drug substance or drug product, from a quality or safety standpoint are additionally provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1A provides Table 1 which details the properties of peptides evaluated. Figure IB provides Table 2 which details the columns assessed. Figure 1C is a series of extracted ion chromatograms (EICs) of individual SSQSILHSSNNNNYLAWFQQKPGQPPK (SEQ ID NO: 6) peptides using a CSH Premier column. Peptides A-G are SEQ ID NO:s 6-12, respectively.

[0010] Figure 2 is a series of EICs of the ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47) peptide group.

[0011] Figure 3 is a series of EICs of the GLEWVAVISYQGNNK (SEQ ID NO: 40) peptide group.

[0012] Figures 4A provides Table 3 which lists native peptide retention time reproducibility determined by 9 injections of 10:1 native:modified synthetic peptide mix. The peptides listed are: HGNFGNSYI (SEQ ID NO: 1), SSQSILHSSNNNNYLAWFQQKPGQPPK (SEQ ID NO: 6), VDNALQSGNSQESVTEQDSK (SEQ ID NO: 13), TTPPVLDSDGSFFLYSK (SEQ ID NO: 16) , IYPTNGYTR (SEQ ID NO: 19), NFHLRPR (SEQ ID NO: 22), CLEWVAVIWYDGSDKYYADSVR (SEQ ID NO: 25), SLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDK (SEQ ID NO: 29), LTVLGGGGDK (SEQ ID NO: 31), GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 33), GLEWVAVISYQGNNK (SEQ ID NO: 40),FNWYVDGVEVHNAK (SEQ ID NO: 45), ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47), and CLEWVAIISDGGYYTYYSDIIK (SEQ ID NO: 49).

[0013] Figure 4B provides Table 4 which lists Peak-to-peak resolution (Rp-p) for each peptide group. The peptides listed (top to bottom) are SEQ ID NOs: 1-51.

[0014] Figure 4C provides Table 5 which shows peptide-specific overall performance of each column. The peptides listed are: HGNFGNSYI (SEQ ID NO: 1), SSQSILHSSNNNNYLAWFQQKPGQPPK (SEQ ID NO: 6), VDNALQSGNSQESVTEQDSK (SEQ ID NO: 13), TTPPVLDSDGSFFLYSK (SEQ ID NO: 16) , IYPTNGYTR (SEQ ID NO: 19), NFHLRPR (SEQ ID NO: 22), CLEWVAVIWYDGSDKYYADSVR (SEQ ID NO: 25), SLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDK (SEQ ID NO: 29), LTVLGGGGDK (SEQ ID NO: 31), GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 33), GLEWVAVISYQGNNK (SEQ ID NO: 40), FNWYVDGVEVHNAK (SEQ ID NO: 45), ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47), and CLEWVAIISDGGYYTYYSDIIK (SEQ ID NO: 49).

[0015] Figure 4D provides Table 6 which lists the observed sequence coverage following tryptic digestion of a representative IgGl mAb. Figures 5A and 5B are exemplary EICs obtained from a dualcolumn LC system, wherein the two columns were connected by an EXP Hand Tight Coupler (Figure 5A) or a Viper SST (Figure 5B).

[0016] Figures 6A-6F are exemplary EICs obtained from an LC system comprising: a single 150-mm CSH Premier column (Figure 6A); a single 150-mm BEH Premier column (Figure 6B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 6C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 6D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 6E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 6F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence HGNFGNSYI, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0017] Figures 7A-7F are exemplary EICs obtained from an LC system comprising: a single 150-mm CSH Premier column (Figure 7A); a single 150-mm BEH Premier column (Figure 7B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 7C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 7D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 7E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 7F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence SSQSILHSSNNNNYLAWFQQKPGQPPK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0018] Figures 8A-8F are exemplary EICs obtained from an LC system comprising: a single 150-mm CSH Premier column (Figure 8A); a single 150-mm BEH Premier column (Figure 8B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 8C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 8D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 8E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 8F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence VDNALQSGNSQESVTEQDSK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0019] Figures 9A-9F are exemplary EICs obtained from an LC system comprising: a single 150-mm CSH Premier column (Figure 9A); a single 150-mm BEH Premier column (Figure 9B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 9C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 9D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 9E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 9F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence TTPPVLDSDGSFFLYSK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0020] Figures 10A-10F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 10A); a single 150-mm BEH Premier column (Figure 10B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 10C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 10D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 10E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 10F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence IYPTNGYTR, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0021] Figures 11A-11F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 11A); a single 150-mm BEH Premier column (Figure 11B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 11C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 11D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure HE); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 11F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence NFHLRPR, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0022] Figures 12A-12F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 12A); a single 150-mm BEH Premier column (Figure 12B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 12C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 12D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 12E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 12F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequenceC[carboxymethyl]LEWVAVIWYDGSDKYYADSVR, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0023] Figures 13A-13F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 13A); a single 150-mm BEH Premier column (Figure 13B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 13C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 13D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 13E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 13F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence SLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0024] Figures 14A-14F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 14A); a single 150-mm BEH Premier column (Figure 14B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 14C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 14D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 14E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 14F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence LTVLGGGGDK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0025] Figures 15A-15F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 15A); a single 150-mm BEH Premier column (Figure 15B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 15C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 15D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 15E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 15F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence GFYPSDIAVEWESNGQPENNYK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0026] Figures 16A-16F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 16A); a single 150-mm BEH Premier column (Figure 16B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 16C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 16D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 16E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 16F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence GLEWVAVISYQGNNK,wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0027] Figures 17A-17F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 17A); a single 150-mm BEH Premier column (Figure 17B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 17C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 17D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 17E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 17F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence FNWYVDGVEVHNAK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0028] Figures 18A-18F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 18A); a single 150-mm BEH Premier column (Figure 18B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 18C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 18D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 18E); two columns: 100 mm CSH Premier column connected to a 100 mm BEHPremier column (Figure 18F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence ASQNVDTNVAWYQQKPGQAPK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0029] Figures 19A-19F are exemplary EICs obtained from an LC system comprising: a single 150- mm CSH Premier column (Figure 19A); a single 150-mm BEH Premier column (Figure 19B); two columns: a 100-mm BEH Premier column connected to a 100-mm CSH Premier column (Figure 19C); two columns: a 50 mm CSH Premier column connected to a 150 mm Acclaim Vanquish column (Figure 19D); two columns: 100 mm CSH Premier column connected to a 50 mm BEH Premier column (Figure 19E); two columns: 100 mm CSH Premier column connected to a 100 mm BEH Premier column (Figure 19F); wherein the sample comprised a 10:1 mixture of unmodified to modified peptide of the peptide family comprising the amino acid sequence C[carboxymethyl]LEWVAIIS GGYYTYYSDIIK, wherein bold underlined letters are possible sites of post translational modification. The peptides are labeled according to the table below:

[0030] Figure 20 is a table listing MCLC systems comprising two RPLC columns that are evaluated as described in Example 5.

[0031] Figure 21A is an illustration of an MCLC system comprising a trap column and a separation column assembly (SCA), wherein the SCA comprises a first reversed-phase liquid chromatography (RPLC) column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column, wherein: (a) the first RPLC column is different fromthe second RPLC column; (b) the first RPLC column is joined to the second RPLC column through a connector; (c) the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and (d) there are no intervening valves or diverters between the first RPLC column and second RPLC column; wherein the MCLC system further comprises a trap column, wherein (i) the trap column is in fluid communication with the first RPLC column, (ii) fluids applied to the trap column flow from the trap column to the first RPLC column, and / or (iii) there are no intervening valves or diverters between the trap column and first RPLC column. The SCA of the MCLC system and is maintained at a temperature greater than the temperature of the trap column (Temp 1 < Temp 2). Blue arrow shows the direction of fluids flowing through the MCLC.

[0032] Figure 21B is an illustration of an MCLC system comprising a trap column and SCA similar to the one in Figure 21A, but additionally comprising an autosampler positioned upstream of the trap column. The sample comprising peptides of a protein is applied to (e.g., injected into) the autosampler and flows to the trap column then through the separation column assembly. The descriptions of the MCLC system of Figure 21A, e.g., descriptions of the first RPLC, connector, second RPLC, and trap column, also apply to the MCLC system of Figure 21B.

[0033] Figure 21C is an illustration of an MCLC system comprising a trap column and a first SCA and a second SCA. Movement of fluids through this MCLC system may be controlled through the action of pumps, switches and valves of this MCLC system. The descriptions of the MCLC system of Figures 21A and 21B, e.g., descriptions of the first RPLC, connector, second RPLC, and trap column, also apply to the MCLC system of Figure 21C.

[0034] Figure 22 is an illustration of control system comprising an autosampler and a singlecolumn SCA lacking a trap column and a second RPLC column.

[0035] Figure 23A and 23B illustrate two-modes of an MCLC system comprising an autosampler, trap column, a first SCA and second SCA, similar to the one in Figure 21C. The MCLC system comprises two 6-port valves (ports shown as numbered circles) and a switch with at least two settings (not shown). A first binary pump causes fluids to move along the path shown with grey arrows that lead to the detector, while a second binary pump causes fluids to move along the path shown with dotted arrows to the waste collection bin. Figure 23A shows the switch in one setting (e.g., the 1-2 position), wherein fluids, pumped by the first binary pump, move from the autosampler to the trap column through the first SCA to the detector, and fluids, pumped by the second binary pump, move through the second SCA to the waste collection bin. Figure 23B shows the switch in another setting (e.g., the 1-6 position), wherein fluids, pumped by the first binary pump, move fromthe autosampler to the trap column through the second SCA to the detector, and fluids, pumped by the second binary pump, move through the first SCA to the waste collection bin. The mobile phase gradient for each of the first binary pump (Pump 1) and the second binary pump (Pump 2), as well, exemplary chromatograms, are shown in Figures 23A and 23B.

[0036] Figure 24 is a table of retention time (RT), FWHM, and RPP values for two different MCLC systems. The peptides listed (top to bottom) are SEQ. ID NOs: 1-51.

[0037] Figure 25 illustrates an MCLC system comprising an autosampler, a trap column, a first SCA and a second SCA, wherein the trap column is in fluid communication with only the first SCA. Fluids applied to the autosampler flow either (1) through the trap column and through the first SCA or (2) through the second SCA.

[0038] Figures 26A-26C are graphs demonstrating the amount (%) of peptides demonstrating oxidation at Met 255 (Figure 26A), Met 431 (Figure 26B), or Met 34 (Figure 26C), as measured using the system of Figure 25, wherein the peptides were separated by the first SCA with trap column (black bars) or the second SCA without a trap column (white bars), wherein the column temperature of the first SCA and second SCA were maintained at a temperature ranging from 50 °C to 80 °C.

[0039] Figures 27A and 27B are graphs demonstrating the amount (%) of peptides demonstrating Asp273-Pro274 clipping (Figure 27A) or Asp88-Pro89 clipping (Figure 27B), as measured using the system of Figure 25, wherein the peptides were separated by the first SCA with trap column (black bars) or the second SCA without a trap column (white bars), wherein the column temperature of the first SCA and second SCA were maintained at a temperature ranging from 50 °C to 80 °C.

[0040] Figures 28A and 28B are graphs demonstrating the amount (%) of peptides demonstrating Asn387 / Asn392 deamidation (Figure 28A) or Asn326 deamidation (Figure 28B), as measured using the system of Figure 25, wherein the peptides were separated by the first SCA with trap column (black bars) or the second SCA without a trap column (white bars), wherein the column temperature of the first SCA and second SCA were maintained at a temperature ranging from 50 °C to 80 °C.

[0041] Figures 29A-29D are graphs depicting the amount (%) of peptides demonstrating oxidation at Met34 (Figure 29A) and Met 431 (Figure 29B) or the amount (%) of peptides demonstrating Asp- Pro clipping at Asp88-Pro89 (Figure 29C) and Asp273-Pro274 (Figure 29D), as measured using the system of Figure 25 as described in Example 9, wherein the peptides were separated by the first SCA with trap column (black bars) or the second SCA without a trap column (white bars), wherein the column temperature of the first SCA and second SCA were maintained at a temperature ranging from 50 °C to 80 °C.DETAILED DESCRIPTION

[0042] The present disclosure provides methods of separating peptides of protein, e.g., peptides of an enzyme digestion. In exemplary embodiments, the method comprises (a) applying a sample comprising peptides (e.g., digested peptides) of a protein to a multi-column liquid chromatography (MCLC) system comprising a first chromatography column in tandem with a second chromatography column, whereby peptides (e.g., digested peptides) of the sample bind to the first chromatography column and / or second chromatography column; and (b) applying a mobile phase to the MCLC system to elute the bound peptides from the first chromatography column and second chromatography column to obtain eluted peptides. As used herein, "separating" or "separation" refers to chromatographically separating or chromatographic separation or separation achieved through chromatography, a technique in which the components of a mixture are separated by their distribution between two phases, a stationary phase and a mobile phase, based on the strength of the physical interactions, e.g., intermolecular interactions, made between the components of the mixture and the two phases. The separation results from repeated sorption / desorption events that occur during the movement of the mixture along the stationary phase in the general direction of the migration of the mobile phase applied to the stationary phase. See, e.g., Poole, "Chromatography", Encyclopedia of Separation Science, pgs. 40-64, Elsevier Science, 2000. Accordingly, in exemplary embodiments, the separation methods of the present invention are chromatographic separation methods. In various embodiments, the separation methods of the present disclosure employ reversed phase liquid chromatography (RPLC) in which a non-polar stationary phase and a polar mobile phase are used to separate molecules, e.g., peptides, based on their hydrophobicity. In various aspects, the presently disclosed separation methods employ reversed phase high- performance liquid chromatography (RP-HPLC) wherein the less hydrophobic molecules which bind to the non-polar stationary phase elute earlier than the more hydrophobic molecules. The theory behind RP-HPLC is detailed in Josie and Kovac, Curr Protoc Protein Sci 61(1): 8.7.1-8.7.22 (2010), https: / / doi.org / 10.1002 / 0471140864.ps0807s61; and Kumar and Kumar, Int J Pharm Sci Res 3(12): 4626-4633 (2012).

[0043] In exemplary embodiments, the methods of the present disclosure employ a multiple column liquid chromatography (MCLC) system, wherein fluids (e.g., sample, mobile phase) applied to the system flow through two or more chromatography columns. In various aspects, the MCLC system comprises two, three, or four chromatography columns. In exemplary aspects, the MCLC system comprises not more than two or three chromatography columns. In various instances, the MCLC system comprises only two chromatography columns. In exemplary instances, the MCLC system comprises three or more chromatography columns, e.g., RPLC columns. In various instances,the MCLC system comprises only three chromatography columns. In exemplary aspects, the chromatography columns of the MCLC system are positioned in tandem with one another. In various aspects, the MCLC system comprises a first chromatography column in tandem with a second chromatography column.

[0044] In exemplary aspects, each of the chromatography columns of the MCLC system differ from one another or at least from one other chromatography column of the MCLC system. In various aspects, the MCLC system comprises a first chromatography column in tandem with a second chromatography column, and the first chromatography column is different from the second chromatography column and / or the first chromatography column is non-identical to the second chromatography column. In exemplary aspects, the first chromatography column differs from the second chromatography column by virtue of its separation performance, e.g., its ability to separate molecules (e.g., peptides) in a mixture (e.g., sample comprising (e.g., enzyme-digested) peptides of a protein). Without being bound by theory, combining different columns having different separation capabilities permits the MCLC system to harness the separation power of all columns of the MCLC system, thereby maximizing the overall separation capabilities of the MCLC system. In various instances, the chromatography columns of the MCLC system differ by column length, inner diameter, and / or composition, and / or by the stationary phase housed within each column. In exemplary aspects, the column length and / or inner diameter of the first chromatography column is different from the column length and / or inner diameter of the second chromatography column. In various aspects, the stationary phase within the first chromatography column differs from the stationary phase of the second chromatography column. In exemplary instances, the stationary phase of the first chromatography column comprises particles that differ from the particles of the stationary phase of the second chromatography column. In some aspects, the particles differ by composition, functional group bound to the particle surface, extent of surface functionalization with functional group, hydrophobicity, pore size, particle size, or a combination thereof.

