Improved sequence variance analysis with ProteoMiner

Abstract

The present invention provides a method and system for identifying host cell protein (HCP) impurities in a sample containing high abundance proteins. The HCP impurities can be enriched using an interacting peptide ligand bound to a solid support. The HCP impurities can be eluted from the solid support. The isolated HCP impurities can be subjected to limited digestion to generate components of the isolated HCP impurities that can then be identified using a mass spectrometer. The present invention also provides a method and system for identifying sequence variant (SV) peptides or proteins in a sample containing high abundance proteins. The SV peptides or proteins can be enriched using an interacting peptide ligand bound to a solid support. The SV peptides or proteins can be eluted from the solid support. The isolated SV peptides or proteins can be subjected to full or limited digestion to generate components of the isolated SV peptides or proteins that can then be identified using a mass spectrometer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The present invention claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 297,822, filed January 10, 2022, U.S. Provisional Patent Application No. 63 / 426,199, filed November 17, 2022, and U.S. Provisional Patent Application No. 63 / 433,106, filed December 16, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] The present invention relates generally to methods for identifying and quantifying low abundance host cell proteins (HCPs) to monitor and control impurities in biopharmaceuticals. The present invention also relates to methods for enriching, identifying, and quantifying amino acid sequence variant (SV) proteins in biopharmaceuticals. [Background technology]

[0003] Recombinant DNA technology has been widely used to produce biopharmaceuticals in host cells. Biopharmaceuticals need to meet very high standards of purity. Therefore, it may be important to monitor any impurities in such biopharmaceuticals at different stages of drug development, manufacturing, storage, and handling. Residual impurities must be at acceptably low levels before clinical trials are conducted. Residual impurities are also a concern for biopharmaceuticals intended for end users. For example, host cell proteins (HCPs) may be present in protein-based biopharmaceuticals that are developed using cell-based systems. The presence of HCPs in the drug product needs to be monitored and may be unacceptable above certain thresholds depending on the product and the specific HCP. Sometimes, even trace amounts of HCPs can cause an immunogenic response.

[0004] Immunoassays have been used to monitor HCP removal using polyclonal anti-HCP antibodies. Immunoassays can provide semi-quantification of total HCP levels in high throughput, but may not be effective for rapidly quantifying individual HCPs. Liquid chromatography-mass spectrometry (LC-MS) methods for monitoring HCP removal have recently emerged. However, the enormous dynamic concentration range of HCPs in the presence of high concentrations of purified antibodies can be a challenge for developing LC-MS methods to monitor HCP removal. In particular, quantifying individual HCPs at very low levels (less than 1 ppm) is challenging.

[0005] It will be appreciated that methods and systems are needed to identify and quantify HCPs in order to monitor and control residual HCPs in drug substances or other products to mitigate safety risks.

[0006] Sequence variants (SVs) resulting from unintended amino acid substitutions in recombinant therapeutic proteins have attracted increasing attention from both regulatory agencies and the biopharmaceutical industry, given their potential impact on efficacy and safety. In well-optimized production systems, such sequence variants are usually present at very low levels in the final product due to the high fidelity of DNA replication and protein biosynthesis processes in mammalian expression systems, such as Chinese Hamster Ovary (CHO) cell lines. However, SV levels may be significantly elevated if the selected production cell line harbors unexpected DNA mutations or if the manufacturing process is not fully optimized, for example, when depletion of certain amino acids occurs in the cell culture medium of the bioreactor. Therefore, it is important to design and implement effective monitoring and control strategies to prevent or minimize possible risks of SVs at the early stages of product and process development. However, there is no established guidance from regulatory agencies or industry-wide consensus for assessing and managing SV risks.

[0007] The biopharmaceutical industry currently targets a common control limit of 0.1% sequence variation of individual amino acids in therapeutic monoclonal antibodies (mAbs), which appears to be the upper limit of natural sequence variation of individual amino acids. However, no sensitive, accurate, and highly precise method exists for detecting SV proteins. For example, three independent laboratories digested NIST standard mAbs, purified NIST mAb tryptic peptides using regular flow surface-charged hybrid (CSH) LC columns, and detected SV NIST mAb tryptic peptides using mass spectrometry (Zhang, et al. 2020). Each of the three laboratories identified 21-23 sequence variations in NIST monoclonal antibodies (mAbs) at rates of 0.01%-0.1%, but the laboratories were in agreement on only 12 sequence variations. There is a need for more reproducible and reliable methods to detect the full array of SV proteins within biopharmaceutical therapeutics, especially with 0.1% sequence variation for individual amino acids as the upper limit for impurities.

[0008] It will be appreciated that methods and systems for identifying and quantifying amino acid sequence variations in biological therapeutics are needed to ensure pharmaceutical safety, consistency, and efficacy. Summary of the Invention

[0009] Identification of HCP impurities in biopharmaceuticals is difficult due to the wide dynamic range of protein concentrations in samples with high complexity.In particular, the presence of at least one high-abundance protein or peptide in a sample, such as a therapeutic protein, poses technical obstacles to the detection, identification, and quantification of ultra-low abundance proteins in the sample.The present application provides a method for identifying HCP impurities in a sample containing high abundance proteins, including an enrichment method to meet the need to enrich low abundance HCPs in therapeutic pharmaceuticals.

[0010] The present disclosure provides a method for identifying and / or quantifying HCP impurities in a sample. In some exemplary embodiments, the method includes: (a) contacting a sample containing at least one high abundance peptide or protein and at least one HCP impurity with a solid support, the solid support being bound to an interacting peptide ligand capable of interacting with the at least one HCP impurity; (b) washing the solid support to provide an eluate containing at least one enriched HCP impurity; (c) subjecting the eluate to enzymatic digestion conditions to generate at least one component of the at least one enriched HCP impurity, the enzymatic digestion conditions not completely digesting all proteins in the eluate; (d) identifying the at least one component of the at least one enriched HCP impurity using a mass spectrometer; and (e) using the identification of the at least one component to identify the at least one enriched HCP impurity.

[0011] In one aspect, the washing step includes a surfactant, the surfactant being a phase transfer surfactant, an ionic surfactant, an anionic surfactant, a cationic surfactant, or a combination thereof. In a particular aspect, the surfactant is sodium deoxycholate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, or a combination thereof. In another aspect, the concentration of the surfactant is about 12 mmol / L. In a particular aspect, the surfactant includes about 12 mmol / L sodium deoxycholate and about 12 mmol / L sodium lauryl sulfate.

[0012] In one embodiment, the concentration of the at least one high abundance peptide or protein is at least about 1000-fold, about 10,000-fold, about 100,000-fold, or about 1,000,000-fold higher than the concentration of the at least one HCP impurity. In another embodiment, the interacting peptide ligand is a library of combinatorial hexapeptide ligands. In yet another embodiment, the at least one high abundance peptide or protein is an antibody, a bispecific antibody, an antibody fragment, an antibody Fab region, an antibody-drug conjugate, a fusion protein, a recombinant protein, a protein pharmaceutical, a biopharmaceutical, or a drug.

[0013] In one embodiment, the enzyme of the enzymatic digestion conditions is trypsin. In a particular embodiment, the enzymatic digestion conditions include trypsin having an enzyme to substrate ratio of less than about 1:200. In another particular embodiment, the enzymatic digestion conditions include trypsin having an enzyme to substrate ratio of about 1:400, about 1:1000, about 1:2500, or about 1:10000. In another embodiment, the at least one concentrated HCP impurity is not subjected to denaturation prior to being subjected to the enzymatic digestion conditions.

[0014] In one embodiment, the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system. In another embodiment, the mass spectrometer can perform LC-MS analysis (liquid chromatography-mass spectrometry) or LC-MRM-MS analysis (liquid chromatography-multiple reaction monitoring-mass spectrometry).

[0015] In one embodiment, the method further includes quantifying the at least one enriched HCP impurity using the mass spectrometer, wherein the detection limit of the at least one enriched HCP impurity is about 0.003-0.006 ppm.

[0016] Existing similar detection methods cannot detect the same array of amino acid sequence mutations within the same NIST mAb standard (Zhang, et al. 2020). A potential explanation could be that unusual amino acid substitutions can occur at any amino acid within the protein's sequence, generating a diverse array of sequence mutations that evade reliable identification. Thus, quantifying the risk associated with SV proteins in general, as well as specific SV proteins or subsets of SV proteins, is not possible using existing methods. The present application presents a method that can identify sequence mutations within the same NIST mAb standard material approximately four times more than multiple previous studies. Furthermore, the disclosed method is particularly suitable for reproducibly identifying amino acid sequence mutations that are likely to affect three-dimensional protein structure. Specifically, the disclosed ProteoMiner™ SV identification method most effectively enriches SV proteins in which an amino acid with physical properties (such as the negatively charged polar side chain of glutamic acid) replaces an amino acid with different physical properties, such as the non-polar hydrophobic side chain of valine.

[0017] The present disclosure provides methods for identifying an SV peptide or protein in a sample, where at least one amino acid of the SV peptide or protein unintentionally differs from a wild-type peptide or protein. In some embodiments, the method includes (a) contacting a sample comprising at least one higher abundance wild-type peptide or protein and at least one SV peptide or protein with a solid support, where the solid support is bound to an interacting peptide ligand capable of interacting with the at least one SV peptide or protein, (b) washing the solid support to provide a first eluate comprising at least one enriched SV peptide or protein, (c) subjecting the first eluate to enzymatic digestion conditions to generate at least one component of the at least one enriched SV peptide or protein, and (d) isolating the at least one enriched SV peptide or protein from the sample. The method includes (a) subjecting the first eluate having the at least one enriched SV peptide or at least one component of the protein to a liquid chromatography system to produce a second eluate having the at least one component of the protein; (e) subjecting the second eluate having the at least one enriched SV peptide or at least one component of the protein to a mass spectrometer; (f) identifying the at least one component of the at least one enriched SV peptide or protein using the mass spectrometer; and (g) using the identification of the at least one component of the at least one enriched SV peptide or protein to identify the at least one enriched SV peptide or protein in the sample.

[0018] In one aspect, the enzymatic digestion conditions are direct digestion.

[0019] In one embodiment, the liquid chromatography system comprises a nanoscale liquid chromatography (nanoLC) column or a regular flow CSH column.

[0020] In one embodiment, the enzymatic digestion conditions do not completely digest all of the proteins in the first eluate.

[0021] In one aspect, the solid support is washed using a detergent, where the detergent is a phase transfer detergent, an ionic detergent, an anionic detergent, a cationic detergent, or a combination thereof.

[0022] In one aspect, the surfactant is sodium deoxycholate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, or a combination thereof.

[0023] In one embodiment, the concentration of the surfactant is about 12 mmol / L.

[0024] In one embodiment, the surfactant comprises about 12 mmol / L sodium deoxycholate and about 12 mmol / L sodium lauryl sulfate.

[0025] In one embodiment, the concentration of the at least one higher abundant wild-type peptide or protein is at least about 1000-fold, about 10,000-fold, about 100,000-fold, or about 1,000,000-fold greater than the concentration of the at least one SV peptide or protein.

[0026] In one embodiment, the interacting peptide ligands are a library of combinatorial hexapeptide ligands.

[0027] In one aspect, the at least one higher abundance wild-type peptide or protein and the at least one SV peptide or protein are an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a recombinant protein, a protein pharmaceutical, a biopharmaceutical, or a drug.

[0028] In one embodiment, the enzyme of the enzymatic digestion conditions is trypsin.

[0029] In one embodiment, the enzymatic digestion conditions include trypsin at an enzyme to substrate ratio of less than about 1:200.

[0030] In one embodiment, the enzymatic digestion conditions include trypsin at an enzyme to substrate ratio of about 1:400, about 1:1000, about 1:2500, or about 1:10000.

[0031] In one embodiment, the at least one enriched SV peptide or protein is not subjected to denaturation prior to being subjected to enzymatic digestion conditions.

[0032] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, and the mass spectrometer is coupled to a liquid chromatography system.

[0033] In one embodiment, the mass spectrometer is capable of performing LC-MS analysis (liquid chromatography-mass spectrometry) or LC-MRM-MS analysis (liquid chromatography-multiple reaction monitoring-mass spectrometry).

[0034] In one aspect, the method further comprises quantifying the at least one enriched SV peptide or protein using a mass spectrometer, wherein the detection limit of the at least one enriched SV peptide or protein is about 0.003-0.006 ppm.

[0035] The present disclosure provides a method for identifying a host cell protein (HCP) impurity in a sample. In some embodiments, the method includes: (a) contacting a sample containing at least one high abundance peptide or protein and at least one HCP impurity with a solid support, the solid support being bound to an interacting peptide ligand capable of interacting with the at least one HCP impurity; (b) washing the solid support to provide an eluate containing at least one enriched HCP impurity; (c) subjecting the eluate to enzymatic digestion conditions to generate at least one component of the at least one enriched HCP impurity, the enzymatic digestion conditions not completely digesting all proteins in the eluate; (d) identifying the at least one component of the at least one enriched HCP impurity using a parallel reaction monitoring-mass spectrometer; and (e) using the identification of the at least one component to identify the at least one enriched HCP impurity.

[0036] In one aspect, the solid support is washed using a detergent, where the detergent is a phase transfer detergent, an ionic detergent, an anionic detergent, a cationic detergent, or a combination thereof.

[0037] In another embodiment, the surfactant is sodium deoxycholate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, or a combination thereof.

[0038] In one embodiment, the concentration of the surfactant is about 12 mmol / L.

[0039] In yet another embodiment, the surfactant comprises about 12 mmol / L sodium deoxycholate and about 12 mmol / L sodium lauryl sulfate.

[0040] In one embodiment, the concentration of the at least one high abundance peptide or protein is at least about 1,000-fold, about 10,000-fold, about 100,000-fold, about 1,000,000-fold, about 10,000,000-fold, about 100,000,000-fold, or about 1,000,000,000-fold higher than the concentration of the at least one HCP impurity.

[0041] In one embodiment, the interacting peptide ligands are a library of combinatorial hexapeptide ligands.

