Size-exclusion chromatography for characterizing host cell proteins

Abstract

The present invention relates generally to methods for identifying and characterizing host cell proteins. In particular, the present invention relates to the use of size exclusion chromatography under non-denaturing or denaturing conditions to enrich samples for host cell proteins and characterize the binding of host cell proteins to a protein of interest.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 314,144, filed February 25, 2022, U.S. Provisional Patent Application No. 63 / 341,381, filed May 12, 2022, and U.S. Provisional Patent Application No. 63 / 426,221, filed November 17, 2022, each of which is incorporated by reference herein.

[0002] Field The present application relates to methods for the identification, characterization, and removal of host cell proteins. [Background technology]

[0003] background High molecular weight (HMW) aggregates in biotherapeutic products pose challenges to drug development, commercial manufacturing, and product stability throughout the product's shelf life. HMW aggregates can form during manufacturing, formulation, and transportation or delivery to patients. The presence of HMW aggregates in biotherapeutic products can affect drug efficacy and increase the risk of adverse immune responses in patients. Therefore, the levels of HMW species in biotherapeutic products are monitored as a critical quality attribute.

[0004] An additional potential mechanism of adverse effects of HMW species in biotherapeutics is the presence of host cell proteins (HCPs). The presence of residual host cell proteins (HCPs) can cause potential safety risks and problems in manufacturing of biopharmaceutical products. Since recombinant DNA technology has been widely used to produce biopharmaceutical products in host cells, impurities need to be removed to obtain biopharmaceutical products with high purity. Any residual impurities after performing purification bioprocesses need to be present at acceptably low levels before conducting clinical studies. Specifically, residual HCPs derived from mammalian expression systems (e.g., Chinese Hamster Ovary (CHO) cells) can compromise the safety, quality, and stability of the product. Even trace amounts of certain HCPs can sometimes cause immunogenic responses or undesirable modifications. Therefore, it is necessary to monitor host cell proteins in medical products and during the manufacturing process.

[0005] HCPs can be present in the final drug substance via several different pathways. For example, HCPs can bind to the biotherapeutic and not be removed during purification. Alternatively, HCPs can be co-purified with the biotherapeutic in an unbound state. Understanding the mechanism of HCP contamination can be useful in process development to prevent further contamination.

[0006] Most monoclonal antibody (mAb) purification techniques involve an initial affinity chromatography step, e.g., Protein A affinity chromatography, followed by several final purification steps. The presence of HCPs in Protein A eluate is mainly due to interactions with column resin / ligand or mAb. HCPs retained after Protein A purification due to column resin interactions can be removed in the subsequent final purification steps. However, HCPs associated with mAb are difficult to remove and may escape the purification process. Identifying co-eluting HCPs and understanding the above interaction mechanisms are important for process development.

[0007] Identification of HCP impurities in biopharmaceutical products is difficult due to the wide dynamic range of protein concentrations in samples with high complexity. Specifically, the presence of at least one high abundance protein or peptide (e.g., therapeutic protein) in a sample poses technical obstacles to the detection, identification, and quantification of very low abundance proteins in the sample. Furthermore, the presence of HCPs in the HMW fraction of biotherapeutic products and their possible contribution to side effects such as instability or immunogenicity are not well characterized.

[0008] Thus, a need exists for methods and systems to identify, characterize, and remove host cell proteins in biotherapeutic products in a highly sensitive and specific manner. Summary of the Invention

[0009] overview Methods have been developed for identifying HCPs using size exclusion chromatography (SEC) as an orthogonal separation method before subjecting a sample containing a protein of interest to liquid chromatography-mass spectrometry (LC-MS). A sample containing a protein of interest and at least one HCP, where the protein of interest is present at a much higher abundance than at least one HCP, can be separated into, for example, high molecular weight (HMW), main, tail, and / or low molecular weight (LMW) fractions. Biotherapeutics such as antibodies, antibody fusion proteins, receptors, or receptor fusion proteins can be much larger than HCPs and can be effectively separated from HCPs based on size. Enrichment of HCPs into fractions depleted of the high abundance protein of interest allows for superior identification of HCPs using LC-MS analysis. The present disclosure describes optimization of SEC-based methods for HCP identification, including exemplary optimized denaturation conditions, digestion conditions, protein loading, SEC fractions, surfactant inclusion, and acid precipitation.

[0010] Methods have also been developed to characterize the binding of HCPs to proteins of interest using SEC. Understanding the binding characteristics of HCPs to proteins of interest is beneficial for understanding the mechanism of host cell protein contamination in samples (e.g., drug substances). HCPs bound to proteins of interest elute earlier in SEC compared to unbound HCPs. Under mild denaturing conditions, for example in 20% acetonitrile, weak HCP binding to proteins of interest is eliminated and HCPs show a shift to lower molecular weight SEC fractions compared to separation under non-denaturing conditions, while strong HCP binding to proteins of interest is largely unaffected. Under strong denaturing conditions, for example in 12 mM sodium lauroyl sarcosinate and 12 mM sodium deoxycholate, all HCP binding to proteins of interest can be effectively eliminated. By comparing the separation profiles of HCPs under non-denaturing and denaturing conditions, the binding characteristics of HCPs to proteins of interest can be established.

[0011] Additionally, methods have been developed for identifying, quantifying, and removing HCP impurities from a sample of interest, such as a biotherapeutic product. Specifically, the present disclosure demonstrates that HCPs of concern can be significantly enriched in the HMW fraction of a biotherapeutic product due to specific interactions with aggregates or multimers of a protein of interest contained in the product. HCPs can be identified and quantified using SEC analysis of the biotherapeutic product, followed by analysis and comparison of the SEC fractions. For example, a drug substance (DS) can be subjected to further sample preparation such as native digestion, SEC analysis, fractionation into HMW, monomeric, and LMW fractions, denaturation, reduction, digestion, and alkylation, and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to identify and quantify HCPs enriched in the HMW fraction of the biotherapeutic product. A profile of the HCPs present in each SEC fraction of a biotherapeutic product DS can be developed to determine whether the product can be improved by isolating and removing the SEC fractions (e.g., by reducing the abundance of HCP impurities of concern). This determination may be made, for example, based on the concentration and / or abundance of the HCPs, the percentage of HCPs that are enriched in a particular SEC fraction, and the known risk of including a particular HCP in the biotherapeutic product. Based on this determination, the process for harvesting and purifying the biotherapeutic product sample (e.g., DS) may be improved by adding an SEC step to remove the HMW fraction containing the enriched HCPs, thereby improving the safety and efficacy of the biotherapeutic product.

[0012] The present disclosure provides a method for identifying HCP impurities in a sample. In some exemplary embodiments, the method includes: (a) subjecting a sample containing at least one protein of interest and at least one HCP impurity to size exclusion chromatography (SEC) analysis to generate a fraction; (b) subjecting the fraction to LC-MS analysis to identify the at least one HCP impurity; Includes.

[0013] In one embodiment, the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0014] In one embodiment, the amount of protein loaded onto the SEC column is about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 1 mg, about 5 mg, about 10 mg, or about 20 mg. In a particular embodiment, the amount of protein loaded onto the SEC column is about 10 mg.

[0015] In one embodiment, the mobile phase for SEC analysis comprises about 10 mM phosphate and about 150 mM NaCl. In another embodiment, the mobile phase for SEC analysis is a denaturing mobile phase. In a further embodiment, the mobile phase for SEC analysis is a non-denaturing mobile phase.

[0016] In one embodiment, the mobile phase for SEC analysis comprises acetonitrile, optionally with a concentration of acetonitrile of about 5% v / v to about 20% v / v, about 10% v / v to about 20% v / v, about 15% v / v to about 20% v / v, about 5% v / v, about 10% v / v, about 15% v / v, or about 20% v / v. In certain embodiments, the concentration of acetonitrile is about 20% v / v.

[0017] In one embodiment, the mobile phase for SEC analysis includes at least one surfactant, and optionally, the concentration of the at least one surfactant is about 6 mM to about 36 mM, about 12 mM, or about 24 mM. In a particular embodiment, the at least one surfactant is a detergent. In a more specific embodiment, the at least one detergent is selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof. In a further particular embodiment, the at least one detergent is sodium deoxycholate and sodium lauroyl sarcosinate, the concentration of sodium deoxycholate is about 12 mM, and the concentration of sodium lauroyl sarcosinate is about 12 mM.

[0018] In one embodiment, the fractions include a high molecular weight (HMW) fraction, a main fraction, and a low molecular weight (LMW) fraction. In a particular embodiment, the fractions further include a tail fraction. In another particular embodiment, the HMW fraction comprises an eluate of about 0.3 column volumes (CV) to about 5 milliabsorbance units (mAU). In a further particular embodiment, the main fraction comprises an eluate of about 5 mAU to about 40 mAU. In yet another particular embodiment, the tail fraction comprises an eluate of about 40 mAU to about 10 mAU, or about 40 mAU to about 3 mAU. In yet another particular embodiment, the LMW fraction comprises an eluate of about 10 mAU to about 1.1 CV, or about 3 mAU to about 1.1 CV.

[0019] In one embodiment, the method further comprises subjecting the fraction to enzymatic digestion prior to the LC-MS analysis of step (b). In a particular embodiment, the enzymatic digestion is limited digestion. In another particular embodiment, the enzymatic digestion is performed by contacting the fraction with trypsin. In a further particular embodiment, the enzymatic digestion is performed by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000. In yet another particular embodiment, the enzyme to protein ratio is about 1:200 for the HMW fraction. In another particular embodiment, the enzyme to protein ratio is about 1:2000 for the main fraction. In a further particular embodiment, the enzyme to protein ratio is about 1:500 for the tail fraction. In yet another particular embodiment, the enzyme to protein ratio is about 1:200 for the LMW fraction.

[0020] In one embodiment, the method further comprises subjecting the fraction to acid precipitation prior to the LC-MS analysis of step (b). In a particular embodiment, the acid precipitation comprises contacting the fraction with about 1% trifluoroacetic acid.

[0021] In one aspect, the liquid chromatography in step (b) comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0022] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to the liquid chromatography system.

[0023] The present disclosure provides additional methods for identifying host cell protein (HCP) impurities in a sample. In some exemplary embodiments, the methods include: (a) subjecting a sample containing at least one protein of interest and at least one HCP impurity to size exclusion chromatography (SEC) analysis to generate a fraction, wherein a mobile phase for the SEC analysis comprises about 12 mM sodium lauroyl sarcosinate and about 12 mM sodium deoxycholate; (b) subjecting the fraction to acid precipitation to produce a detergent-depleted fraction, the acid precipitation comprising contacting the fraction with about 1% trifluoroacetic acid; (c) subjecting the detergent-depleted fraction to buffer exchange to generate a buffer exchanged fraction; (d) subjecting the buffer exchanged fraction to limited digestion to produce peptide digests, the limited digestion comprising contacting the buffer exchanged fraction with trypsin at an enzyme to substrate ratio of about 1:200 to about 1:2000; (e) subjecting the peptide digest to LC-MS analysis to identify the at least one HCP impurity; Includes.

[0024] In one embodiment, the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0025] In one embodiment, the amount of protein loaded onto the SEC column is about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 1 mg, about 5 mg, about 10 mg, or about 20 mg. In a particular embodiment, the amount of protein loaded onto the SEC column is about 10 mg.

[0026] In one embodiment, the fractions include a high molecular weight (HMW) fraction, a main fraction, and a low molecular weight (LMW) fraction. In a particular embodiment, the fractions further include a tail fraction. In another particular embodiment, the HMW fraction comprises an eluate of about 0.3 column volumes (CV) to about 5 milliabsorbance units (mAU). In a further particular embodiment, the main fraction comprises an eluate of about 5 mAU to about 40 mAU. In yet another particular embodiment, the tail fraction comprises an eluate of about 40 mAU to about 10 mAU, or about 40 mAU to about 3 mAU. In yet another particular embodiment, the LMW fraction comprises an eluate of about 10 mAU to about 1.1 CV, or about 3 mAU to about 1.1 CV.

[0027] In one aspect, the liquid chromatography in step (e) comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0028] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system.

[0029] The present disclosure also provides methods for characterizing the binding of HCP impurities to a protein of interest. In some exemplary embodiments, the methods include: (a) obtaining a sample containing a protein of interest and at least one HCP impurity; (b) subjecting the sample to size exclusion chromatography (SEC) analysis using a non-denaturing mobile phase to generate a native fraction; (c) subjecting the sample of (a) to SEC analysis using a denaturing mobile phase to generate a denatured fraction; (d) subjecting the native fraction and the denatured fraction to LC-MS analysis to generate native and denatured separation profiles of the at least one HCP impurity; (e) comparing the native separation profile to the denatured separation profile to characterize binding of the at least one HCP impurity to the protein of interest; Includes.

[0030] In one aspect, the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0031] In one embodiment, the amount of protein loaded onto the SEC column is about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 1 mg, about 5 mg, about 10 mg, or about 20 mg. In a particular embodiment, the amount of protein loaded onto the SEC column is about 10 mg.

[0032] In one embodiment, the mobile phase for SEC analysis comprises about 10 mM phosphate and about 150 mM NaCl, hi another embodiment, the denaturing mobile phase is a mildly denaturing mobile phase.

[0033] In one embodiment, the denaturing mobile phase comprises acetonitrile, optionally with a concentration of acetonitrile of about 5% v / v to about 20% v / v, about 10% v / v to about 20% v / v, about 15% v / v to about 20% v / v, about 5% v / v, about 10% v / v, about 15% v / v, or about 20% v / v. In certain embodiments, the concentration of acetonitrile is about 20% v / v.

[0034] In one embodiment, the fractions include a high molecular weight (HMW) fraction, a main fraction, and a low molecular weight (LMW) fraction. In a particular embodiment, the fractions further include a tail fraction. In another particular embodiment, the HMW fraction comprises an eluate of about 0.3 column volumes (CV) to about 5 milliabsorbance units (mAU). In a further particular embodiment, the main fraction comprises an eluate of about 5 mAU to about 40 mAU. In yet another particular embodiment, the tail fraction comprises an eluate of about 40 mAU to about 10 mAU, or about 40 mAU to about 3 mAU. In yet another particular embodiment, the LMW fraction comprises an eluate of about 10 mAU to about 1.1 CV, or about 3 mAU to about 1.1 CV.

[0035] In one embodiment, the method further comprises subjecting the fraction to enzymatic digestion prior to the LC-MS analysis of step (d). In a particular embodiment, the enzymatic digestion is limited digestion. In another particular embodiment, the enzymatic digestion is performed by contacting the fraction with trypsin. In a further particular embodiment, the enzymatic digestion is performed by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000. In yet another particular embodiment, the enzyme to protein ratio is about 1:200 for the HMW fraction. In another particular embodiment, the enzyme to protein ratio is about 1:2000 for the main fraction. In a further particular embodiment, the enzyme to protein ratio is about 1:500 for the tail fraction. In yet another particular embodiment, the enzyme to protein ratio is about 1:200 for the LMW fraction.

[0036] In one aspect, the liquid chromatography in step (d) comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0037] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to a liquid chromatography system.

[0038] The present disclosure provides a method for identifying HCP impurities in a sample. In some exemplary embodiments, the method includes: (a) combining a sample containing at least one protein of interest and at least one HCP impurity with a dissociation reagent to generate a first combination; (b) subjecting the first combination to acid precipitation to generate a dissociation reagent-depleted fraction; (c) subjecting the dissociation reagent-depleted fraction to liquid chromatography-mass spectrometry analysis to identify the at least one HCP impurity; Includes.

[0039] In one embodiment, the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0040] In one aspect, the sample is incubated in the dissociation reagent for about 5 minutes to about 120 minutes, about 15 minutes, about 30 minutes, about 60 minutes, or about 120 minutes.

[0041] In one aspect, the dissociation reagent comprises at least one surfactant, optionally at a concentration of about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0042] In one embodiment, the at least one surface active agent is a detergent.

[0043] In one embodiment, the at least one surfactant is selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof.

[0044] In one embodiment, the at least one surfactant is sodium lauroyl sarcosinate, and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0045] In one embodiment, the at least one surfactant is sodium deoxycholate and sodium lauroyl sarcosinate, the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM, and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0046] In one embodiment, the at least one detergent is sodium deoxycholate, and the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0047] In one aspect, the method further comprises subjecting the dissociation reagent-depleted fraction to enzymatic digestion prior to analysis by liquid chromatography-mass spectrometry in step (c) to generate a peptide digest.

[0048] In one aspect, the enzymatic digestion is a limited digestion.

[0049] In one embodiment, enzymatic digestion is performed by contacting the dissociation reagent-depleted fraction with trypsin.

[0050] In one aspect, enzymatic digestion is performed by contacting the dissociation reagent-depleted fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000.

[0051] In one embodiment, the enzyme to protein ratio is about 1:200.

[0052] In one embodiment, the method further comprises desalting the peptide digest prior to analysis by liquid chromatography-mass spectrometry in step (c).

[0053] In one aspect, the acid precipitation is incubated for about 5 minutes to about 60 minutes, about 5 minutes, or about 60 minutes.

[0054] In one embodiment, acid precipitation includes contacting the first combination with about 2.5% to about 10% trifluoroacetic acid, about 2.5% trifluoroacetic acid, about 5% trifluoroacetic acid, about 7.5% trifluoroacetic acid, or about 10% trifluoroacetic acid.

[0055] In one aspect, the liquid chromatography includes reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0056] In one aspect, the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is coupled to the liquid chromatography system.