[0045] In exemplary instances, each chromatography column of the MCLC system is joined to another chromatography column by a connector. In various aspects, the first chromatography column is joined to the second chromatography column through a connector. The connector in exemplary aspects comprises two ends each of which attach to a chromatography column. In various instances, each end of the connector attaches to an end of a chromatography column by way of fittings compatible with about 10 to about 32 threaded ports. In exemplary aspects, the inner diameter of each end of the connector is nearly identical to the inner diameter of each end of the chromatography column to be joined by the connector. In various aspects, the connector adds little to no dead volume to the overall volume of the MCLC system. By "dead volume" is meant thevolume or space through which fluids pass upon application to the MCLC system which does not comprise a stationary phase. In various instances, the connector has a length not more than 1 cm. In various aspects, the connector is less than 1 cm, less than 900 mm, less than 800 mm, less than 700 mm, less than 600 mm, less than 500 mm, less than 400 mm, less than 300 mm, less than 200 mm, or less than 100 mm. In various aspects, the length of the connector is less than 75 mm or less than 70 mm. In various instances, the length of the connector is about 50 mm to about 70 mm or about 65 mm.

[0046] In various aspects, the first chromatography column is in fluid communication with the second chromatography column. In various instances, fluids applied to MCLC system, e.g., to the first chromatography column, flow from the first chromatography column to the second chromatography column through the connector. In various instances, there are no intervening valves or diverters between the first chromatography column and second chromatography column. In exemplary aspects, fluids applied to the first chromatography column flow from the first chromatography column to the second chromatography column through the connector without flowing through any valves of diverters. In exemplary instances, flowing from the first chromatography column to the second chromatography column through the connector is the only path for fluids applied to the MCLC system. There are no other alternative paths for fluids applied to the MCLC system, in various aspects. For example, the path of fluids of the MCLC system cannot be diverted through a valve or switch. In exemplary embodiments, all fluids, including the sample and the mobile phase, applied to the MCLC system flow from the first chromatography column to the second chromatography column through the connector. In exemplary aspects, the mobile phase is applied to the first chromatography column and passes through the first chromatography column, the connector and then the second chromatography column, such that peptides eluted from the first chromatography column flow through the second chromatography column.

[0047] Flow, Flow Rate; Pressure, Column Length, and Inner Diameter

[0048] In various aspects, the method comprises chromatographically separating the peptides through ultra-high pressure liquid chromatography (UHPLC), wherein the pressure is greater than or about 900 bar. In various instances, the pressure is greater than 1000 bar, greater than 1100 bar, greater than 1200 bar, or about 1300 bar. Pressure may be influenced by factors including flow rate and parameters of the column, e.g., inner diameter (ID) of the column, column length, particle size of the stationary phase, and the like. In exemplary aspects, the method is carried out with a flow rate of the sample and mobile phase through the MCLC system of about 100 pL / minute to about 1000 pL / minute. In exemplary instances, the flow rate is about 100 pL / minute to about 900 pL / minute, about 100 pL / minute to about 800 pL / minute, about 100 pL / minute to about 700pL / minute, about 100 pL / minute to about 600 pL / minute, about 100 pL / minute to about 500 pL / minute, about 100 pL / minute to about 400 pL / minute, about 100 pL / minute to about 300 pL / minute, about 100 pL / minute to about 200 pL / minute, about 200 pL / minute to about 1000 pL / minute, about 300 pL / minute to about 1000 pL / minute, about 400 pL / minute to about 1000 pL / minute, about 500 pL / minute to about 1000 pL / minute, about 600 pL / minute to about 1000 pL / minute, about 700 pL / minute to about 1000 pL / minute, about 800 pL / minute to about 1000 pL / minute, or about 900 pL / minute to about 1000 pL / minute. In exemplary instances, the flow rate is at least about 200 pL / minute or at least about 250 pL / minute. In various aspects, the flow rate is at least or about 500 pL / minute, at least or about 600 pL / minute, or at least or about 700 pL / minute. In various aspects, the sum of the column lengths of the chromatography columns of the MCLC system is greater than 100 mm and less than about 300 mm. In various aspects, the MCLC system comprises only two chromatography columns and the sum of the column lengths of the two chromatography columns is greater than 150 mm, optionally, greater than 175 mm. In exemplary instances, the sum of the column lengths of the two chromatography columns is less than about 275 mm or less than about 250 mm. The column length of the first chromatography column and / or second chromatography column is about 25 mm to about 200 mm. In some aspects, the column length of the first chromatography column and / or second chromatography column is about 50 mm to about 150 mm. In various aspects, the column length of the first chromatography column or the second chromatography column is 100 mm. In various instances, the inner diameter of the first chromatography column and / or second chromatography column is about 1 mm to about 9 mm, e.g., about 1 mm to about 8 mm, about 1 mm to about 7 mm, about 1 mm to about 6 mm, about 1 mm to about 5 mm, about 1 mm to about 4 mm, about 1 mm to about 3 mm, about 1 mm to about 2 mm, about 2 mm to about 9 mm, about 3 mm to about 9 mm, about 4 mm to about 9 mm, about 5 mm to about 9 mm, about 6 mm to about 9 mm, about 7 mm to about 9 mm, or about 8 mm to about 9 mm.

[0049] Reversed-Phase Liquid Chromatography Column (RPLC)

[0050] In exemplary embodiments, the first chromatography column is a reversed-phase liquid chromatography column (RPLC). In exemplary embodiments, the second chromatography column is a RPLC column. In exemplary embodiments, the method comprises (a) applying a sample comprising (e.g., enzyme-digested) peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column and (b) applying a mobile phase to the MCLC system to elute the bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides. In exemplary aspects, the first RPLC column isdifferent from the second RPLC column, the first RPLC column is joined to the second RPLC column through a connector, the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector, and there are no intervening valves or diverters between the first RPLC column and second RPLC column. In exemplary aspects, the MCLC system comprises only two RPLC columns, e.g., a first RPLC column and a second RPLC column, wherein the only chromatography columns comprised by the MCLC system are the first RPLC column and the second RPLC column. In exemplary aspects, each of the first RPLC column and second RPLC column comprises a non-polar stationary phase. The non-polar stationary phase comprises hydrocarbons, e.g., Cl hydrocarbons, C4 hydrocarbons, C5 hydrocarbons, C8 hydrocarbons, C18 hydrocarbons, and the like, in various aspects. In various instances, the non-polar stationary phase comprises C18 hydrocarbons. In exemplary aspects, the hydrocarbons are linked or bound to the surface of particles of the non-polar stationary phase and the extent of the surface comprising linked hydrocarbons can vary. In various instances, at least about 15% of the surface of the particles of the non-polar stationary phase are linked to a hydrocarbon, e.g., C18 hydrocarbon. In various instances, at least about 25% or at least about 50% or at least about 75% of the surface of the particles of the non-polar stationary phase are linked to a hydrocarbon, e.g., C18 hydrocarbon. In exemplary embodiments, the non-polar stationary phase comprises particles comprising a silica, a hybrid silica, such as, for instance, a polyorganoethoxysilane. In exemplary instances, the particles comprise tetraethoxysilane (TEOS) and bis(triethoxysilyl) ethane (BTEE). In exemplary instances, the particles are any of those described in U.S. Patent Nos. 4,017,528; 6,686,035; 7,723,473; and 7,250,214. In various aspects, the non-polar stationary phase comprises ethylene-bridged-hybrid (BEH) particles. At least one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising a surface charge, in various aspects. In some aspects, one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising charged surface hybrid (CSH) particles. For example, in some cases, the first RPLC column comprises a non-polar stationary phase comprising CSH particles. In various instances, the second RPLC column comprises a nonpolar stationary phase comprising CSH particles. In exemplary aspects, the MCLC system comprises an RPLC column comprising a non-polar stationary phase comprising charged surface hybrid (CSH) particles and an RPLC column comprising a non-polar stationary phase comprising BEH particles. In various aspects, the particles of the non-polar stationary phase have a particle size of about 1.7 pm to 3 pm. In various instances, the particle size is about 1.7 pm to about 2.1 pm. The particles of the non-polar stationary phase in exemplary aspects have a pore size of about 80 A to about 120 A or about 120 A to about 300 A. In various instances, the non-polar stationary phase comprises particlescomprising a pore size of about 120 A to about 160 A. RPLC columns are commercially available through vendors, e.g., Waters (Milford, MA), Tosoh (Portland, OR), ThermoFisher Scientific (Waltham, MA), Agilent (Santa Clara, CA), Pheomenex (Torrance, CA), Hamilton Co. (Reno, NV), Restek (Center County, PA), EMD Millipore (St. Louis, MO), BioRad (Hercules, CA), Hawach Scientific (Xi'an City, PR China), Macherey-Nagel (Duren, Nordrhein-Westfalen, Germany), Halo Columns (Wilmington, DE), Sigma-Aldrich (St. Louis, MO), Develosil (San Diego, CA), Sartorius (Gottingen, Germany), Concise Separations (San Jose, CA), among others. Exemplary RPLC columns include, but are not limited to, those described in Table 2 and Figure 20. For example, the first RPLC column may be selected from Column 1 of Figure 20, and the second RPLC column may be selected from Column 2 of Figure 20.

[0051] Mobile Phase and Elution

[0052] In exemplary embodiments of the presently disclosed methods, a mobile phase is applied to the MCLC system to elute bound peptides from the first chromatography column and second chromatography column to obtain eluted peptides. In exemplary aspects, the method comprises applying a mobile phase to the MCLC system to elute bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides. In various aspects, the mobile phase is applied to the first chromatography column, e.g., the first RPLC column, and the mobile phase flows through the first column, through the connector, and then through the second chromatography column, e.g., second RPLC column. In exemplary aspects, the mobile phase comprises a gradient of a polar organic solvent. In various instances, the mobile phase comprises an increasing concentration gradient of the polar organic solvent. In various instances, the mobile phase comprises only one increasing concentration gradient of the polar organic solvent, and in various aspects, the polar organic solvent increases from 0.09 M to 9 M. In various aspects, the mobile phase comprises only one increasing concentration gradient of the polar organic solvent and in exemplary embodiments, the mobile phase comprises only one organic polar solvent. The mobile phase comprises a polar organic solvent selected from the group consisting of: acetonitrile, methanol, ethanol, propanol, isopropanol, in exemplary instances. In exemplary aspects, the polar organic solvent is acetonitrile. In various instances, the mobile phase comprises a buffer, and in some aspects, the buffer comprises trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), or formic acid. In exemplary instances, the buffer has a pH below 4, e.g., a pH of 2 or 3, or a pH of 2 to 3. In exemplary aspects, the mobile phase has a pH below 4, e.g., a pH of 2 or 3. In various aspects, the pH is about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9. In exemplary instances, the mobile phase comprises a mixture of two mobile phasesolutions, only one of which comprises the polar organic solvent and both mobile phase solutions comprise a buffer. In some instances, both mobile phase solutions comprise less than 1% (v / v) buffer or less than 0.5% (v / v) buffer and in exemplary aspects both mobile phase solutions comprise TFA (pH 2) or formic acid (pH 3). In various aspects, both mobile phase solutions comprise 0.1% TFA (pH 2) or 0.1% formic acid (pH 3). The mobile phase in various instances comprises two mobile phase solutions: Mobile Phase A (MPA) and Mobile Phase B (MPB), wherein Mobile Phase A (MPA) is about 0.1% (v / v) formic acid in water and Mobile Phase B (MPB) is about 0.1% (v / v) formic acid in about 80% (v / v) to 100% (v / v) acetonitrile (ACN) in water.

[0053] The methods of the present disclosure are advantageously faster than conventional methods. In various aspects, the method comprises applying a mobile phase to the MCLC system to elute bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides and this occurs in less than 120 minutes. In various aspects, the method comprises applying a mobile phase to the MCLC system to elute bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides and this occurs in less than 90 minutes or in less than 60 minutes. In exemplary instances, the method comprises applying a mobile phase to the MCLC system to elute bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides and this occurs in 30 minutes or less or 25 minutes or less, such as less than 20 minutes, less than 15 minutes, or less than 10 minutes. In various instances, the mobile phase is applied to the MCLC system according to the following gradient:

[0054] The MS data for the eluted peptides are obtained when % MPA is greater than or equal to50% and % MPB is less than or equal to 50% in exemplary aspects. The MS data for the elutedpeptides are obtained within the first 11.4 minutes of the gradient in exemplary aspects. In various instance, the mobile phase is applied to the MCLC system according to the following gradient:

[0055] High Resolution, Reproducibility, Sensitivity

[0056] The presently disclosed methods are advantageously adept at separating challenging post- translationally modified peptides, such as peptides with a deamidated Asn or an isomerized Asp. Furthermore, the methods of the present disclosure advantageously separate peptides with high resolution, and these results are highly reproducible. The methods achieve high resolution separation of peptides that are, e.g., marked by peak-to-peak resolution (Rp-p) of about 0.5 to about 15. In various aspects, the Rp-p is about 0.8 to about 12.

[0057] In various aspects, the high resolution of the chromatographic separation of the peptides achieved by the present inventive methods is repeatable or reproducible such that there is minimal difference or minimal shift in diastereomer retention times among a given series of method runs. In various instances, the methods exhibit high repeatability in that retention times between replicate injections are highly reproducible. In exemplary aspects, the methods exhibit a separation wherein the maximum retention time variability is less than 1 sec, e.g., less than 0.75 sec. In exemplary aspects, the methods exhibit a separation wherein the maximum retention time is about 0.6 s (RSD < 0.05%). The methods of the present disclosure also are marked by narrow peaks widths (FWHM ranging from 1.2 - 2.6 s), in various aspects.

[0058] The present inventive methods of separating peptides are highly sensitive, such that the methods can detect a small amount of a peptide comprising a challenging PTM. As discussed herein, the methods of the present disclosure detect the deamidation of asparagine and the isomerization of aspartic acid, which PTMs are considerably challenging to detect using convention methods, because the isotopic envelope of a protein comprising such a PTM overlaps with the isotopic envelope of the corresponding unmodified protein (not comprising the PTM). In addition, because aspartic acid and isomerized aspartic acid are isobaric, these species are indistinguishable by conventional mass spectrometry (MS) without specialized fragmentation. While chromatographicresolution of the peptides comprising such modified proteins are typically time-consuming, the methods of the present disclosure overcome such challenges as the methods are adept at detecting challenging PTMs, like deamidated Asn and isoAsp.

[0059] The present inventive methods of separating peptides (e.g., enzyme-digested peptides) employ columns with different separation capabilities. In exemplary embodiments, the columns exhibit different separation capabilities due to the columns comprising different stationary phases. Without being bound by theory, combining different columns having different separation capabilities permits the MCLC system to harness the separation power of all columns of the MCLC system, thereby maximizing the overall separation capabilities of the MCLC system. In exemplary aspects of the present disclosure, more than 50% of the (e.g., enzyme-digested) peptides of the sample bind to the first RPLC column, the second RPLC column, or both. In exemplary aspects of the present disclosure, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of the (e.g., enzyme-digested) peptides of the sample bind to the first RPLC column, the second RPLC column, or both. In exemplary aspects of the present disclosure, nearly all or all of the peptides of the sample bind to the first RPLC column, the second RPLC column, or both. In exemplary aspects of the present disclosure, more than 50% of the (e.g., enzyme-digested) peptides of the sample are retained to the first RPLC column, the second RPLC column, or both. In exemplary aspects of the present disclosure, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of the (e.g., enzyme-digested) peptides of the sample are retained to the first RPLC column, the second RPLC column, or both. In exemplary aspects of the present disclosure, nearly all or all the peptides of the sample are retained to the first RPLC column, the second RPLC column, or both. In exemplary aspects of the present disclosure, nearly all or all the bound or retained peptides elute from the first RPLC column, the second RPLC column, or both. In exemplary embodiments, the methods separate nearly all to all the peptides of the protein, such that nearly all to all of the protein sequence is analyzed for PTMs. In various aspects, the methods are characterized by a high sequence coverage, e.g., the methods analyze a high percentage of the sequence of the protein for PTMs.

[0060] Additional Steps

[0061] The methods disclosed herein, in various aspects, comprise additional steps. In various aspects, the method further comprises analyzing the eluted peptides obtained by mass spectrometry.