[0042] In one embodiment, the at least one high abundance peptide or protein is an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a recombinant protein, a protein pharmaceutical, or a drug.

[0043] In one embodiment, the enzyme of the enzymatic digestion conditions is trypsin.

[0044] In another embodiment, the enzymatic digestion conditions include trypsin at an enzyme to substrate ratio of less than about 1:200.

[0045] In yet another embodiment, the enzymatic digestion conditions comprise trypsin at an enzyme to substrate ratio of about 1:400, about 1:1000, about 1:2500, or about 1:10000.

[0046] In one embodiment, the at least one enriched HCP impurity is not subjected to denaturation prior to being subjected to the enzymatic digestion conditions.

[0047] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, and the mass spectrometer is coupled to a liquid chromatography system.

[0048] In one aspect, the sample includes an internal standard.

[0049] In another embodiment, the internal standard is labeled with a heavy isotope.

[0050] In yet another embodiment, the internal standard is hPLBD2.

[0051] These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments thereof and numerous specific details, is given by way of illustration and not by way of limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention. [Brief description of the drawings]

[0052] [Figure 1] 1 illustrates a workflow of the method of the present invention according to an exemplary embodiment. [Diagram 2] 1 shows the number of HCP and UPS2 proteins identified by the alternative ProteoMiner™ limited digestion method, according to an exemplary embodiment. [Diagram 3] 1 shows the number of HCP and UPS2 proteins identified by the ProteoMiner™ limited digestion method using a range of trypsin to substrate ratios, according to an exemplary embodiment. [Figure 4] 1 shows the number of HCP and UPS2 proteins identified by the ProteoMiner™ limited digestion method using SDC / SLS presented in the range of 2.4 mmol / L to 12 mmol / L according to an exemplary embodiment. [Figure 5A] 1 shows UPS2 proteins identified by the ProteoMiner™ method, the optimized limited digestion method, and the ProteoMiner™ limited digestion method of the present invention, according to exemplary embodiments. [Figure 5B] Same as above. [Figure 6] 1 shows the number of HCPs identified by the optimized ProteoMiner™ limited digestion method of the present invention compared to conventional methods, according to an exemplary embodiment. [Figure 7]1 shows the number of UPS2 proteins identified by the optimized ProteoMiner™ limited digestion method of the present invention compared to conventional methods, according to an exemplary embodiment. [Figure 8] 1 shows the number of NIST monoclonal antibody (mAb) HCPs identified by the optimized ProteoMiner™ limited digestion method of the present invention compared to previously published methods, according to an exemplary embodiment. [Figure 9] 1 illustrates a ProteoMiner™ sample preparation workflow and ultrasensitive quantification method for enhanced detection of host cell proteins, according to an exemplary embodiment. [Figure 10A] 1 shows a comparison of targeted quantitation (PRM) of GLGDVDQLVK from LPL in mAb-1 with mAb-1 and the corresponding recombinant standard spiked at lower limit of quantitation (LLOQ) levels, according to an exemplary embodiment. [Figure 10B] 1 shows a comparison of targeted quantification (PRM) of EFSHITFLTIK from carboxypeptidase in mAb-1 with mAb-1 and corresponding recombinant standards spiked at lower limit of quantification (LLOQ) levels, according to an exemplary embodiment. [Figure 10C] 1 shows a comparison of targeted quantification (PRM) of VNVYTSHSPAGTSVQNLR from LAL in mAb-1 and with the corresponding recombinant standard spiked at lower limit of quantification (LLOQ) levels according to an exemplary embodiment. [Figure 10D] 1 shows a comparison of target quantification (PRM) of VSSLPSVTLK from cathepsin D in mAb-1 and mAb-1 with the corresponding recombinant standard spiked at lower limit of quantification (LLOQ) levels, according to an exemplary embodiment. [Figure 10E] 1 shows a comparison of targeted quantification (PRM) of GVNYASITR from cathepsin Z in mAb-1 with mAb-1 and the corresponding recombinant standard spiked at lower limit of quantification (LLOQ) levels, according to an exemplary embodiment. [Figure 11A]Standard curves for eight peptides according to an exemplary embodiment are shown. Peak area ratios (PAR) were calculated by dividing the peak area selected for each HCP by the peak area of ​​peptides from hPLBD2 from PRM analysis. HCPs were spiked into mAb-1. A list of peptides from HCPs and hPLBD2 is shown in Table 4. [Figure 11B] 1 shows a standard curve of peak area ratios (PAR) of two selected peptides for LAL and human PPT-1 divided by a peptide from hPLBD2 from PRM analysis, according to an exemplary embodiment. LAL and human PPT-1 were spiked into mAb-2. [Figure 12A] 1 shows the degradation profile of PS80 observed for mAb-3 (in the presence of 1.28 ppm LAL and 0.2 ppm LPL) under normal storage conditions (4° C.-8° C.) for up to 6 months based on LC-CAD measurements, according to an exemplary embodiment. [Figure 12B] 1 shows the increased concentration of oleic acid observed in mAb-3 (1.28 ppm LAL and 0.2 ppm LPL present) under normal storage conditions (4° C.-8° C.) based on free fatty acid measurements, according to an exemplary embodiment. [Figure 12C] 1 shows the correlation between daily oleic acid gain under stress conditions (37° C.) and lipase concentrations (LAL and LPL) in mAb-3 through mAb-10 according to an exemplary embodiment. [Figure 13] 1 shows the correlation between daily oleic acid increase under stress conditions (37° C.) and measured CES concentrations in mAb-12 through mAb-17 according to an exemplary embodiment. CES concentrations in mAb-15 through mAb-17 were quantified by comparing the relative abundance of CES to mAb-13 and mAb-14. [Figure 14] 1 shows the correlation between 80% remaining PS under storage conditions (4-8° C.) for mAb-18, according to an exemplary embodiment. [Figure 15A] 1 shows MY cleavage under stress conditions (45° C.) observed for DS-1, DS-2, and DS-3 for up to 6 months according to an exemplary embodiment. [Figure 15B] 1 shows concentrations of Cathepsin D in DS-1, DS-2, and DS-3, according to an exemplary embodiment. [Figure 16] Illustrative embodiments show that potential mechanisms for producing SV proteins may occur during replication, transcription, translation, or a combination thereof. [Figure 17] 1 illustrates the workflow of the enhanced SV protein detection method of the present invention, according to an exemplary embodiment. [Figure 18A-1] 1 shows a table of amino acid substitutions identified in SV NIST mAb using the ProteoMiner™ SV identification method of the present disclosure using a nanoLC column or direct digestion using a nanoLC column, according to an exemplary embodiment. [Figure 18A-2] Same as above. [Figure 18A-3] Same as above. [Figure 18B-1] 1 shows a table of amino acid substitutions identified in SV NIST mAb using the ProteoMiner™ SV identification method of the present disclosure using a nanoLC column or direct digestion using a nanoLC column, according to an exemplary embodiment. [Figure 18B-2] Same as above. [Figure 18B-3] Same as above. [Figure 18C-1] 1 shows a table of amino acid substitutions identified in SV NIST mAb using the ProteoMiner™ SV identification method of the present disclosure using a nanoLC column or direct digestion using a nanoLC column, according to an exemplary embodiment. [Figure 18C-2] Same as above. [Figure 19A] 1 shows a table of enriched SV NIST mAb peptides identified using the ProteoMiner™ SV enrichment method of the present disclosure, according to an exemplary embodiment. [Figure 19B] 1 shows a diagram of NIST mAb amino acid sequence variations enriched using the ProteoMiner™ SV identification method of the present disclosure within the three-dimensional protein structure of the SV NIST mAb, according to an exemplary embodiment. [Figure 19C] 1 shows an alternative view of NIST mAb amino acid sequence mutations enriched using the ProteoMiner™ SV identification method of the present disclosure within the three-dimensional protein structure of the SV NIST mAb, according to an exemplary embodiment. [Figure 19D] 1 shows another alternative view of SV NIST mAb amino acid sequence mutations identified using the ProteoMiner™ SV identification method of the present disclosure within the three-dimensional protein structure of SV NIST mAb, according to an exemplary embodiment. [Figure 20A] Exemplary embodiments show the distinct properties of histidine (e.g., positively charged side chain), asparagine (e.g., polar uncharged side chain), and aspartic acid (e.g., negatively charged side chain) involved in histidine-to-asparagine or aspartic acid sequence mutations that affect the protein structure of SV mAbs enriched by the ProteoMiner™ SV identification method of the present disclosure. [Figure 20B] 1 shows codon sequence variations that can result in histidine to asparagine or aspartic acid sequence variations in SV mAbs enriched by the ProteoMiner™ SV identification method of the present disclosure, according to an exemplary embodiment. [Figure 20C] 1 shows NIST mAb histidine to asparagine or aspartic acid sequence mutations identified using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns or ProteoMiner™ enriched NIST mAb digests run on nanoLC columns according to exemplary embodiments. [Figure 20D] 1 shows MS2 mass spectra of tryptic peptide product ions detected in the eluate from a NIST mAb direct digest run on a regular flow CSH LC column (bottom), and the histidine to asparagine SV tryptic peptide product ion detected in the eluate from a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column (top), according to an exemplary embodiment. [Figure 20E] 1 shows MS2 mass spectra of tryptic peptide product ions detected in the eluate from a NIST mAb direct digest run on a regular flow CSH LC column (bottom), and the histidine to aspartic acid SV tryptic peptide product ion detected in the eluate from a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column (top), according to an exemplary embodiment. [Figure 20F] FIG. 1 shows CHO IgG1 mAb histidine to asparagine or aspartic acid sequence mutations identified using eluates from a CHO IgG1 direct digest run on a regular flow CSH LC column or a ProteoMiner™ enriched CHO IgG1 mAb digest run on a nanoLC column according to an exemplary embodiment. [Figure 21A] Exemplary embodiments show similar properties of serine (e.g., polar, uncharged side chains) and asparagine (e.g., polar, uncharged side chains) that prevent serine-to-asparagine sequence mutations from affecting the protein structure of SV mAbs that are not enriched by the enhanced ProteoMiner™ SV identification method of the present disclosure. [Figure 21B] 1 shows NIST mAb serine to asparagine sequence mutations identified using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns or digested ProteoMiner™ NIST mAb eluates run on nanoLC columns according to exemplary embodiments. [Figure 22A] 1 shows the number of NIST mAb amino acid sequence mutations (SVA>0.01%) identified using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns or digested ProteoMiner™ NIST mAb eluates run on a nanoLC column according to exemplary embodiments. [Figure 22B]1 shows MS2 mass spectra of tryptic peptide product ions detected in the eluate from a NIST mAb direct digest run on a regular flow CSH LC column (bottom), and MS2 mass spectrum of the glycine to aspartic acid SV tryptic peptide product ion (SVA as low as 0.004%) detected in the eluate from a digested ProteoMiner™ NIST mAb run on a nanoLC column (top) according to an exemplary embodiment. [Figure 22C] 1 shows the number of NIST mAb serine, glycine, or valine sequence mutations identified using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns, or digested ProteoMiner™ NIST mAb eluates run on a nanoLC column, according to exemplary embodiments. [Figure 22D] 1 shows the number of NIST mAb serine, glycine, or valine sequence mutations identified by three laboratories using eluates from NIST mAb direct digests run on a regular flow CSH LC column or digested ProteoMiner™ NIST mAb eluates run on a nanoLC column according to an exemplary embodiment. [Figure 22E] 1 shows NIST mAb alanine to threonine, glycine to aspartic acid, serine to asparagine, valine to leucine or isoleucine, arginine to lysine, and lysine to arginine sequence mutations identified by three laboratories using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns, or digested ProteoMiner™ NIST mAb eluates run on nanoLC columns, according to exemplary embodiments. [Figure 23-1]1 shows unsaturated (bottom) and saturated (top) peaks in MS2 mass spectra of tryptic peptide product ions (e.g., VVSVLTVLHQDWLNGK and TTPPVLDSDGSFEYSK) and serine to asparagine SV tryptic peptide product ions (e.g., VVNVLTVLHQDWLNGK and TTPPVLDSDGSFEYNK) detected in the eluate from digested ProteoMiner™ NIST mAb run on a nanoLC column, according to an exemplary embodiment. [Figure 23-2] Same as above. [Figure 24-1] Exemplary embodiments show that using a mass spectrometer to analyze the eluate from a NIST mAb direct digest run on a regular flow CSH LC column produces a larger peak in the MS2 mass spectrum of the tryptic peptide product ion (scan 9602, z=3) than in the MS2 mass spectrum of the cysteine ​​to serine SV tryptic peptide product ion (scan 9515, z=3), whereas using a mass spectrometer to analyze the eluate from a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column produces a smaller peak in the MS2 mass spectrum of the tryptic peptide product ion (scan 59496, z=3) than in the MS2 mass spectrum of the cysteine ​​to serine SV tryptic peptide product ion (scan 59579, z=3). [Figure 24-2] Same as above. [Diagram 25]Illustrated is a mass spectrometer according to an exemplary embodiment that does not produce y ions in the MS2 mass spectrum of serine to leucine or isoleucine SV tryptic peptide product ions using eluate from a NIST mAb direct digest run on a regular flow CSH LC column (scan 14203, z=4), while the mass spectrometer does produce y ions in the MS2 mass spectrum of serine to leucine or isoleucine SV tryptic peptide product ions using eluate from a digested ProteoMiner™ NIST mAb run on a nanoLC column (scan 75616, z=4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Obtaining a high purity biopharmaceutical is important for manufacturing biopharmaceuticals, since residual HCPs may compromise the safety and stability of the product. When producing cell-based recombinant therapeutic antibodies, immunoassays such as enzyme-linked immunosorbent assays (ELISAs) have typically been used to monitor the removal (clearance) of HCPs using polyclonal anti-HCP antibodies during process development. ELISAs can provide semi-quantification of total HCP levels in high throughput. However, although polyclonal anti-HCP antibodies are used in ELISAs to capture, detect, and quantify total HCPs, they may not be effective in quantifying individual HCPs. In particular, some non-immunogenic or weakly immunogenic HCPs may not be detected using ELISAs.