[0057] The present disclosure provides a method for identifying HCP impurities in a sample. In some exemplary embodiments, the method includes: (a) combining a sample containing at least one protein of interest and at least one HCP impurity with a dissociation reagent to generate a first combination; (b) subjecting the first combination to acid precipitation to generate a dissociation reagent-depleted fraction; (c) subjecting the dissociation reagent-depleted fraction to buffer exchange to generate a buffer exchanged fraction; (d) subjecting the buffer exchanged fractions to enzymatic digestion to produce peptide digests; (e) subjecting the peptide digest to liquid chromatography-mass spectrometry analysis to identify the at least one HCP impurity; Includes.

[0058] In one embodiment, the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0059] In another aspect, the dissociation reagent comprises at least one surfactant, optionally at a concentration of about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0060] In yet another embodiment, the at least one surface active agent of interest is a detergent.

[0061] In yet another embodiment, the at least one surfactant is selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof.

[0062] In one aspect, the first combination is incubated for about 5 minutes to about 120 minutes, about 5 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes prior to the acid precipitation in step (b).

[0063] In another embodiment, the at least one surfactant is sodium lauroyl sarcosinate, and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0064] In another embodiment, the at least one surfactant is sodium deoxycholate and sodium lauroyl sarcosinate, the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM, and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0065] In yet another embodiment, the at least one detergent is sodium deoxycholate, and the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

[0066] In one aspect, the enzymatic digestion is a limited digestion.

[0067] In one embodiment, the enzymatic digestion is carried out by contacting the buffer exchanged fraction with trypsin.

[0068] In another aspect, enzymatic digestion is performed by contacting the buffer exchanged fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000.

[0069] In yet another embodiment, the enzyme to protein ratio is about 1:200.

[0070] In one aspect, the method further comprises desalting the peptide digest prior to analysis by liquid chromatography-mass spectrometry in step (e).

[0071] In one aspect, the acid precipitation is incubated for about 5 minutes to about 60 minutes, about 5 minutes, or about 60 minutes.

[0072] In one embodiment, acid precipitation includes contacting the first combination with about 2.5% to about 10% trifluoroacetic acid, about 2.5% trifluoroacetic acid, about 5% trifluoroacetic acid, about 7.5% trifluoroacetic acid, or about 10% trifluoroacetic acid.

[0073] In one aspect, the liquid chromatography includes reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0074] The disclosure further provides a method for producing a biotherapeutic product. In some exemplary embodiments, the method comprises: (a) subjecting a first sample, comprising at least one protein of interest and at least one host cell protein (HCP) impurity, to size exclusion chromatography (SEC) analysis to generate a plurality of fractions; (b) subjecting the plurality of fractions to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to determine the identity and amount of the at least one HCP impurity; (c) using the identity and amount to determine whether the at least one HCP impurity is an impurity of concern in at least one of the plurality of fractions; (d) subjecting a second sample containing the at least one protein of interest and the at least one HCP impurity to SEC analysis to generate a second plurality of fractions; (e) using the determination of step (c) to remove the at least one fraction from the plurality of fractions of step (d) in which the at least one HCP impurity is an impurity of concern to manufacture a biotherapeutic product. Includes.

[0075] In one aspect, the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0076] In one embodiment, the mobile phase for the SEC analysis comprises about 150 mM ammonium acetate.

[0077] In one embodiment, the at least one HCP impurity comprises a lipase, a protease, or a combination thereof, hi another embodiment, the at least one HCP impurity comprises a CC motif chemokine.

[0078] In one aspect, the plurality of fractions comprises a high molecular weight (HMW) fraction, a very high molecular weight (vHMW) fraction, a dimer fraction, a monomer fraction, a low molecular weight (LMW) fraction, a tail fraction, or a combination thereof.

[0079] In one aspect, the fraction in which the at least one HCP impurity is an impurity of concern is a HMW fraction.

[0080] In one embodiment, at least one HCP impurity is present in the fraction at about 1000 parts per million (ppm) to about 10000 ppm, about 1000 ppm, about 2000 ppm, about 3000 ppm, about 4000 ppm, about 5000 ppm, about 6000 ppm, about 7000 ppm, about 8000 ppm, about 9000 ppm, or about 10000 ppm.

[0081] In one embodiment, the percentage of at least one HCP impurity enriched in the HMW fraction is about 30% to about 100%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0082] In one embodiment, the method further comprises subjecting the sample of (a) to native digestion prior to SEC analysis. In a particular embodiment, the native digestion is limited digestion. In another particular embodiment, the native digestion is performed by contacting the sample with trypsin.

[0083] In one aspect, the LC-MS / MS analysis includes reverse phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, Protein A chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0084] In one embodiment, the LC-MS / MS analysis comprises parallel reaction monitoring.

[0085] The present disclosure provides additional methods for producing a biotherapeutic product. In some exemplary embodiments, the methods include subjecting a sample containing a protein of interest, at least one HMW species, and at least one HCP impurity to one or more chromatographic steps that reduce the abundance of the at least one HCP impurity, where the at least one HCP impurity interacts with the at least one HMW species.

[0086] In one embodiment, the interaction between the at least one HCP impurity and the at least one HMW species can be identified by concentrating the at least one HMW species. In a particular embodiment, concentrating comprises subjecting a sample comprising the at least one HMW species and the at least one HCP impurity to SEC. In a more particular embodiment, the method further comprises subjecting the at least one HMW species and the at least one HCP impurity to buffer exchange, native digestion, denaturation, molecular weight filtration, one or more additional chromatographic steps, and / or analysis by mass spectrometry.

[0087] In one aspect, the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

[0088] In one embodiment, the at least one HCP impurity comprises a lipase, a protease, or a combination thereof, hi another embodiment, the at least one HCP impurity comprises a CC motif chemokine.

[0089] In one embodiment, at least one HMW species comprises a dimer, an aggregate, or a combination thereof.

[0090] In one aspect, the one or more chromatography steps include reverse phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, Protein A chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

[0091] 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 and numerous specific details thereof, 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.

[0092] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0093] [Figure 1] FIG 1A shows the degradation of aflibercept over time using SDS-PAGE at 25°C according to an exemplary embodiment. FIG 1B shows the degradation of aflibercept over time using SDS-PAGE at 37°C according to an exemplary embodiment. [Diagram 2]1 shows a SEC analysis of aflibercept drug substance revealing cathepsin D contamination, according to an exemplary embodiment. [Diagram 3] 1 shows SEC chromatograms of mAb1 drug substance spiked with BSA in three different mobile phases according to an exemplary embodiment. [Figure 4] 1 shows the number of HCPs detected in mAb1 drug substance using the method of the present invention with three different mobile phases, according to an exemplary embodiment. [Diagram 5] 1 shows a SEC chromatogram of mAb1 drug substance spiked with BSA at semi-preparative scale according to an exemplary embodiment. [Figure 6] 1 shows a SEC chromatogram of mAb1 drug substance at semi-preparative scale according to an exemplary embodiment. [Figure 7A] 1 shows the amount of protein detected in each SEC fraction from mAb1 drug substance, according to an exemplary embodiment. [Figure 7B] 1 shows UV chromatograms of each SEC fraction from mAb1 drug substance, according to an exemplary embodiment. [Figure 8] 1 shows the number of HCPs detected using various enzyme to substrate ratios in a limited digestion step, according to an exemplary embodiment. [Figure 9] 1 shows the number of HCPs detected in each SEC fraction using either denaturing or limited digestion, according to an exemplary embodiment. [Figure 10] 1 shows UV cutoffs of 10 mAU and 3 mAU in SEC chromatograms according to an exemplary embodiment. [Figure 11] 1 shows the number of HCPs detected in mAb1 drug substance using either a 10 mAU or 3 mAU UV cutoff according to an exemplary embodiment, according to the method of the present invention. [Figure 12] 1 shows SEC chromatograms for mAb1 drug substance using 5 mg, 10 mg, or 20 mg loadings according to an exemplary embodiment. [Figure 13] 1 shows the number of HCPs detected in mAb1 drug substance using the methods of the present invention at various protein loadings, according to an exemplary embodiment. [Figure 14] 1 shows the number of HCPs detected in mAb1 drug substance using the method of the present invention compared to the method described above, according to an exemplary embodiment. [Figure 15A] 1 shows the number of HCPs detected in NISTmAb8671 samples using the methods of the present invention, according to an exemplary embodiment. [Figure 15B] 1 shows the number of HCPs detected in NISTmAb8671 samples using the methods of the present invention compared to published results from the aforementioned methods, according to an exemplary embodiment. [Figure 16A] 1 shows the number of HCPs detected in mAb1 drug substance in each SEC fraction using the method of the present invention, using either native or acetonitrile (ACN) mobile phase, according to an exemplary embodiment. [Figure 16B] 1 shows the overlap of HCPs detected in mAb1 drug substance using the method of the present invention, using either native or acetonitrile mobile phases, according to an exemplary embodiment. [Figure 17] Figure 17A shows the mass of HCPs detected in the mAb1 drug substance of each SEC fraction using the method of the invention with a native mobile phase according to an exemplary embodiment. Figure 17B shows the mass of HCPs detected in the mAb1 drug substance of each SEC fraction using the method of the invention with an acetonitrile mobile phase according to an exemplary embodiment. [Figure 18A] 1 shows HCPs detected in mAb1 drug substance that were found to have strong binding to mAb1 using the methods of the present invention, according to an exemplary embodiment. [Figure 18B] 1 shows HCPs detected in mAb1 drug substance that were found to have no binding to mAb1 using the methods of the present invention, according to an exemplary embodiment. [Figure 18C-1] 1 shows HCPs detected in mAb1 drug substance that were found to have weak binding to mAb1 using the methods of the present invention, according to an exemplary embodiment. [Figure 18C-2] See description of Figure 18C-1. [Figure 19]1 shows the binding profile of a particular HCP of interest in the mAb1 drug substance, according to an exemplary embodiment. [Figure 20] 1 illustrates the use of surfactants to affect the equilibrium of bound and unbound HCPs in a biotherapeutic drug substance, according to an exemplary embodiment. [Figure 21] 1 shows SEC chromatograms of mAb1 drug substance using the method of the present invention with native mobile phase or 12 mM SLS and SDC according to an exemplary embodiment. [Figure 22] 1 illustrates a workflow for a biotherapeutic analysis including an acid precipitation step to remove surfactant, according to an exemplary embodiment. [Figure 23] 1 shows UV chromatograms of the LMW fraction of mAb1 drug substance using either the native mobile phase or surfactant-assisted and acid precipitation process according to an exemplary embodiment. [Figure 24] Figure 24A shows the number of HCPs detected in each SEC fraction using native mobile phase, ACN mobile phase, or surfactant-assisted and acid precipitation processes according to an exemplary embodiment. Figure 24B shows the overlap of HCPs detected in mAb1 drug substance using native mobile phase, ACN mobile phase, or surfactant-assisted and acid precipitation processes according to an exemplary embodiment. [Diagram 25] 1 shows the change in HCP of interest in mAb1 drug substance from the later SEC fraction, showing antibody-bound status, to the earlier SEC fraction, showing unbound status, according to an exemplary embodiment. [Figure 26] 1 illustrates a workflow for biotherapeutic analysis using methods of the present invention for removing surfactants and biotherapeutics, including surfactant-assisted dissociation and acid precipitation, according to an exemplary embodiment. [Figure 27A] FIG. 1 shows protein spectra at 280 nm of a biotherapeutic drug sample after incubation in 6 M urea, 8 M guanidine, 5 mM acetic acid (with heat), 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, or 40 mM n-dodecyl-β-D-maltoside and acid precipitation according to an exemplary embodiment. [Figure 27B]1 shows the percentage of protein remaining in a biotherapeutic sample after incubation in 6 M urea, 8 M guanidine, 5 mM acetic acid (with heat), 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, or 40 mM n-dodecyl-β-D-maltoside and acid precipitation according to an exemplary embodiment. [Figure 28A] FIG. 1 shows protein spectra of biotherapeutic samples at 280 nm after incubating a 500 μL sample containing 5 mg of biotherapeutic in 0 mM, 20 mM, 60 mM, 100 mM, or 120 mM sodium deoxycholate or sodium lauroyl sarcosinate for 2 hours and in 10% trifluoroacetic acid (v / v) for 30 minutes, according to an exemplary embodiment. [Figure 28B] 1 shows total ion chromatograms of biotherapeutic samples obtained using nano liquid chromatography-mass spectrometry after incubating a 500 μL sample containing 5 mg of biotherapeutic with no surfactant (top panel), 60 mM sodium deoxycholate (middle panel), or 60 mM sodium lauroyl sarcosinate (bottom panel) for 2 hours and 10% trifluoroacetic acid (v / v) for 30 minutes, according to an exemplary embodiment. [Figure 29A] FIG. 1 shows protein spectra of biotherapeutic samples at 280 nm after incubating a 500 μL sample containing 5 mg of biotherapeutic in 20 mM, 60 mM, 100 mM, or 120 mM sodium deoxycholate for 2 hours and in 10% trifluoroacetic acid (v / v) for 30 minutes, according to an exemplary embodiment. [Figure 29B] FIG. 1 shows the percentage of protein remaining in a biotherapeutic sample after incubating a 500 μL sample containing 5 mg of biotherapeutic in 20 mM, 60 mM, 100 mM, or 120 mM sodium deoxycholate for 2 hours and in 10% trifluoroacetic acid (v / v) for 30 minutes, according to an exemplary embodiment. [Figure 30A]FIG. 1 shows protein spectra of biotherapeutic samples at 280 nm after incubating a 500 μL sample containing 5 mg of biotherapeutic in 20 mM, 60 mM, 100 mM, or 120 mM sodium lauroyl sarcosinate for 2 hours and in 10% trifluoroacetic acid (v / v) for 30 minutes, according to an exemplary embodiment. [Figure 30B] FIG. 1 shows the percentage of protein remaining in a biotherapeutic sample after incubating a 500 μL sample containing 5 mg of biotherapeutic in 20 mM, 60 mM, 100 mM, or 120 mM sodium lauroyl sarcosinate for 2 hours and in 10% trifluoroacetic acid (v / v) for 30 minutes, according to an exemplary embodiment. [Figure 31A] 1 shows protein spectra of a biotherapeutic sample at 280 nm after incubation in 20 mM sodium deoxycholate for 5 minutes, 15 minutes, 30 minutes, 60 minutes, or 120 minutes, according to an exemplary embodiment. [Figure 31B] 1 shows protein spectra of a biotherapeutic sample at 280 nm after incubation with 20 mM sodium lauroyl sarcosinate for 5 minutes, 15 minutes, 30 minutes, 60 minutes, or 120 minutes, according to an exemplary embodiment. [Figure 31C] 1 shows the percentage of protein remaining in biotherapeutic samples following incubation in 20 mM sodium deoxycholate or 20 mM sodium lauroyl sarcosinate for 5 minutes, 15 minutes, 30 minutes, 60 minutes, or 120 minutes and acid precipitation according to an exemplary embodiment. [Figure 32A] 1 shows protein spectra of biotherapeutic samples at 280 nm after incubation in 20 mM sodium deoxycholate and 0%, 2.5%, 5%, 7.5%, or 10% (v / v) 10% trifluoroacetic acid, according to an exemplary embodiment. [Figure 32B]1 shows protein spectra of biotherapeutic samples at 280 nm after incubation in 20 mM sodium lauroyl sarcosinate and 0%, 2.5%, 5%, 7.5%, or 10% (v / v) 10% trifluoroacetic acid, according to an exemplary embodiment. [Figure 32C] 1 shows the percentage of protein remaining in biotherapeutic drug samples after incubation in 20 mM sodium deoxycholate or 20 mM sodium lauroyl sarcosinate and 0%, 2.5%, 5%, 7.5%, or 10% (v / v) 10% trifluoroacetic acid, according to an exemplary embodiment. [Figure 33A] 1 shows protein spectra of a biotherapeutic sample at 280 nm after 5 or 60 minutes of acid precipitation, according to an exemplary embodiment. [Figure 33B] 1 shows total ion chromatograms of a biotherapeutic sample obtained using nano liquid chromatography-mass spectrometry after 5 minutes or 60 minutes of acid precipitation according to an exemplary embodiment. [Figure 33C] 1 shows the number of HCPs identified in biotherapeutic samples using nano-liquid chromatography-mass spectrometry 5 or 10 minutes after acid precipitation and Protein Metrics' Byonic according to an exemplary embodiment. [Diagram 34]Figure 34A shows the number of HCPs identified in a biotherapeutic sample using nano liquid chromatography-mass spectrometry and Protein Metrics' Byonic after incubation in 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, or both 20 mM sodium deoxycholate and 20 mM sodium lauroyl sarcosinate and acid precipitation according to an exemplary embodiment. Figure 34B shows a Venn diagram and the number of HCPs identified in a biotherapeutic sample using nano liquid chromatography-mass spectrometry and Protein Metrics' Byonic after incubation in 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, or both 20 mM sodium deoxycholate and 20 mM sodium lauroyl sarcosinate and acid precipitation according to an exemplary embodiment. [Figure 35A] FIG. 1 shows the number of HCPs identified in biotherapeutic samples using nano-liquid chromatography-mass spectrometry and Protein Metrics' Byonic after incubation and acid precipitation in 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, or both 20 mM sodium deoxycholate and 20 mM sodium lauroyl sarcosinate according to exemplary embodiments, or existing methods. [Figure 35B] 1 shows total ion chromatograms of a biotherapeutic sample obtained using nano-liquid chromatography-mass spectrometry and Protein Metrics' Byonic after incubation in 40 mM sodium lauroyl sarcosinate and acid precipitation, or limited digestion, molecular weight cut-off filtration, or ProteoMiner methods according to exemplary embodiments. [Figure 35C]FIG. 1 shows the Venn diagram and number of HCPs identified in biotherapeutic samples using incubation in 40 mM sodium lauroyl sarcosinate and acid precipitation, or limited digestion, molecular weight cut-off filtration, or nano-liquid chromatography-mass spectrometry after ProteoMiner methodology and Protein Metrics' Byonic, according to an exemplary embodiment. [Diagram 36] Figure 36A shows the distribution of isoelectric points of HCPs determined only using nano-liquid chromatography-mass spectrometry and Protein Metrics Byonic after incubation in 40 mM sodium lauroyl sarcosinate and acid precipitation, or limited digestion, molecular weight cut-off filtration, and ProteoMiner method according to an exemplary embodiment. Figure 36B shows the distribution of hydropathy of HCPs determined only using nano-liquid chromatography-mass spectrometry and Protein Metrics Byonic after incubation in 40 mM sodium lauroyl sarcosinate and acid precipitation, or limited digestion, molecular weight cut-off filtration, and ProteoMiner method according to an exemplary embodiment. [Figure 37] Figure 37A shows the distribution of molecular weights of HCPs identified only using nano-liquid chromatography-mass spectrometry and Protein Metrics' Byonic after incubation in 40 mM sodium lauroyl sarcosinate and acid precipitation, or limited digestion, molecular weight cut-off filtration, and ProteoMiner method according to an exemplary embodiment. Figure 37B shows the number of HCPs greater than 50 kDa (dark) and less than 50 kDa (light) identified only using nano-liquid chromatography-mass spectrometry and Protein Metrics' Byonic after incubation in 40 mM sodium lauroyl sarcosinate and acid precipitation (outer circle), or limited digestion, molecular weight cut-off filtration, and ProteoMiner method (inner circle) according to an exemplary embodiment. [Figure 38]Figure 38A shows protein spectra at 280 nm of samples of four different biotherapeutic proteins after detergent-assisted dissociation using 40 mM sodium lauroyl sarcosinate and acid precipitation according to an exemplary embodiment, and Figure 38B shows the percentage of protein remaining in samples of four different biotherapeutic proteins after detergent-assisted dissociation using 40 mM sodium lauroyl sarcosinate and acid precipitation according to an exemplary embodiment. [Figure 39] 1 shows several HCPs identified in DS and concentrated HMW samples from various mAbs, according to an exemplary embodiment. [Figure 40-1] 1 shows a heat map of HCPs identified and quantified in DS and enriched HMW samples from various mAbs, according to an exemplary embodiment. [Figure 40-2] See description of Figure 40-1. [Diagram 41] Figure 41A shows a comparison of the theoretical isoelectric points and molecular weights of all HCPs identified in five mAb DS samples, according to an exemplary embodiment. Figure 41B shows a comparison of the theoretical isoelectric points and molecular weights of all HCPs identified in five mAb enriched HMW samples, according to an exemplary embodiment. [Diagram 42] 1 shows a heat map of enriched HMW, enriched dimer, enriched vHMW, and HCPs identified and quantified in DS samples from mAb2, according to an exemplary embodiment. [Diagram 43] 1 shows the distribution of CC motif chemokines in the HMW, monomeric, and LMW fractions of mAb2 according to an exemplary embodiment.The inset shows a SEC chromatogram showing the HMW, monomeric, and LMW fractions of mAb2 according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] Detailed Description High molecular weight (HMW) aggregates in biotherapeutic products pose challenges to drug development, commercial manufacturing, and product stability throughout the product's shelf life. HMW aggregates can form during manufacturing, formulation, and transportation or delivery to patients. The formation of these aggregates can be due to a variety of external factors, such as exposure to interfaces, freeze-thaw cycles, heat and light stress, and agitation stress (Kiese et al., 2008, J Pharm Sci, 97(10):4347-4366; Hawe et al., 2009, Eur J Pharm Sci, 38(2):79-87; Joubert et al., 2011, J Biol Chem, 286(28):25118-25133). The presence of HMW aggregates in biotherapeutic products can affect drug efficacy and increase the risk of adverse immune responses in patients (Ratanji et al., 2014, J Immunotoxicol, 11(2):99-109). Therefore, the levels of HMW species in biotherapeutic products are monitored as a critical quality attribute. Furthermore, various analytical methods have been developed to characterize the biophysical and biochemical properties of HMW aggregates, understand the mechanisms of HMW formation, and evaluate their potential impact on product safety.