[0062] In exemplary aspects, the peptides eluted from the first RPLC column are sent to the mass spectrometer together with the peptides eluted from the second RPLC column. In various instances,the peptides eluted from the first column are not separately injected into the mass spectrometer from the peptides eluted from the second column. In exemplary instances, a peptide comprising a post translational modification elutes separately from the corresponding unmodified peptide comprising the same amino acid sequence but without the post translational modification. In various instances, the method further comprises quantifying the peptides with one or more post translational modifications. Methods of performing mass spectrometry are known in the art. See e.g., International Patent Application Publication No. WO 2022 / 61092, Da Ren, et al., Analytical Biochemistry 392: 12-21 (2009), and Stroobant and de Hoffmann, Mass Spectrometry: Principles and Applications, 3rd Edition, Wiley, Hoboken, NJ, 2007.

[0063] The method in various aspects further comprises preparing the sample comprising (e.g., enzyme-digested) peptides of a protein. For instance, the method comprises incubating the protein with an enzyme to obtain enzyme-digested peptides of the protein. Suitable enzymes for digesting proteins are known in the art. Such enzymes include but are not limited to proteases, e.g., trypsin, chymotrypsin, a protease of AccuMAP™ Modified Trypsin Solution (available from Promega as catalog # V5285), pepsin, elastase, pseudotrypsin, or a combination thereof. In various instances, the method further comprises denaturing and / or alkylated the protein, reducing sulfide bonds of the protein, or a combination thereof. Methods of enzyme digestion and pre-digestion protein processing are described in the prior art, e.g., International Patent Application Publication No. WO 2022 / 61092, and Da Ren et al., 2009, supra.

[0064] In exemplary embodiments, the method comprises obtaining a sample comprising the protein. In various aspects, the protein is a protein of an in-process sample, a drug substance, or drug product. In exemplary aspects, the method comprises obtaining an in-process sample comprising the protein, which in-process sample may be a sample obtained during any one of the steps of a manufacture process. The in-process sample in various aspects is a sample obtained from any one of the upstream steps or downstream steps involved in producing, purifying, and formulating the protein, e.g., a therapeutic protein. Optionally, the downstream steps are any one of those downstream processing steps described herein or known in the art. In exemplary embodiments, the in-process sample is a sample obtained from a cell culture comprising cells expressing the protein. In various aspects, the in-process sample is a sample obtained from a harvest of the cell culture. In exemplary instances, the in-process sample is a sample obtained from the eluate of an affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography which is / are used to purify the protein. In various instances, the in-process sample is obtained from a step leading to a drug substance or drug product comprising the protein. When the sample is an in-process sample, themethods of the present disclosure are useful for analyzing a protein during or post-manufacturing. The methods of the present disclosure are thus useful for evaluating the manufacturing process of the protein and can be used to identify problematic steps or parts of the manufacturing process.

[0065] The sample may comprise, for example, a finished or polished drug substance. The sample in various instances, is a drug product that has undergone filling. When the sample is a sample of a drug product, the methods of the present disclosure are useful for analyzing the finished drug product, as in, for example, a lot of the drug product. The presently disclosed methods may be utilized as part of a lot release assay to ensure the protein does not comprise any PTMs that could impact the safety or efficacy of the drug product.

[0066] Additional Features of the MCLC System and Methods of Using the Same

[0067] In exemplary embodiments of the present inventive methods or present inventive MCLC systems, the MCLC system comprises three or more chromatography columns, e.g., RPLC columns. In various instances, the MCLC system comprises only three chromatography columns, e.g., three RPLC columns. In various aspects, one of the chromatography columns is a trap column. In various instances, the MCLC system further comprises a trap column. In various aspects, the MCLC system comprises a trap column and a separation column assembly (SCA) comprising one or more chromatography columns (e.g., RPLC columns), and the trap column is positioned upstream of the separation column assembly. See, e.g., the MCLC system of Figure 21A. As used herein, the term "trap column" is synonymous with "pre-column" and refers to a chromatography column that binds to select components of the sample applied to the MCLC system for purposes of retaining, and typically concentrating these select components prior to contact with the separation column assembly. Without being limited by theory, by retaining analytes (e.g., peptide) before they together flow to a higher-temperature separation column assembly, a trap column may minimize the time that the analytes spends on the higher-temperature separation column assembly, minimizing temperature-induced modifications to the analyte, such as oxidation, deamidation, clipping and the like. As used herein, the term "separation column assembly" or "SCA" refers to the one or more chromatography (e.g., RPLC) columns that bind(s) to select components of the sample for purposes of chromatographically separating the components based on a particular property of the select components, e.g., hydrophobicity, charge, size. In exemplary instances, the trap column binds to peptides (e.g., enzyme-digested peptides) of the sample and, when the sample is applied to the trap column, components of the sample that are not peptides flow through the trap column, and, thus, the trap column concentrates the peptides prior to reaching the separation column assembly. In various aspects, the MCLC system comprises a trap column positioned upstream of a separation column assembly comprising one or more RPLC columns, and the trap column binds to the peptidesof the sample, which concentrates the peptides prior to separation based on hydrophobicity, which separation occurs in the separation column assembly. In exemplary aspects, each of the trap column and the one or more columns of the separation column assembly comprises a non-polar stationary phase. In exemplary instances, each of the trap column and the one or more columns of the separation column assembly is an RPLC column. In various aspects, the separation column assembly comprises a first RPLC column in tandem with a second RPLC column, and in various aspects, the first RPLC column is different from the second RPLC column. In exemplary aspects, the first RPLC column differs from the second RPLC column by virtue of its separation performance, e.g., its ability to separate molecules (e.g., peptides) in a mixture (e.g., sample comprising enzyme-digested peptides of a protein). In various instances, the RPLC columns of the separation column assembly of the MCLC system differ by column length, inner diameter, and / or composition, and / or by the stationary phase housed within each column. In exemplary instances, each chromatography column of the separation column assembly of the MCLC system is joined in fluid communication to another chromatography column by a connector. In various aspects, the first RPLC column is joined to the second RPLC column through a connector. In various aspects, the first RPLC column, second RPLC column, and the connector of the separation column assembly are any of those described herein. In various aspects, the first RPLC column is in fluid communication with the second RPLC column. In various instances, fluids applied to the separation column assembly flow from the first RPLC column to the second RPLC column through the connector. In various instances, there are no intervening valves or diverters between the first RPLC column and second RPLC column of the separation column assembly. In exemplary aspects, fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector without flowing through any valves of diverters. In exemplary instances, flowing from the first RPLC column to the second RPLC column through the connector is the only path for fluids applied to the separation column assembly. There are no other alternative paths for fluids applied to the separation column assembly, in various aspects. In exemplary embodiments, all fluids, including the sample and the mobile phase, applied to the separation column assembly flow from the first RPLC column to the second RPLC column through the connector. In exemplary aspects, the mobile phase is applied to the first RPLC column and passes through the first RPLC column, the connector and then the second RPLC column, such that peptides eluted from the first RPLC column flow through the second RPLC column. In various instances, the trap column is in fluid communication with the first RPLC column of the separation column assembly, and, in various aspects, fluids applied to the trap column flow from the trap column to the first RPLC column. In exemplary aspects, there are no intervening valves or diverters between the trap column and first RPLC column. In alternative aspects, there are intervening valvesor diverters between the trap column and first RPLC column. In various instances, the trap column has a column length that is less than 10% the column length of either the first RPLC column or the second RPLC column. The column length of the trap column is in various aspects about 2 mm to about 10 mm, e.g., about 2 mm to about 9 mm, about 2 mm to about 8 mm, about 2 mm to about 7 mm, about 2 mm to about 6 mm, about 2 mm to about 5 mm, about 2 mm to about 4 mm, about 2 mm to about 3 mm, about 3 mm to about 10 mm, about 4 mm to about 10 mm, about 5 mm to about 10 mm, about 6 mm to about 10 mm, about 7 mm to about 10 mm, about 8 mm to about 10 mm, or about 9 mm to about 10 mm. The column length of the trap column is in various aspects about 2 mm to about 7 mm, e.g., about 2 mm to about 6 mm, about 2 mm to about 5 mm, about 2 mm to about 4 mm, about 2 mm to about 3 mm, about 3 mm to about 7 mm, about 4 mm to about 7 mm, about 5 mm to about 7 mm, or about 6 mm to about 7 mm. In various instances, the column length of the trap column is about 3 mm to about 6 mm, about 4 mm to about 5 mm, or about 5 mm. In various aspects, the sum of the column lengths of the RPLC columns of the separation column assembly is greater than or about 200 mm. In various aspects, the sum of the column lengths of the RPLC columns of the separation column assembly is greater than or about 250 mm. In exemplary aspects, the trap column comprises a non-polar stationary phase and in exemplary instances the non-polar stationary phase is the same as the non-polar stationary phase of the first RPLC column or the second RPLC column of the separation column assembly. In various aspects, the trap column is maintained at a column temperature that is less than the column temperature of the first RPLC column and / or the second RPLC column. In exemplary instances, the column temperature of the trap column is less than 50 °C and / or the column temperature of the first RPLC column and second RPLC column is above 50 °C, or the column temperature of the trap column is less than 40 °C and / or the column temperature of the first RPLC column and second RPLC column is above 50 °C, for example 51 °C - 90 °C. In exemplary instances, the column temperature of the trap column is greater than about 15 °C, greater than about 20 °C, greater than or about 25 °C, for example 20 °C to 39 °C. In various aspects, the column temperature of the trap column is greater than about 30 °C or greater than about 35 °C. In various aspects, the column temperature of the trap column is about 37 °C. In various aspects, the column temperature of the first RPLC column and second RPLC column of the separation column assembly is above 60 °C or above 70 °C, optionally about 80 °C. In various aspects, when the MCLC system comprises a trap column and a separation column assembly, the mobile phase comprises Mobile Phase A (MPA) and Mobile Phase B (MPB), wherein Mobile Phase A (MPA) is about 0.1% (v / v) formic acid in water and Mobile Phase B (MPB) is about 0.1% (v / v) formic acid in about 80% (v / v) to 100% (v / v) acetonitrile (ACN) in water and / or the mobile phase is applied to the MCLC system for less than 20 minutes, less than 15 minutes or less than 13 minutes. Invarious aspects, eluted peptides are obtained within the first 12.5 minutes of the gradient. In various aspect, the mobile phase is applied according to the following gradient:

[0068] In exemplary embodiments, the MCLC system comprises an autosampler and the sample comprising peptides (e.g., enzyme digested peptides) of a protein is applied to the autosampler. In exemplary embodiments, the MCLC system comprises a trap column positioned downstream of the autosampler, and fluids (e.g., sample, mobile phase) applied to the autosampler flow to the trap column. See, e.g., Figure 21B. The trap column may be any of the trap columns described herein. For example, the trap column may (a) have a column length that is less than 10% the column length of either the first RPLC column or the second RPLC column; and / or (b) have a column length less than 7 mm, optionally, about 5 mm. If the MCLC system does not comprise a trap column, fluids (e.g., sample, mobile phase) applied to the autosampler flow to the separation column assembly, e.g., the first RPLC column thereof.

[0069] In various aspects of the present disclosure, the MCLC system further comprises two separation column assemblies and / or comprises a first SCA of RPLC columns and a second SCA of RPLC columns, wherein each of the first SCA and second SCA comprises a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column, wherein, for each of the first SCA and second SCA,: (i) the first RPLC column is different from the second RPLC column; (ii) the first RPLC column is joined to the second RPLC column through a connector; (iii) the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column. See, e.g., Figure 21 C. In certain aspects, the RPLC columns of the first SCA are identical to the RPLC columns of the second SCA. In various aspects, the MCLC system further comprises a trap column, wherein (a) the trap column is in fluid communication with the first RPLC column of the first SCA or the second SCA, (b) fluids applied to the trap column flow from the trap column to the first RPLC column of the first SCA or second SCA, (c) there are no intervening valves or diverters between the trap column and first RPLC column, and / or (d) the trap column comprises a non-polar stationary phase that is the same as one of theRPLC columns of the first SCA and / or second SCA. See, e.g, Figure 21C. In exemplary instances, the column temperature of the trap column is less than the column temperature of RPLC columns of the first SCA and / or second SCA. For instance, the column temperature of the trap column is less than 40 °C (for example 21 °C - 39 °C) or 50 °C (for example 21 °C - 49 °C), and / or the column temperature of the RPLC columns of the first SCA and / or second SCA is above 50 °C (for example 51 °C - 90 °C). In various aspects, the column temperature of trap column, the first RPLC column and second RPLC column are as described herein. For example, the column temperature of the trap column is greater than 20 °C, for example 21 °C - 49 °C, 21 °C - 40 °C, or 21 °C - 39 °C, and / or the column temperature of the RPLC columns of the first SCA and / or second SCA is above 60 °C, for example 61 °C - 90 °C or 61 °C - 80 °C. In various aspects, the column temperature of the first RPLC column and second RPLC column is above 70 °C or about 80 °C. The MCLC system in various aspects further comprises an autosampler and the sample comprising peptides of a protein is applied to the autosampler. See, e.g., Figure 21C. In various aspects, the MCLC system further comprises two or more (e.g., 2, 3, 4, 5) binary pumps. In exemplary aspects, the binary pumps move fluids through the MCLC system. In various aspects, a first binary pump causes fluids to move through the first SCA while a second binary pump causes fluids to move through the second SCA, wherein the first binary pump causes fluids to move to a detector (e.g., UV and / or mass spectrometer) for analysis, and the second binary pump causes fluids to move to a waste collection bin for column reconditioning. In various aspects, the first binary pump causes fluids to move to a detector (e.g., UV and / or mass spectrometer) for analysis, and the second binary pump causes fluids to move to a waste collection bin for column reconditioning, and actions of the first binary pump and second binary pump allow for column reconditioning of an SCA and analysis of peptides eluted from an SCA to occur simultaneously. In various instances, the MCLC further comprises a switch with at least two settings and a series of valves, wherein fluids move through a series of valves controlled by the switch. In various aspects, the switch in one setting allows fluids to move from the autosampler to the trap column through the first SCA to the detector, and the switch in another setting allows fluids to move through the first SCA to the waste collection bin. An exemplary MCLC system comprising two separation column assemblies, a trap column, two binary pumps, a switch and a series of valves are shown in Figure 23A and B. In exemplary embodiments, the MCLC system comprises two 6-port valves and a switch with at least two settings, wherein a first binary pump causes fluids to move along a first path that leads to the detector, while a second binary pump causes fluids to move along a second path to the waste collection bin, and, when the switch is in a first setting, fluids, pumped by the first binary pump, move from the autosampler to the trap column through the first SCA to the detector, and fluids, pumped by the second binary pump, move through the second SCA to thewaste collection bin, and, when the switch is in a second setting, fluids, pumped by the first binary pump, move from the autosampler to the trap column through the second SCA to the detector, and fluids, pumped by the second binary pump, move through the first SCA to the waste collection bin.

[0070] The present disclosure provides a method of separating peptides of a protein, wherein, in exemplary embodiments, the method comprises applying a sample comprising peptides (e.g., enzyme-digested peptides) of a protein to a MCLC system comprising a trap column and one or more separation column assemblies, which MCLC system may be any of those described herein. In various aspects, the trap column is maintained at a column temperature lower than the column temperature of the RPLC columns of the separation column assemblies. In various aspects, the sum of the column lengths of the RPLC columns of the separation columns are greater than or about 200 mm or greater than or about 250 mm, for example 200 mm - 500 mm, while the column length of the trap column is less than 10 mm, for example 1 mm-10 mm. In various aspects, the trap column comprises a non-polar stationary phase which is the same as that of one of the RPLC columns of the separation column assembly, whereas each of the RPLC columns of the separation column assembly have non-identical non-polar stationary phases. In various aspects, the method of separating peptides further comprises analyzing the eluted peptides by mass spectrometry for post- translational modifications, such that the present disclosure also provides a method of analyzing a protein for post-translational modifications.