[0054] To identify and quantify HCPs, several complementary approaches, such as one-dimensional / two-dimensional (1D / 2D) PAGE or liquid chromatography (LC)-coupled tandem mass spectrometry (LC-MS / MS), have been used to monitor HCPs. However, the wide dynamic concentration range of HCPs in the presence of high concentrations of purified antibodies can be a major challenge in developing LC-MS methods to monitor the removal of HCP impurities. Mass spectrometry (MS) alone lacks the ability to detect low-abundance targets such as low ppm levels of HCPs in the presence of high concentrations of therapeutic antibodies due to their wide dynamic concentration range, which can be over six orders of magnitude higher than the HCP impurities. To overcome this issue, one strategy is to separate co-eluting peptides prior to MS analysis by adding another dimension of separation, such as 2D-LC and / or ion mobility, in combination with data-dependent or data-independent acquisition to increase the separation efficiency.

[0055] Huang et al. (Huang et al., A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies, Anal. Chem. 2017, May 16;89(10):5436-5444) describe a sample preparation method using trypsin digestion for shotgun proteomic characterization of HCP impurities in antibody samples. Huang's sample preparation method keeps the antibody nearly intact while the HCPs are digested. Compared to traditional trypsin digestion sample preparation, Huang's approach can reduce the dynamic range of HCP detection using mass spectrometry by 1-2 orders of magnitude. As demonstrated by HCP spiking experiments, Huang's approach can detect 0.5 ppm HCPs with molecular weights above 60 kDa, such as rPLBL2. For example, using Huang's approach, 60 mouse HCP impurities were detected in RM 8670 (NIST mAb, NIST monoclonal antibody standard, expressed in a mouse cell line, obtained from the National Institute of Standards and Technology, Gaithersburg, MD).

[0056] Doneanu et al. (Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography, Anal. Chem. 2015 Oct 20;87(20):10283-10291) report the detection of low abundance HCP impurities down to 1 ppm in antibody samples using a liquid chromatography-mass spectrometry (LC-MS) method. Doneanu's approach includes using a novel charge surface-modified C18 stationary phase to mitigate column saturation challenges, incorporating traveling wave ion mobility separation of co-eluting peptide precursors, and improving the fragmentation efficiency of low abundance HCP peptides by correlating the collision energy used for precursor fragmentation with the mobility drift time. HCP impurities can be identified at 10-50 ppm using 2D-HPLC (2D high performance liquid chromatography) in combination with ion mobility mass spectrometry analysis. However, the cycle time of 2D-LC or 2D-HPLC may be very long.In addition, these methods may not be sensitive enough for low-level HCP analysis, for example, less than 10 ppm.Other approaches to identify HCP impurities include sample preparation to enrich HCP by removing antibodies in the sample, such as using affinity purification or limited digestion to remove antibodies.In addition, another common approach is to capture HCP using polyclonal antibodies.

[0057] Analytical techniques required to identify HCP impurities encounter the challenge of dealing with approximately one million times more matrix molecules than analytes (e.g., HCPs or HCP peptides) due to the extremely high sample complexity. HCP impurities are most often present at low levels, such as 1-100 ppm, in protein biopharmaceuticals, making it challenging to concentrate HCPs to levels compatible with detection. Without knowing the identity and properties of the HCP, it can be very difficult to develop a general sample preparation procedure to enrich HCPs (or HCP peptides) or remove the matrix background (Doneanu et al.).

[0058] Chen et al. (Chen et al., Improved host cell protein analysis in monoclonal antibody products through ProteoMiner, Anal. Biochem. 2020 Dec. 1; 610: 113972) describe a method for enriching HCPs using interacting peptide ligands, specifically ProteoMiner™ beads. The method of the present invention improves upon the previously described ProteoMiner™ method of enrichment, identification, and quantification of HCPs.

[0059] The present application provides methods for enriching HCPs using interacting peptide ligands (e.g., combinatorial ligand libraries). In some exemplary embodiments, ProteoMiner™ beads (Bio-Rad Laboratories, Inc., Hercules, Calif.), a combinatorial hexapeptide library immobilized on beads, are used to enrich HCPs. When the peptide ligand-bound beads are applied to a sample containing various protein species, each protein species can bind to its interacting peptide ligands. HCPs bind to interacting peptide ligands primarily through hydrophobic forces in combination with some weaker interaction forces (e.g., ionic interactions and hydrogen bonds).

[0060] Since the number of interacting peptide ligands corresponding to each protein species in the combinatorial ligand library is limited, the protein species with high abundance may saturate its interacting peptide ligands by being present in excess. The limited number of corresponding interacting peptide ligands can be easily saturated in the presence of an excess of high abundance proteins. The excess of high abundance proteins that cannot bind to the interacting peptide ligands can be washed off the beads. Since the amount of low abundance proteins in the sample is relatively low compared to the high abundance proteins, the low abundance proteins may not saturate the corresponding interacting peptide ligands. Thus, the low abundance proteins can be relatively enriched compared to the high abundance proteins. After performing the enrichment process, the protein concentration of a wide dynamic range can be significantly reduced, allowing the detection of low abundance proteins.

[0061] The broad dynamic range of protein concentrations can be further reduced using limited digestion: reducing the ratio of digestive enzyme to substrate and performing the digestion reaction on natively folded proteins instead of denatured proteins results in incomplete digestion of proteins in the sample, disproportionately reducing the presence of peptides corresponding to highly abundant proteins in the sample, thus reducing the dynamic range of protein concentrations.

[0062] The HCP enrichment method of the present application can enrich and detect medium and low abundance proteins by reducing the amount of high abundance proteins. The HCP enrichment method of the present application also meets the need to enrich low abundance HCP impurities in pharmaceutical or other samples of interest.

[0063] In some exemplary embodiments, the sample is treated with ProteoMiner™ beads to reduce the amount of therapeutic proteins present in high abundance and enrich for low abundance HCP impurities. The HCP enriched sample is then subjected to proteomic analysis. This procedure can enrich low abundance HCP impurities and simultaneously reduce the level of therapeutic proteins. It can successfully reduce the dynamic concentration range between HCP and protein drug, allowing for the detection of low abundance HCP impurities. The detection limit of HCP impurities using the HCP enrichment method of the present application is about 0.003-0.006 ppm.

[0064] In some exemplary embodiments, the disclosure provides a method of identifying and / or quantifying a host cell protein (HCP) impurity in a sample, the method comprising: contacting a sample comprising at least one high abundance peptide or protein and at least one HCP impurity with a solid support, the solid support being bound to an interacting peptide ligand capable of interacting with the at least one HCP impurity; washing the solid support to provide an eluate comprising at least one enriched HCP impurity; subjecting the eluate to enzymatic digestion conditions to generate at least one component of the at least one enriched HCP impurity, the enzymatic digestion conditions being limited digestion that does not completely digest all proteins in the eluate; identifying and / or quantifying the at least one component of the at least one enriched HCP impurity using a mass spectrometer; and using the identification and / or quantification of the at least one component to identify and / or quantify the at least one enriched HCP impurity.

[0065] In some exemplary embodiments, phase transfer detergents (PTS) such as sodium deoxycholate (SDC) and sodium lauryl sulfate (SLS) are used to elute HCPs from ProteoMiner™ beads. SDC is an ionic detergent that is particularly useful for disrupting and dissociating protein interactions. Ionic detergents have a charged hydrophilic head group and can be either negatively (anionic) or positively (cationic) charged. SLS is an anionic detergent. Anionic detergents such as SLS or sodium dodecylbenzenesulfonate are sodium salts of sulfonated long chain, alcohols or hydrocarbons.

[0066] In some exemplary embodiments, the elution buffer for eluting HCPs from ProteoMiner™ beads comprises an ionic surfactant, an anionic surfactant, a cationic surfactant, a phase transfer surfactant, or a combination thereof. In one aspect, the elution buffer comprises SDC, SLS, or sodium dodecylbenzenesulfonate. In one aspect, the elution buffer comprises a PTS buffer, comprising 12 mmol / L SDC (sodium deoxycholate), 12 mmol / L SLS (sodium lauroyl sarcosinate), 10 mmol / L TCEP (tris(2-carboxyethyl)phosphine, a reducing agent), and 30 mmol / L CAA (chloroacetamide).

[0067] Trace amounts of certain HCPs may cause immune responses or toxic biological activity after drug injection. The presence of residual HCPs in biopharmaceuticals has been a concern with regard to drug safety and has led to an increased demand for the development of methods and systems to identify and characterize HCP impurities in biopharmaceuticals. There is an unmet need to identify and monitor individual HCPs for risk assessment in therapeutic protein products.

[0068] The present disclosure provides methods and systems for fulfilling the aforementioned needs by providing methods and systems for identifying and quantifying HCPs in drug substances to monitor and control residual HCPs in drug substances to mitigate safety risks. The exemplary embodiments disclosed herein fulfill the aforementioned needs and long felt needs.

[0069] In addition to HCPs, sequence variants (SVs) resulting from unintended amino acid substitutions are another product quality attribute of concern in drug development and manufacturing. Such SVs have been shown to exist in both native and recombinant proteins and are thought to be caused by many mechanisms, including DNA mutations during replication, as well as transcription and translation errors during the protein biosynthesis process.

[0070] Due to the high fidelity of biological systems evolved to prevent the occurrence of such spontaneous errors, SVs are usually present at very low levels (less than 0.1%) in native biological proteins. However, during the development of therapeutic protein drugs, the objective is to increase protein titers and process productivity to reduce the cost of goods to meet global demand and expand patient access. This has led to the widespread use of so-called enhanced bioreactor manufacturing systems designed to maximize cell density and specific productivity of the target therapeutic protein during the cell culture process. Such enhanced production systems can impose higher than normal expression mechanical stresses on the production cell line. If not fully optimized, elevated levels of SVs can be generated in the protein product. In addition, to further increase the product titer, cell line development usually undergoes multiple rounds of selection with increasing selective stress to find the most productive cell clones. This selection process can potentially introduce DNA mutations into the cell line. If not properly screened, unexpected high levels of SVs can occur in the final drug product.

[0071] Given these concerns about how elevated SVs affect drug product quality, both industry and regulatory agencies have begun to focus on SVs. Over the past decade, significant effort and resources have been dedicated across the industry to better understand the causes of and control of SVs in biologics development. As a result of these collective efforts, several control strategies have been developed to optimally monitor and mitigate SV issues during product and process development. As expected, these proposed strategies have highlighted the importance of multi-assay, multi-tiered SV screening approaches to guide process development from initial cell line selection through small-scale cell culture process development and scale-up confirmation. Together, these strategies have provided a useful, industry-wide framework and high-level guidance toward the goal of establishing some general best practices regarding SV control.

[0072] However, clarity and consensus across the industry remains lacking on a variety of important aspects, including, for example, 1) the selection and combination use of multiple SV-related analytical techniques (e.g., next-generation sequencing-based DNA or RNA sequencing, liquid chromatography (LC)-mass spectrometry (MS) / MS, surrogate amino acid analysis); 2) the selection of the step(s) and degree for implementing SV monitoring and control during product and process development, taking into account both the effectiveness of the overall control strategy and the development timeline; 3) proper assessment of SV risk with respect to product safety and efficacy; 4) determination of reasonable SV control limits or tolerance levels in process development and final drug product; and 5) reporting of SV data in regulatory submissions.

[0073] To fill some of these knowledge gaps, the results of a survey of industry practices regarding SV analysis and control in biological development were recently published by the International Consortium for Innovation and Quality in Pharmaceutical Development (Zhang, et al. 2020). In the survey, one of the most important questions is what level of SV has individual companies set as an action limit (or control target) for product and process development. The problem is that no reliable method exists for reproducibly detecting the same set of SV mAbs in a sample. For example, in a previous study, the performance of an LC-MS method for the detection of SV NIST mAbs was fully characterized and evaluated for two independent laboratories performing similar analyses (Zhang, et al. 2020). All three laboratories were able to detect and identify low levels of SV in the range of 0.01-0.1%, but the sets of SVs identified by the three laboratories did not completely overlap with each other. Although each of the three laboratories identified a similar number of SVs (e.g., 21–23) with the NIST mAb, only 12 of these were commonly identified across all three laboratories, suggesting that there is a large method-based variability in the detection of low-level SVs.

[0074] The present application provides methods to enhance the detection limit of SV proteins, particularly mAbs, with or without enrichment, using interacting peptide ligands (e.g., combinatorial ligand libraries). In some exemplary embodiments, ProteoMiner™ beads (Bio-Rad Laboratories, Inc., Hercules, Calif.), a combinatorial hexapeptide library immobilized on beads, are used to improve the detection limit of SV mAbs (e.g., the resolution at which SV mAbs can be detected). In some exemplary embodiments, ProteoMiner™ beads can enrich for SV mAbs where amino acid substitutions affect the mAb protein structure. When peptide ligand-bound beads are applied to a sample containing various protein species, each protein species can bind to its interacting peptide ligand. SV proteins bind to interacting peptide ligands primarily by hydrophobic forces in combination with some weaker interaction forces (e.g., ionic interactions and hydrogen bonds).

[0075] The high abundance non-SV protein species and its corresponding low abundance SV protein species may bind to the same interacting peptide ligand. The affinity of the low abundance SV protein species for the peptide ligand may be equal to the affinity of the corresponding non-SV protein species for the same peptide ligand. Alternatively, the affinity of the low abundance SV protein species for the peptide ligand may be higher or lower than the affinity of the corresponding non-SV protein species for the same peptide ligand. Excessive high abundance non-SV proteins that cannot bind to the interacting peptide ligand may be washed off the beads. Thus, the detection limit of the low abundance SV protein species may be relatively improved compared to the high abundance non-SV protein species. After improving the detection limit of the low abundance SV protein species, a wide dynamic range of protein concentrations may be significantly reduced, allowing the detection of low abundance SV proteins.