[0095] Recent studies have shown that the immunogenicity of HMW species is related to modifications of the primary structure. For example, oxidized species incorporated into aggregates pose a high immunogenicity risk (Filipe et al., 2012, mAbs, 4(6):740-752). Therefore, multiple analytical techniques are also used to study the primary structure and post-translational modifications (PTMs) of HMW species during the drug development process. The PTMs of HMW species, such as glycosylation, glycation, deamidation, and oxidation, were evaluated and compared with those of drug substances (DS).

[0096] Another potential critical quality attribute in biotherapeutic products is the presence of host cell proteins (HCPs). HCPs are process-related impurities introduced during antibody production from mammalian cell lines and must be controlled to appropriate levels to ensure product safety and efficacy. The composition and abundance of HCPs at each step of the manufacturing process and in the final drug substance depends on many factors: the host expression system (e.g., Escherichia coli (E. coli) with approximately 4,300 genes compared to Chinese Hamster Ovary (CHO) cells with approximately 30,000 genes), the mode of expression (e.g., cytoplasm compared to medium), the physiochemical properties of the biotherapeutic (e.g., hydrophobicity, charge, and structure), and the purification process (e.g., protein A chromatography, ion exchange chromatography, hydrophobic interaction chromatography, or filtration).

[0097] HCP impurities in biotherapeutic products can potentially cause many problems. HCPs can jeopardize patient safety, for example, HCP PLBD2 can induce dose-dependent immune responses, and host cell cytokines such as MCP-1 or TGF-β1 can cause toxicity. They can also compromise product quality and efficacy. For example, cathepsin D can cause drug degradation, while lipases can cause degradation of polysorbates, which are common excipients that contribute to drug stability. Recent studies have demonstrated that low levels of HCPs can reduce product fragmentation (Robert et al., 2009, Biotechnol Bioeng, 104(6):1132-1141; Dick et al., 2008, Biotechnol Bioeng, 100(6):1132-1143; Luo et al., 2019, Biotechnol Prog, 35(1):e2732), immunogenic responses (Fischer et al., 2017, AAPS J, 19(1):254-263; Jawa et al., 2016, AAPS J, 18(6):1439-1452), and formulation changes (Chiu et al., 2017, Biotechnol Bioeng, 114(5):1006-1015; McShan et al., 2016, PDA J Pharm Sci Technol,70(4):332-345, Zhang et al.,2020,J Pharm Sci,109(11):3300-3307, Zhang et al.,2021,J Pharm Sci,110(12):3866-3873). For example, trace amounts of lipase can degrade polysorbate 20 and polysorbate 80, thereby causing aggregation of medical products and affecting the shelf life of the products (Chiu et al., McShan et al., Zhang et al. 2020, Zhang et al. 2021). To guide process development, it is important to use analytical methods to evaluate important process parameters and critical quality attributes (CQAs) of biotherapeutic and biotherapeutic candidates.Therefore, sensitive analytical methods are needed to monitor downstream process activities and remove low levels of HCPs to avoid adverse clinical events. The present disclosure provides novel improvements to analytical methods for assessing HCPs in biotherapeutic products.

[0098] Subpopulations of residual HCPs are often co-purified with biotherapeutics throughout the purification process. Identification of HCPs is difficult, at least in part, due to the relatively high abundance of biotherapeutics, which poses technical obstacles to the detection of low-abundance proteins in samples. To ameliorate this problem, various approaches have been developed, such as compressing the dynamic range of protein concentrations of samples using ProteoMiner beads or using immunoassays to specifically enrich for HCPs, but each has logistical, technical, or analytical shortcomings.

[0099] Traditionally, enzyme-linked immunosorbent assay (ELISA) and Western blotting have been used for HCP analysis in biotherapeutics. However, these methods are unlikely to detect HCPs that induce weak or no immune responses. In addition, ELISA does not usually indicate the identity of individual HCPs. Because the risks posed by individual HCP species are different, methods that can provide information about individual HCPs should be implemented as orthogonal strategies for risk mitigation (Bracewell et al., 2015, Biotechnol Bioeng, 112(9):1727-1737; Abiri et al., 2018, PLoS ONE, 13(3)e0193339).

[0100] Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS)-based proteomics has become a commonly used orthogonal HCP analysis strategy after recent advances in instrumentation and workflow improvements. LC-MS / MS for HCP analysis not only allows for the identification and quantification of individual HCPs, but also mitigates the risk of lack of coverage of specific HCPs in polyclonal antibody reagents used in ELISA HCP assays. Furthermore, LC-MS / MS-based HCP analysis can enable understanding of downstream processes and provide guidance for removal of abundant or problematic HCPs.

[0101] Most monoclonal antibody (mAb) purification techniques involve an initial affinity chromatography step, e.g., Protein A affinity chromatography, followed by several final purification steps. The presence of HCPs in Protein A eluate is mainly due to interactions with column resin / ligand or mAb. HCPs retained after Protein A purification due to column resin interactions can be removed in the subsequent final purification steps. However, HCPs associated with mAb are difficult to remove and may escape the purification process. Identifying co-eluting HCPs and understanding the above interaction mechanisms are important for process development.

[0102] The main challenge in identifying HCPs in final drug substances (DS) by MS is the large dynamic range of concentrations, which can be overcome by adding additional dimensions in the separation, such as 2D-LC (Yang et al., 2018, Anal Chem, 90(22):13365-13372; Farrell et al., 2015, Anal Chem, 87(18):9186-9193) or ion mobility (Doneanu et al., 2015, Anal Chem, 87(20):10283-10291). Other strategies include protein A depletion (Madsen et al., 2015, mAbs, 7(6):1128-1137; Thompson et al., 2014, Rapid Commun Mass Spectrom, 28(8):855-860; Johnson et al., 2020, Anal Chem, 92(15):10478-10484) or native digestion (Huang et al., 2017, Anal Chem, 89(10):5436-5444) to remove therapeutic proteins in the sample preparation step (Madsen et al., Huang et al.). In addition, chromatographic separations can be performed to separate HCPs and therapeutic proteins prior to sample preparation. Examples of these methods include HCP analysis of reversed-phase HPLC fractionation (Bomans et al., 2013, PLoS ONE, 8(11):e81639), strong cation exchange (SCX) fractionation (Soderquist et al., 2015, Biotechnol Prog, 31(4):983-989), and hydrophilic interaction liquid chromatography (HILIC) fractionation (Wang et al., 2020, Anal Chem, 92(15):10327-10335).

[0103] Many characterization methods focus on protein product aggregates as a causative mechanism for product issues associated with HMW species, but little profiling of HCP impurities in HMW fractions has been performed (Xu et al., 2021, J Pharm Sci, 110(10):3403-3409). Thus, there is a need for characterization of HCPs in biotherapeutic products, specifically, their distribution in size-separated fractions.

[0104] The examples presented below demonstrate some surprising discoveries that are important for the development and manufacture of biotherapeutic products. Surprisingly, it has been found that size exclusion chromatography (SEC) analysis methods can be used to identify HCPs with comparable or superior sensitivity and specificity compared to alternative methods. Even more surprisingly, it has been found that SEC analysis using a denaturing mobile phase can be used to characterize the binding of HCPs to a protein of interest, and can generate a binding profile of all detected HCPs in a sample, compared to a non-denaturing mobile phase. Additionally, it has been found that a surfactant-assisted method involving acid precipitation can further enhance the detection, identification, and characterization of HCPs.

[0105] Relatedly, it has been found that the HMW fraction of a biotherapeutic product may contain high abundance HCPs that, rather than eluting at early SEC retention times based on their own size, preferentially interact with certain HMW species. It has also been surprisingly found that samples such as total biotherapeutic product DS that contain unacceptably high abundance HCPs may have a high percentage of those HCPs in the LMW or HMW fraction. Finally, it has been found that the identity and amount of HCPs in the total drug substance and HMW fractions vary significantly between different biotherapeutic products, even using the same purification process. These findings illuminate potential mechanisms of adverse activity in HMW species of biotherapeutic products that may contain high abundance HCPs in a manner specific to the particular product. These findings also demonstrate the utility of a novel approach to biotherapeutic product manufacturing that involves determining whether the HMW fraction of a particular product contains high abundance problematic HCPs, and if so, using SEC to remove HCPs that interact with HMW species, thereby improving product quality (e.g., stability and immunogenicity).

[0106] The present disclosure provides methods for identifying, quantifying, and characterizing HCPs in a sample (e.g., drug substance or HMW fraction of a biotherapeutic product), as well as methods for removing said HCPs to improve the manufacturing process of a biotherapeutic product. The examples presented below illustrate the use of native digestion to characterize HCPs in enriched HMW from, for example, drug substance (DS). More HCPs were identified in the enriched HMW fraction than in the total DS across the five studied mAbs, thus demonstrating that SEC can potentially be used as a fractionation strategy to enhance the detection of HCPs. Several frequently identified and problematic HCPs were present at higher levels in the enriched HMW fraction than in the total DS, thus indicating the possibility that certain HCPs preferentially interact with HMW species in biotherapeutic products. The most abundant HCP from mAb1, a CC motif chemokine, was substantially enriched in the HMW fraction. Therefore, further studies were performed on the HCP profiles of the enriched dimer and enriched very HMW (vHMW) fractions to identify fractions associated with CC motif chemokines. The association of CC motif chemokines with mAb1 was specifically attributable to mAb1 dimers.Finally, HMW species were removed from mAb1 by SEC and MS quantification was performed to determine CC motif chemokine levels in the HMW, monomeric, and LMW fractions.

[0107] 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, specific methods and materials are now described.

[0108] The terms "a" and "an" 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 meant to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0109] As used herein, the terms "protein" and "protein of interest" may include any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, generally known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues linked through peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. 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 of skill in the art. A protein may include one or more polypeptides to form a single functional biomolecule. Proteins may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins of interest may include 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 any of the bispecific antibodies. Proteins can be produced using recombinant cell-based production systems such as the insect baculovirus system, yeast systems (e.g., Pichia species), 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., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 Biotechnology and Genetic Engineering Reviews ). 147-176 (2012), the entire teachings of which are incorporated herein by reference. 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), and the like. myc-epitopes, fluorescent labels and other dyes, etc. Proteins can be classified based on composition and solubility, thus including simple proteins such as globular proteins, fibrous proteins, complex proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, lipoproteins, etc., and derived proteins such as primary derived proteins, secondary derived proteins, etc. Non-limiting examples of proteins or pharmaceutical protein products can include recombinant proteins, antibodies, bispecific antibodies, multispecific antibodies, antibody fragments, monoclonal antibodies, fusion proteins, scFv, and combinations thereof.

[0110] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried in a recombinant expression vector introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., an IgG1 or IgG4 immunoglobulin), or the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0111] 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 more conserved regions, called framework regions (FR), and interspersed regions of hypervariability, called complementarity determining regions (CDR). Each VH and VL is composed of three complementarity determining regions and four framework regions, 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 framework regions 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 complementarity determining regions. As used herein, the term "antibody" also includes antigen-binding fragments of an intact antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically accessible, 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 intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, 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 can be sequenced and manipulated using chemical or molecular biology techniques, for example to place one or more variable and / or constant domains in a suitable configuration, to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids.

[0112] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, 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 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 variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. In some exemplary embodiments, an antibody fragment comprises 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 can 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 can be wholly or partially synthetically produced. Antibody fragments can optionally include single chain antibody fragments. Alternatively, or additionally, antibody fragments can include multiple chains linked together, for example, by disulfide bonds. Antibody fragments can optionally include multimolecular complexes. Functional antibody fragments typically include at least about 50 amino acids, more typically include at least about 200 amino acids.

[0113] The term "bispecific antibody" includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally include 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 will generally be at least one to two orders of magnitude lower, 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 target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, 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.

[0114] A typical bispecific antibody has two heavy chains, each with three heavy chain complementarity determining regions 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 epitopes bound by the heavy chain antigen binding regions, or can associate with each heavy chain and bind one or two 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 usually being smaller than Fc-containing IgG and IgG-like bispecific molecules. IgG-like bispecific antibodies (bsAbs) include various formats such as, but are not limited to, triomabs, knobs into holes IgG (KiH IgG), crossMabs, 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. Various 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 and Mingju Hao, Bispecific Antibodies and Their Applications, 8 Journal of Hematology & Oncology 130; Dafne Mueller and Roland E. Kontermann, Bispecific Antibodies, Handbook of Therapeutic Antibodies 265-310 (2014), the teachings of 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 with chemical crosslinkers, and genetic approaches using recombinant DNA technology.

[0115] As used herein, a "multispecific antibody" refers to an antibody that has binding specificities for at least two different antigens. Such molecules typically bind only two antigens (e.g., 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.

[0116] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can 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 can 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.

[0117] As used herein, the term "host cell proteins" (HCPs) includes proteins derived from host cells in the production of recombinant proteins. Host cell proteins can be process-related impurities that can originate from the manufacturing process and can 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 and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables. In some exemplary embodiments, the types of host cell proteins in the composition can be at least two. In some exemplary embodiments, the host cell proteins can bind to the biotherapeutic. In some exemplary embodiments, the host cell proteins can bind strongly, weakly, or not at all to the biotherapeutic. In some exemplary embodiments, a host cell protein may preferentially bind to a particular form of the biotherapeutic, e.g., an aggregate, a multimer, a dimer, a monomer, a post-translationally modified form, a truncated form, or a fragment of the biotherapeutic.

[0118] The presence of host cell proteins in a biotherapeutic product may be considered to be a higher or lower risk based on several measurable factors. One such factor is the concentration or abundance (amount) of the HCP impurity in the biotherapeutic product. At low enough abundance, the HCP may not have a discernible effect, as measured, for example, by ELISA or mass spectrometry. The level at which the HCP may pose a significant risk (which may be considered an unacceptable level in the product and monitored as a critical quality attribute (CQA)) may depend on the specific identity of the HCP. Certain HCPs may be known to pose a risk at certain levels, for example, depending on the level of enzymatic activity of the HCP, which is an enzyme.