[0071] The present disclosure further provides any of the MCLC system described herein. In various aspects, the MCLC system comprises a trap column and one or more separation column assemblies. In various aspects, the trap column is maintained at a column temperature lower than the column temperature of the RPLC columns of the separation column assemblies. In various aspects, the sum of the column lengths of the RPLC columns of the separation columns are greater than or about 200 mm or greater than or about 250 mm, for example 200 mm - 500 mm, while the column length of the trap column is less than 10 mm, for example 1 mm-10 mm. In various aspects, the trap column comprises a non-polar stationary phase which is the same as that of one of the RPLC columns of the separation column assembly, whereas each of the RPLC columns of the separation column assembly have non-identical non-polar stationary phases.

[0072] Proteins

[0073] In exemplary embodiments, the protein is a therapeutic protein. As used herein, the term "therapeutic protein" refers to any molecule, which may be naturally-occurring or engineered or synthetic, comprising at least one polypeptide chain, which, when administered to a subject, is intended for achieving a therapeutic effect for treatment of a disease or medical condition.Therapeutic proteins can include antigen binding proteins such as antibodies and proteins comprising antibody fragments, as well as growth factors and enzymes. In exemplary instances, the protein, e.g., therapeutic protein, comprises one or more domains of an antibody, e.g., antibody domains, immunoglobulin domains. As used herein, the term "antibody" which is synonymous with "immunoglobulin", refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions, as described in, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 4thed., Elsevier Science Ltd. / Garland Publishing, 1999. For example, an antibody may be an IgG which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and varies among other antibodies that bind to different antigens. The constant region of the antibody functions to recruit cells and molecules of the immune system. The variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4thed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0074] The general structure and properties of CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0075] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, lgG2, lgG3, and lgG4. IgM has subclasses, including, but not limited to, IgMl and lgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constantregion. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgGl, lgG2, lgG3 or lgG4. In exemplary aspects, the therapeutic protein comprises one or more antibody heavy chains.

[0076] An antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab')? fragment and a pFc' fragment. In exemplary aspects of the present disclosure, the fusion protein of the present disclosure comprises an antigen binding antibody fragment. As used herein, the term "antigen binding antibody fragment" or "antigen-binding fragment" or "antigen-binding portion" refers to a portion of an antibody that is capable of binding to the antigen of the antibody. In exemplary instances, the antigen binding antibody fragment comprises a Fab fragment or a F(ab')? fragment. Optionally, the protein of which the digested peptides are separated comprises an Fc domain and / or a Fab fragment.

[0077] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as "antibody protein products". Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs and VHH / VH (discussed below). The smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble, flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are VHH / VH of camelid heavy chain Abs as well as single domain Abs (sdAb). The building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ~15 amino acid residues. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically activepeptide grafted onto an Fc domain. Peptibodies are well-described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012). Other antibody protein products include a single chain antibody (SCA); a diabody; a triabody; a tetrabody; bispecific or trispecific antibodies, and the like. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97- 106 (2015). In various instances, the protein, e.g., therapeutic protein, of which the digested peptides are se is an antibody or comprises a single chain variable (scFv) domain or is a bispecific antibody.

[0078] In exemplary embodiments, the therapeutic protein comprises an antibody heavy chain variable region and / or an antibody light chain variable region. The therapeutic protein is an antibody in various aspects. In exemplary instances, the therapeutic protein is an antigen-binding antibody fragment, for example, an scFv or scFab. In various aspects, the protein is a protein of an in-process sample, a drug substance, or drug product. In exemplary instances, the therapeutic protein is any one of the therapeutic proteins described herein at "Therapeutic Proteins".

[0079] Post Translational Modifications (PTMs)

[0080] The method of the present disclosure is particularly useful at separating peptides comprising a PTM from peptides lacking a PTM, and in various aspects, the method separates peptides comprising a particular type of PTM from peptides comprising a different type of PTM or comprising a different combination of PTMs. For example, the methods in various instances separate a peptide comprising an amino acid sequence comprising an Asn and / or Gin which is subject to deamidation, and a Met, Trp, and / or His which are subject to oxidation, wherein multiple species of the peptide comprising no PTMs, all possible PTMs, or a combination of PTMs (e.g., deamidated Asn without oxidated Met, oxidated Met without deamidated Met, are separated by the method. The method of the present disclosure in exemplary aspects separate peptides comprising a deamidated Asn and / or an isomerized Asp. In various aspects of the present disclosure, the method separates peptide comprising any one or more PTMs selected from amino acid mutations, amino acid misincorporations, Pro peptide bonds, and / or any PTMs listed in the table below.

[0081] Therapeutic Proteins

[0082] The following disclosure is provided merely to illustrate therapeutic proteins of the present invention and not in any way to limit its scope. The therapeutic protein may be any of the following therapeutic proteins: CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; including those that interfere with receptor binding. HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor. Cell adhesion molecules, for example, LFA-I, Mol, pl50, 95, VLA-4, ICAM-I, VCAM, and alpha v / beta 3 integrin. Growth factors, such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-I -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF- a and TGF-P, including TGF-pi, TGF-P2, TGF-P3, TGF- P4, or TGF- P 5, insulin-like growth factors-l and -II (IGF-I and IGF-II), des(l-3)-IG F-l (brain IGF-I), and osteoinductive factors. Insulins and insulin-related proteins, including insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins. Coagulation and coagulation- related proteins, such as, among others, factor VIII, tissue factor, von Willebrands factor, protein C, alpha-l-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t- PA"), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens. Colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM-CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor (c-fms). Receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors ("TPO-R," "c-mpl"), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c-Kit, and other receptors. Receptor ligands, including,for example, OX40L, the ligand for the 0X40 receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A- chain, relaxin B-chain, and prorelaxin; interferons and interferon receptors, including for example, interferon-a, -p, and -y, and their receptors. Interleukins and interleukin receptors, including IL-I to IL-33 and IL-I to IL-33 receptors, such as the IL-8 receptor, among others. Viral antigens, including an AIDS envelope viral antigen. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, and activin. Integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatins, TALL proteins, including TALL-I, amyloid proteins, including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins ("TSLP"), RANK ligand ("RANKL" or "OPGL"), c-kit, TNF receptors, including TNF Receptor Type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing. The therapeutic protein in some aspects, is a protein which binds to any one of the aforementioned proteins.

[0083] Additional exemplary therapeutic proteins include Activase® (Alteplase); alirocumab, Aranesp® (Darbepoetin-alfa), Epogen® (Epoetin alfa, or erythropoietin); Avonex® (Interferon P-la); Bexxar® (Tositumomab); Betaseron® (Interferon-P); bococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see US8080243); Campath® (Alemtuzumab); Dynepo® (Epoetin delta); Velcade® (bortezomib); MLN0002 (anti-a4P7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex® (Epoetin alfa); Erbitux® (Cetuximab); evolocumab; Genotropin® (Somatropin); Herceptin® (Trastuzumab); Humatrope® (somatropin [rDNA origin] for injection); Humira® (Adalimumab); Infergen® (Interferon Alfacon-1); Natrecor® (nesiritide); Kineret® (Anakinra), Leukine® (Sargamostim); LymphoCide® (Epratuzumab); BenlystaTM (Belimumab); Metalyse® (Tenecteplase); Mircera® (methoxy polyethylene glycol-epoetin beta); Mylotarg® (Gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia® (certolizumab pegol); SolirisTM (Eculizumab); Pexelizumab (Anti-C5 Complement); MEDI-524 (Numax®); Lucentis® (Ranibizumab); Edrecolomab (,Panorex®); Trabio® (lerdelimumab); TheraCim hR3 (Nimotuzumab); Omnitarg (Pertuzumab, 2C4); Osidem® (IDM-I); OvaRex® (B43.13); Nuvion® (visilizumab); Cantuzumab mertansine (huC242-DMI); NeoRecormon® (Epoetin beta); Neumega® (Oprelvekin); Neulasta® (Pegylated filgastrim, pegylated G-CSF, pegylated hu-Met-G-CSF); Neupogen® (Filgrastim); Orthoclone OKT3® (Muromonab-CD3), Procrit® (Epoetin alfa); Remicade® (Infliximab), Reopro® (Abciximab), Actemra® (anti-IL6 ReceptormAb), Avastin® (Bevacizumab), HuMax-CD4 (zanolimumab), Rituxan® (Rituximab); Tarceva® (Erlotinib); Roferon-A®-(lnterferon alfa-2a); Simulect® (Basiliximab); StelaraTM (Ustekinumab); Prexige® (lumiracoxib); Synagis® (Palivizumab); 146B7-CHO (anti-IL15 antibody, see US7153507), Tysabri® (Natalizumab); Valortim® (MDX-1303, anti-B. anthracis Protective Antigen mAb);ABthraxTM; Vectibix® (Panitumumab); Xolair® (Omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portion of human IgGI and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRI fused to IgGI Fc), Zenapax® (Daclizumab); Zenapax® (Daclizumab), Zevalin® (Ibritumomab tiuxetan), Zetia (ezetimibe), Atacicept (TACI-lg), anti-a4P7 mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti- CD23 mAb (lumiliximab); BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); SimponiTM (Golimumab); Mapatumumab (human anti-TRAIL Receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (Volociximab, anti-a5pi integrin mAb); MDX-010 (Ipilimumab, anti-CTLA-4 mAb and VEGFR-I (IMC-18F1); anti-BR3 mAb; anti-C. difficile Toxin A and Toxin B C mAbs MDX-066 (CDA-I) and MDX-1388); anti-CD22 dsFv-PE38 conjugates (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibodies; anti-TSLP receptor antibody (US8101182); anti-TSLP antibody designated as A5 (US7982016); (anti-CD3 mAb (NI-0401);Adecatumumab (MT201, anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAbs); MDX-1333 (anti- IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti- CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxinl mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb; anti-sclerostin antibodies (see, US8715663 or US7592429) anti-sclerostin antibody designated as Ab-5 (US8715663 or US7592429); anti- ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF Receptor mAb (CAM-3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNa mAb); anti-IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); anti-l L12 / I L23p40 mAb (Briakinumab); anti-IL-23pl9 mAb (LY2525623); anti- 1113 mAb (CAT-354); anti-IL-17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC); anti-IL5 Receptor mAb; anti-integrin receptors mAb (MDX-018, CNTO 95); anti-l PIO Ulcerative Colitis mAb (MDX- 1100); anti- LLY antibody; BMS-66513; anti-Mannose Receptor / hCGP mAb (MDX-1307); anti-mesothelin dsFv- PE38 conjugate (CAT-5001); anti-PdlmAb (MDX-1 106 (ONO- 4538)); anti-PDGFRa antibody (IMC- 3G3); anti-TGFP mAb (GC-1008); anti-TRAIL Receptor-2 human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR / Flt-1 mAb; anti- ZP3 mAb (HuMax-ZP3); NVS Antibody #1; NVS Antibody #2; and an amyloid-beta monoclonal antibody.

[0084] Examples of therapeutic proteins suitable for the methods include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab,altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, bemarituzumab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maridebart cafraglutide, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide,secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox. Antibodies also include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezepelumab, and trastuzumab, and antibodies selected from Table G of International Patent Application Publication No. W02022 / 061092.

[0085] Applications

[0086] The methods of the present disclosure are thus useful for detecting post translational modifications (PTM) in a therapeutic protein, which forms, for example, during manufacture or upon storage, transportation, or other post-manufacture event. As the method can detect structural changes of a therapeutic protein in a drug product, the methods are advantageous for evaluating product quality, manufacturing process steps, and overall stability and safety of a drug product.

[0087] Accordingly, the present disclosure provides methods of analyzing a protein for post- translational modifications, methods of detecting PTMs in a therapeutic protein, and methods of monitoring for post translational modification of proteins. In exemplary embodiments, the method comprises (a) applying a sample comprising peptides (e.g., enzyme-digested peptides) of a protein to a multi-column liquid chromatography (MCLC) system comprising a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; (b) applying a mobile phase to the MCLC system to elute the bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides; and (c) analyzing the eluted peptides by mass spectrometry for peptides comprising post-translational modifications. In exemplary aspects, the first RPLC column is different from the second RPLC column. In exemplary instances, the first RPLC column is joined to the second RPLC column through a connector. In various aspects, the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector. In various instances, there are no intervening valves or diverters betweenthe first RPLC column and second RPLC column. In various instances, only two chromatography columns are comprised by the MCLC system: the first RPLC column and the second RPLC column. In exemplary instances, the methods of analyzing a protein for post-translational modifications and methods of detecting PTMs in a therapeutic protein comprise separating peptides in accordance with any of the teachings herein relating to the presently disclosed methods of separating peptides.

[0088] The present disclosure further provides methods of characterizing a drug substance or drug product, or a process preparing the drug substance or drug product, from a quality or safety standpoint. In exemplary embodiments, the method comprises separating peptides in accordance with any of the presently disclosed methods of separating peptides.

[0089] MCLC Systems

[0090] The present disclosure also provides multi-column liquid chromatography (MCLC) systems. In exemplary embodiments, the MCLC system comprises a first reversed-phase liquid chromatography (RPLC) column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column. In exemplary aspects, the MCLC system comprises only two RPLC columns, wherein the only chromatography columns comprised by the MCLC system are the first RPLC column and the second RPLC column. In various aspects, the sum of the column lengths of the two RPLC columns is greater than 150 mm, optionally, greater than 175 mm. For example, the sum of the column lengths of the two RPLC columns may be 150 mm - 250mm, or 175 mm - 250 mm. In exemplary aspects, the sum of the column lengths of the two RPLC columns is less than about 250 mm. In exemplary instances, the connector is less than 100 mm in length. In exemplary aspects, the connector is less than 75 mm in length, optionally, about 45 mm to about 70 mm. The column length of the first RPLC column and / or second RPLC column is about 25 mm to about 200 mm or about 50 mm to about 150 mm, in exemplary instances. In various aspects, the inner diameter of the first RPLC column and / or second RPLC column is about 1 mm to about 9 mm. In various instances, each of the first RPLC column and second RPLC column comprises a non-polar stationary phase. In various aspects, the non-polar stationary phase comprises hydrocarbons, including for example, C18 hydrocarbons. In exemplary aspects, the non-polar stationary phase comprises particles having a particle size of about 1.7 pm to about 3 pm or about 1.7 pm to about 2.1 pm, and, in various instances, the particles have a pore size of about 120 A to about 300 A orabout 120 A to about 160 A. In exemplary embodiments, the non-polar stationary phase comprises particles comprising tetraethoxysilane (TEOS) and bis(triethoxysilyl) ethane (BTEE). In various aspects, the non-polar stationary phase comprises ethylene-bridged-hybrid (BEH) particles. In some instances, at least one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising a surface charge. In exemplary aspects, one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising charged surface hybrid (CSH) particles. In various instances, the first RPLC column comprises a non-polar stationary phase comprising CSH particles. In various instances, the second RPLC column comprises a non-polar stationary phase comprising CSH particles. In various aspects, the first RPLC column comprises CSH particles and the second RPLC column comprises BEH particles. In various aspects, the first RPLC column comprises BEH particles and the second RPLC column comprises CSH particles. The MCLC system in various instances is any of those described herein, e.g., any of the dual-column systems described herein, e.g., one described in Figure 20.