[0076] The wide dynamic range of protein concentrations can be further reduced using limited digestion: Reducing the ratio of digestive enzyme to substrate and performing the digestion reaction on natively folded proteins instead of denatured proteins can result in incomplete digestion of proteins in the sample, which disproportionately reduces the presence of peptides corresponding to high abundance proteins in the sample and can reduce the dynamic range of protein concentrations.

[0077] The detection limit of low-abundance SV protein species can be further enhanced using nanoflow LC (nanoLC), which can improve MS2 spectra by increasing the signal of SV peptide product ions derived from SV proteins and allowing the formation of more y ions.

[0078] The enhanced SV protein detection method of the present application can increase the detection limit of SV proteins by reducing the amount of high abundance non-SV proteins. The method of increasing the detection limit of SV proteins of the present application can also meet the need to enrich low abundance SV proteins in therapeutic pharmaceuticals.

[0079] In some exemplary embodiments, the sample is treated with ProteoMiner™ beads to reduce the amount of therapeutic proteins present in high abundance and enhance the detection of low-abundance SV therapeutic proteins, with or without enrichment. The sample is then subjected to proteomic analysis. This procedure can enrich low-abundance SV therapeutic proteins and simultaneously reduce the levels of non-SV therapeutic proteins. It can successfully reduce the dynamic concentration range between SV and non-SV protein drugs, allowing the detection of low-abundance SV proteins. The enhanced SV protein detection method of the present application can detect amino acid substitutions occurring in approximately 0.003% of proteins.

[0080] In some exemplary embodiments, the disclosure provides a method for identifying a sequence variant (SV) peptide or protein in a sample, where at least one amino acid of the SV peptide or protein unintentionally differs from a wild-type peptide or protein, comprising: (a) contacting a sample comprising at least one higher abundant wild-type peptide or protein and at least one SV peptide or protein with a solid support, where the solid support is bound to an interacting peptide ligand capable of interacting with the at least one SV peptide or protein; (b) washing the solid support to provide a first eluate comprising at least one enriched SV peptide or protein; and (c) enzymatically digesting the first eluate to produce at least one component of the at least one enriched SV peptide or protein. (d) subjecting the first eluate having the at least one component of the at least one enriched SV peptide or protein to elution conditions in a liquid chromatography system to produce a second eluate having the at least one component of the at least one enriched SV peptide or protein; (e) subjecting the second eluate having the at least one component of the at least one enriched SV peptide or protein to a mass spectrometer; (f) identifying the at least one component of the at least one enriched SV peptide or protein using a mass spectrometer; and (g) using the identification of the at least one component of the at least one enriched SV peptide or protein to identify the at least one enriched SV peptide or protein in the sample.

[0081] In some exemplary embodiments, a phase transfer detergent (PTS) is used to elute SV proteins from ProteoMiner™ beads. In some exemplary embodiments, the elution buffer for eluting SV proteins from ProteoMiner™ beads comprises an ionic detergent, an anionic detergent, a cationic detergent, a phase transfer detergent, or a combination thereof. In one aspect, the elution buffer comprises SDC, SLS, or sodium dodecylbenzenesulfonate. In one aspect, the elution buffer comprises a PTS buffer and comprises 12 mmol / L SDC (sodium deoxycholate), 12 mmol / L SLS (sodium lauroyl sarcosinate), 10 mmol / L TCEP (tris(2-carboxyethyl)phosphine, a reducing agent), and 30 mmol / L CAA (chloroacetamide).

[0082] Trace amounts of certain SV proteins can cause immune responses or toxic biological activity after drug injection. The presence of SV proteins in biopharmaceuticals has been a concern for drug safety, leading to an increased demand for the development of methods and systems for identifying and characterizing SV proteins in biopharmaceuticals. There is an unmet need to identify and monitor SV proteins for risk assessment of their presence in therapeutic protein products.

[0083] The present disclosure provides methods and systems for fulfilling the aforementioned needs by providing methods and systems for identifying and quantifying SV proteins in drug substances to monitor and control them to mitigate safety risks. The exemplary embodiments disclosed herein fulfill the aforementioned needs, as well as other long felt needs.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, certain methods and materials are described herein.

[0085] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variations as understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included. As used herein, the terms "include," "includes," and "including" are intended to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0086] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer having covalently linked amide bonds. A protein comprises one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer consisting of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. A "synthetic peptide or polypeptide" refers to a peptide or polypeptide that does not occur in nature. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those skilled in the art. A protein may comprise one or more polypeptides to form a single functional biomolecule.

[0087] As used herein, the term "therapeutic protein" includes any of the following proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies.

[0088] In another exemplary embodiment, the protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. The protein of interest may include any of biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins may be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation" (Darius Ghaderi et al., 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), which is incorporated by reference in its entirety). In some exemplary embodiments, the proteins include modifications, adducts, and other covalently linked moieties. These modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescent labels, and other dyes.Proteins may be classified based on composition and solubility, and thus may include simple proteins, such as globular proteins and fibrous proteins; conjugate proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

[0089] In one embodiment, the at least one high abundance peptide or protein in the method of the invention is an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical, or a drug.

[0090] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried on a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein can be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein can be an antibody of an isotype selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., IgG1) or, alternatively, the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).

[0091] The term "antibody" as used herein includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains (CH1, CH2, and CH3). Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. The "antigen-binding portion" of an antibody, the "antigen-binding fragment" of an antibody, and similar terms as used herein include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the antibody variable domains and, optionally, the constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized.The DNA may be manipulated using sequencing and chemical or molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids.

[0092] As used herein, an "antibody fragment" includes a portion of an intact antibody (e.g., an antigen-binding or variable region of an antibody). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the heavy and light chain variable regions of an immunoglobulin, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the light and heavy chain variable regions of an immunoglobulin are linked by a peptide linker. In some exemplary embodiments, an antibody fragment includes sufficient amino acid sequence of a parent antibody that the fragment binds to the same antigen as the parent antibody, and in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments may be produced by any means. For example, antibody fragments may be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments may be wholly or partially produced synthetically. Antibody fragments may optionally include single chain antibody fragments. Alternatively or additionally, antibody fragments may include multiple chains linked together, for example, by disulfide linkages. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments usually contain at least about 50 amino acids, more usually at least about 200 amino acids.

[0093] The term "bispecific antibody" (bsAb) includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, each of which specifically binds to a different epitope, either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). When a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two, or three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.

[0094] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen binding specificity but can associate with each heavy chain, or can associate with each heavy chain and bind one or more of the epitopes bound by the heavy chain antigen binding region, or can associate with each heavy chain and allow binding of one or both of the heavy chains to one or both epitopes. bsAbs can be divided into two major classes, those with an Fc region (IgG-like) and those that lack an Fc region, the latter being usually smaller than Fc-containing IgG and IgG-like bispecific molecules. IgG-like bsAbs can have different formats such as, but not limited to, triomabs, knob-into-hole IgG (kih IgG), crossMab, orth-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or dual acting Fab (DAF), IgG-single chain Fv (IgG-scFv), or κλ bodies. Different non-IgG-like formats include tandem scFv, diabody formats, single chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), which are incorporated herein in their entirety). Methods for producing bsAbs include, but are not limited to, quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation including chemical crosslinkers, and genetic approaches utilizing recombinant DNA technology.

[0095] As used herein, the term "multispecific antibody" refers to an antibody that has specificity for at least two different antigens. Such molecules usually bind only two antigens (i.e., bispecific antibodies, bsAbs), but antibodies with additional specificities, such as trispecific antibodies and KIH trispecifics, can also be addressed by the systems and methods disclosed herein.

[0096] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies may be derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) by any means available or known in the art. Monoclonal antibodies useful in the present disclosure may be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0097] As used herein, the term "host cell proteins" (HCPs) includes proteins derived from host cells. Host cell proteins may be process-related impurities derived from the manufacturing process and may include three major categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins and nucleic acids (host cell genome, vector, or total DNA) derived from the host organism. Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical treatment reagents (e.g., cyanogen bromide, guanidine, oxidizing agents, and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other eluates. In some exemplary embodiments, the types of HCP process-related impurities in the composition may be at least two.

[0098] In some exemplary embodiments, the sample may comprise at least one high abundance protein or peptide and at least one HCP. In some exemplary embodiments, the concentration of the at least one high abundance protein or peptide may be at least about 1000 times, about 10,000 times, about 100,000 times, or about 1,000,000 times higher than the concentration of the at least one HCP. Another way to express relative concentration is, for example, parts per million (ppm). When using ppm to describe the concentration of a low abundance protein or peptide, such as an HCP, in a sample containing a high abundance protein or peptide, such as a therapeutic protein, it should be understood that the ppm is measured relative to the concentration of the high abundance protein or peptide. In some exemplary embodiments, the concentration of the at least one HCP may be less than about 1000 ppm, less than about 100 ppm, less than about 10 ppm, or less than about 1 ppm.

[0099] As used herein, the term "sequence variant protein" (SV protein) includes any protein with unintentionally substituted amino acids. For example, as seen in Figure 15A and / or Figure 15B, unintentional amino acid substitutions in SV proteins can result from at least one DNA mutation in the coding sequence, a transcription error from DNA to mRNA, a translation error from mRNA to protein sequence, or a combination thereof. As noted in the literature, due to the finite fidelity in DNA replication and protein biosynthesis processes, unintentional amino acid substitutions naturally occur in a spontaneous manner in any natural biological system. However, in normal biological systems, the probability of such spontaneous errors occurring is very low, occurring within 10 minutes during DNA replication. -11 -10 -8 , 10 during mRNA transcription -6 -10 -4 , 10 during protein translation -5 -10 -4 In prokaryotic systems such as E. coli, translation errors can increase the size of an SV by up to 10 compared to its native form (Figure 16). -3, or 0.1% higher. Being naturally occurring events, these very low levels of SV resulting from transcription or translation errors are usually unavoidable and can therefore be considered as biological noise in protein expression. Suboptimal recombinant protein production systems can elevate SV proteins, for example, by including rare codon sequences or as a result of amino acid depletion.

[0100] In some exemplary embodiments, a sample may contain at least one high abundance non-SV protein or peptide and at least one SV protein. In some exemplary embodiments, the concentration of the at least one high abundance non-SV protein or peptide may be at least about 1000 times, about 10,000 times, about 100,000 times, or about 1,000,000 times higher than the concentration of the at least one SV protein. Another way to express relative concentration is, for example, parts per million (ppm). When ppm is used to describe the concentration of a low abundance protein or peptide, such as an SV protein, in a sample containing a high abundance non-SV protein or peptide, such as a therapeutic protein, it is understood that the ppm is measured relative to the concentration of the high abundance non-SV protein or peptide. In some exemplary embodiments, the concentration of at least one SV protein may be less than about 1000 ppm (e.g., 0.1%), less than about 100 ppm (e.g., 0.01%), less than about 10 ppm (e.g., 0.001%), or less than about 1 ppm (e.g., 0.0001%).

[0101] Although the present disclosure is primarily directed to HCPs and SVs, it should be understood that the methods and systems of the present invention can be used to identify and quantitate any low abundance peptide or protein in a sample.

[0102] As used herein, a "protein pharmaceutical" or "biopharmaceutical" includes an active ingredient that may be fully or partially biological in nature. In one embodiment, a protein pharmaceutical may include a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof. In another embodiment, a protein pharmaceutical may include a recombinant, engineered, modified, mutated, or truncated version of a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof.

[0103] As used herein, a "sample" may be obtained from any step of a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in downstream processing, drug substance (DS), or drug product (DP), including the final formulated product. In some specific exemplary embodiments, the sample may be selected from any step of downstream processing of clarification, chromatographic purification, or filtration. In some specific exemplary embodiments, the drug product may be selected from a manufactured drug product in clinical, transportation, storage, or handling.

[0104] As used herein, the term "solid support" may include any surface capable of binding proteins or peptides. Non-limiting examples of solid supports may include affinity resins, beads, and coated plates or microplates. The solid support may be bound to a molecule capable of binding to a protein or peptide, including an affinity reagent, an antigen-binding molecule, or an interacting peptide ligand. In some exemplary embodiments, the solid support comprises a bead bound to an interacting peptide ligand. In some exemplary embodiments, the solid support comprises a ProteoMiner™ bead.

[0105] In some exemplary embodiments, samples may be prepared prior to LC-MS analysis. Preparation steps may include denaturation, alkylation, dilution, and digestion.

[0106] As used herein, the term "protein alkylating agent" or "alkylating agent" refers to an agent used to alkylate specific free amino acid residues in a protein. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

[0107] As used herein, "protein denaturation" or "denaturation" may refer to a process that changes the three-dimensional shape of a molecule from its native state. Protein denaturation may be performed using a protein denaturant. Non-limiting examples of protein denaturants include heat, high or low pH, reducing agents such as DTT, or exposure to chaotropic agents. Several chaotropic agents may be used as protein denaturants. Chaotropic solutes increase the entropy of a system by disrupting intramolecular interactions mediated by non-covalent forces such as hydrogen bonding, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroyl sarcosine, urea, and salts thereof.

[0108] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to perform the digestion of proteins in a sample using a suitable hydrolysis agent, e.g., enzymatic digestion or non-enzymatic digestion. Digestion of proteins into their constituent peptides can generate "peptide digests," which can be further analyzed using peptide mapping analysis.

[0109] As used herein, the term "digestive enzyme" refers to any of a number of different agents that can perform protein digestion.Non-limiting examples of hydrolytic agents that can perform enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoprotease (Lys-C), endoprotease Asp-N (Asp-N), endoprotease Arg-C (Arg-C), endoprotease Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin degrading enzyme (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or biologically active fragments or homologues thereof, or combinations thereof. For a recent review discussing available techniques for protein digestion, see Switazar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzer, Martin Giera & Wilfried MANiessen, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077(2013)).