[0119] In this context, the significance of the presence of an HCP may depend on the function of that HCP, specifically in relation to the components of the biotherapeutic product. For example, HCP lipases that may or are known to degrade polysorbates present in the biotherapeutic product of interest may be closely monitored. HCP lipases may have a low threshold for the amount of HCP impurities that can be tolerated in the biotherapeutic product. Other HCPs of particular concern may be, for example, proteases that may or are known to degrade proteins of interest in the biotherapeutic product, or immunogenic HCPs that may or are known to cause an immune response when administered to a subject. Using the methods of the present invention, one of skill in the art can assess the abundance, distribution, and / or identity of an HCP impurity in relation to the biotherapeutic product of interest to determine whether the HCP impurity is an HCP impurity of concern, and based on that determination, the impurity can be removed using chromatography or other separation methods when manufacturing the biotherapeutic product.

[0120] In some exemplary embodiments, the sample may include 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, 10,000 times, 100,000 times, or 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 (e.g., HCP) in a sample containing a high abundance protein or peptide (e.g., 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.

[0121] In some exemplary embodiments, the sample may include at least one protein of interest and at least one HCP. In some exemplary embodiments, the sample may include at least one protein of interest and at least one HCP, where the protein of interest is larger in size than the HCP. In some exemplary embodiments, the sample may include at least one protein of interest and at least one HCP, where the protein of interest is larger in size than the HCP, such that they can be separated using SEC. The protein of interest may be, for example, greater than 50 kilodaltons (kDa), greater than 100 kDa, greater than 150 kDa, about 50 kDa to about 250 kDa, about 100 kDa to about 250 kDa, about 100 kDa to about 200 kDa, about 150 kDa to about 250 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, about 250 kDa, about 260 kDa, about 270 kDa, about 280 kDa, about 290 kDa, about 300 kDa, about 310 kDa, about 320 kDa, about 330 kDa, about 340 kDa, about 350 kDa, about 360 kDa, about 370 kDa, about 380 kDa, about 390 kDa, about 400 kDa, about 400 kDa, about 40 a, approx. 130kDa, approx. 140kDa, approx. 150kDa, approx. 160kDa, approx. 170kDa, approx. 180kDa, approx. 190kDa, approx. 200kDa, approx. 210kDa, approx. 290kDa, approx. 300kDa, approx. 310kDa, approx. 320kDa, approx. 330kDa, approx. 340kDa, approx. 350kDa, approx. 360kDa, approx. 370kDa, approx. kDa, about 460 kDa, about 470 kDa, about 480 kDa, about 490 kDa, about 500 kDa, about 510 kDa, about 520 kDa, about 530 kDa, about 540 kDa, about 550 kDa, about 560 kDa, about 570 kDa, about 580 kDa, about 590 kDa, or about 600 kDa.The host cell protein may be about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 ...16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, 0 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, Approximately 240kDa, approximately 250kDa, approximately 260kDa, approximately 270kDa, approximately 280kDa, approximately 290kDa, approximately 300kDa, approximately 310kDa, approximately 320kDa, approximately 33 0kDa, about 340kDa, about 350kDa, about 360kDa, about 370kDa, about 380kDa, about 390kDa, about 400kDa, about 410kDa, about 420kDa , about 430 kDa, about 440 kDa, about 450 kDa, about 460 kDa, about 470 kDa, about 480 kDa, about 490 kDa, about 500 kDa, about 510 kDa, about 520 kDa, about 530 kDa, about 540 kDa, about 550 kDa, about 560 kDa, about 570 kDa, about 580 kDa, about 590 kDa, or about 600 kDa.

[0122] As used herein, a "protein pharmaceutical product", "biopharmaceutical product" or "biopharmaceutical" comprises an active ingredient that may be fully or partially biological in nature. In one aspect, a protein pharmaceutical product may comprise 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 aspect, a protein pharmaceutical product may comprise 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.

[0123] Proteins, pharmaceutical protein products, biopharmaceutical products, or biotherapeutic drugs may be produced from mammalian cells. Mammalian cells may be of human or non-human origin and include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their derivatives (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells, and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells, or derivatives thereof), fibroblasts from any tissue or organ, including but not limited to:Heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph glands, tonsils, tonsils, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 551 ... 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS -3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BAL B cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, 2071 strain (mouse L) cells, LM strain (mouse L) cells, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells, or derivatives thereof).

[0124] 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.

[0125] In some exemplary embodiments, a sample containing a protein of interest may be prepared prior to LC-MS analysis. Preparation steps may include denaturation, alkylation, dilution, digestion, precipitation, centrifugation, buffer exchange, and desalting. In some exemplary embodiments, precipitation may be mediated by acid. In some preferred exemplary embodiments, precipitation may be mediated by trifluoroacetic acid.

[0126] 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.

[0127] 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 can 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 can 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, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroyl sarcosine, urea, and salts thereof. In some exemplary embodiments, a denaturant may be used in the mobile phase in a chromatographic analysis. In some exemplary embodiments, the denaturant used in the mobile phase may be acetonitrile. In some exemplary embodiments, the modifier used in the mobile phase may be a surfactant.

[0128] 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 digestion (e.g., enzymatic or non-enzymatic digestion) of proteins in a sample using an appropriate hydrolysis agent. Digestion of proteins into constituent peptides can produce "peptide digests" that can be further analyzed using peptide mapping analysis.

[0129] As used herein, the term "digestive enzyme" refers to any of a number of different agents capable of effecting the digestion of proteins. Non-limiting examples of hydrolytic agents capable of performing enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase 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 homologs 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, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077(2013)).

[0130] Conventional methods use digestive enzymes under conditions and concentrations sufficient to completely digest all proteins in a sample prior to LC-MS analysis. In the present disclosure, it has been found that the identification and characterization of HCPs can be improved by limited digestion, meaning that the digestive enzymes are used under conditions such that the proteins in the sample are not completely digested. In some exemplary embodiments, the ratio of digestive enzyme to substrate (e.g., the ratio of enzyme to protein when the enzyme is a protease and the substrate is a protein or mixture of proteins) 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 less than 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 1:100 to about 1:10000, about 1:200 to about 1:2000, about 1:200, about 1:400, about 1:500, about 1:1000, about 1:2000, about 1:2500, or about 1:10000.

[0131] 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. Reduced peptide mapping, a traditional method of protein analysis, involves protein reduction prior to LC-MS analysis. In contrast, non-reduced peptide mapping omits the sample preparation step of reduction in order to preserve endogenous disulfide bonds.

[0132] As used herein, the term "dissociation reagent" refers to a class of molecules that can modulate protein-protein interactions, stabilize the molecules in an unbound state, or both. In some exemplary embodiments, the dissociation reagent can destabilize the interaction between at least one protein of interest and at least one HCP. In some exemplary embodiments, the dissociation reagent can include a surface active agent, a detergent, or both.

[0133] As used herein, the term "surfactant" refers to a class of molecules that contain a hydrophobic "head" domain and a hydrophilic "tail" domain. Surfactants can be used, for example, to modulate solvent and solute interactions or to modulate protein-protein interactions. Surfactants can be, for example, phase transfer surfactants, ionic surfactants, anionic surfactants, cationic surfactants, or combinations thereof.

[0134] An exemplary type of surface active agent useful in the methods of the invention is a surfactant. Non-limiting examples of surfactants include anionic surfactants (e.g., salts of deoxycholic acid, 1-heptanesulfonic acid, N-lauryl sarcosine, lauryl sulfate, 1-octane sulfonic acid, taurocholic acid, and sodium lauroyl sarcosinate (SLS)), cationic surfactants (e.g., benzalkonium chloride, cetylpyridinium, methylbenzethonium chloride, and decamethonium bromide), zwitterionic surfactants (e.g., alkyl betaines, alkyl amido alkyl betaines, N-dodecyl-N,N-dimethyl-3-ammonium, etc.), and / or cyclic surfactants (e.g., cyclic alkyl betaines, alkyl amido alkyl betaines, N-dodecyl-N,N-dimethyl-3-ammonium, etc.). o-1-propanesulfonate, and phosphatidylcholine), and non-ionic detergents (e.g., n-decyl α-D-glucopyranoside, n-decyl β-D-maltopyranoside, n-dodecyl β-D-maltoside, n-octyl β-D-glucopyranoside, sorbitan esters, n-tetradecyl β-D-maltoside, Triton, Nonidet-P-40, poloxamer 188, and any of the Tween group of detergents), sodium lauryl sulfate (SLS), and sodium deoxycholate (SDC). The detergent may be a combination of multiple surface active agents. The detergent may be denaturing or non-denaturing with respect to protein structure. In some exemplary embodiments, the preferred detergent is a denaturing detergent. In some exemplary embodiments, the preferred detergent is SLS, SDC, or a combination thereof.

[0135] As used herein, the term "liquid chromatography" refers to a process in which a biological / chemical mixture carried by a liquid can be separated into multiple components as a result of differences in the distribution of the components as they flow (or flow) through 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, a sample containing at least one protein or peptide digest of interest can be subjected to any one or combination of the above-mentioned chromatographic methods. Analytes separated using chromatography are characterized by unique retention times that reflect the speed at which the analytes pass through the chromatography column. Analytes can be compared using chromatograms that plot retention time on one axis and measured signal on another axis, where the measured signal can be generated, for example, from UV or fluorescence detection.

[0136] Size Exclusion Chromatography (SEC) or gel filtration relies on the separation of components as a function of molecular size. Separation depends on the time a substance spends in the porous stationary phase compared to the time in the fluid. The probability that a molecule will be present in the pores will depend on the size of the molecule and the pores. In addition, the ability of a substance to penetrate the pores is determined by the diffusion mobility of the macromolecule, which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all, and for very small macromolecules the probability of penetration is close to 1. Components with larger molecular size will pass through the stationary phase faster, while components with smaller molecular size will have a longer path length through the pores of the stationary phase and will therefore be retained in the stationary phase for a longer time.

[0137] Analytes eluting from a SEC column can be separated into multiple fractions based on elution time. For example, analytes that elute earlier than the functional form of the protein of interest (e.g., monomeric form) can be broadly classified as high molecular weight (HMW) species. The HMW fraction can be further subdivided, for example, into an ultra-high molecular weight (vHMW) fraction and a dimer fraction (representing the elution time of the dimer of the protein of interest). Analytes that elute later than the functional form of the protein of interest can be broadly classified as low molecular weight (LMW) species and further subdivided into an LMW fraction and a later tail fraction.

[0138] The chromatographic material may include a size-exclusion material, which may be a resin or a membrane. The matrix used for size-exclusion is preferably an inert gel medium, which may be a complex of cross-linked polysaccharides, e.g., cross-linked agarose and / or dextran in the form of spherical beads. The degree of cross-linking determines the size of the pores present in the swollen gel beads. Molecules above a certain size do not enter the gel beads and therefore pass through the chromatographic bed the fastest. Smaller molecules (e.g., surfactants, proteins, DNA, etc.), which enter the gel beads to various degrees depending on their size and shape, are delayed as they pass through the bed. Thus, molecules are generally eluted in order of decreasing molecular size.

[0139] Porous chromatography resins suitable for size-exclusion chromatography of viruses can be made of dextrose, agarose, polyacrylamide, or silica, which have different physical properties. Combinations of polymers can also be used. The most commonly used is available from Amersham Biosciences under the trade name "SEPHADEX". Other size-exclusion supports from different construction materials are also suitable, such as Toyopearl 55F (polymethacrylate, Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, Calif.).

[0140] As used herein, the term "mass spectrometer" includes an instrument capable of recognizing and measuring the exact mass of a specific molecular species. The term is meant to include any molecular detector capable of characterizing a polypeptide or peptide. A mass spectrometer includes three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to generate gas phase ions. Analyte atoms, molecules, or clusters are transferred to the gas phase and ionized simultaneously (as in electrospray ionization) or through a separate process. The choice of ion source depends on the application.

[0141] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" includes techniques that use multiple stages of mass selection and mass separation to obtain structural information of sample molecules. The prerequisite is that after an initial mass selection step, the sample molecules are transferred to the gas phase and ionized such that fragments are formed in a predictable and controllable manner. MS / MS or MS 2 First, the precursor ion (MS 1 ) and fragment it to obtain meaningful information. Tandem MS is successfully performed with a 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, but hybrids also exist in which a tandem-in-time analyzer is spatially or spatially coupled with a tandem-in-space analyzer. A tandem-in-space mass analyzer 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 the product ions are sent to another analyzer for m / z separation and data collection. In tandem-in-time, mass analyzer ions generated in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0142] The peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational or other modifications. They can be used to characterize proteins by correlating experimental and theoretical MS / MS data, the latter generated from potential peptides in protein sequence databases. This characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequence determination, protein de novo sequencing, localization of post-translational modifications, or identification of post-translational modifications, or comparability analysis, or a combination thereof.

[0143] In some exemplary aspects, the mass spectrometer may operate with nanoelectrospray or nanospray. The term "nanoelectrospray" or "nanospray" as used herein refers to electrospray ionization at very low solvent flow rates, often without external solvent delivery, typically hundreds of nanoliters of sample solution per minute or less. The electrospray injection setup 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 solvent delivery system to perform chromatographic separation of a mixture prior to analysis by a mass spectrometer.

[0144] In some exemplary embodiments, mass spectrometry may be performed under native conditions. As used herein, the term "native conditions" may include performing mass spectrometry under conditions that preserve non-covalent interactions in the analyte. For a detailed review of native MS, see review: Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0145] 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 being 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 databases 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).

[0146] The present disclosure provides a method for identifying HCP impurities in a sample. In some exemplary embodiments, the method includes: (a) subjecting a sample containing at least one protein of interest and at least one HCP impurity to size exclusion chromatography (SEC) analysis to generate a fraction; (b) subjecting the fraction to LC-MS analysis to identify the at least one HCP impurity; Includes.

[0147] In some exemplary embodiments, the amount of protein loaded onto a SEC column can be about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg.

[0148] In some exemplary embodiments, the mobile phase for SEC analysis may include acetonitrile. In some exemplary embodiments, the concentration of acetonitrile may be about 5% v / v to about 20% v / v, about 10% v / v to about 20% v / v, about 15% v / v to about 20% v / v, about 5% v / v, about 6% v / v, about 7% v / v, about 8% v / v, about 9% v / v, about 10% v / v, about 11% v / v, about 12% v / v, about 13% v / v, about 14% v / v, about 15% v / v, about 16% v / v, about 17% v / v, about 18% v / v, about 19% v / v, or about 20% v / v.

[0149] In some exemplary embodiments, the mobile phase for SEC analysis may include at least one surfactant. In some exemplary embodiments, the concentration of the at least one surfactant is about 6 mM to about 36 mM, about 10 mM to about 20 mM, about 12 mM to about 24 mM, about 10 mM to about 14 mM, about 20 mM to about 28 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, It can be about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, or about 36 mM.

[0150] In some exemplary embodiments, the at least one surface active agent may be a detergent. In some exemplary embodiments, the sample may be incubated in a detergent. In some exemplary embodiments, the sample may be incubated in a detergent prior to acid precipitation. In some exemplary embodiments, the time the sample is incubated in the detergent may be about 5 minutes to about 120 minutes, about 15 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 45 minutes to about 60 minutes, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, or about 120 minutes. In some exemplary embodiments, the at least one detergent may be selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof. In some exemplary embodiments, the concentration of sodium deoxycholate is about 6 mM to about 120 mM, about 20 mM to about 120 mM, about 20 mM to about 60 mM, about 40 mM to about 60 mM, about 6 mM to about 36 mM, about 10 mM to about 20 mM, about 12 mM to about 24 mM, about 10 mM to about 14 mM, about 20 mM to about 28 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 50 mM, about 55 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 M, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 3 5mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43,mM, about 44mM, about 45mM , about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM.In some exemplary embodiments, the concentration of sodium lauroyl sarcosinate is about 6 mM to about 120 mM, about 20 mM to about 120 mM, about 20 mM to about 60 mM, about 40 mM to about 60 mM, about 6 mM to about 36 mM, about 10 mM to about 20 mM, about 12 mM to about 24 mM, about 10 mM to about 14 mM, about 20 mM to about 28 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 50 mM, about 55 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43,mM, about 44mM, about 45m M, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM.

[0151] In some exemplary embodiments, the method may further include subjecting the fraction to enzymatic digestion prior to LC-MS analysis. In some exemplary embodiments, the enzymatic digestion may be performed by contacting the fraction with trypsin. In some exemplary embodiments, the enzymatic digestion may be performed by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:100 to about 1:2000, about 1:50, about 1:100, about 1:150, about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900, about 1:1000, or about 1:2000.

[0152] In some exemplary embodiments, the method may further include subjecting the fraction to acid precipitation prior to LC-MS analysis. In some exemplary embodiments, the acid precipitation may include contacting the fraction with trifluoroacetic acid. In some exemplary embodiments, the acid precipitation may include centrifugation. In some exemplary embodiments, the concentration of trifluoroacetic acid can be about 0.5% to about 20%, about 1% to about 10%, about 0.5% to about 5%, about 1% to about 2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.

[0153] In some exemplary embodiments, the method may further include subjecting the fraction to buffer exchange. In some exemplary embodiments, the steps of the method may be in the order of SEC analysis, acid precipitation, buffer exchange, digestion, and LC-MS analysis. In some exemplary embodiments, the steps of the method may be in the order of SEC analysis, buffer exchange, digestion, and LC-MS analysis. In some exemplary embodiments, the steps of the method may be in the order of SEC analysis, buffer exchange, digestion, and LC-MS analysis. In some exemplary embodiments, the steps of the method may be in the order of acid precipitation, buffer exchange, digestion, and analysis by liquid chromatography-mass spectrometry. In some exemplary embodiments, the steps of the method may be in the order of acid precipitation, buffer exchange, digestion, desalting, and analysis by liquid chromatography-mass spectrometry. In some exemplary embodiments, the acid precipitation may include centrifugation.