[0091] Exemplary Embodiments

[0092] The following is a listing of exemplary embodiments of the present disclosure.1. A method of separating peptides of of a protein, said method comprising: a. applying a sample comprising peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first reversed-phase liquid chromatography (RPLC) column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein: the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column; and b. applying a mobile phase to the MCLC system to elute the bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides.2. The method of embodiment 1, wherein peptides eluted from the first RPLC column flow through the second RPLC column.The method of embodiment 1 or 2, wherein the sample is applied to the first RPLC column and flows from the first RPLC column to the second RPLC column through the connector. The method of any one of embodiments 1 to 3, wherein the mobile phase is applied to the first RPLC column and flows from the first RPLC column to the second RPLC column through the connector. The method of any one of the preceding embodiments, wherein the flow rate of the sample and mobile phase through the MCLC system is about 100 pL / minute to about 1000 pL / minute. The method of embodiment 5, wherein the flow rate is at least about 200 pL / minute. The method of embodiment 6, wherein the flow rate is at least or about 250 pL / minute The method of embodiment 7 , wherein the flow rate is at least or about 600 pL / minute. The method of embodiment 8, wherein the flow rate is at least or about 700 pL / minute. The method of any one of the preceding embodiments, wherein the MCLC system comprises only two RPLC columns, wherein the only chromatography columns comprised by the MCLC system are the first RPLC column and the second RPLC column. The method of embodiment 10, wherein the sum of the column lengths of the two chromatography columns is greater than 150 mm, optionally, greater than 175 mm. The method of embodiment 11, wherein the sum of the column lengths of the two chromatography columns is less than about 250 mm. The method of any one of the preceding embodiments, wherein the connector is less than 100 mm in length. The method of embodiment 13, wherein the connector is less than 75 mm in length, optionally, about 45 mm to about 70 mm. The method of any one of the preceding embodiments, wherein the column length of the first RPLC column and / or second RPLC column is about 25 mm to about 200 mm. The method of any one of the preceding embodiments, wherein the column length of the first RPLC column and / or second RPLC column is about 50 mm to about 150 mm. The method of any one of the preceding embodiments, wherein the inner diameter of the first RPLC column and / or second RPLC column is about 1 mm to about 9 mm. The method of any one of the preceding embodiments, wherein each of the first RPLC column and second RPLC column comprises a non-polar stationary phase. The method of embodiment 18, wherein the non-polar stationary phase comprises hydrocarbons.The method of embodiment 19, wherein the non-polar stationary phase comprises C18 hydrocarbons. The method of any one of embodiments 18 to 20, wherein the non-polar stationary phase comprises particles having a particle size of about 1.7 pm to about 3 pm. The method of embodiment 21, wherein the particle size is about 1.7 pm to about 2.1 pm. The method of any one of the preceding embodiments, wherein the non-polar stationary phase comprises particles having a pore size of about 120 A to about 300 A. The method of embodiment 23, wherein the pore size is about 120 A to about 160 A. The method of any one of embodiments 18 to 24, wherein the non-polar stationary phase comprises particles comprising tetraethoxysilane (TEOS) and bis(triethoxysilyl) ethane (BTEE). The method of embodiment 25, wherein the non-polar stationary phase comprises ethylene-bridged-hybrid (BEH) particles. The method of any one of embodiments 18 to 26, wherein at least one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising a surface charge. The method of embodiment 27, wherein one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising charged surface hybrid (CSH) particles. The method of embodiment 28, wherein the first RPLC column comprises a non-polar stationary phase comprising CSH particles. The method of embodiment 28, wherein the second RPLC column comprises a non-polar stationary phase comprising CSH particles. The method of any one of the preceding embodiments, wherein the first RPLC column comprises CSH particles and the second RPLC column comprises BEH particles. The method of any one of embodiments 1 to 30, wherein the first RPLC column comprises BEH particles and the second RPLC column comprises CSH particles. The method of any one of the preceding embodiments, comprising chromatographically separating the peptides through ultra-high pressure liquid chromatography (UHPLC), wherein the pressure is greater than 900 bar. The method of any one of the preceding embodiments, wherein the mobile phase comprises a gradient of a polar organic solvent. The method of embodiment 34, wherein the mobile phase comprises an increasing concentration gradient of the polar organic solvent.The method of embodiment 35, wherein the mobile phase comprises only one increasing concentration gradient of the polar organic solvent. The method of any one of the preceding embodiments, wherein the mobile phase comprises only one organic polar solvent. The method of any one of the preceding embodiments, wherein mobile phase comprises a polar organic solvent selected from the group consisting of: acetonitrile, methanol, ethanol, propanol, and isopropanol. The method of any one of the preceding embodiments, wherein the mobile phase comprises a buffer. The method of embodiment 39, wherein the buffer comprises trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), or formic acid. The method of embodiment 39 or 40, wherein the buffer has a pH below 4. The method of embodiment 41, wherein the buffer has a pH of 2 to 3. The method of any one of the preceding embodiments, wherein the mobile phase comprises a mixture of two mobile phase solutions, only one of which comprises the polar organic solvent and both mobile phase solutions comprise a buffer. The method of embodiment 43, wherein both mobile phase solutions comprise less than 1% (v / v) buffer or less than 0.5% (v / v) buffer. The method of 44, wherein both mobile phase solutions comprise TFA (pH 2) or formic acid (pH 3). The method of embodiment 45, wherein both mobile phase solutions comprise 0.1% TFA (pH 2) or 0.1% formic acid (pH 3). The method of any one of the preceding embodiments, wherein the mobile phase comprises Mobile Phase A (MPA) and Mobile Phase B (MPB), wherein Mobile Phase A (MPA) is about 0.1% (v / v) formic acid in water and Mobile Phase B (MPB) is about 0.1% (v / v) formic acid in about 80% (v / v) to 100% (v / v) acetonitrile (ACN) in water. The method of any one of the preceding embodiments, wherein (b) occurs in less than 120 minutes. The method of any one of the preceding embodiments, wherein (b) occurs in less than 90 minutes. The method of embodiment 49, wherein (b) occurs in less than 60 minutes. The method of embodiment 50, wherein (b) occurs in 30 minutes or less or 25 minutes or less.The method of any one of the preceding embodiments, comprising applying the mobile phase to the MCLC system according to the following gradient:The method of embodiment 52 or 55, wherein eluted peptides are obtained within the first 11.4 minutes of the gradient. The method of embodiments 1 to 51, wherein (b) occurs in 15 minutes or less or 10 minutes or less. The method of embodiment 54, comprising applying the mobile phase to the MCLC system according to the following gradient:The method of any one of the preceding embodiments, further comprising analyzing the eluted peptides obtained in (b) by mass spectrometry. The method of embodiment 56, wherein the peptides eluted from the first RPLC column are sent to a mass spectrometer together with the peptides eluted from the second RPLC column and / or the peptides eluted from the first column are not separately injected into the mass spectrometer from the peptides eluted from the second column.The method of any one of the preceding embodiments, wherein a peptide comprising a post translational modification elutes separately from the corresponding unmodified peptide comprising the same amino acid sequence but without the post translational modification. The method of any one of the preceding embodiments, further comprising quantifying the peptides with one or more post translational modifications. The method of any one of the preceding embodiments, further comprising preparing the sample comprising peptides of a protein. The method of embodiment 60, comprising incubating the protein with an enzyme to obtain enzyme-digested peptides of the protein. The method of any one of the preceding embodiments, further comprising denaturing and / or alkylated the protein, reducing sulfide bonds of the protein, or a combination thereof. The method of any one of the preceding embodiments, comprising obtaining a sample comprising the protein. The method of any one of the preceding embodiments, wherein the protein is a protein of an in-process sample, a drug substance, or drug product. The method of any one of the preceding embodiments, wherein the protein is a therapeutic protein. The method of embodiment 65 wherein the therapeutic protein comprises an antibody heavy chain variable region and / or an antibody light chain variable region. The method of embodiment 66, wherein the therapeutic protein is an antibody. The method of embodiment 66, wherein the therapeutic protein comprises an antigenbinding antibody fragment. The method of embodiment 68, wherein the antigen-binding antibody fragment is an scFv or scFab. The method of any one of the preceding embodiments, wherein the protein comprises a post translational modification selected from the group consisting of Asn or Gin deamidation, Asp isomerization, oxidation of Met, Trp, or His, deamination of Glu, Ser, Gly, glycation of Lys, hydroxylysine, hydroxyproline, glycosylation of Asn, Ser, or Thr, cyclization of an N-terminal Gin or Gly, fragmentation or clipping of Asp, Pro peptide bonds, amino acid mutations, and amino acid misincorporations. The method of any one of the preceding embodiments, wherein the protein comprises a deamidated Asn and / or an isomerized Asp.The method of any one of embodiments 1-9 and 11-71, wherein the MCLC system comprises three or more RPLC columns. The method of any one of embodiments 1-9 and 11-72, further comprising a trap column, wherein (a) the trap column is in fluid communication with the first RPLC column, (b) fluids applied to the trap column flow from the trap column to the first RPLC column, and / or (c) there are no intervening valves or diverters between the trap column and first RPLC column. The method of embodiment 73, wherein the trap column has a column length that is less than 10% the column length of either the first RPLC column or the second RPLC column. The method of embodiment 73 or 74, wherein the column length of the trap column is about 2 mm to about 7 mm, optionally, about 2 mm to about 5 mm. The method of any one of embodiments 73 to 75, wherein the trap column comprises a nonpolar stationary phase that is the same as the non-polar stationary phase of the first RPLC column or the second RPLC column. The method of any one of embodiments 73-76, wherein the trap column is maintained at a column temperature that is less than the column temperature of the first RPLC column and / or the second RPLC column. The method of any one of embodiments 73-77, wherein the column temperature of the trap column is less than 40 °C and / or the column temperature of the first RPLC column and second RPLC column is above 50 °C. The method of embodiment 78, wherein the column temperature of the trap column is about 37 °C. The method of embodiment 78 or 79, wherein the column temperature of the first RPLC column and second RPLC column is above 60 °C or above 70 °C. The method of embodiment 80, wherein the column temperature of the first RPLC column and second RPLC column is about 80 °C. The method of any one of embodiments 72-81, wherein the mobile phase comprises Mobile Phase A (MPA) and Mobile Phase B (MPB), wherein Mobile Phase A (MPA) is about 0.1% (v / v) formic acid in water and Mobile Phase B (MPB) is about 0.1% (v / v) formic acid in about 80% (v / v) to 100% (v / v) acetonitrile (ACN)in water. The method of any one of embodiments 72-82, wherein (b) occurs in less than 20 minutes. The method of any one of embodiments 72-83, wherein (b) occurs in less than 15 minutes or less than 13 minutes. The method of any one of the preceding embodiments, comprising applying the mobile phase to the MCLC system according to the following gradient:The method of embodiment 85, wherein eluted peptides are obtained within the first 12.5 minutes of the gradient. The method of any one of the preceding embodiments, wherein the MCLC system comprises an autosampler and the sample comprising the peptides of a protein is applied to the autosampler which is in fluid communication with the MCLC system. The method of embodiment 87, wherein the MCLC system comprises a trap column positioned downstream of the autosampler, and fluids applied to the autosampler flow to the trap column. The method of embodiment 87 or 88, wherein the trap column (a) has a column length that is less than 10% the column length of either the first RPLC column or the second RPLC column; (b) has a column length less than 7 mm, optionally, about 5 mm. The method of any one of the preceding embodiments, wherein the MCLC system comprises a first separation column assembly (SCA) and a second SCA, wherein each of the first SCA and the second SCA comprises a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein, for each of the first SCA and second SCA,:(i) the first RPLC column is different from the second RPLC column;(ii) the first RPLC column is joined to the second RPLC column through a connector;(iii) the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and(iv) there are no intervening valves or diverters between the first RPLC column and second RPLC column. The method of embodiment 90, wherein the RPLC columns of the first SCA are the same as the RPLC columns of the second SCA.The method of embodiment 90 or 91, wherein the MCLC system comprises a trap column, wherein (a) the trap column is in fluid communication with the first RPLC column of the first SCA or the second SCA, (b) fluids applied to the trap column flow from the trap column to the first RPLC column of the first SCA or second SCA, (c) there are no intervening valves or diverters between the trap column and first RPLC column, and / or (d) the trap column comprises a non-polar stationary phase that is the same as one of the RPLC columns of the first SCA and / or second SCA. The method of embodiment 92, wherein the column temperature of the trap column is less than the column temperature of RPLC columns of the first SCA and / or second SCA. The method of embodiment 92 or 93, wherein the column temperature of the trap column is less than 40 °C and / or the column temperature of the RPLC columns of the first SCA and / or second SCA is above 50 °C. The method of embodiment 94, wherein the column temperature of the trap column is greater than 20 °C and / or the column temperature of the RPLC columns of the first SCA and / or second SCA is above 60 °C. The method of embodiment 94 or 95, wherein the column temperature of the first RPLC column and second RPLC column is above 70 °C or about 80 °C. The method of any one of embodiments 90-96, wherein the MCLC system comprises an autosampler and the sample comprising the peptides of a protein is applied to the autosampler which is in fluid communication with the MCLC system. The method of any one of embodiments 90-97, wherein the MCLC system comprises two or more binary pumps. The method of embodiment 98, wherein the binary pumps move fluids through the MCLC system. . The method of embodiment 99, wherein a first binary pump causes fluids to move through the first SCA while a second binary pump causes fluids to move through the second SCA, wherein the first pump causes fluids to move to the detector, and the second binary pump causes fluids to move to the waste collection bin. . The method of any one of embodiments 97-100, wherein the MCLC system comprises a switch with at least two settings and a series of valves, wherein fluids move through a series of valves controlled by the switch. . The method of embodiment 101, wherein the switch in one setting allows fluids to move from the autosampler to the trap column through the first SCA to the detector, andthe switch in another setting allows fluids to move through the first SCA to the waste collection bin. . A method of analyzing a protein for post-translational modifications, said method comprising separating peptides of a protein according to any one of the methods of embodiments 1-102 and analyzing the eluted peptides by mass spectrometry for post- translational modifications. . A method of analyzing a protein for post-translational modifications, said method comprising: a. applying a sample comprising peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein: the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column; b. applying a mobile phase to the MCLC system to elute bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides, and c. analyzing the eluted peptides by mass spectrometry for post-translational modifications. . The method of embodiment 104, wherein the peptides eluted from the first RPLC column are injected into a mass spectrometer together with the peptides eluted from the second RPLC column. . A multi-column liquid chromatography (MCLC) system as described in any one of embodiments 1-105. . A multi-column liquid chromatography (MCLC) system comprising a first reversed- phase liquid chromatography (RPLC) column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein:the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column. . The MCLC system of embodiment 107, comprising only two RPLC columns, wherein the only chromatography columns comprised by the MCLC system are the first RPLC column and the second RPLC column. . The MCLC system of embodiment 107 or 108, wherein the sum of the column lengths of the two RPLC columns is greater than 150 mm, optionally, greater than 175 mm.. The MCLC system of embodiment 109, wherein the sum of the column lengths of the two RPLC columns is less than about 250 mm. . The MCLC system of any one of embodiments 107-110, wherein the connector is less than 100 mm in length. . The MCLC system of embodiment 111, wherein the connector is less than 75 mm in length, optionally, about 45 mm to about 70 mm. . The MCLC system of any one of embodiments 107-112, wherein the column length of the first RPLC column and / or second RPLC column is about 25 mm to about 200 mm.. The MCLC system of any one of embodiments 107-113, wherein the column length of the first RPLC column and / or second RPLC column is about 50 mm to about 150 mm.. The MCLC system of any one of embodiments 107-114, wherein the inner diameter of the first RPLC column and / or second RPLC column is about 1 mm to about 9 mm. . The MCLC system of any one of embodiments 107-115, wherein each of the first RPLC column and second RPLC column comprises a non-polar stationary phase. . The MCLC system of embodiment 116, wherein the non-polar stationary phase comprises hydrocarbons. . The MCLC system of embodiment 117, wherein the non-polar stationary phase comprises C18 hydrocarbons. . The MCLC system of any one of embodiments 116 to 118, wherein the non-polar stationary phase comprises particles having a particle size of about 1.7 pm to about 3 pm.. The MCLC system of embodiment 119, wherein the particle size is about 1.7 pm to about 2.1 pm.. The MCLC system of any one of embodiments 107-120, wherein the non-polar stationary phase comprises particles having a pore size of about 120 A to about 300 A.. The MCLC system of embodiment 121, wherein the pore size is about 120 A to about 160 A. . The MCLC system of any one of embodiments 116 to 122, wherein the non-polar stationary phase comprises particles comprising tetraethoxysilane (TEOS) and bis(triethoxysilyl) ethane (BTEE). . The MCLC system of embodiment 123, wherein the non-polar stationary phase comprises ethylene-bridged-hybrid (BEH) particles. . The MCLC system of any one of embodiments 116 to 124, wherein at least one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising a surface charge. . The MCLC system of embodiment 125, wherein one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising charged surface hybrid (CSH) particles. . The MCLC system of embodiment 126, wherein the first RPLC column comprises a non-polar stationary phase comprising CSH particles. . The MCLC system of embodiment 126, wherein the second RPLC column comprises a non-polar stationary phase comprising CSH particles. . The MCLC system of any one of embodiments 107-128, wherein the first RPLC column comprises CSH particles and the second RPLC column comprises BEH particles.. The MCLC system of any one of embodiments 107 to 128, wherein the first RPLC column comprises BEH particles and the second RPLC column comprises CSH particles.. The MCLC system of any of embodiments 107 to 130, comprising the RPLC columns of Figure 20. . The MCLC system of any one of the preceding embodiments, further comprising a first SCA of RPLC columns and a second SCA of RPLC columns, an autosampler, a trap column, and two or more binary pumps. . The MCLC system of embodiment 132, wherein the first SCA is identical to the second SCA. . The MCLC system of embodiment 132 or 133, wherein a first binary pump causes fluids to move through one of the first SCA and second SCA to a detector for mass spectrometry analysis and a second binary pump causes fluids to move through one of the first SCA and second SCA to a waste collection bin for column reconditioning.135. The MCLC system of embodiment 134, wherein a first binary pump causes fluids to move from the autosampler to the trap column to the first column of the first SCA or the second SCA and to the detector for mass spectrometry analysis, and a second binary pump causes fluids to move from the first SCA or second SCA to the waste collection bin for column reconditioning.136. The MCLC system of any one of embodiments 132-135, wherein the column temperature of the trap column is less than the column temperature of RPLC columns of the first SCA and / or second SCA.