[0110] Conventional methods use digestive enzymes under conditions and concentrations sufficient to completely digest all proteins in a sample prior to LC-MS analysis. The present disclosure surprisingly finds that the identification and quantification of low-abundance proteins such as HCPs can be improved by limited digestion, meaning that digestive enzymes are used under conditions such that the proteins in a sample are not completely digested. In some exemplary embodiments, proteins are subjected to digestion without prior denaturation, meaning that "native digestion" is performed on naturally folded proteins. In some exemplary embodiments, the ratio of digestive enzyme to substrate is selected to ensure limited digestion. In some exemplary embodiments, the ratio of digestive enzyme to substrate is less than about 1:100, less than about 1:200, less than about 1:300, less than about 1:400, less than about 1:500, less than about 1:600, less than about 1:700, less than about 1:800, less than about 1:900, less than about 1:1000, less than about 1:2000, less than about 1:3000, less than about 1:4000, less than about 1:5000, less than about 1:6000, less than about 1:7000, less than about 1:8000, less than about 1:9000, less than about 1:10000, about 1:400, about 1:1000, about 1:2500, or about 1:10000.

[0111] As used herein, the term "protein reducing agent" or "reducing agent" refers to an agent used for the reduction of disulfide bridges in proteins. Non-limiting examples of protein reducing agents used to reduce proteins are dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof.

[0112] As used herein, the term "liquid chromatography" refers to a process in which a biological and / or chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some embodiments, the sample or eluate can be subjected to any one of the above-mentioned chromatographic methods or a combination thereof.

[0113] As used herein, the term "mass spectrometer" includes devices that can identify specific molecular species and measure their exact mass. The term is meant to include any molecular detector that can characterize a polypeptide or peptide. A mass spectrometer can include three main parts: an ion source, a mass spectrometer, and a detector. The role of the ion source is to form gas phase ions. Analyte atoms, molecules, or clusters are transferred to the gas phase and may be ionized simultaneously (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application.

[0114] The mass spectrometer may be coupled to a liquid chromatography-multiple reaction monitoring system. More generally, the mass spectrometer may be capable of analysis by selected reaction monitoring (SRM), including sequential reaction monitoring (CRM) and parallel reaction monitoring (PRM).

[0115] As used herein, "multiple reaction monitoring" or "MRM" refers to a mass spectrometry-based technique that can accurately quantify small molecules, peptides, and proteins in complex matrices with high sensitivity, specificity, and wide dynamic range (Paola Picotti & Ruedi Aebersold, Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions, 9 NATURE METHODS 555-566 (2012)). MRM can typically be performed using a triple quadrupole mass spectrometer, where precursor ions corresponding to selected small molecules / peptides are selected in the first quadrupole, and fragment ions of the precursor ions are selected for monitoring in the third quadrupole (Yong Seok Choi et al., Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimers disease biomarker candidates, 930 JOURNAL OF CHROMATOGRAPHY B 129-135 (2013)).

[0116] SRM / MRM / selected ion monitoring (SIM) is a method used in tandem mass spectrometry in which ions of a particular mass are selected in the first stage of a tandem mass spectrometer, and ion products of the fragmentation reaction of the precursor ion are selected for detection in the second mass spectrometer stage. Examples of triple quadrupole mass spectrometers (TQMS) that can perform MRM / SRM / SIM include, but are not limited to, the QTRAP® 6500 system (Sciex), the QTRAP® 5500 system (Sciex), the triple QTriple Quad 6500 system (Sciex), the Agilent® 6400 series triple quadrupole LC / MS system, and the Thermo Scientific® TSQ™ triple quadrupole system.

[0117] In addition to MRM, a selection of peptides can also be quantified through parallel reaction monitoring (PRM). PRM is an application of SRM with parallel detection of all transitions in a single analysis using a high-resolution mass spectrometer. PRM offers high selectivity, high sensitivity, and high throughput to quantify selected peptides (Q1) and therefore proteins. Multiple peptides can be specifically selected for each protein. PRM methodology can use the quadrupole of the mass spectrometer to isolate target precursor ions, fragment the target precursor ions in a collision cell, and then detect the resulting product ions in an Orbitrap mass analyzer. PRM can use quadrupole time-of-flight (QTOF) or hybrid quadrupole orbital trap (QOrbitrap) mass analyzers to perform peptide and / or protein identification. Examples of QTOF include, but are not limited to, the Triple TOF 6600 system (Sciex), the Triple TOF 5600 system (Sciex), the X500R QTOF system (Sciex), the 6500 Series Accurate Mass Quadrupole Time of Flight (Q-TOF) (Agilent), and the Xevo G2-XS QT Quadrupole Time of Flight Mass Spectrometer (Waters). Examples of QObitrap include, but are not limited to, the Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific) and the Orbitrap Fusion™ Tribrid™ (Thermo Scientific).

[0118] Non-limiting advantages of PRM include eliminating most interferences, providing greater precision and attomolar limits of detection and quantification, enabling definitive confirmation of peptide identity through spectral library matching, reducing assay development time since no target transitions need to be pre-selected, and ensuring UHPLC-compatible data acquisition speeds through spectral multiplexing and advanced signal processing.

[0119] The mass spectrometer in the method or system of the present application can be, for example, an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, and the mass spectrometer can be coupled to a liquid chromatography system, and the mass spectrometer can perform LC-MS (liquid chromatography-mass spectrometry) or LC-PRM-MS (liquid chromatography-parallel reaction monitoring-mass spectrometry) analysis. In some exemplary embodiments, peptide identification is performed using PRM-MS.

[0120] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" includes techniques in which structural information of a sample molecule is obtained by using multiple stages of mass selection and mass separation. The prerequisite is that after the first mass selection step, the sample molecule is converted into the gas phase and ionized so that fragments are formed in a predictable and controllable manner. MS / MS, or MS2, can be performed by first selecting and isolating a precursor ion (MS1) and then fragmenting it to obtain meaningful information. Tandem MS has been successfully performed with a wide variety of analyzer combinations. Which analyzer to combine for a particular application can be determined by many different factors, such as size, cost, and availability as well as sensitivity, selectivity, and speed. The two main categories of tandem MS methods are tandem-in-space and tandem-in-time, although hybrids also exist, in which a tandem-in-time analyzer is connected in space or with a tandem-in-space analyzer. A tandem-in-space mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A specific m / z separation function can be designed such that ions are selected in one section of the instrument, dissociated in an intermediate region, and then the product ions are transmitted to another analyzer for m / z separation and data collection. In tandem-in-time, mass analysis ions produced in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0121] The peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational modifications. They can be used to characterize proteins by correlating experimental and theoretical MS / MS data, the latter generated from possible peptides in protein sequence databases. Characterization includes, but is not limited to, sequencing the amino acids of protein fragments, sequencing the protein, de novo sequencing the protein, localizing or identifying post-translational modifications, or comparability analysis, or a combination thereof.

[0122] In some exemplary aspects, the mass spectrometer may operate with nanoelectrospray or nanospray. As used herein, the term "nanoelectrospray" or "nanospray" refers to electrospray ionization at very low solvent flow rates (typically a few hundred nanoliters of sample solution per minute or less), often without the use of external solvent delivery. The electrospray injector that forms the nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small amounts of sample (analyte) solution over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and a solvent delivery system to perform chromatographic separation of the mixture prior to analysis by the mass spectrometer.

[0123] As used herein, the term "database" refers to a compiled collection of protein sequences that may be present in a sample, for example in the form of FASTA format files. The relevant protein sequences may be derived from the cDNA sequences of the species studied. Public databases that may be used to search for relevant protein sequences included, for example, databases hosted by Uniprot® or Swiss-prot®. The databases may be searched using what are referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search uninterpreted MS / MS spectra against all possible sequences in the database(s) and provide interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools are Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (www.thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (www.proteinmetrics.com / products / byonic), or Sequest (fields.scripps.edu / sequest).

[0124] The present invention is not limited to any of the above-mentioned protein(s), therapeutic protein(s), antibody(s), recombinant protein(s), host cell protein(s), sequence variant protein(s), protein pharmaceutical(s), sample(s), solid support(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH range(s) or value(s), temperature(s), or concentration(s). It is understood that any of the proteins, therapeutic proteins, antibodies, recombinant proteins, host cell proteins, sequence mutated proteins, protein pharmaceuticals, sample, solid support, protein alkylating agent, protein denaturant, protein reducing agent, digestive enzyme, chromatographic method, mass spectrometer, database, bioinformatics tool, pH, temperature, or concentration may be selected by any suitable means.

[0125] The present invention may be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. EXAMPLES

[0126] Materials and Methods for Examples 1-3 material ProteoMiner™ Protein Enrichment Kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). ProteoMiner™ technology is a sample preparation tool for compressing the dynamic range of protein concentrations in biological samples. A large library of combinatorial hexapeptide ligands was immobilized on beads to capture a variety of proteins. The ProteoMiner™ spin column contained 500 μl of bead slurry (4% beads, 20% (v / v) EtOH in water) with a sedimentation bead volume of 20 μl. The wash buffer of the kit contains 50 mL of PBS (phosphate buffer saline, 150 mmol / L NaCl, 10 mmol / L NaH2PO4, pH 7.4). The kit's elution buffer contains lyophilized urea CHAPS (8 M urea, 2% CHAPS; CHAPS detergent is 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate). The kit's rehydration buffer contains 5% acetic acid.

[0127] Chromatography solvents that were LC-MS grade were purchased from Thermo Fisher Scientific (Waltham, MA). Monoclonal antibodies were produced by Regeneron (Tarrytown, NY). Sodium deoxycholate (SDC), sodium lauroyl sarcosinate (SLS) and chloroacetamide (CAA) were purchased from Sigma-Aldrich (St. Louis, MO). Tris-(2-carboxyethyl)phosphine (TCEP) was purchased from Thermo Fisher Scientific. RM 8670 (NIST mAb, NIST monoclonal antibody standard, expressed in a mouse cell line) was obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD).

[0128] Protein Enrichment using the ProteoMiner™ Protein Enrichment Kit The proteins in the samples were enriched using the ProteoMiner™ Protein Enrichment Kit. A small-scale ProteoMiner™ cartridge was used for five experiments. The ProteoMiner™ beads were washed twice with 200 μL of wash buffer provided in the kit. The beads were resuspended in 200 μL of water and 40 μL of bead slurry was transferred to a tube for performing one experiment. The mAB DS or NIST mAb was diluted in water and the pH of the solution was then adjusted to pH 6 using 25 mmol / L of sodium acetate pH 4.0. The sample was added to the ProteoMiner™ bead slurry and incubated at room temperature with rotation for 2 hours. The sample mixture was then loaded into the tip with a frit. The supernatant was removed by centrifugation at 1000×g for 1 minute. The beads were then washed by adding 100 μL of wash buffer to the tip and then centrifuged three times at 200×g for 1 minute. Finally, the concentrated proteins were eluted using 10 μL of PTS buffer (12 mmol / L SDC, 12 mmol / L SLS, 10 mmol / L TCEP, and 30 mmol / L CAA), followed by centrifugation at 200 × g for 1 min three times.

[0129] For the optimized ProteoMiner limited digestion method of the present invention, the collected eluate containing the enriched proteins was reduced. The reduced proteins were digested overnight at 28° C. with an enzyme-to-substrate ratio of 1:400 to obtain a solution containing a peptide mixture. The peptide mixture was then subjected to reduction, denaturation, and alkylation.

[0130] The solution containing the peptide mixture was acidified using 10 μL of 10% TFA to precipitate SDC and SLS. The solution containing the peptide mixture was then centrifuged at 14,000 rcf for 20 minutes. The supernatant containing the peptide mixture was then desalted using a GL-Tip GC desalting tip and dried using a SpeedVac®.

[0131] LC-MS / MS analysis The desalted peptide mixture obtained from the ProteoMiner™ limited digest enrichment was dried and resuspended in 30 μL of 0.1% formic acid (FA) solution. 5 μL of the solution containing the peptide mixture was injected into a low-flow liquid chromatography system (e.g., UltiMate™ 3000 RSLCnano system (Thermo Fisher Scientific)) coupled to a Q-Exactive HFX mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a 25 cm C18 column (0.075 mm inner diameter, 2.0 μm, 100 Å, Thermo Fisher Scientific). The mobile phase buffer contained 0.1% FA in ultrapure water (Buffer A) and the elution buffer contained 0.1% FA in 80% acetonitrile (ACN) (Buffer B). Peptides were eluted using a 100 min linear gradient from 2% to 25% buffer B at a flow rate of 300 nL / min. The mass spectrometer was operated in data-dependent mode. The 10 most intense ions were each subjected to full MS scans at resolution 120,000 (automatic gain control (AGC) target 3e6, maximum injection time 60 ms, m / z 375-1500) and higher energy collision dissociation (HCD) fragmentation with a normalized collision energy (NCE) of 27% for MS / MS events at resolution 30,000 (AGC target 1e5, maximum injection time 60 ms, m / z 200-2000). MS proteomics data were deposited in the ProteomeXchange Consortium under project accession number PXD016194 via the JPOST repository.

[0132] Example 1. Comparison of digestion methods The aforementioned ProteoMiner™ method (Chen et al.) for HCP identification ("direct digestion method") was compared with various alternative techniques for optimizing the detection of host cell proteins (HCPs) and other low-abundance proteins in a sample containing at least one high-abundance protein or peptide. The direct digestion method includes the steps of contacting a sample containing at least one high-abundance protein or peptide with a solid support such as beads, where an interacting peptide ligand is bound to the solid support, and HCP impurities can be bound to the interacting peptide ligand, e.g., ProteoMiner™ beads; washing the solid support with a solution containing a detergent to concentrate HCP impurities and provide an eluate; subjecting the eluate to denaturation, alkylation, and reduction; subjecting the denatured, alkylated, and reduced eluate to an enzymatic digestion reaction to generate components of enriched HCP impurities; identifying the components of enriched HCP impurities using a mass spectrometer; and identifying the enriched HCP impurities using the identified components.

[0133] An alternative approach is the on-bead native digestion method, in which the HCP impurities are subjected to enzymatic digestion before being eluted from the solid support. Another alternative approach is the "elution mild denaturing digestion", or "limited digestion", in which the HCP impurities are eluted and subjected to limited digestion using a lower ratio of digestive enzyme to substrate, reduced, denatured, and alkylated, and then analyzed using a mass spectrometer. An exemplary workflow of the limited digestion method is shown in Figure 1.