[0154] The present disclosure also provides methods for characterizing the binding of HCP impurities to a protein of interest. In some exemplary embodiments, the methods include: (a) obtaining a sample containing a protein of interest and at least one HCP impurity; (b) subjecting the sample to size exclusion chromatography (SEC) analysis using a non-denaturing mobile phase to generate a native fraction; (c) subjecting said sample of (a) to SEC analysis using a denaturing mobile phase to generate a denatured fraction; (d) subjecting the native fraction and the denatured fraction to LC-MS analysis to generate native and denatured separation profiles of the at least one HCP impurity; (e) comparing the native separation profile to the denatured separation profile to characterize binding of the at least one HCP impurity to the protein of interest; Includes.

[0155] In some exemplary embodiments, the amount of protein loaded onto a SEC column can be about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg.

[0156] In some exemplary embodiments, the denaturing mobile phase for SEC analysis may include acetonitrile. In some exemplary embodiments, the concentration of acetonitrile may be about 5% v / v to about 20% v / v, about 10% v / v to about 20% v / v, about 15% v / v to about 20% v / v, about 5% v / v, about 6% v / v, about 7% v / v, about 8% v / v, about 9% v / v, about 10% v / v, about 11% v / v, about 12% v / v, about 13% v / v, about 14% v / v, about 15% v / v, about 16% v / v, about 17% v / v, about 18% v / v, about 19% v / v, or about 20% v / v.

[0157] In some exemplary embodiments, the denaturing mobile phase for SEC analysis may include at least one surfactant. In some exemplary embodiments, the concentration of the at least one surfactant is about 6 mM to about 120 mM, about 20 mM to about 120 mM, about 20 mM to about 60 mM, about 40 mM to about 60 mM, about 6 mM to about 36 mM, about 10 mM to about 20 mM, about 12 mM to about 24 mM, about 10 mM to about 14 mM, about 20 mM to about 28 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43,mM, about 44mM, about 45m M, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM.

[0158] In some exemplary embodiments, the at least one surface active agent may be a surfactant. In some exemplary embodiments, the at least one surfactant may be selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof. In some exemplary embodiments, the concentration of sodium deoxycholate is about 6 mM to about 120 mM, about 20 mM to about 120 mM, about 20 mM to about 60 mM, about 40 mM to about 60 mM, about 6 mM to about 36 mM, about 10 mM to about 20 mM, about 12 mM to about 24 mM, about 10 mM to about 14 mM, about 20 mM to about 28 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 50 mM, about 55 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 M, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 3 5mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43,mM, about 44mM, about 45mM , about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM.In some exemplary embodiments, the concentration of sodium lauroyl sarcosinate is about 6 mM to about 120 mM, about 20 mM to about 120 mM, about 20 mM to about 60 mM, about 40 mM to about 60 mM, about 6 mM to about 36 mM, about 10 mM to about 20 mM, about 12 mM to about 24 mM, about 10 mM to about 14 mM, about 20 mM to about 28 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 50 mM, about 55 mM, about 50 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43,mM, about 44mM, about 45m M, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM.

[0159] In some exemplary embodiments, the method may further include subjecting the fraction to enzymatic digestion prior to LC-MS analysis. In some exemplary embodiments, the enzymatic digestion may be performed by contacting the fraction with trypsin. In some exemplary embodiments, the enzymatic digestion may be performed by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:50, about 1:100, about 1:150, about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900, about 1:1000, or about 1:2000.

[0160] In some exemplary embodiments, the method may further include subjecting the fraction to acid precipitation prior to LC-MS analysis. In some exemplary embodiments, the acid precipitation may include contacting the fraction with trifluoroacetic acid. In some exemplary embodiments, the concentration of trifluoroacetic acid can be about 0.5% to about 20%, about 1% to about 10%, about 0.5% to about 5%, about 1% to about 2%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.

[0161] In some exemplary embodiments, the method may further include subjecting the fraction to buffer exchange. In some exemplary embodiments, the method may further include desalting the peptide digest after digestion and before subjecting the peptide digest to analysis by liquid chromatography-mass spectrometry, or both. In some exemplary embodiments, the steps of the method may be in the order of SEC analysis, acid precipitation, buffer exchange, digestion, and LC-MS analysis. In some exemplary embodiments, the steps of the method may be in the order of SEC analysis, buffer exchange, digestion, and LC-MS analysis. In some exemplary embodiments, the steps of the method may be in the order of SEC analysis, buffer exchange, digestion, and LC-MS analysis.

[0162] The disclosure further provides a method for producing a biotherapeutic product. In some exemplary embodiments, the method comprises: (a) subjecting a first sample, comprising at least one protein of interest and at least one host cell protein (HCP) impurity, to size exclusion chromatography (SEC) analysis to generate a plurality of fractions; (b) subjecting the plurality of fractions to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to determine the identity and amount of the at least one HCP impurity; (c) using the identity and amount to determine whether the at least one HCP impurity is an impurity of concern in at least one of the plurality of fractions; (d) subjecting a second sample containing the at least one protein of interest and the at least one HCP impurity to SEC analysis to generate a second plurality of fractions; (e) using the determination of step (c) to remove the at least one fraction from the plurality of fractions of step (d) in which the at least one HCP impurity is an impurity of concern to manufacture a biotherapeutic product. Includes.

[0163] In some exemplary embodiments, the protein of interest may be an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product. In some exemplary embodiments, a sample may contain multiple proteins of interest.

[0164] In some exemplary embodiments, the at least one HCP impurity may include a lipase, a protease, or a combination thereof. In some exemplary embodiments, the at least one HCP impurity may include a CC motif chemokine, a carboxypeptidase, a beta-hexosaminidase, an inter-alpha trypsin inhibitor heavy chain H5, a lipoprotein lipase, a peptidyl-prolyl cis-trans isomerase, a cathepsin L1, an annexin, a legumain, a complement C1r-A subcomponent, a cornifin A, a peroxiredoxin, a sialic acid O-acetyl esterase, a glutathione S-transferase mu 6, a G protein-coupled receptor 56, a cathepsin Z, an annexin, a lipase, a metalloproteinase inhibitor 1, a clusterin, a fructose niobium ionomerase, a phosphodiester ... The proteins may include phosphate aldolase, fatty acid binding protein, putative phospholipase B-like 2, acid ceramidase, cathepsin D, connective tissue growth factor, procollagen C-endopeptidase enhancer 1, neogenin, CD166 antigen, intercellular adhesion molecule 1, leucine-rich repeat transmembrane protein FLRT3, oncostatin-M specific receptor subunit beta, N-acetylglucosamine-6-sulfatase, ethanolamine-phosphate cytidylyltransferase-like protein, vasorin, tyrosine protein phosphatase non-receptor type 11, beta-hexosaminidase subunit alpha, or a combination thereof.

[0165] In some exemplary embodiments, the plurality of fractions may comprise a high molecular weight (HMW) fraction, a very high molecular weight (vHMW) fraction, a multimer fraction, a dimer fraction, a monomer fraction, a low molecular weight (LMW) fraction, a tail fraction, or a combination thereof.

[0166] In some exemplary embodiments, the fraction in which the at least one HCP impurity is an impurity of concern may be a HMW fraction, a total HMW fraction, a vHMW fraction, a dimer fraction, or a LMW fraction.

[0167] In some exemplary embodiments, the HCPs may be enriched primarily in the dimer fraction, hi some exemplary embodiments, the HCPs may be enriched primarily in the vHMW fraction.

[0168] In some exemplary embodiments, the HCPs are present in the fraction at about 1 ppm to about 10,000 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 20 ppm, about 30 ppm, about 40 ppm, about 50 ppm, about 60 ppm, about 70 ppm, about 80 ppm, about 90 ppm, about 100 ppm, about 200 ppm, about 300 ppm, about 400 ppm, about 500 ppm, about 600 ppm, about 700 ppm, about 800 ppm, about 900 ppm, about 1000 ppm, about 1100 ppm, about 1200 ppm, about 1 It may be present at about 300 ppm, about 1400 ppm, about 1500 ppm, about 1600 ppm, about 1700 ppm, about 1800 ppm, about 1900 ppm, about 2000 ppm, about 2100 ppm, about 2200 ppm, about 2300 ppm, about 2400 ppm, about 2500 ppm, about 3000 ppm, about 3500 ppm, about 4000 ppm, about 4500 ppm, about 5000 ppm, about 5500 ppm, about 6000 ppm, about 6500 ppm, about 7000 ppm, about 7500 ppm, about 8000 ppm, about 8500 ppm, about 9000 ppm, about 9500 ppm, or about 10000 ppm.

[0169] In some exemplary embodiments, HCPs are present in the sample at about 1 ppm to about 10,000 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 20 ppm, about 30 ppm, about 40 ppm, about 50 ppm, about 60 ppm, about 70 ppm, about 80 ppm, about 90 ppm, about 100 ppm, about 200 ppm, about 300 ppm, about 400 ppm, about 500 ppm, about 600 ppm, about 700 ppm, about 800 ppm, about 900 ppm, about 1000 ppm, about 1100 ppm, about 1200 ppm, about 1300 ppm, about 1400 ppm, about 1500 ppm, about 1600 ppm, about 1700 ppm, about 1800 ppm, about 1900 ppm, about 2000 ppm, about 2100 ppm, about 2200 ppm, about 2300 ppm, about 2400 ppm, about 2500 ppm, about 2600 ppm, about 2700 ppm, about 2800 ppm, about 2900 ppm, about 3000 ppm, about 3100 ppm, about 3200 ppm, about 3300 ppm, about 3400 ppm, about 3500 ppm, about 3600 ppm, about 3700 ppm, about 3800 ppm, about 3900 ppm, about 40 ... An impurity may be determined to be a concern if it is present at about 400 ppm, about 1500 ppm, about 1600 ppm, about 1700 ppm, about 1800 ppm, about 1900 ppm, about 2000 ppm, about 2100 ppm, about 2200 ppm, about 2300 ppm, about 2400 ppm, about 2500 ppm, about 3000 ppm, about 3500 ppm, about 4000 ppm, about 4500 ppm, about 5000 ppm, about 5500 ppm, about 6000 ppm, about 6500 ppm, about 7000 ppm, about 7500 ppm, about 8000 ppm, about 8500 ppm, about 9000 ppm, about 9500 ppm, or about 10000 ppm.

[0170] In some exemplary embodiments, an HCP may be determined to be an impurity of concern if it is a lipase, a protease, and / or immunogenic.

[0171] In some exemplary embodiments, the percentage of HCPs found in the HMW fraction can be about 1% to about 100%, about 20% to about 95%, about 50% to about 90%, about 60% to about 80%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%.

[0172] In some exemplary embodiments, the sample may be subjected to sample preparation prior to SEC analysis. In some exemplary embodiments, these sample preparation steps may include buffer exchange, addition of at least one internal standard at a known concentration, reduction, alkylation, filtration, deglycosylation, acidification, or a combination thereof.

[0173] In some exemplary embodiments, the samples may be subjected to sample preparation prior to LC-MS / MS analysis. In some exemplary embodiments, these sample preparation steps may include addition of at least one internal standard at a known concentration, drying (e.g., using a SpeedVac), concentration, denaturation, reduction, alkylation, digestion, acidification, or combinations thereof.

[0174] The present invention relates to any of the above-mentioned protein(s), protein(s), antibody(s), host cell protein(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), sample(s), surface active agent(s), detergent(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s), and may be used in any manner. It is understood that the protein(s), protein(s), antibody(es), host cell protein(s), protein alkylating agent(s), protein denaturing agent(s), protein reducing agent(s), digestive enzyme(s), sample(s), surfactant(s), detergent(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH, temperature(s), or concentration(s) may be selected by any suitable means.

[0175] The present invention may be more fully understood by reference to the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0176] Example 1. Degradation of biotherapeutic drugs As shown in Figure 1A, aflibercept was produced at two different manufacturing facilities and the stability of the final drug substance was evaluated using an accelerated stability study at 25 °C. Degradation was indicated by non-reducing band 1 (NR-1) fragment formation. This study revealed that aflibercept processed at facility 2 degraded faster than facility 1. As shown in Figure 1B, a confirmatory study was performed by incubating aflibercept from each facility at 37 °C for 2 weeks and visualizing degradation over time using SDS-PAGE. The results of the initial study were confirmed.

[0177] To investigate the cause of degradation of the biotherapeutic drug, drug substance samples from site 2 were subjected to size exclusion chromatography (SEC) analysis to detect contaminating HCPs, as shown in Figure 2. Cathepsin D, a protease known to cause drug degradation, was detected in aflibercept samples from site 2.

[0178] Example 2. Size Exclusion Chromatography Method for Identification of Host Cell Proteins HCPs may be present in drug substances by binding to the biotherapeutic at some point during or throughout the purification process, and / or by co-purification in an unbound form. To determine the mechanism of HCP contamination in samples, a SEC-based HCP characterization method was developed and designed as an orthogonal separation method to enrich HCPs based on size. SEC has not previously been reported as an analytical method for the identification of HCPs in process development.

[0179] Analytical scale separation of analytes from an exemplary therapeutic protein sample, mAb1, was performed as follows. The concentration of the mAb was 25 mg / mL. An exemplary chromatography system suitable for this method is a Waters Acquity UPLC equipped with a Waters Xbridge Protein BEH200Å SEC (7.8×300 mm, 3.5 μm) column with a fraction collector. 500 μg of sample was injected in duplicate. Spiked-in standards used included BSA (66.5 kDa) and angiotensin (1 kDa). Mobile phases tested included native (non-denaturing) mobile phase, denaturing mobile phase with 2 M urea, and denaturing mobile phase with 20% acetonitrile.

[0180] As shown in Figure 3, SEC analysis of mAb1 was performed using the three mobile phases listed, including BSA as a standard. Based on these results, three distinct fractions were identified and collected for further analysis: a high molecular weight (HMW) fraction, a main fraction containing the mAb peak, and a low molecular weight (LMW) fraction. As shown in Figure 4, the collected SEC fractions were further subjected to LC-MS analysis to compare the number of HCPs identified in each fraction using each mobile phase. In all cases, most HCPs were enriched in the LMW fraction. Based on the total number of HCPs detected, denaturing conditions with 20% acetonitrile were selected for further optimization.

[0181] Example 3. Optimization of a size-exclusion chromatography method for the identification of host cell proteins Based on the results of the analytical scale SEC method described above, the protein loading amount was increased to create a semi-preparative scale SEC method. An exemplary chromatography system suitable for this method is an AKTA Pure 25 chromatography system with a Cytiva Superdex 200 Increase 10 / 300 GL (8.6 μm) column. As shown in Figure 5, the feasibility of the semi-preparative scale method was tested using spiked-in BSA as a standard. Based on the optimization of the method, the final semi-preparative SEC parameters include loading 5 mg of protein (e.g., mAb1) using a denaturing mobile phase containing 20% ​​acetonitrile, a flow rate of 0.5 mL / min, and a fractionation of 0.25 mL / well. As shown in Figure 6 , the fractions of interest from the SEC chromatogram were defined as the HMW fraction from 0.3 column volumes (CV) to the 5 milliabsorbance units (mAU) front, the main fraction from the 5 mAU front to the 40 mAU end, the tail fraction from the 40 mAU front to the 10 mAU end, and the LMW fraction from the 10 mAU front to 1.1 CV.

[0182] The amount of protein in each fraction was estimated using re-injection, as shown in Figure 7A and Figure 7B. Each fraction was injected again on the SEC to confirm the purity of the fraction. Approximately 95% of the mAb was enriched in the main fraction. Approximately 5% of the sample was lost during buffer exchange. HCP was expected to be enriched in the HMW, tail, and LMW fractions.

[0183] The method was further optimized using limited digestion, which has previously been shown to be useful for HCP analysis. To generate limited digestion, the enzyme to protein (substrate) ratio must be optimized. Figure 8 shows the limited digestion test using a range of enzyme to protein ratios. The optimized enzyme to protein ratios selected were 1:200 for the HMW fraction, 1:2000 for the main fraction, 1:500 for the tail fraction, and 1:200 for the LMW fraction. As shown in Figure 9, the optimized limited digestion step was compared to the denaturing digestion step combined with the SEC method of the present invention and was found to show an improved ability to identify HCPs.

[0184] Further optimization of the method of the present invention includes testing to define the tail and LMW fractions at different UV mAU values, which impacts the depletion of antibody drug product from the tested fractions and the sensitivity of HCP detection. As shown in Figure 10, a cutoff of 10 mAU and a cutoff of 3 mAU were evaluated. As shown in Figure 11, the number of HCPs identified using each cutoff was compared. The cutoff of 3 mAU was found to be more effective and was selected for use in the optimized method of the present invention.

[0185] The loading amount of the semi-preparative scale method was further optimized by testing increasing loading amounts of protein. Figure 12 shows the SEC chromatograms of the method using 5 mg protein (small blue peak), 10 mg protein (medium green peak), or 20 mg protein (large black peak). As shown in Figure 13, the number of HCPs identified using loading amounts of 1, 5, 10, or 20 mg was compared. A positive correlation was found between loading amount and the number of HCPs detected, but the maximum loading amount of 20 mg resulted in distorted peak shapes due to overloading. Therefore, 10 mg was selected as the optimized loading amount. A higher overall loading amount can be achieved by loading 10 mg per run.