[0093] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLESEXAMPLE 1

[0094] This example describes high-throughput characterization of therapeutic proteins by peptide mapping using ultra-high pressure liquid chromatography (UHPLC) mass spectrometry (MS).

[0095] Introduction

[0096] Monitoring of chemical liabilities that may potentially affect safety and / or efficacy is of critical importance to the development of therapeutic proteins. While peptides containing many modifications are readily differentiated from native peptides by modification-induced mass shifts, monitoring Asn deamidation and Asp isomerization (isoAsp) is considerably more challenging. Chromatographic resolution of these peptides is a time limiting factor for rapid peptide mapping. To address this bottleneck, rapid, ultra-high pressure LC-MS (UHPLC-MS) methods were utilized to assess separation of peptides containing these challenging attributes.

[0097] Methods

[0098] Fifty-one synthetic peptides of 14 peptide families (i.e., peptide groups) were obtained from Anaspec (Fremont, CA). Each peptide group comprised unmodified or native peptides which lacked any post translational modifications (PTMs) as well as post translationally modified versions thereof, wherein the post translationally modified peptide version comprised the same amino acid sequence as the unmodified peptide but also comprised a post-translational modification (PTM). Table 1 of Figure 1A lists the amino acid sequence of each synthetic peptide of the peptide families. Site(s) for possible PTMs are shown in red. Table 1 also lists the type of PTM that is possible at thered amino acid residue, the number of residues for each peptide in the family, as well as the hydrophobicity score and molecular weight.

[0099] Samples comprising a single peptide, a peptide mixture, or a digested IgGl were prepared for UHPLC-MS analysis. Samples comprising a single peptide of Table 1 were individually injected to determine the elution order of the peptides within the peptide family. Samples comprising a mixture of peptides of a peptide family were also assessed. The sample comprised a mixture of unmodified peptides and post translationally modified versions thereof at a 10:1 ratio. In a separate study, a digested therapeutic IgGl protein was evaluated. The IgGl protein was denatured, reduced, alkylated, and digested with trypsin prior to UHPLC-MS analysis.

[0100] In this study, multiple columns were evaluated using a Thermo Vanquish LC with a flow rate set at 700 pL / min and the column temperature set at 50°C. The Thermo Vanquish LC interfaced with a Thermo Q. Exactive MS. Data were processed by Skyline [1] and MassAnalyzer [2], Control methods employed a BEH column at 250 pL / min flow rate and column temperature of 50°C. Details of the columns evaluated in this study, including column length, inner diameter of column (LD.), and particle size and pore size of the stationary phase of the column, are listed in Table 2 of Figure IB.

[0101] Peptide Elution Order

[0102] Samples comprising a single peptide of a peptide family (as shown in Table 1) were individually injected to determine the elution order of the peptides within the peptide family. Figure 1C shows an overlay of the chromatograms obtained with samples comprising a single peptide.

[0103] Differentiating Separation

[0104] Each of Figures 2 and 3 is a series of extracted ion chromatograms (EICs) obtained with different columns using the sample comprising the peptide mixture. In Figure 2, EICs for a first peptide family are shown, and, in Figure 3, EICs for a different peptide family are provided. As shown in Figure 2, only a single peak is observed using the Waters CSH Premier column. The modified peptide is not observed with CSH Premier, whereas peaks for modified and unmodified peptides are seen using all other columns, e.g., Waters BEH, Waters BEH Premier. Figure 3 shows that for a different peptide family from the same mixture, the Waters CSH Premier column separated more deamidated species, relative to the other columns. These results support that peptides can be uniquely resolved based on the particular column used.

[0105] Retention Time (RT) Reproducibility

[0106] The retention times for all peptides of a given peptide family were averaged. The range of retention times was determined by subtracting the highest retention time from the shortestretention time and the range was recorded in seconds. % RSD was calculated by dividing the standard deviation of the retention time by the average retention time and multiplying by 100%. These data are presented in Table 3. As shown in Table 3, the RTs were highly reproducible regardless of column. The % Relative Standard Deviation (RSD) was less than 1.5%.

[0107] Peak-to-Peak Resolution

[0108] Peak-to-peak resolution (Rp-p) was used to evaluate separation of each modified peptide relative to the corresponding unmodified peptide and these values are listed in Table 4 of Figure 4B. Rp-p for each peptide family is listed in Table 4. Rp-p > 0.83 was considered as good; 0.61 < R-pp < 0.83 was considered as intermediate; and Rp-p < 0.61 was considered as poor. While separation was consistently good for many peptides, other peptides showed differentiation among the different columns (e.g. ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47) , SLSLSP(GGGGS)6DK (SEQ ID NO: 29)).

[0109] Column Overall Performance

[0110] While the CSH Premier column showed the best overall performance, some individual peptide groups (e.g. NFHLRPR (SEQ ID NO: 22), IYPTNGYTR (SEQ ID NO: 19)) were better resolved using other columns. Table 5 of Figure 4C shows the peptide-specific overall performance of each column.

[0111] Sequence Coverage

[0112] Analysis of a tryptic digestion of representative IgGl mAb showed comparable sequence coverage regardless of which column was used. Coverage was also comparable to that observed with a control method. Retention of short peptides will be further assessed in future studies. Table 6 of Figure 4D shows the observed sequence coverage following tryptic digestion of a representative IgGl mAb.

[0113] Conclusions

[0114] High retention time reproducibility was observed for all columns (RSD<1.5%). Retention time shifts <1.2s were observed in replicate injections on some columns.

[0115] Regardless of column, Rp-p values for most peptide families using rapid LC were comparable to / better than those observed using standard flow rates.

[0116] CSH Premier column showed best overall performance with synthetic peptide mixture, though some peptide families were better resolved with other columns.

[0117] Sequence coverage of a digested IgGl mAb using rapid LC was comparable to control method.

[0118] These results demonstrate feasibility of leveraging high flow / high pressure LC conditions to address difficulties associated with identifying challenging chemical liabilities encountered during development of therapeutic proteins.

[0119] This approach can increase peptide map throughput by a factor of 10.

[0120] References

[0121] 1. MacLean et. al. Bioinformatics. 2010, 26, p966-968.

[0122] 2. Zhang, Z., Anal. Chem. 2009, 81(20), 8354-8364.

[0123] 3. Krokhin et al. Mol. Cell Proteomics 2004, 3, p908-919.EXAMPLE 2

[0124] This example provides additional information about the studies described in Example 1.

[0125] Sample Preparation

[0126] Synthetic peptides listed in Table 1 were prepared by AnaSpec (Fremont, CA) and provided as lyophilized aliquots. Each peptide was solubilized in 10% dimethyl sulfoxide in water to a concentration of 1 mg / mL. Each peptide was then diluted to 200 pM with water to create a peptide-specific stock solution. Stock solutions were then used to make samples comprising a single peptide or comprising a peptide mixture.

[0127] Samples comprising a single peptide: A series of samples comprising only one of the peptides listed in Table 1 was made. Each peptide-specific stock solution was diluted to 4 pM with water, and this solution was then diluted to 2 pM with 8 M guanidine HCI, 250 mM acetate, pH 4.7. A 20 pL injection consisted of 40 pmol of the peptide.

[0128] Samples comprising a peptide mixture: Samples comprising a peptide mixture of unmodified and modified peptides were made. The peptide mixture comprised all 14 of the unmodified peptides of Table 1 and all 37 post-translationally modified peptides of Table 1 at a 10:1 unmodified peptide:modified peptide ratio. The peptide mixture was made by combining a volume from each of the 200 pM stock solutions described above so that the peptide mixture consisted of 40 pM of each of the 14 unmodified peptides and 4 pM of each of the 37 modified peptides. This solution was then diluted 1:1 with 8 M guanidine HCI, 250 mM acetate, pH 4.7 A 20 pL injection consisted of 40 pmol of each of the 14 unmodified peptide and 4 pmol of each of the 37 post- translationally modified peptides.

[0129] Samples comprising IgGl digestion peptides were prepared by as described in Da Ren, et al., Analytical Biochemistry 392: 12-21 (2009).

[0130] Peptide Separation by UHPLC

[0131] Each sample was applied to a single column fitted within a ThermoFisher Scientific Vanquish™ high throughput liquid chromatography (LC) system which interfaced with a ThermoFisher Scientific Q. Exactive™ Mass Spectrometer. The samples were applied to the column which was maintained at 50°C for ultra high pressure liquid chromatography (UHPLC). The column flow rate was set at 700 pL / min, the injection volume was 20 pL (needle wash with water), and the ALS temp was 4 °C. Gradient elution was carried out with two mobile phase solutions: Mobile Phase A (MPA), which was 0.1% (v / v) formic acid in water and Mobile Phase B (MPB), which was 0.1% (v / v) formic acid in 90% (v / v) acetonitrile (ACN), 10% (v / v) water. Gradient elution was carried out with MPA and MPB according to Table 7. Detection was performed with a Thermo Q-Exactive Plus mass spectrometer. Data were collected from 3.6 min - 11.4 min. For synthetic peptides, data were acquired in Full MS mode with MSI resolution of 35,000, an AGC target of 1E6, a maximum IT of 120 ms, and a scan range of 200-2000 m / z. Skyline v. 23.1.0.455 (599e74ad2) was used to extract retention times and full width at half max (FWHM) values for each sample. For digested peptides, MS data were acquired in data-dependent mode with MSI resolution of 35,000, MSI AGC target of 1E6, MSI maximum IT of 120 ms, MSI scan rage of 200-2000 m / z, MS2 resolution of 17,500, MS2 AGC target of 1E5, MS2 maximum IT of 120ms, loop count of 3, MS2 isolation window of 2.0 m / z, and dynamic exclusion of 1.0 s. MSMS were not collected for species with undefined charge states or charge states greater than 8. Digested peptides were processed with MassAnalyzer v. 6.03.TABLE 7

[0132] Samples comprising a single peptide were applied as described above to determine the peptide's retention time for each column. Retention times (minutes) for each peptide wererecorded. Full Width at Half Max (FWHM) was derived using Skyline and peak to peak resolution (RPP or Rp-p) was calculated using the following equation:Rp-p = 1.18 * [(Retention Time 2-Retention Time 1) / (FWHM1 +FWHM2)]

[0133] An Rp-p greater than 0.83 was considered as "good", an Rp-p less than 0.83 but greater than 0.61 < R-pp < 0.83 was considered as "intermediate", and an Rp-p less than 0.61 was considered as "poor". This protocol was carried out for each of the columns listed in Table 2 and the retention time (RT), FWHM, and Rp-p (RPP) for each column are provided in Table 4.

[0134] The number of peaks achieved by the indicated column are summarized in Table 8.TABLE 8

[0135] Control Method

[0136] As a control, a method utilizing a BEH column at a flow rate of 250 pL / min was used. The column temperature was maintained at 50°C. The mobile phase comprised two mobile phase solutions: Mobile Phase A (MPA), which was 0.1% (v / v) formic acid in water and Mobile Phase B (MPB), which was 0.1% (v / v) formic acid in acetonitrile. Gradient elution was carried out with MPA and MPB according to Table 9. As the flow rate was low, this was not UHPLC. MS data were collected from 10-78 min. For the first lOmin, flow was diverted to waste to remove sample prep- related reagents. From 78-123min, the flow was diverted to waste while to column was washed to minimize carryover and equilibrated for the next injection.TABLE 9

[0137] IgGl Analysis

[0138] Samples comprising IgGl digestion peptides were applied to each of the columns of Table 2 as essentially described above. The % listed in Table 6 was determined by MassAnalyzer. Briefly, the sequence coverage describes the percentage of the amino acid sequence of a protein corresponding to peptides identified by the search algorithm. For example, if a protein sequence consisted of 100 amino acid residues and peptides identified in a peptide map experiment consist of a total of 90 unique residues, then the sequenced coverage would be 90%.EXAMPLE 3

[0139] The example demonstrates use of a dual-column LC system.

[0140] The results achieved from the single-column LC studies described in Examples 1-3 suggest that the CSH Premier column demonstrated the best overall performance. However, this column demonstrated a poor performance for the ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47) peptide family, and it is possible that the column may demonstrate poor performance with other peptide families not tested here. Whereas the CSH Premier column demonstrated poor performance for the ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47) peptide family, other columns, including but not limited to the BEH Premier column and the Vanquish Acclaim column, performed very well on this peptide family. It was hypothesized that combining one of these two columns in tandem with the CSH Premier could lead to an overall better performance compared to that achieved with only one column.

[0141] Studies purposed for testing a dual-column LC system were carried out. In an initial study, a BEH Premier column (100mm) was coupled to a CSH Premier column (100mm) using an EXP® Hand Tight Column Connector (Product No. 15-20-04128, Optimize Technologies, Oregon City, OR) and fitted into the Vanquish UHPLC instrument interfaced with the MS as described in Examples 1 and 2. Samples prepared as described in Example 2 were applied to the dual-column LC system for UHPLC as essentially described in Example 2, except that the column temperature was set to 70 °C. As shown in Figure 5A, fluctuations in pressure were observed with this dual-column LC system.

[0142] To address this issue, a different column fitting was used in place of the EXP® Hand Tight Column Connector. In this study, a Viper stainless steel 0.100 pm x 65 mm fitting (Product No.6040.2207, ThermoFisher Scientific, Waltham, MA) was used to join the BEH Premier column (100mm) to the CSH Premier column (100mm). The Viper fitting differed in length (65 mm) compared to the EXP® Hand Tight Column Connector (50 mm). As shown in Figure 5B, the pressure fluctuations observed with the dual-column LC system comprising the EXP® Hand Tight Column Connector were not observed in this study using the Viper fitting. The Viper fitting was used in subsequent studies involving dual column LC systems.EXAMPLE 4

[0143] This example describes a study evaluating peptide separation achieved using different dual column LC systems.

[0144] The results from Examples 1 and 2 suggested that two columns may lead to better performance compared to that achieved with only one column. However, the use of two columns could lead to issues that outweigh the advantages of using two columns. For example, connecting columns introduces a dead volume between the two columns, and dead volumes are known contributors to peak broadening and decreased sensitivity. Additionally, multiple column chemistries may, in some circumstances, contribute to poor chromatographic resolution. For example, if peptide A elutes before peptide B in the first column but peptide B before peptide A in the second column, when the two columns are combined in tandem, the resulting chromatogram may have a smeared profile with poorer resolution than either individual column.

[0145] Additional dual-column LC systems were made using the Viper stainless steel fitting to connect the two columns. The performance of each dual-column systems was compared to the performance of single-column systems. A summary of the column systems evaluated in this study is provided in Table 10.TABLE 10All columns had an Inner Diameter of 2.1 mm.

[0146] Samples comprising synthetic peptides (samples comprising a single peptide and samples comprising a peptide mixture) were prepared as essentially described in Example 2.