[0134] These three alternative techniques were compared based on their ability to identify HCPs in monoclonal antibody drug substance (mAb DS) samples and to identify spike-in UPS2 proteins in mAb DS samples. UPS2 is a commercially available proteomics standard that contains 48 human proteins with a wide dynamic range of concentrations spanning several orders of magnitude. As shown in Table 1 and Figure 2, ProteoMiner™ limited digestion was the most sensitive approach. [Table 1]

[0135] Example 2. Parameter optimization Based on the effectiveness of the ProteoMiner™ method of limited digestion for the identification of low abundance proteins, as shown in Example 1, the method was further optimized. The method was performed with decreasing ratios of trypsin enzyme:substrate for digestion to compare the sensitivity of HCP and UPS2 protein identification. The method used an enzyme:substrate ratio of 1:20. As shown in Table 2 and Figure 3, an enzyme:substrate ratio of 1:400 was found to be the most effective. [Table 2]

[0136] Without being bound by theory, it is believed that more limited digestion may result in a disproportionate reduction in the digestion of high abundance proteins or proteins in the sample, further reducing the dynamic range of digested peptides and allowing more effective measurement of low abundance proteins. Subjecting a protein sample to native digestion without denaturation contributes to more limited digestion, as does subjecting the protein sample to a lower digestive enzyme to substrate ratio (Huang et al.).

[0137] The method of the present invention was further optimized by comparing a range of denaturing reagent concentrations. As shown in Table 3 and Figure 4, a SLS / SDC concentration of 12 mmol / L was found to be the most effective for identifying HCP and UPS2 proteins. [Table 3]

[0138] These optimized conditions were used for further experiments.

[0139] Example 3. Case study using optimized ProteoMiner™ method Compared to the previously described ProteoMiner™ method, the optimized method described in Example 2 was used with UPS2 spiked into the mAb DS sample. The optimized method of the present invention had superior efficacy in identifying low abundance UPS2 proteins compared to the previously described method, as shown in Figures 5A and 5B. The UPS2 column on the right side of Tables 1-3 represents the UPS2 standard that was directly digested without being spiked into the mAb DS as a control for the detection limit of the instrument.

[0140] Both methods identified all spike-in proteins at levels between 1 and 4 ppm. At levels between 0.1 and 1 ppm, the ProteoMiner™ method identified 8 / 8 spike-in proteins, while the optimized ProteoMiner™ limited digestion method identified 7 / 8. At levels between 0.01 and 0.07 ppm, the ProteoMiner™ method identified 3 / 8 spike-in proteins, while the optimized ProteoMiner™ limited digestion method identified 8 / 8. At levels between 0.001 and 0.006 ppm, the ProteoMiner™ method identified 0 / 8 spike-in proteins, while the optimized ProteoMiner™ limited digestion method identified 3 / 8.

[0141] The optimization method of the present invention was further compared with the conventional method using mAb DS as a sample with or without spike-in UPS2. The conventional methods compared include immunoprecipitation, filtration, limited digestion alone, and the aforementioned ProteoMiner™ method. For mAb DS without spike-in UPS2, the optimization method of the present invention was more effective in identifying HCPs than any other method, as shown in Figure 6. For mAb DS with spike-in UPS2, the optimization method of the present invention was more effective in identifying UPS2 proteins than any other method, as shown in Figure 7. In Figure 7, the first column represents the total number of UPS2 proteins identified, and the second column represents the number of UPS2 proteins identified at 0.1875 ppm out of a total of 8. The optimization method of the present invention identified 8 / 8 UPS2 proteins at this concentration.

[0142] The optimization method of the present invention was further compared with the above-mentioned method using NIST mAb standard as sample. The conventional methods compared include normal digestion, native digestion, native digestion using ProA beads, and field asymmetric ion mobility spectrometry (FAIMS), filtration, and the above-mentioned ProteoMiner™ method. The number of HCPs identified in NIST mAb samples using the method of the present invention was compared with the number of HCPs identified using conventional methods according to previous publications. As shown in Figure 8, the method of the present invention was more effective in identifying HCPs in NIST mAb samples than any of the previously published methods.

[0143] Materials and Methods for Examples 4 to 6 material Chromatography solvents were LC-MS grade and purchased from Thermo Fisher Scientific (Waltham, MA). mAbs and spike-in CHO proteins were produced by Regeneron (Tarrytown, NY). Sodium deoxycholate (SDC), sodium lauroyl sarcosinate (SLS), iodoacetamide, urea, 10× Tris-buffered saline, ammonium acetate, oleic acid, and oleic acid were used. -13 C 18 (CAS number 287100-82-7) was purchased from Sigma-Aldrich (St. Louis, MO). Ultra-purified PS80 was purchased from Croda (East Yorkshire, UK). Dithiothreitol was purchased from Thermo Fisher Scientific. Human palmitoyl protein thioesterase 1 (hPPT1) and human LPL were purchased from Abcam. CHO LAL, CHO complement component 1r (C1r-A), CHO acid ceramidase (ASAH1), CHO beta-2-microglobulin, CHO carboxypeptidase, CHO cathepsin D, and CHO cathepsin Z were synthesized in-house by Regeneron Pharmaceuticals.

[0144] Preparation of internal standards and standard curves Eight recombinant proteins (LAL, LPL, C1r-A, ASAH1, beta-2-microglobulin, carboxypeptidase, cathepsin D, and cathepsin Z) were dissolved in water to a final concentration of 100 ng / μL as stock solutions. The stock solutions were further diluted to 1 ng / μL and 10 ng / μL and spiked into antibody matrix (mAb-1) at concentrations of 0.05 ppm, 0.1 ppm, 0.5 ppm, 1 ppm, 2 ppm, and 5 ppm to prepare a standard curve. QC proteins were prepared from separate stocks of recombinant protein mixture (7 ng / μL cathepsin Z, 17 ng / μL cathepsin D, 35 ng / μL LAL, 87 ng / μL carboxypeptidase, and 175 ng / μL LPL) and spiked into mAb-1 to obtain 0.2 ppm cathepsin Z, 0.5 ppm cathepsin D, 1 ppm LAL, 2.5 ppm carboxypeptidase, and 5 ppm LPL. Heavy isotope labeled putative phospholipase B-like 2 (hPLBD2) was diluted to 5 ng / μL and spiked into each sample at 5 ppm.

[0145] Stock solutions of the two recombinant proteins (LAL and hPPT1) were prepared at 1 ng / μL and 5 ng / μL and spiked into the antibody matrix (mAb-2) at concentrations of 0.1 ppm, 0.5 ppm, 1 ppm, 2 ppm, 5 ppm, 10 ppm, and 20 ppm to prepare a standard curve. Heavy isotope labeled hPLBD2 was diluted to 5 ng / μL and spiked into each sample at 5 ppm. The same antibody matrix was used to measure PPT-1 and LAL in mAb-18 and mAb-19.

[0146] Preparation of samples by PMLD method Host cell proteins were first enriched by ProteoMiner enrichment coupled with limited digestion (PMLD). ProteoMiner beads were washed sequentially with wash buffer and water, then suspended in water. A total of 15 mg of mAb was diluted or concentrated to 50 mg / mL in water, adjusted to pH 6, and added into the ProteoMiner bead slurry. Each sample was incubated rotating at room temperature for 2.5 hours, then loaded into a home-made tip with a 9.5 mm pore size frit. The beads were then washed, and the enriched proteins were eluted three times by adding 10 μL of elution buffer (12 mmol / L SDC and 12 mmol / L SLS). The collected eluate was then further prepared by modified limited digestion by adding 75 ng trypsin, then digested overnight at 28° C. The digested samples were reduced at 90° C. for 20 minutes and alkylated at room temperature for another 20 minutes. The peptide mixture was acidified to pH 2-3 with 10% TFA to precipitate the mAb, SDC, and SLS in the acidic solution. The mixture was then centrifuged at 14,000 rcf for 10 min. The peptide-containing supernatant was collected, desalted with a GL-Tip GC desalting tip, dried, and resuspended in 0.1% FA for nano-LC-MS / MS analysis.

[0147] Non-targeted nanoLC-MS / MS and targeted parallel reaction monitoring analysis The peptide mixture was injected into an UltiMate™ 3000 RSLCnano system coupled to an Orbitrap Exploris® 480 mass spectrometer (Thermo Fisher Scientific). The peptide mixture was loaded onto a 20 cm x 0.075 mm Acclaim PepMap 100 C18 trap column (Thermo Fisher Scientific) for desalting, and then separated on a 25 cm x 75 μm ID x 1.7 μm C18 integrated column (CoAnn Technologies). Peptides were separated with a 150 min linear gradient of 2% to 32% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL / min. An Orbitrap Exploris® 480 mass spectrometer (Thermo Fisher Scientific) operated in data-dependent mode was used for untargeted HCP detection. For targeted PRM detection, each sample was analyzed under PRM with an isolation window of 2 m / z. In all experiments, a full mass spectrum at resolution 60,000 (normalized AGC target (%) 300, maximum injection time 20 ms, m / z 380-1600) to m / z 200 was followed by a time-scheduled PRM scan at resolution 15,000 (normalized AGC target (%) 100, maximum injection time 60 ms). HCD was used with an NCE of 30%.

[0148] Data analysis Mass spectrometry raw files were searched against UniProt Cricetulus Griseus (version 2020) without redundant entries using Byonic software (version 4.1.10). Mass tolerance was set at 10 ppm and fragment mass tolerance was set at 20 ppm. Search criteria included static carbamidomethylation of cysteine ​​(+57.0214 Da) and variable modification of oxidation of methionine residues (+15.9949 Da). Database searches were performed on tryptic digests with up to two missing cleavages. HCPs were positively identified when at least two unique peptides were found. PRM data were manually curated within Skyline (version 21.1).

[0149] PS80 Degradation Profiling of mAb-3 by 2DLC-CAD PS80 degradation profiling was performed. mAb-3 containing 0.1% PS80 was diluted to 0.004% in water and injected into a 2D HPLC-CAD system. PS80 was retained on an Oasis® Max column (2.1×20 mm, 30 mm), separated by an Acquity® BEH C4 column (2.1×50 mm, 1.7 mm), and detected with a Corona Ultra CAD detector.

[0150] Accelerated hydrolysis of PS80 in formulated pharmaceutical products Accelerated hydrolysis of PS80 in mAb-3 to mAb-15 was performed. 13 C 18 was added to the mAb to a final concentration of 1 μg / mL, and a 10% PS80 stock solution was also added to the mAb to a final concentration of 1% PS80. All samples were incubated at 37° C. for 3 or 5 days, and 10 μL of each sample was collected for oleic acid quantification before and after incubation.

[0151] Quantitative determination of oleic acid in mAb Oleic acid released from PS80 degradation in mAb-3 stability samples was quantified by LC-MRM. 13 C 18 90 μL of extraction buffer containing (80% IPA / 20% MeOH) was added to 10 μL of each mAb-3 stability sample, mixed and incubated at room temperature for 1 h. Proteins were then precipitated by centrifugation at 14,000 rcf for 30 min at 25° C., and 40 μL of oleic acid-containing supernatant was transferred to a 96-well plate for LC-MRM analysis. Oleic acid released by accelerated PS80 hydrolysis from mAb-3 to mAb-15 was analyzed by LC-MRM analysis using the internal standard oleic acid-extraction buffer added to mAb-3 to mAb-15 samples before and after incubation. 13 C 18Oleic acid and oleic acid- were quantified by LC-MRM in a similar manner, except that the oleic acid-containing oleic acid mixture contained 80% IPA / 20% MeOH without oleic acid. 13 C 18 was quantified by monitoring peaks 281.2 / 281.2 and 299.2 / 299.2 using an Agilent 6495 QQQ mass spectrometer equipped with an Agilent 1290 Infinity® UHPLC (Agilent, Wilmington, Germany). Peak integration was performed in Skyline.

number

[0152] MY clipping measurement by intact mass analysis of DS-1 to DS-3 Clipping between the amino acid residues methionine and tyrosine in DS (MY clipping) was identified and quantified by intact mass analysis. DS samples were reduced with a mixture of 5 μg DS (0.25 μg / μL) and 4 μL 5× rapid PNGase buffer (New England Biolabs) and incubated at 80° C. for 10 min. Deglycosylation was performed by adding 1 μL rapid PNGase (New England Biolabs) to the mixture and incubating at 50° C. for 25 min. Then, 2 μg of each sample was injected into the LCMS system and separated by reversed-phase chromatography using a BioResolve® RP mAb polyphenyl column (2.7 μm, 2.1 mm×50 mm) and detected by a Waters G2S mass spectrometer. MS data was analyzed with Waters' MassLynx® software.

[0153] Example 4. Identification of HCPs by PMLD Untargeted HCP Profiling ProteoMiner enrichment with limited digestion (PMLD) and ProteoMiner enrichment with targeted PRM analysis were used to quantify HCPs at sub-ppm levels. This enrichment-based targeted quantification further improved detection down to 0.06 ppm with high accuracy and precision compared to other mass spectrometry-based quantification methods. The low detection limits were important for risk assessment: 0.13 ppm hepatic carboxylesterase (CES), 2.48 ppm lysosomal acid lipase (LAL), 1.46 ppm palmitoyl protein thioesterase 1 (PPT-1), and 0.5 ppm cathepsin D were found to have a negative impact on drug stability, whereas 0.5 ppm lipoprotein lipase (LPL), 0.04 ppm CES, 0.8 ppm LAL, 0.49 ppm PPT-1, and 0.3 ppm cathepsin D were found to be safe allowing the drug to maintain a shelf life of 2-3 years.