[0186] The ability of the method of the present invention to detect HCP was compared with the above-mentioned method.As shown in Figure 14, mAb1 sample was subjected to HCP analysis using immunoprecipitation, limited digestion, molecular weight cutoff filtration, ProteoMiner beads, ProteoMiner with limited digestion, or the SEC limited digestion method of the present invention.The method of the present invention was found to be more effective than most of the methods previously reported.

[0187] Further comparisons were made using the protein standard NIST mAb8671. As shown in Figure 15A, a total of 544 HCPs were identified in the NIST mAb sample using the method of the present invention. As shown in Figure 15B, the results were compared to previous data from NIST mAb analysis using the method described above and found to be equally valid.

[0188] Example 4. Size Exclusion Chromatography Method for Host Cell Protein Binding Analysis The advantage of the method of the present invention is the ability to analyze HCPs based on size due to the separation mechanism of SEC. In addition to obtaining information about the size of the protein itself, this provides information about the binding properties of the protein, since a protein bound to another protein will show an apparent change in size. This distinction can be evaluated using a comparison of a protein separated with a native mobile phase, which preserves protein-protein interactions, and the same protein separated with a mildly denaturing mobile phase, such as 20% acetonitrile, which eliminates weak protein-protein interactions without destroying strong protein-protein interactions.

[0189] The sensitivity of the method of the present invention, carried out in two different mobile phases, was evaluated using the UPS2 standard. UPS2 is a commercially available proteomics standard that contains 48 human proteins at concentrations with a wide dynamic range spanning several orders of magnitude. mAb1 was fractionated using the optimized conditions described above with one of two different mobile phases (either the native non-denaturing mobile phase or the denaturing mobile phase with 20% acetonitrile). Table 1 shows the number of UPS2 proteins detected in each condition at each concentration level. Ppm is given as molar ratio.

[0190] [Table 1]

[0191] As shown in Figure 16A, the number of HCPs from each fraction of the mAb1 sample detected by the method of the present invention was compared using either the native mobile phase or the 20% acetonitrile mobile phase. In the HMW and main fractions, more HCPs were detected using the native mobile phase, while in the tail and LMW fractions, more HCPs were detected using the 20% acetonitrile mobile phase. Figure 16B shows the overlap of the total HCPs detected in each condition.

[0192] The HCPs identified in each condition were further characterized by molecular weight, as shown in Figures 17A and 17B. Overall, there was a trend for molecular weight to decrease from the HMW fraction to the LMW fraction, consistent with the separation mechanism of SEC. However, the HMW fraction also contained proteins with lower molecular weights than expected (e.g., less than 100 kDa). This suggests that binding to mAb1 may result in shorter retention on the SEC column.

[0193] The apparent sizes of each HCP identified by SEC under native (non-denaturing) or denaturing conditions were compared to generate a binding profile of all detected HCPs, as shown in Table 2.

[0194] [Table 2] TIFF2025510508000003.tif215166TIFF2025510508000004.tif206170TIFF202 5510508000005.tif189170TIFF2025510508000006.tif181170TIFF20255105080 00007.tif199169TIFF2025510508000008.tif215167TIFF2025510508000009.t if185169TIFF2025510508000010.tif190169TIFF2025510508000011.tif163166

[0195] Comparison of the SEC separation of each HCP under each condition can be used to estimate the binding properties of each HCP to the biotherapeutic. For example, as shown in FIG. 18A, HCPs found in the HMW fraction under both native and denaturing conditions can be inferred to have relatively strong binding to mAb1 that could not be dissociated by mild denaturation with 20% acetonitrile. As shown in FIG. 18B, HCPs found in the LMW fraction under both conditions can be inferred to have effectively no binding to mAb1. As shown in FIG. 18C, HCPs found in the HMW fraction under native conditions but shifted to the previous fraction under denaturing conditions can be inferred to have relatively weak binding to mAb1 that was disrupted by 20% acetonitrile. HCPs found in the mixture of HMW and other fractions under both conditions can be inferred to have relatively weak binding to mAb1 that was not affected by 20% acetonitrile. Finally, HCPs found primarily in the HMW fraction under denaturing conditions but not under native conditions can be inferred to have binding that is re-equilibrated by acetonitrile.

[0196] This analysis can be used to generate a binding profile of a particular HCP of interest for a given biotherapeutic. Figure 19 shows the binding profile of an HCP of interest in mAb1 drug substance. For example, it was found that cathepsin D binds to mAb1 during the purification process, but this interaction is weak and can be disrupted using 20% ​​acetonitrile. This HCP binding profile analysis can be used to inform the process development of any biotherapeutic.

[0197] Example 5. Detergent-assisted size-exclusion chromatography method for the analysis of host cell proteins Based on the information provided by the HCP binding profile analysis described above, a detergent-assisted dissociation method was developed to enrich HCPs from biotherapeutic samples.

[0198] As shown in Figure 20, HCPs may exist in equilibrium between bound and unbound states of the biotherapeutic. To disrupt this equilibrium of bound states and stabilize the unbound states, thereby preventing HCPs from co-purifying with the biotherapeutic, various surfactants were tested as part of the biotherapeutic manufacturing process. As shown in Figure 21, SEC separation profiles of mAb1 samples were generated using either a native mobile phase or a mobile phase containing 12 mM sodium lauroyl sarcosinate (SLS) and 12 mM sodium deoxycholate (SDC). A shift in retention time was observed, indicating partial denaturation of the antibody and / or formation of micelles / liposomes.

[0199] High levels of surfactants can adversely affect digestion and MS analysis. Surfactants were found to be difficult to remove during buffer exchange. As shown in FIG. 22, an optimized sample preparation method was developed to solve the problem of effective surfactant removal before digestion and MS analysis. An acid precipitation step using about 1% trifluoroacetic acid (TFA) was used to remove surfactants before the buffer exchange step (which successfully removed SLS and SDC). This is referred to as the "surfactant-assisted" or "surfactant-assisted" SEC method of the present invention.

[0200] The effect of acid precipitation on HCP enrichment and analysis was further evaluated. As shown in Figure 23, acid precipitation resulted in partial removal of mAb1. As shown in Figures 24A and 24B, the ability of the optimized surfactant-assisted method using SLS and SDC together with acid precipitation to detect HCPs in mAb1 samples was compared to the method using native or ACN mobile phase. A higher number of HCPs were detected using the surfactant-assisted method compared to using native or ACN mobile phase.

[0201] As shown in Table 3, the sensitivity of the method was tested using the UPS2 standard, and the surfactant-assisted method (SLS+SDC) was shown to be more sensitive than methods using native or ACN mobile phases. Without being bound by theory, it is possible that the acid precipitation step preferentially precipitates mAb1 compared to HCPs due to the larger size of mAb1, thereby further enriching the sample for HCPs. This effect would be expected for any large protein of interest (e.g., an antibody or antibody fusion protein).

[0202] [Table 3]

[0203] As shown in FIG. 25, the surfactant-assisted method shifted the HCPs of interest from the HMW fraction to the LMW fraction, demonstrating successful disruption of HCP binding to the biotherapeutic agent and improved enrichment and analysis of the HCPs.

[0204] Example 6. Optimization of detergent-assisted acid precipitation method for host cell protein analysis FIG. 26 shows the workflow of the surfactant-assisted acid precipitation method used to enrich and identify HCPs in biotherapeutic samples. In the acid precipitation step, the biotherapeutic samples were incubated with surfactant followed by acid and centrifuged to remove the surfactant and biotherapeutic. Ultraviolet absorption at 280 nm (UV A280) was used to determine the protein concentration of the samples after acid precipitation. This served as an indication of the effectiveness of the acid precipitation parameters. The sample buffer was then exchanged and the biotherapeutic samples were digested, desalted, and analyzed using nano-liquid chromatography-mass spectrometry and Protein Metrics' Byonic. The clarity of the spectra and identification of host cell proteins in the total ion chromatogram also served as an indication of the effectiveness of the acid precipitation parameters.

[0205] The effect of various dissociation reagents on proteins in biotherapeutic samples subjected to acid precipitation was evaluated. The detergents sodium deoxycholate, sodium lauroyl sarcosinate, and n-dodecyl-β-D-maltoside can be used as dissociation reagents. Figures 27A and 27B show that acid precipitation depleted 99.92%, 99.98%, and 11.38% of proteins in biotherapeutic samples incubated in 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, and 40 mM n-dodecyl-β-D-maltoside, respectively. Additionally, 6 M urea, 8 M guanidine, and 5 mM acetic acid (heated) can be used as dissociation reagents. Figures 27A and 27B show that acid precipitation depleted 16.6%, 10.02%, and 15.58% of protein in biotherapeutic samples incubated in 6 M urea, 8 M guanidine, and 5 mM acetic acid (heated), respectively. These data indicate that sodium deoxycholate and sodium lauroyl sarcosinate are unique surfactants that can be used in conjunction with acid precipitation to deplete protein in biotherapeutic samples.

[0206] The effect of sodium deoxycholate and sodium lauroyl sarcosinate concentration on protein depletion in biotherapeutic samples subjected to acid precipitation was examined. Each biotherapeutic sample was 500 μL and contained 5 mg of biotherapeutic. It was incubated for 2 hours in 0 mM, 20 mM, 60 mM, 100 mM, or 120 mM sodium deoxycholate or sodium lauroyl sarcosinate and 30 minutes in 10% acid (v / v). Figure 28 shows that acid precipitation more effectively depleted protein in biotherapeutic samples incubated in sodium deoxycholate or sodium lauroyl sarcosinate. Figures 29 and 30 show that incubating the biotherapeutic samples in 10% acid (v / v) for 30 minutes can deplete 99.5% of protein in the biotherapeutic samples after incubation for 2 hours in 40 mM sodium deoxycholate and 40 mM sodium lauroyl sarcosinate, respectively.

[0207] The effect of incubation time of sodium deoxycholate and sodium lauroyl sarcosinate on protein depletion in biotherapeutic samples subjected to acid precipitation was assayed. Each biotherapeutic sample was incubated in 20 mM sodium deoxycholate or 20 mM sodium lauroyl sarcosinate for 5, 15, 30, 60, or 120 minutes prior to acid precipitation. Figure 31 shows that the longer the sample was incubated in 20 mM sodium deoxycholate or 20 mM sodium lauroyl sarcosinate, the more effectively the proteins depleted by acid precipitation were depleted, especially for incubation times up to about 60 minutes, with more than 99.5% of the proteins being depleted.

[0208] The effect of the pH of the acid precipitation on protein depletion in biotherapeutic samples was examined. Each biotherapeutic sample was incubated in 20 mM sodium deoxycholate or 20 mM sodium lauroyl sarcosinate and 0%, 2.5%, 5%, 7.5%, or 10% (v / v) 10% trifluoroacetic acid. Figure 32 shows that lowering the pH of the acid precipitation using a higher percentage (v / v) of 10% trifluoroacetic acid more effectively depleted protein, especially for percentages (v / v) of 10% trifluoroacetic acid up to about 7.5%, and using more than 7.5% (v / v) of 10% trifluoroacetic acid for precipitation depleted more than 99.5% of protein.

[0209] The effect of acid incubation time on protein depletion in biotherapeutic protein samples was evaluated. Each biotherapeutic sample was incubated in acid for 5 minutes or 60 minutes. Figures 33A and 33B show that incubating the biotherapeutic sample in acid for more than 5 minutes did not more effectively deplete protein in the sample. Furthermore, Figure 33C shows that incubating the biotherapeutic sample in acid for more than 5 minutes did not allow for the identification of more HCPs.

[0210] The effect of incubating biotherapeutic samples in sodium deoxycholate and sodium lauroyl sarcosinate on the identification of HCPs using surfactant-assisted acid precipitation was assayed. Each biotherapeutic sample was incubated in 40 mM sodium deoxycholate, 40 mM sodium lauroyl sarcosinate, or both 20 mM sodium deoxycholate and 20 mM sodium lauroyl sarcosinate, and subjected to acid precipitation, followed by identification of HCPs using nano liquid chromatography-mass spectrometry and Protein Metrics' Byonic. Figure 34 shows that surfactant-assisted acid precipitation identified 109, 103, and 43 HCPs in biotherapeutic samples incubated in 40 mM sodium lauroyl sarcosinate, 20 mM sodium deoxycholate and both 20 mM sodium deoxycholate, and 40 mM sodium deoxycholate, respectively. These data show that incubation of biotherapeutic samples in sodium lauroyl sarcosinate allows for the identification of most HCPs using surfactant-assisted acid precipitation, followed by incubation of biotherapeutic samples in both sodium deoxycholate and sodium lauroyl sarcosinate, and sodium deoxycholate, respectively. In comparison, Figure 35 shows that the methods of ProteoMiner with limited digestion, ProteoMiner, molecular weight cutoff filtration, limited digestion, and immunoprecipitation identified 199, 113, 63, 53, and 32 HCPs, respectively, in the same biotherapeutic samples, and 104 of the HCPs were not identified using surfactant-assisted acid precipitation after incubation in 40 mM sodium lauroyl sarcosinate. These data suggest that HCPs identified using surfactant-assisted acid precipitation after incubation in sodium lauroyl sarcosinate may have unique properties that prevent detection using other methods.

[0211] The HCPs identified using surfactant-assisted acid precipitation after incubation in 40 mM sodium lauroyl sarcosinate were characterized. Figure 36 shows that the HCPs identified using surfactant-assisted acid precipitation after incubation in 40 mM sodium lauroyl sarcosinate were generally more acidic and hydrophilic than the HCPs identified using limited digestion, molecular weight cutoff filtration, and ProteoMiner methods. Additionally, Figure 37 shows that the HCPs identified using surfactant-assisted acid precipitation after incubation in 40 mM sodium lauroyl sarcosinate were generally larger (e.g., greater than 50 kDa) than the host cell proteins identified using limited digestion, molecular weight cutoff filtration, and ProteoMiner methods. These data suggest that the properties of HCPs identified using detergent-assisted acid precipitation after incubation in 40 mM sodium lauroyl sarcosinate may affect their detection or depletion using other HCP identification methods. For example, Figure 38 shows that incubation of four different biotherapeutic samples in 40 mM sodium lauroyl sarcosinate and acid precipitation resulted in 92.22%, 99.3%, 99.92%, and 99.98% protein depletion. These data suggest that the efficacy of protein depletion in biotherapeutic samples using 40 mM sodium lauroyl sarcosinate and acid precipitation depends on the physiochemical properties of the biotherapeutic, and that the efficacy of protein depletion may affect the detection of HCPs.

[0212] Example 7. HCP profiling in enriched HMW fractions To further characterize HCP contaminants in recombinant protein samples (e.g., monoclonal antibody drug substances), a novel method was developed to identify, characterize, and remove HCPs from the enriched HMW fraction of the drug substance. This example illustrates the workflow of an exemplary method taught in the subsequent examples.

[0213] Materials. Monoclonal antibodies DS and concentrated HMW were produced in-house. 10 KDa filters were purchased from Pall (New York, NY) and 3K Amicon ultracentrifugal filters were obtained from MilliporeSigma (Burlington, MA). Dithiothreitol (DTT) and iodoacetamide (IAM) were purchased from Thermo Scientific (Rockford, IL). Sequencing grade modified trypsin was purchased from Promega (Madison, WI). Tris-HCl buffer (pH 7.5) was purchased from Invitrogen (Carlsbad, CA). Urea and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO). PNGase F was purchased from New England Biolabs (Ipswich, MA). LC-MS grade 0.1% formic acid in water and 0.1% formic acid in acetonitrile were obtained from Honeywell (Charlotte, NC).

[0214] Trypsin digestion of DS and enriched HMW species. Enriched HMW species were generated in-house through preparative SEC of DS. HCP analysis of mAb and enriched HMW fractions was performed using a native digestion approach similar to previously published methods (Huang et al.) with optimized enzyme-to-substance ratios and spiked recombinant protein internal standards. Briefly, all samples were buffer-exchanged into water using 3KDa centrifugal filters. The concentration of each sample was measured by nanodrop after buffer exchange. 2mg of each sample was then spiked with two internal protein standards, recombinant human IL33 and heavy isotope-labeled CHO putative phospholipase B-like 2 (PLBD2), at known concentrations. Samples were then digested with trypsin (enzyme:substrate ratio of 1:1000 w / w) overnight at 37°C. After digestion, disulfide bonds were reduced by adding 2μL of 25mg / mL DTT and incubated at 90°C for 20 minutes. The reduced samples were further alkylated by adding 2 μL of 0.25 M IAM for 30 min at room temperature in the dark. The final digest was filtered through a 10 KDa MW molecular weight cutoff filter. The flow-through was acidified with 5 μL of 10% formic acid and collected for analysis.

[0215] Fraction collection. Enriched dimer, monomer, and low molecular weight fractions from DS were prepared through SEC. PNGase F was added to 100 μg of DS sample for deglycosylation (1:5 w / w enzyme to protein ratio) for 1 hour. Proteins were separated and collected on a Waters ACQUITY UPLC system equipped with an ACQUITY UPLC Protein BEH SEC column (200 Å, 1.7 μm, 4.6 mm×300 mm) and operated with an isocratic flow of 0.2 mL / min using a mobile phase of 150 mM ammonium acetate. The UV detector was set at 280 nm wavelength.

[0216] Trypsin digestion of fractionated samples. Each fraction was spiked with 0.01 μg of heavy isotope-labeled PLBD2 as an internal standard. All samples were dried in a SpeedVac instrument and then reconstituted in 20 μL of denaturation buffer consisting of 0.1 M Tris-HCl (pH 7.5), 8 M urea, and 10 mM DTT. Samples were denatured and reduced at 56° C. for 30 min, then alkylated with 50 mM IAM for 30 min at room temperature in the dark. Samples were then digested with 100 μL of 20 ng / μL trypsin and incubated overnight at 37° C. Digestion was quenched by adding 5 μL of acetic acid.