[0147] UHPLC was performed using a Thermo Vanquish, as essentially described in Example 2. Column temperatures are as indicated in Table 10. 20 pL of each sample was injected onto column. Gradient elution was carried out with two mobile phase solutions: Mobile Phase A which consisted of 0.1% formic acid in water, and Mobile Phase B which consisted of 0.1% formic acid, 90% acetonitrile, 10% water. Gradient elution was carried out following the gradient of Table 11. Flow was diverted to waste from 0-3.6 min and from 11.4 min to 24.9 min. Following completion of a sequence, the system and column were flushed with 80% methanol, 20% water for 1 h at 400 pL / min.TABLE 11

[0148] Separated peptides were detected with a Thermo Q-Exactive Plus mass spectrometer.Data were collected from 3.6 min - 11.4 min. Data were acquired in Full MS mode with MSI resolution of 35,000, an AGC target of 1E6, a maximum IT of 120 ms, and a scan range of 200-2000 m / z. Skyline v 23.1.0.455 (599e74ad2) was used to extract retention times and full width at half max (FWHM) values for each sample. Control methods are as described in Example 2.

[0149] Exemplary results are provided in Figures 6A-19F as follows:TABLE 12

[0150] As shown in the Figures 6A-19F, high resolution separation of peptides of the peptide mixture sample comprising both post-translationally modified and unmodified peptides was achieved using the dual column LC systems.

[0151] Scores were assigned to each column system per peptide family as shown in Tables 13 and 14, wherein "++" means best observed separation for a peptide family, "+" means intermediate separation for a peptide family and means poor separation for a peptide family. Summed scores are equal to 2*(# "++" peptide families) + 1*(# "+" peptide families) + 0*(# peptide families).TABLE 13S* is the SEQ ID NO:TABLE 14S* is the SEQ ID NO:

[0152] In some cases, the order of the column made a difference (Table 13). For example, the ASQNVDTNVAWYQQKPGQAPK (SEQ ID NO: 47) peptide family was separated with higher resolution when the BEH Premier column was positioned before the CSH Premier column, although the Dual Column LC System wherein the CSH Premier column is positioned before the BEH Premier column exhibited a better overall performance (11 ++ scores).

[0153] These results suggest that using two columns in tandem provides higher resolution results for more peptides. A dual column LC system resolves a higher number of peptides and their modified versions compared to a single column LC system.

[0154] The results presented herein indicate that combining columns in tandem can be used to leverage strengths of each column with respect to difficult-to-resolve peptides while at the sametime maintaining narrow peak widths. This enables up to a lOx increase in peptide map throughput while maintaining comparable or improved performance relative to the control method.EXAMPLE 5

[0155] This example describes a study that evaluates a variety of MCLC systems.

[0156] The study described in Example 5 is carried out with different combinations of two RPLC columns, including those described in Figure 20, as well as a control system comprising a single column. The performance of the dual column systems is evaluated and their performance as to separating the peptide mixtures described in Example 5 are compared to the control system.EXAMPLE 6

[0157] This example describes high-throughput peptide mapping of therapeutic proteins with a dual binary pump, in-tandem dual column system, and the improved separations of chemical liabilities thereof.

[0158] It is useful to monitor chemical liabilities that may affect the safety and / or efficacy of therapeutic proteins, such as antibodies. While most modifications (e.g. oxidation) are easily detected by mass shifts, tracking Asn deamidation and Asp isomerization (isoAsp) is more challenging. Without being limited by theory, the isotopic envelope of a deamidated peptide overlaps with the unmodified peptide, and Asp and isoAsp are isobaric, making them indistinguishable by conventional MS without specialized fragmentation. In view of these limitations of conventional MS, chromatographic resolution may be used for identification and accurate quantitation, but may also be rate-limiting. To address these challenges, in this example multiple C18 columns with different selectivity values and particle chemistries were linked in tandem, and this column setup was combined with rapid, ultra-high pressure LCMS methods to separate peptides with these challenging attributes.

[0159] 51 synthetic peptides corresponding to 14 peptide families were obtained from Anaspec, and these peptides were mixed in a 10:1 unmodified:modified peptide ratio. An IgGl antibody was also reduced, alkylated, and enzymatically digested with trypsin. LCMS analysis was performed using a Thermo Vanquish HPLC system with dual binary pumps interfaced with a Thermo Q-Exactive MS. Using different combinations of Waters CSH and BEH columns connected in tandem, synthetic peptides and digested IgGl were evaluated using compressed 12.5 min method (l%-10% acetonitrile in 0.3 min, 10% B-50% acetonitrile in 10.4 min) at 620 pL / min. Dual binary pumps doubled throughput by enabling column switching and offline washing and equilibration. Data were processed using Skyline and MassAnalyzer software.

[0160] The optimized UHPLC configuration in this Example comprises the following three features: 1) two binary pumps, 2) peptides were captured on 5mm CSH pre-column "peptide trap" column or "trap column" at 37 °C to minimize method-induced degradation, 3) two tandem column pairs (total four columns) at 80 °C were used for efficient separation. Two Waters columns with different particle chemistries were combined in tandem (e.g. 100mm CSH followed by 150mm BEH) at 80 °C for pseudo-2D chromatography. Using ultra high-pressure LCMS conditions with column pressure exceeding 1000 bar, all modified peptides were resolved from the corresponding unmodified peptide for 13 of the 14 peptide families (93%). Tryptic peptides from some therapeutic proteins may contain multiple residues vulnerable to these modifications, and one residue may be involved in target binding domain while others are not; differentiating susceptibility to degradation at each residue is necessary. 41 unique peaks were observed for the mixture of 51 peptides, indicating that a high percentage (80%) of peptides with multiple modification sites were mutually resolved compared to 38 of 51 (75%) using a convention 2 h, single-column method at standard pressure. 73 of the 87 peak-to-peak resolution measurements (84%) obtained for this sample set were > 0.61 (10% valley). LCMS data of a digested therapeutic protein yielded 97.6% and 96.3% sequence coverage for the heavy chain and light chain, respectively, compared to 93.3% and 92.6% using a conventional method. Conventional peptide methods typically devote a significant amount of time to column washing and column equilibration (>50%) to minimize carryover; these timeconsuming steps were taken offline by incorporating a second binary pump. These optimized high- throughput LCMS conditions permitted data acquisition for >100 injections per day with improved sequence coverage and chemical liability separations compared to conventional methods, addressing a critical bottleneck and yielding timeline reductions during the development of therapeutic proteins.Supplemental Content

[0161] Additional information about the study described above is provided below.

[0162] A. Sample Preparation

[0163] Samples comprising synthetic peptides (samples comprising a single peptide and samples comprising a peptide mixture) were prepared as essentially described in Example 2. The sequences of the synthetic peptides used in the study of Example 6, as well as the domain(s) of the antigen binding protein(s) (e.g., BiTE, mAb, fusion) from which each peptides originates, are listed in Table 15. Briefly, peptides were prepared by AnaSpec (Fremont, CA) and provided as lyophilized aliquots. Each peptide was solubilized in 10% dimethyl sulfoxide in water to a concentration of 1 mg / mL. Each peptide was then diluted to 200 pM with water to create a peptide-specific stock solution.Stock solutions were then used to make samples comprising a single peptide or comprising a peptide mixture.TABLE 15

[0164] Samples comprising a single peptide: A series of samples comprising only one of the peptides listed in Table 15 was made. Each peptide-specific stock solution was diluted to 4 pM with water, and this solution was then diluted to 2 pM with 8 M guanidine HCI, 250 mM acetate, pH 4.7. A 20 pL injection consisted of 40 pmol of the peptide.

[0165] Samples comprising a peptide mixture: Samples comprising a peptide mixture of unmodified and modified peptides were made. The peptide mixture comprised all 14 of the unmodified peptides of Table 15 and all 37 post-translationally modified peptides of Table 15 at a 10:1 unmodified peptide:modified peptide ratio. The peptide mixture was made by combining a volume from each of the 200 pM stock solutions described above so that the peptide mixture consisted of 40 pM of each of the 14 unmodified peptides and 4 pM of each of the 37 modified peptides. This solution was then diluted 1:1 with 8 M guanidine HCI, 250 mM acetate, pH 4.7 A 20 pL injection consisted of 40 pmol of each of the 14 unmodified peptide and 4 pmol of each of the 37 post-translationally modified peptides.

[0166] Samples comprising IgGl digestion peptides were prepared by as described in Da Ren, et al., Analytical Biochemistry 392: 12-21 (2009).

[0167] B. Peptide Separation by UHPLC and Mass Spectrometry Analysis

[0168] Each sample (comprising either a single peptide or a peptide mixture) was applied to a multiple column (MC) LC system fitted within a ThermoFisher Scientific Vanquish™ high throughput liquid chromatography (HPLC) system interfaced with a ThermoFisher Scientific Q Exactive™ Mass Spectrometer for ultra-high pressure liquid chromatography (UHPLC) and mass spectrometry analysis. The HPLC system comprised an MCLC system comprising an autosampler with a 5-port valve, two binary pumps, two 6-port valves, and two identical separation column assemblies (SCA), each SCA comprising the components described in Table 16 and Figure 21C:TABLE 16

[0169] For comparison purposes, UHPLC-MS of the samples were carried out on a single-column control LC system (shown in Figure 22).

[0170] Each sample was injected into the autosampler, wherein each injection had an injection volume of 20 pL (needle wash with water). The trap column (Column 1) was maintained at 37 °C through a MonoSLEEVE controller and 5cm HotSleeve+ column heater, and the columns of theseparation column assemblies were maintained at 80 °C through the instrument column assembly. Peptides were captured on the trap column at lower temperature to minimize artifactual thermal degradation, and as the organic composition of the mobile phase increased, peptides were released. These peptides were then further resolved by the columns of the SCAs at higher temperature. Two 6-port valves in the column assembly were used to determine the flow path of the two SCAs. For odd-numbered injections of a sequence, the 6-port valve was set to the 1-2 position. Binary Pump 1 (BP1) directed peptides from the autosampler to the 1stSCA, and these peptides were then separated with an analytical gradient. Simultaneously, Binary Pump 2 (BP2) washed and equilibrated the2ndSCA with a reconditioning gradient. See Figure 23A. For even-numbered injections of a sequence, the 6-port valve was set to position 6-1. In this valve configuration, binary pump 1 directed peptides from the autosampler to the 2ndSCA, and binary pump 2 reconditioned the 1stSCA with the reconditioning gradient. See Figure 23B. The same analytical and reconditioning gradients were used for both odd- and even-numbered injections of the sequence (Figures 23A-23B). The operation of the two SCAs using two binary pumps improved throughput 2-fold. The column flow rate was set at 620 pL / min and the ALS temp was 4 °C. Pressure was in the range of 900 - 1220 bar. Gradient elution was carried out with two mobile phase solutions: Mobile Phase A which consisted of 0.1% formic acid in water, and Mobile Phase B which consisted of 0.1% formic acid, 90% acetonitrile, 10% water. Gradient elution was carried out with BP1 set to follow the analytical gradient shown in Figures 23A and 23B and BP2 set to follow the reconditioning gradient shown in Figures 23A-23B. Flow from BP1 (analytical pump) was diverted to waste from 0-1.8 min, and all flow from BP2 (reconditioning pump) was sent to waste. Following completion of a sequence, the system and column were flushed with 80% methanol, 20% water for 1 h at 400 pL / min.

[0171] Detection of separated peptides was performed with a Thermo Q-Exactive Plus mass spectrometer. Data were collected from 1.8 min - 12.5 min Data were acquired in Full MS mode with MSI resolution of 35,000, an AGC target of 1E6, a maximum IT of 120 ms, and a scan range of 200-2000 m / z. Skyline v 23.1.0.455 (599e74ad2) was used to extract retention times and full width at half max (FWHM) values for each sample. Control methods are as described in Example 2.

[0172] C. Retention Time (RT), Full Width at Half Max (FWHM), and Peak To Peak Resolution (RPP or Rp-p)

[0173] Samples comprising a single peptide were applied as described above to determine the peptide's retention time for each column. Retention times (minutes) for each peptide were recorded. Full Width at Half Max (FWHM) was derived using Skyline and peak to peak resolution (RPP or Rp-p) was calculated using the following equation:Rp-p = 1.18 * [(Retention Time 2-Retention Time 1) / (FWHM1 +FWHM2)]

[0174] An Rp-p greater than 0.83 was considered as "good", an Rp-p less than 0.83 but greater than 0.61 < R-pp < 0.83 was considered as "intermediate", and an Rp-p less than 0.61 was considered as "poor". The retention time (RT), FWHM, and Rp-p (RPP) for each column are provided in Figure 24.

[0175] The number of peaks achieved by the indicated column are summarized in Table 17.TABLE 17*Values accord to a second iteration of this experiment for the peptide family. An earlier iteration erroneously excluded one peptide from the peptide mixture of the family, and the experiment was repeated. S** is SEQ ID NO:

[0176] Taken together, these results suggest that the MCLC of Configuration 1 demonstrated "good" peak to peak resolution (Rp-p) for all peptides, except for two, while Configuration 2 demonstrated "good" Rp-p for all but 3 peptides, and the % observed peaks out of the total number were greater than that using the single-column control (74.5%).

[0177] D. IgGl Analysis

[0178] Samples comprising IgGl digestion peptides were applied to each MCLC system as essentially described above. The % listed in Table 18 was determined by MassAnalyzer. Briefly, the sequence coverage describes the percentage of the amino acid sequence of a protein correspondingto peptides identified by the search algorithm. For example, if a protein sequence consisted of 100 amino acid residues and peptides identified in a peptide map experiment consist of a total of 90 unique residues, then the sequenced coverage would be 90%. As shown in Table 18, the MCLC of Configuration 1 demonstrated a sequence coverage greater than 95% for both the heavy chain and light chain. Similar results were achieved with the MCLC of Configuration 2 (Table 18).TABLE 18EXAMPLE 7

[0179] This example describes the use of a trap column in a peptide separation method.

[0180] To assess the impact of capturing peptides on a trap column at lower temperature, a trypsin-digested IgGl (as described in Example 6) was analyzed using an MCLC system comprising two single column separation column assemblies, each comprising a 150 mm Waters ACQUITY Premier Peptide CSH C18 column (Product No. 186009489), wherein only one SCA comprised a trap column (5 mm Xselect CSH C18 Vanguard column (Product No. 186005303)) upstream of the column of the SCA. An illustration of the MCLC system with the two SCAs and one trap column is shown in Figure 25. The trap column was maintained at a lower temperature than the columns of the SCAs, and the column temperature of the SCA columns was the same for each run. Several runs were carried out with the SCA column temperature set to 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C.The mobile phase was applied to the system according to the gradient in Table 7.

[0181] Exemplary results are shown in Figures 26A-26C, Figures 27A-27B, and Figures 28A-28B. The amount of site-specific oxidation as measured with (dark bars) or without (light bars) a trap column is shown in Figure 26A (Met255 Oxidation), Figure 26B (Met431 Oxidation), and Figure 26C (Met34 Oxidation). The amount of site-specific clipping as measured with (dark bars) or without (light bars) a trap column is shown in Figure 27A (Asp273-Pro274 Clip) and Figure 27B (Asp99-Pro89 Clip), while the amount of site-specific deamidation as measured with (dark bars) or without (light bars) a trap column is shown in Figure 28A (Asn387 / Asn392 Deamidation) and Figure 28B (Asn 326 Deamidation). As shown in these Figures 26C, 27A, and 27B without the trap column, there was an analytical column temperature-dependent effect on the amount of modification detected, including M34 oxidation, D273-P374 clips, D88-P89 clips. The trap column mitigated these effects andobtained more consistent measurements across different column 2+3 temperatures. See Figures 26A-26C, 27A-27B, and 28A-28B. Many attributes increased in abundance as the column temperature increased when a trap column was not leveraged. Attribute levels are mostly consistent across the range of temperatures evaluated when an in-line trap column was included.

[0182] Taken together, these results support that the peptide separation with a trap column demonstrated lower amounts of artifactual degradation across all temperatures analyzed, compared to peptide separation without a trap column. These results further support additional UHPLC-MS studies using a trap column.EXAMPLE 8

[0183] This example describes a study that evaluates a variety of MCLC systems employed in an HPLC system interfacing with a mass spectrometer for ultra-high pressure liquid chromatography (UHPLC) and mass spectrometry analysis.