[0154] The polysorbate (PS) degradation ability of different PS-degrading enzymes (PSDEs) is usually evaluated by comparing their activities in spike-in experiments with recombinant proteins. However, the observed lipase activity of recombinant proteins may not represent the activity of endogenous proteins. For example, PS degradation observed from putative phospholipase B-like 2 (PLBD2) may be due to impurities rather than PLBD2 itself. Since neither recombinant CHO LPL nor endogenous LPL below 1.5 ppm exhibit lipase activity, it remains questionable whether LPL can degrade PS. ProteoMiner enrichment with limited digestion (PMLD) and ProteoMiner enrichment with targeted PRM analysis provided a quantification method for the comparison of lipase activity of PSDEs through the correlation between lipase activity in DP and endogenous PSDE concentration. The main cause of PS degradation can be estimated according to the correlation between PSDE and lipase activity.

[0155] HCP profiling using PMLD was performed for several in-house mAbs to determine the most appropriate matrix for PRM method development. The PMLD workflow is shown in Figure 9. The detection limit of PMLD was as low as 0.002 ppm. mAb-1 was selected as the matrix for the standard curve and QC because it showed minimal interference (≦10% LLOQ level) for eight spike-in recombinant CHO protein standards, as shown in Figures 10A-E. mAb-2 was selected as the matrix for the standard curves of PPT-1 and LAL because it was the same mAb as mAb-18 and mAb-19 but did not contain PPT-1 or LAL. mAb-3 contained various levels of HCP and was therefore used to assess intra-run reproducibility. mAb-4 to mAb-16 were antibodies with different levels of lipase activity that were used to access biologically relevant concentrations of lipase or esterase. The lipase responsible for PS80 degradation in mAb-3 to mAb-10 was LAL. The esterase responsible for PS80 degradation in mAb-11 to mAb-17 was CES, and the two lipases responsible for PS80 degradation in mAb-18 and mAb-19 were LAL and PPT-1. DS-1 to DS-3 are fusion proteins used to assess biologically relevant concentrations of cathepsin D.

[0156] Example 5. Development of a PMLD-PRM method for HCP quantification Table 4 shows the tryptic peptides selected for each HCP for targeted PRM quantification. These peptides were selected because they had high MS signal intensity, no or low post-translational modifications, no missing cleavage, and were unique to each CHO HCP. hPLBD2 served as a common internal standard for all HCP quantifications. Peak area ratios (PARs) of the selected HCP peptides were obtained by dividing the peak area of ​​the hPLBD2 peptide by the peak area of ​​the HCP peptide. The PARs were used to construct a calibration curve for quantification. The relative enrichment of each individual HCP by the PMLD method was comparable to the relative enrichment of hPLBD2 in the same mAb sample, as ProteoMiner is a non-biased enrichment method. Indeed, Figure 11A shows the PRM-based quantification of all eight HCPs in mAb-1 in the range of 0.05–5 ppm, with regression coefficients (R ) greater than 0.99. 2 ) followed a linear regression equation with a mean mean of 0.99. This finding suggested that the dynamic range of the PMLD enrichment method followed by targeted PRM analysis is suitable for quantification of HCPs ranging from sub-ppm to low ppm levels. PMLD-PRM analysis was performed on human PPT-1 and LAL ranging from 0.1 to 20 ppm in mAb-2 to test whether PMLD-PRM analysis could be applied to a wider range of HCPs. Figure 11B shows that the regression coefficients (R 2 ) and linear regression lines were observed for both human PPT-1 and LAL. [Table 4]

[0157] The PMLD-PRM method was evaluated for within-run reproducibility and quantification precision using a mixture of five QC standards spiked into mAb-1 with triplicate replicates. A mixture of five QC peptides was prepared with different concentrations of five HCPs: 0.2 ppm cathepsin Z, 0.5 ppm cathepsin D, 1 ppm LAL, 2.5 ppm carboxypeptidase, and 5 ppm LPL. The concentration of each QC standard was selected based on its abundance in mAb-1 to minimize interference in the scans shown in Figures 10A-10E and to be biologically relevant to the quality of the drug product. As shown in Table 5, the precision of detection of the QC peptides of the five HCPs in the range of 0.2 ppm-5 ppm was within 85%-111% of the theoretical value, and the variation between triplicate samples was less than 12%. The quantification results were based on peptides of each HCP enriched from mAb-1, and each spiked sample was analyzed in triplicate. The lower limits of quantification (LLOQ) ranged from 0.06 ppm to 0.66 ppm. [Table 5]

[0158] Intra-run reproducibility was assessed using mAb-3 that contained HCPs at various levels ranging from 0.03 ppm to 4.24 ppm. Three biological replicates of mAb-3 were prepared and analyzed separately on three different days to quantitatively evaluate six HCPs, the results of which are shown in Table 6. Precision was within 25% for all HCPs below 0.5 ppm and within 20% for HCPs above 0.5 ppm. Quantitative results were based on peptides of each HCP enriched from mAb-3, and each sample was analyzed in triplicate. [Table 6]

[0159] Example 6. Biologically relevant concentrations of selected HCPs Sub-ppm levels of lipases or esterases cause PS degradation in pharmaceutical products during long-term storage. Table 6 and Figure 12A show that 1.28 ppm of LAL was detected in mAb-3 resulting in 20% PS80 degradation, and Figure 12B shows that 23 μg / mL of oleic acid was released within 6 months at 4-8 °C. Figure 12C shows that LAL ranging from 0.1 ppm in mAb-3 to 3.5 ppm in mAb-10 was accurately quantified using PMLD. The regression coefficient (R 2 ) was greater than 0.98. The LAL concentration in mAb-4 was determined to be 0.1 ppm by PMLD method. Figure 12C shows that 0.1 ppm LAL in mAb-4 did not cause any visible PS80 degradation within 4 days at 37 °C. Therefore, the low detection limit of 0.1 ppm LAL aids in accurate estimation of the potential adverse effect of LAL on PS80 degradation and may be used to predict the potential shelf life of pharmaceutical products. LPL at concentrations less than 0.5 ppm was detected in all eight samples from mAb3 to mAb10. Figure 12C shows that the degradation of PS80 did not correlate with the concentration of LPL in mAb-3 to mAb-10, and R 2 was 0.19, thus suggesting that LPL had no effect on PS80. Figure 12C also shows that 0.5 ppm LAL released 1.45 μg / mL of oleic acid per day from PS80 degradation at 37°C.

[0160] CES is an esterase that can degrade PS80 when present in low abundance. For example, 20 ppm of CES results in complete depletion of monoesters from PS80 species at 4-8 °C within 24 h. The concentration of CES in mAb-11 was determined to be 2.3 ppm by quantification by native digestion coupled with MRM. In mAb-12, the same mAb as mAb-11 but obtained by a different purification step, CES was not detected by PMLD and no PS80 degradation was observed. mAb-13 was formulated with a mixture of mAb-11 and mAb-12 in a ratio of 1:9, and the concentration of CES in mAb-13 was determined to be 0.23 ppm. mAb-14 was formulated with a mixture of mAb-11 and mAb-12 in a ratio of 1:49, and the concentration of CES in mAb-14 was determined to be 0.046 ppm. mAb-12 to mAb-17 were enriched by PMLD, and the absolute abundance of CES in each sample was calculated as the relative abundance to mAb-13 and mAb-14. Figure 13 shows that a correlation between the increase in oleic acid concentration per day and the CES concentration was established. Even 0.1 ppm of CES was estimated to lead to an increase of 5.4 μg / mL of oleic acid per day under accelerated degradation conditions (37 °C).

[0161] PPT-1 and LAL were found to be possible causes of PS80 degradation in both mAb-18 and mAb-19. In contrast to mAb-3, where only LAL, not LPL, was responsible for PS80 degradation, both PPT-1 and LAL played important roles in PS80 degradation. Table 4 shows that quantification of CHO PPT-1 was performed with peptide ETIPLQESTLYTEDR due to the lack of recombinant CHO PPT-1, whereas the calibration curve was made with human PPT-1 peptide ETIPLQETSLYTQDR. Considering the difference of only a single amino acid residue between the 15 residues, the ionization efficiency was not expected to change substantially between these two peptides.

[0162] Table 7 shows the quantification results of PPT-1 and LAL in mAb-18 and mAb-19, as well as the measurement of released oleic acid after incubation at 37° C. 1.8 ppm PPT-1 and 0.39 ppm LAL were detected in mAb-18, thus resulting in an increase of 8.57 μg / mL oleic acid per day, respectively, whereas 1.1 ppm PPT-1 and 0.34 ppm LAL were found in mAb-19, resulting in an increase of 5.28 μg / mL oleic acid per day under accelerated degradation conditions (37° C.). Based on the results of mAb-3 in Figure 12A, Figure 12B, and / or Figure 12C, 0.5 ppm LAL was found to degrade approximately 1.45 μg / mL oleic acid per day at 37° C. It was estimated that 1 ppm of PPT-1 induced an increase in oleic acid of approximately 4 μg / mL per day. [Table 7]

[0163] Figure 14 shows that 71.6% PS80 degradation was observed after incubating mAb-18 for 36 months at 4-8 °C. Subvisible and visible particles are formed after 61% degradation of PS80. Although particle formation may vary with different protein formulations, this observation was used to estimate particle formation time. Using the results from the mAb-18 stability study, it was estimated that approximately 7.3 μg / mL of oleic acid was released per day under accelerated degradation conditions (37 °C), resulting in 61% PS80 degradation with subsequent particle formation at 36 months. Therefore, it was estimated that 1.8 ppm of PPT-1, 0.14 ppm of CES, or 2.5 ppm of LAL would likely cause particle formation in DP throughout the long-term stability study. The relative lipase activities among the different PSDEs were also compared based on the enzyme oleate increment per ppm per day, which was calculated to be 2.9, 54.7, and 4.1 μg / mL per ppm per day for LAL, CES, and PPT-1, respectively. Thus, the PS degradation abilities of these three enzymes were ranked as CES>PPT-1>LAL.

[0164] Lipases and esterases are not the only types of high-risk HCPs that must be quantified at sub-ppm levels. Figure 15A shows that during high stability testing of drug substances DS-1, DS-2, and DS-3, cathepsin D caused clipping between amino acid residues methionine and tyrosine of more than 12%, 4%, and 0.2%, respectively, within 6 months under stress conditions at 45°C. Using PMLD-PRM quantification analysis, cathepsin D concentrations in DS-1, DS-2, and DS-3 were determined to be 1.5 ppm, 1.1 ppm, and 0.3 ppm cathepsin D, respectively (Figure 15B). At concentrations as low as 1.1 ppm or higher, cathepsin D cleaves proteins, but at 0.3 ppm, protein cleavage was shown to be negligible.

[0165] Example 7. Use of ProteoMiner™ to increase the detection limit of SV NIST mAb The ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure improves upon the ProteoMiner™ method for HCP identification described by Chen et al. to identify sequence variations within the SV NIST mAb, as shown in FIG. 17. The ProteoMiner SV enrichment method includes contacting a sample containing at least one higher abundance protein or peptide whose amino acid sequence has not been unintentionally altered (e.g., wild-type or recombinant) with a solid support, such as beads, where an interacting peptide ligand is bound to the solid support, and where the SV NIST mAb can bind to the interacting peptide ligand, e.g., ProteoMiner™ beads; washing the solid support with a solution containing a detergent to enrich for the SV NIST mAb and provide an eluate; subjecting the eluate to denaturation, alkylation, and reduction; subjecting the denatured, alkylated, and reduced eluate to an enzymatic digestion reaction to generate components of the enriched SV NIST mAb (e.g., direct digestion); identifying the components of the enriched SV NIST mAb using a mass spectrometer; and using the identified components to identify amino acid substitutions in the enriched SV NIST mAb.

[0166] The disclosed ProteoMiner™ method for enhanced detection of SV proteins, nanoLCs, or combinations thereof allows for detection of amino acid substitutions within the SV NIST mAb, including alanine to glutamic acid, proline, threonine or valine, cysteine ​​to glycine, serine or tyrosine, aspartic acid to glutamic acid, glutamic acid to aspartic acid or valine, phenylalanine to serine, tyrosine or leucine or isoleucine, glycine to aspartic acid, glutamic acid or serine, histidine to asparagine, aspartic acid or tyrosine, isoleucine to arginine, lysine to arginine, leuc ... The amino acid substitutions included arginine to arginine, glutamine, phenylalanine or proline, methionine to threonine, leucine or isoleucine, proline to alanine, histidine, leucine or serine, arginine to lysine, serine to asparagine, phenylalanine, proline, threonine, leucine or isoleucine, threonine to alanine, asparagine, isoleucine or serine, valine to alanine, glutamine, methionine, leucine or isoleucine, tryptophan to serine, and thymine to aspartic acid, cysteine ​​or phenylalanine. Cells highlighted in red indicate amino acid substitutions in SV peptides enriched using the disclosed ProteoMiner™ method for enhanced detection of SV proteins, nanoLCs, or combinations thereof.

[0167] Example 8. Use of ProteoMiner™ to enrich SV NIST mAbs FIG. 19A shows a table of amino acid sequence mutations in SV NIST mAb enriched using the ProteoMiner™ method for enhanced detection of SV proteins, nanoLCs, or combinations thereof of the present disclosure. The amino acid sequence mutations in the enriched SV NIST mAb generally resulted in the substitution of amino acids with one set of physical characteristics for amino acids with a different set of physical characteristics. Such substitutions were achieved in the three-dimensional protein structure of SV NIST mAb enriched using the ProteoMiner™ method for enhanced detection of SV proteins, nanoLCs, or combinations thereof of the present disclosure. FIG. 19B, FIG. 19C, and FIG. 19D show diagrams of NIST mAb amino acid sequence mutations identified and enriched using the ProteoMiner™ SV identification method of the present disclosure in the three-dimensional protein structure of SV NIST mAb, according to an exemplary embodiment.