[0217] LC-MS / MS analysis. The digested samples were analyzed on a Waters ACQUITY UPLC system interfaced with a Thermo Scientific Q Exactive Plus mass spectrometer. The UPLC system was equipped with a Waters CSH C18 column (1.7 μm particle size, 2.1 mm × 150 mm). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 0.25 mL / min, and a linear LC gradient was set as follows: 0.1% B from 0 to 5 min, 32% B from 85 min, 40% B from 90 min, 90% B from 95 to 105 min, and 0.1% B from 106 to 125 min. Mass spectral data acquisition was performed using the top 10 DDA method. The MS1 scan range was 300-1500 m / z at 70k resolution (m / z 400). The MS / MS isolation window was set at 3 m / z and the nominal collision energy (NCE) was set at 28. The minimum automatic gain control (AGC) was set at 5e4 and the maximum IT was 300 ms.

[0218] Directly digested samples were analyzed on an UltiMate 3000 RSLCnano system (Thermo Scientific) connected to a Q-Exactive HFX mass spectrometer (Thermo Scientific). The RSLCnano system was equipped with an Acclaim PepMap 100 75 μm × 2 cm trap column and an Acclaim PepMap 75 μm × 25 cm C18 analytical column. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in 80% acetonitrile. The flow rate was 300 nL / min and a linear LC gradient was set as follows: 3% B from 0 to 5 min, 25% B from 40 min, 37% B from 48 min, 95% B from 53 to 58 min, and 3% B from 58 to 60 min. Each sample was analyzed in a full mass scan followed by parallel reaction monitoring (PRM) mode. A single full mass scan was set at 120k resolution, AGC target 1e6, max IT 60ms, and scan range 350-2000m / z. PRM was set at 30K resolution with an isolation window of 3m / z, NCE set to 27, AGC 2e5, max IT 150ms.

[0219] Data analysis. Raw files were searched against the Uniprot Cricetulus griseus proteome database using SEQUEST and Mascot (Matrix Science) via Proteome Discoverer 2.2 (Thermo Scientific). Precursor mass tolerance was set to 10 ppm and fragment ion mass tolerance was set to 0.02 Da. Trypsin was set as the digestion enzyme. Methionine oxidation was set as the dynamic modification and cysteine ​​alkylation was set as the static modification. Proteins with a minimum of two unique peptides detected and with high quality MS / MS spectra and peptide lengths >6 amino acids were filtered as true positives. Skyline was used for peak integration and further analysis.

[0220] For native digested samples, heavy isotope-labeled PLBD2 was used as a qualitative control to verify digestion efficiency and HCP signal intensity. The relative abundance of each identified protein was quantified by averaging the peak areas of the top 2-3 peptides of each host cell protein and dividing by the average abundance from the top 3 peptides of recombinant human IL33 spiked-in with internal standard at 50 ppm (micromoles of standard to moles of antibody). All results were calculated as molar ratio ppm.

[0221] For samples analyzed by PRM, the relative abundance of each protein was quantified by averaging the product ion areas of peptides for each host cell protein and dividing by the average abundance of the top three peptides of heavy isotope-labeled PLBD2 spiked in at 0.01 μg of internal standard for each sample. Results were calculated in fmol.

[0222] Experimental Design HCP analysis was performed on five in-house generated mAbs and their enriched HMW species. The DS and enriched HMW species were subjected to buffer exchange and concentrations were determined prior to sample analysis. Sample preparation was performed using the optimized native digestion method introduced by Huang in 2017. The native digestion method is adopted in many HCP workflows in industry as it is simple and effective in reducing the dynamic range of the analytes. Briefly, a small amount of trypsin was added to the samples without a denaturation step. The antibodies were minimally digested under native conditions, whereas the HCPs were preferentially digested. After digestion, the samples were denatured with a heating step and the undigested antibodies were removed by precipitation. The HCP peptides were injected into an LC-MS / MS instrument for analysis with minimal matrix interference. The ratio of the top three most abundant peptides from the HCPs to the spiked-in standards was used to calculate the levels of HCPs in molar ratio ppm (micromoles of HCP to moles of antibody). Reporting the molar ratio between HCP and antibody allows for a fair comparison by taking into account the broad molecular weight distribution of HCPs present in the samples.

[0223] Example 8. HCP profiling of DS and enriched HMW species from five mAbs The number of HCPs (>0.1 ppm) identified across all samples using the workflow described in Example 7 is shown in Figure 39. For each mAb, the blue bar on the left represents the number of HCPs identified from DS, and the red bar on the right represents the number of HCPs identified from concentrated HMW. For all five mAbs, more HCPs were detected from concentrated HMW than from DS. A total of 110, 110, 105, 100, and 101 HCPs were identified in the concentrated HMW fractions from mAb2 to mAb6, respectively. In contrast, only 26, 68, 76, 64, and 58 HCPs were identified from mAb2-mAb6 DS. The total number of HCPs identified from HMW differed between samples. This indicates the molecular dependency of HCP enrichment in HMW. Notably, for mAb2, the number of HCPs identified was 4 times higher in concentrated HMW than in DS.

[0224] The 25 most frequently identified HCPs and their estimated abundance in ppm molar ratio are listed in Figure 40. HCPs are listed in order of abundance of HMW species in mAb2. The heatmap is color-coded by HCP abundance ranging from 0 ppm (blue, undetected) to >100 ppm (red).

[0225] Most of the commonly identified HCPs were preferentially enriched in HMW for all five mAbs. For example, CC motif chemokines were the predominant HCPs in mAb2 at 37.4 ppm and were identified in enriched HMW at higher abundance than DS across all five mAbs. mAb2 HMW showed significantly higher CC motif chemokine levels (1738.5 ppm) than other mAb HMW fractions (20.9-61.6 ppm). Inhibitor of metalloproteinases 1 (TIMP1) was a frequently identified HCP from different processing steps (Singh et al., 2020, Biotechnology Prog, 36(2):e2936; Park et al., 2017, Sci Rep, 10(7):44246), with levels of 0.2 ppm in mAb2 DS and 11.4 ppm in mAb4 DS. In enriched HMW, the abundance of TIMP1 was 3-13 times higher than that in DS. β-hexosaminidase, which is reported to be associated with N-glycan degradation when present at levels of hundreds of ppm (Li et al., 2021, Biotechnol Prog, 37(3):e3128), was not detected in most mAb DS samples but was at elevated levels in enriched HMW. Other frequently identified examples of HCPs include cornifin-A, peroxiredoxin, and complement C1r-A subcomponent, which had higher abundance in enriched HMW than in DS.

[0226] Lipases are a problematic group of host cell proteins that can degrade polysorbates, thus inducing the formation of aggregates and affecting the shelf life of medical products (Chiu et al.; McShan et al.; Zhang et al. 2020; Zhang et al. 2021). Sialic acid-O-acetyl esterase, lipoprotein lipase, and other lipases showed higher abundance in concentrated HMW than in DS, while PLBD2 was below the detection limit across all five mAbs and was present at trace levels in concentrated HMW. Overall lipase levels were below 3 ppm in the five mAbs, thus indicating that the purification process effectively removed this group of HCPs.

[0227] Protease enzyme activity is involved in protein degradation, inducing the formation of small fragments, which may affect product quality. Cathepsin L1, which has been reported to induce fragmentation of mAbs (Luo et al.), was detected at significantly higher levels in concentrated HMW than in DS for all five mAbs. Notably, the level of cathepsin L1 in DS was less than 0.8 ppm for all mAb DS. Carboxypeptidase, another protease that cleaves both lysine and arginine at the C-terminus, was mainly detected in concentrated HMW for mAb2. Cathepsin D, which adversely affects product stability by causing mAb fragmentation and particle formation (Bee et al., 2015, Biotechnol Prog, 31(5):1360-1369), was not detected in DS and was less than 0.3 ppm in concentrated HMW.

[0228] Previous studies have shown that HCPs circumvent multiple purification processes and exist as "hitchhiker" proteins in the final medical product (Ranjan et al., 2019, Biotechnol Bioeng, 116(7):1684-1697). These HCPs are likely to have strong specific or non-specific interactions with mAbs (Bee et al., 2017, Biotechnol Prog, 33(1):140-145) and cannot be removed by the final purification step based on their chemical properties. The present disclosure shows that certain HCPs specifically or non-specifically associate with mAb HMW species, since the abundance of HCPs in concentrated HMW was substantially higher than the abundance in DS. In addition, these results show that the interaction between HCPs and HMW species is highly molecular dependent, since the abundance of the same HCP varies between different mAbs.

[0229] Example 9. Characterization of host cell proteins in concentrated HMW Determining which specific properties of HCPs may contribute to enrichment in the high molecular weight fraction of biotherapeutic products is important to inform process development. Thus, the predicted isoelectric point (pI) and molecular weight (MW) distributions of all HCPs across the analyzed enriched HMW of all five studied mAbs are shown in Figure 41B and compared to the properties of HCPs from the DS of all five studied mAbs (shown in Figure 41A). It was assumed that the high MW HCPs would co-elute with the HMW mAb species. However, most HCPs that were enriched in the HMW fraction had a MW between 10 kDa and 100 kDa, lower than the MW of the mAbs. Thus, the majority of HCPs were retained in the HMW fraction due to interactions with the HMW mAb species, rather than their own size. The pI distributions of HCPs identified from HMW were comparable to those identified from DS.

[0230] As mentioned previously, CC motif chemokines had the highest relative abundance in mAb2, being 46-fold more abundant in the enriched HMW than in DS. CC motif chemokines belong to the chemokine family and play a role in immune and inflammatory responses. Based on the known characteristics and measured abundance of this HCP, it was determined that the relatively high levels of CC motif chemokines in the mAb2 DS could be a safety concern. To understand how this HCP is retained in the final DS, the chemical properties of the CC motif chemokines and the detailed purification steps of mAb2 were examined.

[0231] CC motif chemokines are small HCPs with a molecular weight of 15.8 kDa and an estimated pI of 9.16. The mAb can be first purified by affinity chromatography to capture the mAb, wash away process impurities, and effectively remove HCPs that are not bound to the mAb or resin. In the following final purification step, acidic HCPs in the Protein A eluate attach to the AEX ligand, which is positively charged at neutral pH, and are removed. Basic HCPs, such as CC motif chemokines, are positively charged at neutral pH and co-elute with the mAb in the flow-through mode of AEX purification. In the HIC purification step, CC motif chemokines are likely to flow-through with the mAb, while very hydrophobic impurities bind to the column. Therefore, CC motif chemokines, when present in the Protein A eluate, cannot be effectively removed by conventional mAb purification steps based on their chemical properties. For HCPs such as CC motif chemokines, further analysis and potentially further purification steps may be required for optimal production of biotherapeutic products.

[0232] Example 10. Comparison of CC motif chemokines in dimer, vHMW, and concentrated HMW from mAb1 The CC motif chemokine had a higher abundance in mAb2 than in other products. Thus, we demonstrate that the presence of this HCP is likely due to a specific interaction with mAb2 and not with the resin. Because the CC motif chemokine was substantially more abundant in the concentrated HMW than in DS, it is possible that an association occurs between the HMW species and this HCP. The HMW fraction contains heterogeneous species, including dimers and vHMW. To determine which specific HMW species are involved in the interaction with the CC motif chemokine, further studies were investigated using additional SEC fractionation of mAb2.

[0233] The mAb2 enriched HMW was further fractionated by another round of SEC to obtain enriched dimer and enriched vHMW fractions. The 20 most abundant HCPs identified from these three fractions are shown in Figure 42. HCPs are listed in order of abundance in the enriched HMW of mAb2. The heatmap is color-coded by HCP levels ranging from 0 ppm (blue, undetected) to over 100 ppm (red).

[0234] A total of 123 HCPs were identified from the dimer fraction compared to 110 and 102 HCPs in the enriched total HMW and vHMW fractions. CC motif chemokine abundance in all three HMW fractions was substantially higher than that in DS, and the levels differed between fractions. The concentrations of CC motif chemokines were 934.5 ppm, 1738.5 ppm, and 7733.9 ppm in the enriched vHMW, total HMW, and dimer fractions, respectively. The dimer fraction had the highest levels of CC motif chemokines, eight-fold higher than the enriched vHMW. Meanwhile, CC motif chemokines in the enriched vHMW fraction were nearly half as abundant as in the enriched total HMW fraction. This finding indicated that CC motif chemokines are most likely associated with dimers rather than vHMW species or total DS.

[0235] In addition to CC motif chemokines, most of the detected HCPs, such as carboxypeptidases and β-hexosaminidases, were specifically enriched in the dimeric fraction, whereas other HCPs, such as connective tissue growth factor, were substantially higher in the vHMW fraction than in the dimeric fraction.

[0236] Example 11. SEC fractionation and PRM analysis Since CC motif chemokines were enriched in the mAb2 HMW fraction, it was reasoned that removal of HMW species may facilitate the clearance of this HCP from the DS. To verify this hypothesis, mAb2 DS was fractionated by SEC. As shown in the inset of Figure 43, HMW species, monomers, and LMW species were obtained after SEC fractionation. The SEC fraction and mAb2 DS were subjected to trypsin digestion and PRM analysis. The level of CC motif chemokines was estimated by comparison with the spiked-in internal standard. Figure 43 shows the distribution of CC motif chemokines in different fractions.

[0237] Approximately 63.8 fmol of CC motif chemokine was present in the unfractionated DS. After SEC column purification, the CC motif chemokine level was reduced to 2.4 fmol in the monomer fraction, which corresponds to 3.2% of the total CC motif chemokine in the three fractions. Interestingly, the LMW fraction contained 25.3% of the CC motif chemokine, which may represent a dissociated species. This finding indicated that under SEC conditions, the CC motif chemokine was partially dissociated and existed in an unbound form. The fraction volume fractionated for LMW was approximately 6-fold higher than that of the monomer, and the level of CC motif chemokine was approximately 8-fold higher in LMW than in DS. The CC motif chemokine present in mAb2 DS may have been in a dissociated form after SEC fractionation. Despite potential dissociation, the levels of most CC motif chemokines were significantly enriched in the HMW fraction, at 53.2 fmol or 71.5% of total HCP.

[0238] These results indicate that HCPs present at worryingly high abundances, such as the CC motif chemokines in the mAb2 DS, as shown in Figure 40, may have specific interactions with HMW species such as antibody dimers, and therefore may be enriched in the HMW fraction of the DS, as shown in Figure 42. Using SEC and LC-MS / MS, it is possible to assess the distribution and abundance of problematic HCPs, determine the specific antibody species involved in the undesired interactions, and use SEC fractionation to substantially remove the problematic HCPs from the DS, as shown in Figure 43. Thus, the present disclosure provides a novel method for improving the manufacturing of biotherapeutic products by specifically removing HCPs that may otherwise cause product instability or immunogenicity.

[0239] HMW species are product-associated variants that may affect the efficacy and safety of therapeutic protein products. Immunogenicity assays have shown that aggregates induce immune responses to FVIII (Reipert et al., 2007, Br J Haematol, 136(1):12-15; Purohit and Balasubramanian, 2008, J Pharm Sci, 95(2):358-371), recombinant human growth hormone (Fradkin and Randolph, 2009, J Pharm Sci, 98(9):3247-3264), and IgG (Joubert et al., 2012, J Biol Chem, 287(30):25266-25279). The mechanism by which HMW species cause immunogenicity is unclear. The above studies demonstrate that high levels of HCPs may be present in the HMW fraction of biotherapeutic product DS. Thus, immunogenicity may also be induced by HCPs in addition to or instead of the HMW therapeutic protein species itself.

[0240] In this study, we performed HCP analysis of HMW fractions separated by SEC. Similar to other chromatographic separation methods, SEC fractionation of HMW resulted in the detection of a higher number of HCPs. Where applicable, SEC can be incorporated as an alternative HCP enrichment strategy for HCP analysis to facilitate the detection of low-abundance HCPs. Unlike HILIC chromatography, which denatures HCPs under high organic gradients, SEC maintains the native state of HCPs and is easily combined with native digestion. Thus, detection sensitivity can be further improved by using native digestion after HCP enrichment.

[0241] Studying the association between HCPs and mAbs is difficult because the level of HCPs is very low compared to that of medical products. In previous studies, cross-interaction chromatography (Levy et al., 2013, Biotechnol Bioeng, 111(5):904-912; Aboulaich et al., 2014, Biotechnol Bioeng, 30(5):1114-1124; Zhang et al., 2016, Biotechnol Prog, 32(3):708-717) or surface plasmon resonance (Bee et al. 2017) have been applied to determine the binding activity between mAbs and HCPs. The results described in this disclosure indicate that the association between HCPs and HMW species can explain why certain HCPs are co-purified with DS. Although the mechanism of interaction remains unclear, these findings suggest a new direction for studying the association of mAbs and HCPs. More importantly, removal of HMW species from the DS can significantly reduce the levels of certain HCPs and provide information to facilitate the development of downstream purification processes.Hence, the HCP profiling of HMW species disclosed herein expands the knowledge of HCPs present in mAb preparations and their mechanisms of interaction, aiding in the understanding of HMW species, immunogenicity, HCP identification, and HCP removal for the overall development of therapeutic protein drugs.

Claims

1. 1. A method for identifying host cell protein (HCP) impurities in a sample, comprising: a) subjecting a sample containing at least one protein of interest and at least one HCP impurity to size exclusion chromatography (SEC) analysis to generate a fraction; b) subjecting said fraction to LC-MS analysis to identify said at least one HCP impurity; A method comprising:

2. 2. The method of claim 1, wherein the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

3. 2. The method of claim 1, wherein the amount of protein loaded onto the SEC column is about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 1 mg, about 5 mg, about 10 mg, or about 20 mg.

4. The method of claim 3, wherein the amount of protein loaded onto the SEC column is about 10 mg.

5. 10. The method of claim 1, wherein the mobile phase for the SEC analysis comprises about 10 mM phosphate and about 150 mM NaCl.