[0184] The study described in Example 6 is carried out with different combinations of three RPLC columns, including those described in Table 20, as well as a control system comprising a single column (Figure 22). The performance of the MCLC systems is evaluated, and their performance as to separating the peptide mixtures described in Example 6 are compared to the control system, as well as the MCLC systems of Tablel6 and Figure 21.TABLE 20

[0185] The study is carried out in three rounds wherein the column temperatures of Column 1 is varied. Column temperatures for each column in Rounds 1-3 are noted in Table 21.TABLE 21

[0186] Additional studies are carried out to explore the type of trap column (Column 1). In these studies, Column 1 of the MCLC systems of Configurations 1-4 is replaced with a 5 mm Xselect HSS T3 Vanguard column (Product No. 186008757) to arrive at Configurations 5-8. See Table 22.TABLE 22

[0187] The study is carried out in three rounds wherein the column temperature of Column 1 is varied. Column temperatures for each column in Rounds 5-7 are noted in Table 23.TABLE 23EXAMPLE 9

[0188] This example describes the use of a trap column in a peptide separation method.

[0189] A sample of a trypsin-digested IgGl (as described in Example 6) was analyzed using the MCLC system described in Example 7 and Figure 25. This study was essentially a repeat of that described in Example 7 , except that columns of the MCLC system at the time this study was carried out were more worn (having been used for more than 150 runs), relative to when the study ofExample 7 was carried out. As in the study of Example 7 , the trap column was maintained at a lower temperature than the columns of the SCAs, and the column temperature of the SCA columns was the same for each run. Several runs were carried out with the SCA column temperature set to 50°C, 60°C, 70°C, or 80°C. The mobile phase was applied to the system according to the gradient in Table 7.

[0190] Exemplary results are shown in Figures 29A-29D. The amount of site-specific oxidation as measured with (light bars) or without (dark bars) a trap column is shown in Figures 29A-29B. The amount of site-specific clipping as measured with (light bars) or without (dark bars) a trap column is shown in Figure 29C and Figure 29D. As shown in these figures, without the trap column, there was an analytical column temperature-dependent effect on the amount of modification detected. The trap column mitigated these effects and obtained more consistent measurements across different SCA column temperatures. Many attributes increased in abundance as the column temperature increased when a trap column was not leveraged. Attribute levels are mostly consistent across the range of temperatures evaluated when an in-line trap column was included.

[0191] Taken together, these results support that the peptide separation with a trap column demonstrated lower amounts of artifactual degradation across all temperatures analyzed, compared to peptide separation without a trap column. These results further support additional UHPLC-MS studies using a trap column.

[0192] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms including the indicated component(s) but not excluding other elements (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0193] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0194] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0195] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein.Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A method of separating peptides of a protein, said method comprising: a. applying a sample comprising peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first reversed-phase liquid chromatography (RPLC) column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein: the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column; and b. applying a mobile phase to the MCLC system to elute the bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides.

2. The method of claim 1, wherein (a) peptides eluted from the first RPLC column flow through the second RPLC column, (b) the sample is applied to the first RPLC column and flows from the first RPLC column to the second RPLC column through the connector, and / or (c) the mobile phase is applied to the first RPLC column and flows from the first RPLC column to the second RPLC column through the connector3. The method of claim 1 or 2, wherein the flow rate of the sample and mobile phase through the MCLC system is about 100 pL / minute to about 1000 pL / minute, optionally, at least about 200 pL / minute, at least or about 250 pL / minute, at least or about 600 pL / minute, or at least or about 700 pL / minute.

4. The method of any one of the preceding claims, wherein the MCLC system comprises only two RPLC columns, wherein the only chromatography columns comprised by the MCLC system are the first RPLC column and the second RPLC column.

5. The method of claim 4, wherein the sum of the column lengths of the two chromatography columns is greater than 150 mm, optionally, greater than 175 mm, optionally, about 250 mm.

6. The method of any one of the preceding claims, wherein the connector is less than 100 mm in length, optionally, less than 75 mm in length, optionally, about 45 mm to about 70 mm.

7. The method of any one of the preceding claims, wherein the column length of the first RPLC column and / or second RPLC column is about 25 mm to about 200 mm, optionally, about 50 mm to about 150 mm, and / or the inner diameter of the first RPLC column and / or second RPLC column is about 1 mm to about 9 mm.

8. The method of any one of the preceding claims, wherein each of the first RPLC column and second RPLC column comprises a non-polar stationary phase, optionally, a non-polar stationary phase comprising hydrocarbons, optionally, C18 hydrocarbons.

9. The method of claim 8, wherein the non-polar stationary phase comprises particles having a particle size of about 1.7 pm to about 3 pm, optionally, about 1.7 pm to about 2.1 pm , and / or a pore size of about 120 A to about 300 A, optionally about 120 A to about 160 A.

10. The method of claim 8 or 9, wherein the non-polar stationary phase comprises particles comprising tetraethoxysilane (TEOS) and bis(triethoxysilyl) ethane (BTEE).

11. The method of claim 10, wherein the non-polar stationary phase comprises ethylene- bridged-hybrid (BEH) particles.

12. The method of any one of claims 8 to 11, wherein at least one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising a surface charge.

13. The method of claim 12, wherein one of the first RPLC column and second RPLC column comprises a non-polar stationary phase comprising charged surface hybrid (CSH) particles.

14. The method of any one of the preceding claims, wherein the first RPLC column comprises CSH particles and the second RPLC column comprises BEH particles or the first RPLC column comprises BEH particles and the second RPLC column comprises CSH particles.

15. The method of any one of the preceding claims, comprising chromatographically separating the peptides through ultra-high pressure liquid chromatography (UHPLC), wherein the pressure is greater than 900 bar.

16. The method of any one of the preceding claims, wherein the mobile phase comprises a gradient of a polar organic solvent, optionally, wherein the mobile phase comprises an increasing concentration gradient of the polar organic solvent.

17. The method of claim 16, wherein the mobile phase comprises only one increasing concentration gradient of the polar organic solvent.

18. The method of any one of the preceding claims, wherein the mobile phase comprises only one organic polar solvent.

19. The method of any one of the preceding claims, wherein the mobile phase comprises a polar organic solvent selected from the group consisting of: acetonitrile, methanol, ethanol,propanol, and isopropanol and / or the mobile phase comprises a buffer, optionally, a buffer comprising trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), or formic acid.

20. The method of claim 19, wherein the buffer has a pH below 4, optionally, a pH of 2 to 3.

21. The method of any one of the preceding claims, wherein the mobile phase comprises a mixture of two mobile phase solutions, only one of which comprises the polar organic solvent and both mobile phase solutions comprise a buffer.

22. The method of claim 21, wherein both mobile phase solutions comprise less than 1% (v / v) buffer or less than 0.5% (v / v) buffer, optionally, wherein both mobile phase solutions comprise TFA (pH 2) or formic acid (pH 3).

23. The method of claim 22, wherein both mobile phase solutions comprise 0.1% TFA (pH 2) or 0.1% formic acid (pH 3).

24. The method of any one of the preceding claims, wherein the mobile phase comprises Mobile Phase A (MPA) and Mobile Phase B (MPB), wherein Mobile Phase A (MPA) is about 0.1% (v / v) formic acid in water and Mobile Phase B (MPB) is about 0.1% (v / v) formic acid in about 80% (v / v) to 100% (v / v) acetonitrile (ACN) in water.

25. The method of any one of the preceding claims, wherein (b) occurs in less than 120 minutes or less than 90 minutes or less than 60 minutes.

26. The method of claim 25, wherein (b) occurs in 30 minutes or less or 25 minutes or less.

27. The method of any one of the preceding claims, comprising applying the mobile phase to the MCLC system according to the following gradient:

28. The method of claim 27 or 30, wherein eluted peptides are obtained within the first 11.4 minutes of the gradient.

29. The method of claims 1 to 26, wherein (b) occurs in 15 minutes or less or 10 minutes or less.

30. The method of claim 29, comprising applying the mobile phase to the MCLC system according to the following gradient:

31. The method of any one of the preceding claims, further comprising analyzing the eluted peptides obtained in (b) by mass spectrometry.

32. The method of claim 31, wherein the peptides eluted from the first RPLC column are sent to a mass spectrometer together with the peptides eluted from the second RPLC column and / or the peptides eluted from the first column are not separately injected into the mass spectrometer from the peptides eluted from the second column.

33. The method of any one of the preceding claims, wherein a peptide comprising a post translational modification elutes separately from the corresponding unmodified peptide comprising the same amino acid sequence but without the post translational modification.

34. The method of any one of the preceding claims, further comprising (i) quantifying the peptides with one or more post translational modifications, (ii) preparing the sample comprising peptides of a protein, optionally, comprising incubating the protein with an enzyme to obtain enzyme-digested peptides of the protein, and / or (iii) denaturing and / or alkylated the protein, reducing sulfide bonds of the protein, or a combination thereof.

35. The method of any one of the preceding claims, wherein the method comprises obtaining a sample comprising the protein and / or wherein the protein is a protein of an in-process sample, a drug substance, or drug product.

36. The method of any one of the preceding claims, wherein the protein is a therapeutic protein, optionally, comprising an antibody heavy chain variable region and / or an antibody light chain variable region.

37. The method of claim 36, wherein the therapeutic protein is an antibody or comprises an antigen-binding antibody fragment, optionally, an scFv or scFab.

38. The method of any one of the preceding claims, wherein the protein comprises a post translational modification selected from the group consisting of Asn or Gin deamidation, Aspisomerization, oxidation of Met, Trp, or His, deamination of Glu, Ser, Gly, glycation of Lys, hydroxylysine, hydroxyproline, glycosylation of Asn, Ser, or Thr, cyclization of an N-terminal Gin or Gly, fragmentation or clipping of Asp, Pro peptide bonds, amino acid mutations, and amino acid misincorporations.

39. The method of any one of the preceding claims, wherein the protein comprises a deamidated Asn and / or an isomerized Asp.

40. The method of any one of claims 1-3 and 5-39, wherein the MCLC system comprises three or more RPLC columns.

41. The method of any one of claims 1-3 and 5-40, further comprising a trap column, wherein (a) the trap column is in fluid communication with the first RPLC column, (b) fluids applied to the trap column flow from the trap column to the first RPLC column, and / or (c) there are no intervening valves or diverters between the trap column and first RPLC column.

42. The method of claim 41, wherein the trap column has a column length that is less than 10% the column length of either the first RPLC column or the second RPLC column.

43. The method of claim 41 or 42, wherein the column length of the trap column is about 2 mm to about 7 mm, optionally, about 2 mm to about 5 mm.

44. The method of any one of claims 41 to 43, wherein the trap column (i) comprises a nonpolar stationary phase that is the same as the non-polar stationary phase of the first RPLC column or the second RPLC column and / or (ii) is maintained at a column temperature that is less than the column temperature of the first RPLC column and / or the second RPLC column.

45. The method of any one of claims 41-44, wherein the column temperature of the trap column is less than 40 °C and / or the column temperature of the first RPLC column and second RPLC column is above 50 °C.

46. The method of claim 45, wherein the column temperature of the trap column is about 37 °C and / or wherein the column temperature of the first RPLC column and second RPLC column is above 60 °C or above 70 °C, optionally, about 80 °C.

47. The method of any one of claims 40-46, wherein the mobile phase comprises Mobile Phase A (MPA) and Mobile Phase B (MPB), wherein Mobile Phase A (MPA) is about 0.1% (v / v) formic acid in water and Mobile Phase B (MPB) is about 0.1% (v / v) formic acid in about 80% (v / v) to 100% (v / v) acetonitrile (ACN)in water.

48. The method of any one of claims 40-47, wherein (b) occurs in less than 20 minutes, optionally, less than 15 minutes or less than 13 minutes.

49. The method of any one of the preceding claims, comprising applying the mobile phase to the MCLC system according to the following gradient:optional ly, wherein eluted peptides are obtained within the first 12.5 minutes of the gradient.

50. The method of any one of the preceding claims, wherein the MCLC system comprises an autosampler and the sample comprising the peptides of a protein is applied to the autosampler which is in fluid communication with the MCLC system.

51. The method of claim 50, wherein the MCLC system comprises a trap column positioned downstream of the autosampler, and fluids applied to the autosampler flow to the trap column.

52. The method of claim 50 or 51, wherein the trap column (a) has a column length that is less than 10% the column length of either the first RPLC column or the second RPLC column; (b) has a column length less than 7 mm, optionally, about 5 mm.

53. The method of any one of the preceding claims, wherein the MCLC system comprises a first separation column assembly (SCA) and a second SCA, wherein each of the first SCA and the second SCA comprises a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein, for each of the first SCA and second SCA,:(v) the first RPLC column is different from the second RPLC column;(vi) the first RPLC column is joined to the second RPLC column through a connector;(vii) the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and(viii) there are no intervening valves or diverters between the first RPLC column and second RPLC column.

54. The method of claim 53, wherein the RPLC columns of the first SCA are the same as the RPLC columns of the second SCA.

55. The method of claim 53 or 54, wherein the MCLC system comprises a trap column, wherein (a) the trap column is in fluid communication with the first RPLC column of the first SCA or the second SCA, (b) fluids applied to the trap column flow from the trap column to the first RPLC column of the first SCA or second SCA, (c) there are no intervening valves or diverters between the trap column and first RPLC column, and / or (d) the trap column comprises a non-polar stationary phase that is the same as one of the RPLC columns of the first SCA and / or second SCA.

56. The method of claim 55, wherein the column temperature of the trap column is less than the column temperature of RPLC columns of the first SCA and / or second SCA.

57. The method of claim 55 or 56, wherein the column temperature of the trap column is less than 40 °C and / or the column temperature of the RPLC columns of the first SCA and / or second SCA is above 50 °C.

58. The method of claim 57, wherein the column temperature of the trap column is greater than 20 °C and / or the column temperature of the RPLC columns of the first SCA and / or second SCA is above 60 °C.

59. The method of claim 57 or 58, wherein the column temperature of the first RPLC column and second RPLC column is above 70 °C or about 80 °C.

60. The method of any one of claims 53-59, wherein the MCLC system comprises an autosampler and the sample comprising the peptides of a protein is applied to the autosampler which is in fluid communication with the MCLC system and / or the MCLC system comprises two or more binary pumps.

61. The method of claim 60, wherein the binary pumps move fluids through the MCLC system, optionally, wherein a first binary pump causes fluids to move through the first SCA while a second binary pump causes fluids to move through the second SCA, wherein the first pump causes fluids to move to the detector, and the second binary pump causes fluids to move to the waste collection bin.

62. The method of claim 60 or 61, wherein the MCLC system comprises a switch with at least two settings and a series of valves, wherein fluids move through a series of valves controlled by the switch.

63. The method of claim 62, wherein the switch in one setting allows fluids to move from the autosampler to the trap column through the first SCA to the detector, and the switch in another setting allows fluids to move through the first SCA to the waste collection bin.

64. A method of analyzing a protein for post-translational modifications, said method comprising separating peptides of a protein according to any one of the methods of claims1-63 and analyzing the eluted peptides by mass spectrometry for post-translational modifications.

65. A method of analyzing a protein for post-translational modifications, said method comprising: a. applying a sample comprising peptides of a protein to a multi-column liquid chromatography (MCLC) system comprising a first RPLC column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein: the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; and there are no intervening valves or diverters between the first RPLC column and second RPLC column; b. applying a mobile phase to the MCLC system to elute bound peptides from the first RPLC column and second RPLC column to obtain eluted peptides, and c. analyzing the eluted peptides by mass spectrometry for post-translational modifications. optionally, wherein the peptides eluted from the first RPLC column are injected into a mass spectrometer together with the peptides eluted from the second RPLC column.

66. A multi-column liquid chromatography (MCLC) system as described in any one of claims 1-65.

67. A multi-column liquid chromatography (MCLC) system comprising a first reversed-phase liquid chromatography (RPLC) column in tandem with a second RPLC column, whereby peptides of the sample bind to the first RPLC column and / or second RPLC column; wherein: the first RPLC column is different from the second RPLC column; the first RPLC column is joined to the second RPLC column through a connector; the first RPLC column is in fluid communication with the second RPLC column and fluids applied to the first RPLC column flow from the first RPLC column to the second RPLC column through the connector; andthere are no intervening valves or diverters between the first RPLC column and second RPLC column.

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