[0168] For example, Figure 20A shows a positively charged histidine substituted with a negatively charged aspartic acid or a polar, uncharged asparagine in SV NIST mAb enriched using the ProteoMiner™ method for enhanced detection of SV proteins, nanoLC, or combinations thereof of the present disclosure. Figure 20B shows possible codon sequences for histidine, aspartic acid, and asparagine, and shows that a point mutation (e.g., one mutant DNA) can result in the substitution of a histidine codon for an aspartic acid or asparagine codon. Figure 20C shows NIST mAb histidine to asparagine or aspartic acid sequence mutations identified using eluates from NIST mAb direct digests subjected to regular flow CSH LC or nanoLC columns or ProteoMiner™ enriched NIST mAb digests subjected to nanoLC columns, according to exemplary embodiments. FIG. 20C also shows that the ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure enriched SV NIST mAbs with histidine 227, 271, or 313 substitutions, or combinations thereof, for aspartic acid or asparagine.

[0169] Figure 20D shows MS2 mass spectra of tryptic peptide product ions detected in the eluate from a direct digest of NIST mAb run on a regular flow CSH LC column (bottom) and MS2 mass spectra of histidine to aspartic acid SV tryptic peptide product ions detected in the eluate from a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column (top) according to an exemplary embodiment. Figure 20E shows MS2 mass spectra of tryptic peptide product ions detected in the eluate from a direct digest of NIST mAb run on a regular flow CSH LC column (bottom) and MS2 mass spectra of histidine to aspartic acid SV tryptic peptide product ions detected in the eluate from a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column (top) according to an exemplary embodiment. Similarly, the ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure may enrich for CHO IgG1 mAbs with sequence mutations from histidine to asparagine or aspartic acid. Figure 20F shows that the ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure enriched for CHO IgG1 mAbs with substitutions of histidine 432 or 436 for asparagine, or combinations thereof. Figure 20F also shows that the ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure enriched for CHO IgG1 mAbs with substitutions of histidine 227, 271, 288, 432, or 436 for aspartic acid, or combinations thereof.

[0170] Example 9. ProteoMiner™ enriches SV NIST mAbs with altered three-dimensional protein structures. Amino acid sequence mutations in SV NIST mAbs that were not enriched using the disclosed ProteoMiner™ method for enhanced detection of SV proteins generally resulted in the substitution of amino acids with a set of physical characteristics for amino acids with the same set of physical characteristics. Such substitutions did not affect the three-dimensional protein structure of SV NIST mAbs that were enriched using the disclosed ProteoMiner™ method for enhanced detection of SV proteins, nanoLCs, or combinations thereof.

[0171] For example, Figure 21A shows a polar uncharged serine substituted with a polar uncharged asparagine in SV in SV NIST mAb that was not enriched using the ProteoMiner™ method for enhanced detection of SV proteins, nanoLC, or combinations thereof of the present disclosure. Figure 21B shows a NIST mAb serine to asparagine sequence mutation identified using eluates from NIST mAb direct digests run on a regular flow CSH LC or nanoLC column or ProteoMiner™ eluted NIST mAb digests run on a nanoLC column, according to an exemplary embodiment.

[0172] Example 10. ProteoMiner™ increases the number of SVs detected compared to direct digestion and regular flow LC or nanoLC As shown in Figure 22A, the number of NIST mAb amino acid sequence mutations identified using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns (SVA>0.01%) was less than the ProteoMiner™-eluted NIST mAb digests run on nanoLC columns. Although the ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure can enrich for certain SV proteins, the method generally enhances detection of SV proteins even without enrichment. Furthermore, nanoLC improved the sensitivity for detecting SV proteins. FIG. 22B shows MS2 mass spectra of tryptic peptide product ions detected in the eluate from a NIST mAb direct digest run on a regular flow CSH LC column (bottom), and MS2 mass spectra of glycine to aspartic acid SV tryptic peptide product ions (SVA as low as 0.004%) detected in the eluate from a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column (top), according to an exemplary embodiment.

[0173] To further evaluate the ProteoMiner™ method for enhanced detection of SV proteins of the present disclosure, serine, glycine, and valine SV NIST mAbs were used. Figure 22C shows the number of NIST mAb serine, glycine, and valine sequence variants identified using eluates from NIST mAb direct digests run on regular flow CSH LC or nanoLC columns or ProteoMiner™ enriched NIST mAb digests run on nanoLC columns, according to exemplary embodiments. Figure 22C shows that approximately 50% more SVs can be detected using ProteoMiner™ eluted NIST mAb digests run on nanoLC columns than direct digests run on r nanoLC columns or direct digests run on regular flow CSH LC columns. Figure 22D shows the number of NIST mAb serine, glycine, or valine sequence mutations identified by three laboratories using eluates from a NIST mAb direct digest run on a regular flow CSH LC column or a ProteoMiner™ enriched NIST mAb digest run on a nanoLC column according to an exemplary embodiment. Figure 22D shows that about 85% of SVs were detected using the disclosed ProteoMiner™ method for enhanced detection of SV proteins, and about 92.3% of SVs were detected using the disclosed ProteoMiner™ method for enhanced detection of SV proteins, nanoLC, or a combination thereof.

[0174] 22E shows NIST mAb alanine to threonine, glycine to aspartic acid, serine to asparagine, valine to leucine or isoleucine, arginine to lysine, and lysine to arginine sequence mutations identified by three laboratories using eluates from a NIST mAb direct digest run on a regular flow CSH LC column, or a NIST mAb direct digest run on a nanoLC column enriched with ProteoMiner™ according to an exemplary embodiment. The ProteoMiner™ method for enhanced detection of SV proteins or a nanoLC column of the present disclosure detected 15 of the 17 SV NIST mAbs detected by running a NIST mAb direct digest run on a regular flow CSH LC column. Furthermore, FIG. 22E shows that of the 17 SV NIST mAbs detected by subjecting the NIST mAb direct digest to a regular-flow CSH LC column, one showed a higher SV percentage using nanoLC, whereas the others showed similar relative abundance.

[0175] Example 11. NanoLC may cause overestimation of SVA 23 shows unsaturated (bottom) and saturated (top) peaks in the MS2 mass spectra of tryptic peptide product ions (e.g., VVSVLTVLHQDWLNGK and TTPPVLDSDGSFEYSK) and serine to asparagine SV tryptic peptide product ions (e.g., VVNVLTVLHQDWLNGK and TTPPVLDSDGSFEYNK) detected in the eluate from the digested ProteoMiner™ NIST mAb eluate run on a nanoLC column according to an exemplary embodiment. Saturated peaks in the MS2 mass spectra of SV and non-SV peptide product ions can obscure the relative abundance of SV and non-SV proteins, resulting in an overestimation of the relative abundance of SVA.

[0176] Example 12. NanoLC can improve MS2 spectra NanoLC can improve MS2 spectra by increasing the signal. For example, FIG. 24 shows that using a mass spectrometer to analyze eluates from a NIST mAb direct digest run on a regular flow CSH LC column produces a larger peak in the MS2 mass spectrum of the tryptic peptide product ions (scan 9602, z=3) than in the MS2 mass spectrum of the cysteine ​​to serine SV tryptic peptide product ions (scan 9515, z=3). Conversely, using a mass spectrometer to analyze eluates from a digested ProteoMiner™ NIST mAb eluate run on a nanoLC column, according to an exemplary embodiment, produces a smaller peak in the MS2 mass spectrum of the tryptic peptide product ions (scan 59496, z=3) than in the MS2 mass spectrum of the cysteine ​​to serine SV tryptic peptide product ions (scan 59579, z=3).

[0177] NanoLC can also improve MS2 spectra by allowing the generation of y ions. For example, FIG. 25 shows that the mass spectrometer does not generate y ions in the MS2 mass spectrum of serine to leucine or isoleucine SV tryptic peptide product ions using eluate from a NIST mAb direct digest run on a regular flow CSH LC column (scan 14203, z=4). Conversely, the mass spectrometer, according to an exemplary embodiment, generates y ions in the MS2 mass spectrum of serine to leucine or isoleucine SV tryptic peptide product ions using eluate from a digested ProteoMiner™ NIST mAb eluate run on a nanoLC column (scan 75616, z=4).

Claims

1. 1. A method for identifying host cell protein (HCP) impurities in a sample, comprising: (a) contacting a sample containing at least one high abundance peptide or protein and at least one HCP impurity with a solid support, wherein the solid support is bound to an interacting peptide ligand capable of interacting with the at least one HCP impurity; (b) washing the solid support to provide an eluate comprising at least one enriched HCP impurity; (c) subjecting the eluate to enzymatic digestion conditions to produce at least one component of the at least one enriched HCP impurity, wherein the enzymatic digestion conditions do not completely digest all proteins in the eluate; and (d) identifying said at least one component of said at least one enriched HCP impurity using a mass spectrometer; (e) using said identification of said at least one component to identify said at least one enriched HCP impurity.

2. 10. The method of claim 1, wherein the solid support is washed using a detergent, wherein the detergent is a phase transfer detergent, an ionic detergent, an anionic detergent, a cationic detergent, sodium deoxycholate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, or a combination thereof.

3. 3. The method of claim 2, wherein the surfactant comprises about 12 mmol / L sodium deoxycholate and about 12 mmol / L sodium lauryl sulfate.

4. 2. The method of claim 1, wherein the concentration of the at least one high abundance peptide or protein is at least about 1000-fold, about 10,000-fold, about 100,000-fold, or about 1,000,000-fold greater than the concentration of the at least one HCP impurity.

5. The method of claim 1 , wherein the interacting peptide ligands are a library of combinatorial hexapeptide ligands.

6. 2. The method of claim 1, wherein the at least one high abundance peptide or protein is an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a recombinant protein, a protein pharmaceutical, or a drug.

7. 2. The method of claim 1, wherein the enzyme of the enzymatic digestion conditions is trypsin at an enzyme to substrate ratio of less than about 1:200, about 1:400, about 1:1000, about 1:2500, or about 1:10000.

8. 10. The method of claim 1, wherein the at least one enriched HCP impurity is not subjected to denaturation prior to being subjected to the enzymatic digestion conditions.

9. 2. The method of claim 1, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, and the mass spectrometer is coupled to a liquid chromatography system, and the mass spectrometer is capable of performing LC-MS analysis (liquid chromatography-mass spectrometry) or LC-MRM-MS analysis (liquid chromatography-multiple reaction monitoring-mass spectrometry).

10. 10. The method of claim 9, further comprising quantifying the at least one concentrated HCP impurity using the mass spectrometer, wherein the detection limit of the at least one concentrated HCP impurity is about 0.003 to 0.006 ppm.

11. 1. A method for identifying a sequence variant (SV) peptide or protein in a sample, wherein at least one amino acid of the SV peptide or protein unintentionally differs from a wild-type peptide or protein, said method comprising: (a) contacting a sample containing at least one higher abundance wild-type peptide or protein and at least one SV peptide or protein with a solid support, wherein the solid support is bound to an interacting peptide ligand capable of interacting with the at least one SV peptide or protein; (b) washing the solid support to provide a first eluate comprising at least one enriched SV peptide or protein; (c) subjecting the first eluate to enzymatic digestion conditions to produce at least one component of the at least one enriched SV peptide or protein; (d) subjecting the first eluate having the at least one component of the at least one enriched SV peptide or protein to a liquid chromatography system to produce a second eluate; (e) subjecting the second eluate to a mass spectrometer; (f) identifying said at least one component of said at least one enriched SV peptide or protein using a mass spectrometer; (g) using said identification of said at least one component to identify said at least one enriched SV peptide or protein in said sample.

12. 12. The method of claim 11, wherein the enzymatic digestion conditions are direct digestion.

13. 13. The method of claim 12, wherein the liquid chromatography system comprises a nanoscale liquid chromatography (nanoLC) column or a regular flow charged surface hybrid (CSH) column.

14. 14. The method of claim 13, wherein the enzymatic digestion conditions do not completely digest all of the proteins in the first eluate.

15. 15. The method of claim 14, wherein the solid support is washed using a detergent, wherein the detergent is a phase transfer detergent, an ionic detergent, an anionic detergent, a cationic detergent, sodium deoxycholate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, or a combination thereof.

16. 16. The method of claim 15, wherein the surfactant comprises about 12 mmol / L sodium deoxycholate and about 12 mmol / L sodium lauryl sulfate.

17. 15. The method of claim 14, wherein the concentration of the at least one more highly abundant wild-type peptide or protein is at least about 1000-fold, about 10,000-fold, about 100,000-fold, or about 1,000,000-fold greater than the concentration of the at least one SV peptide or protein.

18. 15. The method of claim 14, wherein the interacting peptide ligands are a library of combinatorial hexapeptide ligands.

19. 15. The method of claim 14, wherein the at least one higher abundance wild-type peptide or protein and the at least one SV peptide or protein are an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a recombinant protein, a protein pharmaceutical, or a drug.

20. 15. The method of claim 14, wherein the enzyme of the enzymatic digestion conditions is trypsin at an enzyme to substrate ratio of less than about 1:200, about 1:400, about 1:1000, about 1:2500, or about 1:10000.

21. 15. The method of claim 14, wherein the at least one enriched SV peptide or protein is not subjected to denaturation prior to being subjected to the enzymatic digestion conditions.

22. 15. The method of claim 14, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system, and the mass spectrometer is capable of performing LC-MS analysis (liquid chromatography-mass spectrometry) or LC-MRM-MS analysis (liquid chromatography-multiple reaction monitoring-mass spectrometry).

23. 15. The method of claim 14, further comprising quantifying the at least one enriched SV peptide or protein using the mass spectrometer, wherein the detection limit of the at least one enriched SV peptide or protein is about 0.003 to 0.006 ppm.

24. 1. A method for identifying host cell protein (HCP) impurities in a sample, comprising: (a) contacting a sample containing at least one high abundance peptide or protein and at least one HCP impurity with a solid support, wherein the solid support is bound to an interacting peptide ligand capable of interacting with the at least one HCP impurity; (b) washing the solid support to provide an eluate comprising at least one enriched HCP impurity; (c) subjecting the eluate to enzymatic digestion conditions to produce at least one component of the at least one enriched HCP impurity, wherein the enzymatic digestion conditions do not completely digest all proteins in the eluate; and (d) identifying said at least one component of said at least one enriched HCP impurity using a parallel reaction monitoring-mass spectrometer; (e) using said identification of said at least one component to identify said at least one enriched HCP impurity.

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