6. The method of claim 1 , wherein the mobile phase for the SEC analysis is a denaturing mobile phase.

7. The method of claim 1 , wherein the mobile phase for the SEC analysis is a non-denaturing mobile phase.

8. 7. The method of claim 6, wherein the mobile phase comprises acetonitrile, and optionally the concentration of the acetonitrile is about 5% v / v to about 20% v / v, about 10% v / v to about 20% v / v, about 15% v / v to about 20% v / v, about 5% v / v, about 10% v / v, about 15% v / v, or about 20% v / v.

9. 9. The method of claim 8, wherein the concentration of acetonitrile is about 20% v / v.

10. 7. The method of claim 6, wherein the mobile phase comprises at least one surfactant, and optionally, the concentration of the at least one surfactant is from about 6 mM to about 36 mM, about 12 mM, about 24 mM, or about 40 mM.

11. The method of claim 10 , wherein the at least one surface-active agent is a surfactant.

12. 12. The method of claim 11, wherein the at least one surfactant is selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof.

13. 13. The method of claim 12, wherein the at least one surfactant is sodium deoxycholate and sodium lauroyl sarcosinate, the concentration of sodium deoxycholate being about 12 mM and the concentration of sodium lauroyl sarcosinate being about 12 mM.

14. 2. The method of claim 1, wherein the fractions include a high molecular weight (HMW) fraction, a main fraction, and a low molecular weight (LMW) fraction.

15. The method of claim 14 , wherein the fractions further comprise a tail fraction.

16. 16. The method of claim 15, wherein the HMW fraction comprises from about 0.3 column volumes (CV) to about 5 milliabsorbance units (mAU) of eluate.

17. The method of claim 15, wherein the main fraction comprises an eluate of about 5 mAU to about 40 mAU.

18. 16. The method of claim 15, wherein the tail fraction comprises from about 40 mAU to about 10 mAU, or from about 40 mAU to about 3 mAU of eluate.

19. 16. The method of claim 15, wherein the LMW fraction comprises from about 10 mAU to about 1.1 CV, or from about 3 mAU to about 1.1 CV of eluate.

20. 2. The method of claim 1, further comprising subjecting said fractions to enzymatic digestion prior to the LC-MS analysis of step (b).

21. 21. The method of claim 20, wherein the enzymatic digestion is a limited digestion.

22. 21. The method of claim 20, wherein the enzymatic digestion is carried out by contacting the fraction with trypsin.

23. 21. The method of claim 20, wherein the enzymatic digestion is carried out by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000.

24. 24. The method of claim 23, wherein the enzyme to protein ratio is about 1:

200.

25. 10. The method of claim 1, further comprising subjecting said fractions to acid precipitation prior to said LC-MS analysis in step (b).

26. 26. The method of claim 25, wherein the acid precipitation comprises contacting the fraction with about 1% trifluoroacetic acid.

27. 2. The method of claim 1(b), wherein the liquid chromatography comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

28. The method of claim 1 , wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

29. 1. A method for identifying host cell protein (HCP) impurities in a sample, comprising: a) subjecting a sample containing at least one protein of interest and at least one HCP impurity to size exclusion chromatography (SEC) analysis to generate a fraction, wherein a mobile phase for said SEC analysis comprises about 12 mM sodium lauroyl sarcosinate and about 12 mM sodium deoxycholate; b) subjecting the fraction to acid precipitation to produce a detergent-depleted fraction, the acid precipitation comprising contacting the fraction with about 1% trifluoroacetic acid; c) subjecting the detergent-depleted fraction to buffer exchange to generate a buffer exchanged fraction; d) subjecting the buffer exchanged fraction to limited digestion to produce a peptide digest, said limited digest comprising contacting the buffer exchanged fraction with trypsin at an enzyme to substrate ratio of about 1:200 to about 1:2000; e) subjecting said peptide digest to LC-MS analysis to identify said at least one HCP impurity; A method comprising:

30. 30. The method of claim 29, wherein the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

31. 30. The method of claim 29, wherein the amount of protein loaded onto the SEC column is about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 1 mg, about 5 mg, about 10 mg, or about 20 mg.

32. 30. The method of claim 29, wherein the amount of protein loaded onto the SEC column is about 10 mg.

33. 30. The method of claim 29, wherein the fractions comprise a high molecular weight (HMW) fraction, a main fraction, and a low molecular weight (LMW) fraction.

34. 34. The method of claim 33, wherein the fractions further comprise a tail fraction.

35. 35. The method of claim 34, wherein the HMW fraction comprises from about 0.3 column volumes (CV) to about 5 milliabsorbance units (mAU) of eluate.

36. 35. The method of claim 34, wherein the main fraction comprises an eluate of about 5 mAU to about 40 mAU.

37. 35. The method of claim 34, wherein the tail fraction comprises an eluate of about 40 mAU to about 10 mAU, or about 40 mAU to about 3 mAU.

38. 35. The method of claim 34, wherein the LMW fraction comprises from about 10 mAU to about 1.1 CV, or from about 3 mAU to about 1.1 CV of eluate.

39. 30. The method of claim 29(e), wherein the liquid chromatography comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

40. 30. The method of claim 29, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

41. 1. A method for characterizing the binding of host cell protein (HCP) impurities to a protein of interest, comprising: a) obtaining a sample containing a protein of interest and at least one HCP impurity; b) subjecting the sample to size exclusion chromatography (SEC) analysis using a non-denaturing mobile phase to generate a native fraction; c) subjecting the sample of (a) to SEC analysis using a denaturing mobile phase to generate a denatured fraction; d) subjecting the native and denatured fractions to LC-MS analysis to generate native and denatured separation profiles of the at least one host cell protein impurity; e) comparing the native separation profile to the denatured separation profile to characterize the binding of the at least one HCP impurity to the protein of interest; A method comprising:

42. 42. The method of claim 41, wherein the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

43. 42. The method of claim 41, wherein the amount of protein loaded onto the SEC column is about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 8 mg to about 12 mg, about 1 mg, about 5 mg, about 10 mg, or about 20 mg.

44. 44. The method of claim 43, wherein the amount of protein loaded onto the SEC column is about 10 mg.

45. 42. The method of claim 41 , wherein the mobile phase comprises about 10 mM phosphate and about 150 mM NaCl.

46. 42. The method of claim 41 , wherein the denaturing mobile phase is a mildly denaturing mobile phase.

47. 42. The method of claim 41, wherein the denaturing mobile phase comprises acetonitrile, optionally at a concentration of about 5% v / v to about 20% v / v, about 10% v / v to about 20% v / v, about 15% v / v to about 20% v / v, about 5% v / v, about 10% v / v, about 15% v / v, or about 20% v / v.

48. 48. The method of claim 47, wherein the concentration of acetonitrile is about 20% v / v.

49. 42. The method of claim 41, wherein the fractions comprise a high molecular weight (HMW) fraction, a main fraction, and a low molecular weight (LMW) fraction.

50. 50. The method of claim 49, wherein the fractions further comprise a tail fraction.

51. 51. The method of claim 50, wherein the HMW fraction comprises from about 0.3 column volumes (CV) to about 5 milliabsorbance units (mAU) of eluate.

52. 51. The method of claim 50, wherein the main fraction comprises an eluate of about 5 mAU to about 40 mAU.

53. 51. The method of claim 50, wherein the tail fraction comprises from about 40 mAU to about 10 mAU, or from about 40 mAU to about 3 mAU of eluate.

54. 51. The method of claim 50, wherein the LMW fraction comprises from about 10 mAU to about 1.1 CV, or from about 3 mAU to about 1.1 CV of eluate.

55. 42. The method of claim 41, further comprising subjecting said fractions to enzymatic digestion prior to said LC-MS analysis in step (d).

56. 56. The method of claim 55, wherein the enzymatic digestion is a limited digestion.

57. 56. The method of claim 55, wherein the enzymatic digestion is carried out by contacting the fraction with trypsin.

58. 56. The method of claim 55, wherein the enzymatic digestion is carried out by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000.

59. 59. The method of claim 58, wherein the enzyme to protein ratio is about 1:

200.

60. 41(d) The method of claim 41(d), wherein the liquid chromatography comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

61. 42. The method of claim 41, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

62. 1. A method for identifying host cell protein (HCP) impurities in a sample, comprising: a) combining a sample containing at least one protein of interest and at least one HCP impurity with a dissociation reagent to generate a first combination; b) subjecting said first combination to acid precipitation to generate a dissociation reagent-depleted fraction; c) subjecting said dissociation reagent-depleted fraction to liquid chromatography-mass spectrometry analysis to identify said at least one HCP impurity; A method comprising:

63. 63. The method of claim 62, wherein the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

64. 63. The method of claim 62, wherein the sample is incubated in the dissociation reagent for about 5 minutes to about 120 minutes, about 15 minutes, about 30 minutes, about 60 minutes, or about 120 minutes.

65. 65. The method of claim 64, wherein the dissociation reagent comprises at least one surfactant, and optionally the concentration of the at least one surfactant is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

66. 66. The method of claim 65, wherein the at least one surface active agent is a surfactant.

67. 67. The method of claim 66, wherein the at least one surfactant is selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof.

68. 67. The method of claim 66, wherein the at least one surfactant is sodium lauroyl sarcosinate and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

69. 67. The method of claim 66, wherein the at least one surfactant is sodium deoxycholate and sodium lauroyl sarcosinate, and the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM, and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

70. 67. The method of claim 66, wherein the at least one surfactant is sodium deoxycholate and the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

71. 67. The method of claim 66, further comprising subjecting said dissociation reagent-depleted fraction to enzymatic digestion prior to said liquid chromatography-mass spectrometry analysis in step (c) to produce peptide digests.

72. 72. The method of claim 71, wherein the enzymatic digestion is a limited digestion.

73. 72. The method of claim 71, wherein the enzymatic digestion is carried out by contacting the dissociation reagent-depleted fraction with trypsin.

74. 72. The method of claim 71, wherein said enzymatic digestion is carried out by contacting said dissociation reagent-depleted fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000.

75. 75. The method of claim 74, wherein the enzyme to protein ratio is about 1:

200.

76. 72. The method of claim 71, further comprising desalting said peptide digests prior to said liquid chromatography-mass spectrometry analysis in step (c).

77. 63. The method of claim 62, wherein the acid precipitation is incubated for about 5 minutes to about 60 minutes, about 5 minutes, or about 60 minutes.

78. 78. The method of claim 77, wherein the acid precipitation comprises contacting the first combination with about 2.5% to about 10% trifluoroacetic acid, about 2.5% trifluoroacetic acid, about 5% trifluoroacetic acid, about 7.5% trifluoroacetic acid, or about 10% trifluoroacetic acid.

79. 63. The method of claim 62, wherein the liquid chromatography comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

80. 63. The method of claim 62, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or an orbitrap-based mass spectrometer, and the mass spectrometer is connected to a liquid chromatography system.

81. 1. A method for identifying host cell protein (HCP) impurities in a sample, comprising: a) combining a sample containing at least one protein of interest and at least one HCP impurity with a dissociation reagent to generate a first combination; b) subjecting said first combination to acid precipitation to generate a dissociation reagent-depleted fraction; c) subjecting said dissociation reagent-depleted fraction to buffer exchange to generate a buffer exchanged fraction; d) subjecting the buffer exchanged fraction to enzymatic digestion to produce a peptide digest; e) subjecting said peptide digests to liquid chromatography-mass spectrometry analysis to identify said at least one HCP impurity; A method comprising:

82. 82. The method of claim 81, wherein the at least one protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

83. 82. The method of claim 81, wherein the dissociation reagent is at least one surfactant, and optionally, the concentration of the at least one surfactant is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

84. 84. The method of claim 83, wherein the at least one surface active agent is a surfactant.

85. 85. The method of claim 84, wherein the at least one surfactant is selected from the group consisting of sodium deoxycholate, sodium lauroyl sarcosinate, and combinations thereof.

86. 86. The method of claim 85, wherein the first combination is incubated for about 5 minutes to about 120 minutes, about 5 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes prior to the acid precipitation of step (b).

87. 85. The method of claim 84, wherein the at least one surfactant is sodium lauroyl sarcosinate and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

88. 85. The method of claim 84, wherein the at least one surfactant is sodium deoxycholate and sodium lauroyl sarcosinate, and the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM, and the concentration of sodium lauroyl sarcosinate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

89. 85. The method of claim 84, wherein the at least one surfactant is sodium deoxycholate and the concentration of sodium deoxycholate is about 20 mM to about 120 mM, about 20 mM, about 40 mM, about 60 mM, about 100 mM, or about 120 mM.

90. 82. The method of claim 81 , wherein the enzymatic digestion is a limited digestion.

91. 82. The method of claim 81 , wherein the enzymatic digestion is carried out by contacting the fraction with trypsin.

92. 82. The method of claim 81, wherein the enzymatic digestion is carried out by contacting the fraction with a digestive enzyme at an enzyme to protein ratio of about 1:100 to about 1:2000, about 1:200 to about 1:2000, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:1000, or about 1:2000.

93. 93. The method of claim 92, wherein the enzyme to protein ratio is about 1:

200.

94. 82. The method of claim 81, further comprising desalting said peptide digests prior to said liquid chromatography-mass spectrometry analysis in step (e).

95. 82. The method of claim 81, wherein the acid precipitation is incubated for about 5 minutes to about 60 minutes, about 5 minutes, or about 60 minutes.

96. 82. The method of claim 81, wherein the acid precipitation comprises contacting the first combination with about 2.5% to about 10% trifluoroacetic acid, about 2.5% trifluoroacetic acid, about 5% trifluoroacetic acid, about 7.5% trifluoroacetic acid, or about 10% trifluoroacetic acid.

97. 82. The method of claim 81, wherein the liquid chromatography comprises reverse phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

98. 1. A method for producing a biotherapeutic product, comprising: (a) subjecting a first sample, comprising at least one protein of interest and at least one host cell protein (HCP) impurity, to size exclusion chromatography (SEC) analysis to generate a plurality of fractions; (b) subjecting the plurality of fractions to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to determine the identity and amount of the at least one HCP impurity; (c) using the identity and amount to determine whether the at least one HCP impurity is an impurity of concern in at least one of the plurality of fractions; (d) subjecting a second sample comprising the at least one protein of interest and the at least one HCP impurity to SEC analysis to generate a second plurality of fractions; (e) using said determination of step (c) to remove said at least one fraction from said plurality of fractions of step (d) in which said at least one HCP impurity is an impurity of concern to manufacture a biotherapeutic product; A method comprising:

99. 99. The method of claim 98, wherein the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

100. 99. The method of claim 98, wherein the mobile phase for the SEC analysis comprises about 150 mM ammonium acetate.

101. 99. The method of claim 98, wherein the at least one HCP impurity comprises a lipase, a protease, or a combination thereof.

102. 99. The method of claim 98, wherein the at least one HCP impurity comprises a C-C motif chemokine.

103. 99. The method of claim 98, wherein the plurality of fractions comprises a high molecular weight (HMW) fraction, a very high molecular weight (vHMW) fraction, a dimer fraction, a monomer fraction, a low molecular weight (LMW) fraction, a tail fraction, or a combination thereof.

104. 99. The method of claim 98, wherein the fraction in which the at least one HCP impurity is an impurity of concern is a HMW fraction.

105. 99. The method of claim 98, wherein the at least one HCP impurity is present in the fraction from about 1,000 parts per million (ppm) to about 10,000 ppm, about 1,000 ppm, about 2,000 ppm, about 3,000 ppm, about 4,000 ppm, about 5,000 ppm, about 6,000 ppm, about 7,000 ppm, about 8,000 ppm, about 9,000 ppm, or about 10,000 ppm.

106. 99. The method of claim 98, wherein the percentage of the at least one HCP impurity enriched in the HMW fraction is about 30% to about 100%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

107. 99. The method of claim 98, further comprising subjecting the sample of (a) to native digestion prior to SEC analysis.

108. 108. The method of claim 107, wherein the native digestion is a limited digestion.

109. 108. The method of claim 107, wherein the native digestion is carried out by contacting the sample with trypsin.

110. 99. The method of claim 98, wherein the LC-MS / MS analysis comprises reverse phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, Protein A chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

111. 99. The method of claim 98, wherein the LC-MS / MS analysis comprises parallel reaction monitoring.

112. 1. A method for producing a biotherapeutic product, comprising: subjecting a sample containing a protein of interest, at least one HMW species, and at least one HCP impurity to one or more chromatographic steps that reduce the abundance of said at least one HCP impurity. wherein said at least one HCP impurity interacts with said at least one HMW species.

113. 113. The method of claim 112, wherein the interaction of the at least one HCP impurity with the at least one HMW species can be identified by concentrating the at least one HMW species.

114. 114. The method of claim 113, wherein said concentrating comprises subjecting a sample containing said at least one HMW species and said at least one HCP impurity to SEC.

115. 115. The method of claim 114, further comprising subjecting the at least one HMW species and the at least one HCP impurity to buffer exchange, native digestion, denaturation, molecular weight filtration, one or more additional chromatography steps, and / or analysis by mass spectrometry.

116. 113. The method of claim 112, wherein the protein of interest is an antibody, a bispecific antibody, a monoclonal antibody, a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product.

117. 113. The method of claim 112, wherein the at least one HCP impurity comprises a lipase, a protease, or a combination thereof.

118. 113. The method of claim 112, wherein said at least one HCP impurity comprises a C-C motif chemokine.

119. 113. The method of claim 112, wherein the at least one HMW species comprises a dimer, an aggregate, or a combination thereof.

120. 113. The method of claim 112, wherein the one or more chromatography steps comprise reverse phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, Protein A chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, mixed mode chromatography, or a combination thereof.

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