Host cell protein analysis of aav using proteominer technology

Abstract

The present disclosure provides methods for enriching, identifying and / or characterizing at least one host cell protein (HCP) impurity in a sample containing a viral vector. HCP impurities can be enriched by utilizing a bead-based peptide ligand library. The enriched HCP may then be subjected to enzymatic digestion, producing a peptide that can be identified by liquid chromatography-mass spectrometry (LC-MS) analysis to specifically identify and / or characterize the at least one HCP impurity.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to U.S. Patent Application No. 63 / 534,637, filed August 25, 2023, entitled “HOST CELL PROTEIN ANALYSIS OF AAVUSING PROTEOMINER TECHNOLOGY,” the disclosure of which is hereby incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which is submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy created on August 5, 2024, is named 086939_500702_SL.xml and is 11 kilobytes in size. Background Technology

[0005] Biopharmaceutical products must meet very high purity standards. Therefore, monitoring for any impurities in such biopharmaceutical products at different stages of drug development, manufacturing, storage, and handling is crucial. Residual impurities should be at acceptable low levels prior to clinical trials. Residual impurities are also a concern for biopharmaceutical products intended for end-users. For example, host cell proteins (HCPs) may be present in biopharmaceuticals developed using cell-based systems. The presence of HCPs in drug products should be monitored, and exceeding certain thresholds may be unacceptable, depending on the product and the specific HCP. Sometimes, even trace amounts of HCPs can induce immunogenic responses in end-users.

[0006] Despite significant efforts to detect hepatic cytokines (HCPs) in antibody drugs, information regarding HCPs in gene therapy products remains limited and has not been widely integrated into host cell engineering or purification processes. HCPs are proteins produced by the host organism but retained in the drug product (DP). After multiple rigorous purification steps in the process, the concentration of HCPs in the DP is typically low. However, despite their low abundance, HCPs are still considered a critical quality attribute (CQA) due to the potential risks associated with low-abundance residual HCPs, including but not limited to immunogenicity, protein cleavage, and effects on excipient stability.

[0007] Adeno-associated virus (AAV) is the most widely used viral vector for in vivo gene therapy applications. AAV exhibits low immunogenicity and enables long-term, stable gene expression. The use of AAV in gene therapy has created a need for analytical methods to monitor and characterize these products. Process-related and product-related impurities should be monitored to ensure product quality and process consistency.

[0008] Extensive research has been conducted on host cell proteins (HCPs) in monoclonal antibodies (mAbs). Significant efforts have been made in the removal, monitoring, identification, and quantification of HCPs in mAb drug products. Unlike the broad range of HCP analysis methods employed for mAb products, methods for analyzing HCPs in gene therapy products using mass spectrometry (MS) are limited. Most HCP identification in gene therapy products has been performed by direct digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). SDS-PAGE or 2D-PAGE coupled with intragel digestion and LC-MS / MS has also been applied to separate HCPs from capsid proteins, allowing for the detection of low-abundance HCPs. No enrichment methods have been reported to reduce the dynamic range between AAV capsid protein peptides and HCP peptides.

[0009] One of the main challenges of HCP analysis in gene therapy is the limited availability of products. HCP enrichment methods for mAb drugs typically require at least 1 mg of mAb to accumulate sufficient HCP for MS detection; however, the quantity of gene therapy products available for HCP analysis (such as AAV materials) is usually limited to 10 µg or less. Therefore, some methods (such as molecular weight cutoff) cannot be applied to separate HCP from AAV capsid proteins because low-abundance HCPs are mostly adsorbed by the filter, with very little flowing through the membrane. Furthermore, Pluronic F-68, a commonly used detergent in AAV products, is difficult to remove by filtration, and the MS signal from the detergent interferes with the MS detection of HCP peptides. Anti-HCP polyclonal antibodies that can be used to immunocapture HCP from mAb drugs have proven insufficient to recognize all HCP from AAV products. Additionally, capsid proteins do not contain disulfide bonds, so limiting the amount of trypsin does not prevent capsid protein digestion, and therefore, HCP analysis in AAV materials cannot be improved by restrictive digestion.

[0010] It should be understood that methods for enriching, identifying, and characterizing HCPs in gene therapy products are needed to monitor and control residual HCPs in active pharmaceutical ingredients or other products to mitigate safety risks. Summary of the Invention

[0011] This disclosure provides a method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising: (a) contacting a sample containing at least one HCP impurity with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby generating a slurry; (b) washing the slurry containing at least one HCP impurity to remove unbound material; (c) eluting bound HCP to generate an enriched HCP sample; (d) subjecting the enriched HCP to enzymatic digestion conditions to generate a peptide digest; and (e) subjecting the peptide digest to tandem mass spectrometry (MS / MS) analysis to enrich, identify, and / or characterize the at least one HCP impurity.

[0012] In one respect, an exemplary solid support having a peptide ligand library attached thereto is ProteoMiner™ beads.

[0013] In one aspect, enriched HCP is subjected to a denaturing agent to produce a denatured sample. In a specific aspect, the denaturing agent includes heat, high pH, ​​low pH, reducing agents, or ionizing agents.

[0014] In one aspect, the enriched HCP is subjected to an alkylating agent to produce an alkylated sample. In a specific aspect, the alkylating agent includes iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.

[0015] In one aspect, the enriched HCP is subjected to a reducing agent to produce a reduced sample. In one specific aspect, the reducing agent includes dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), or combinations thereof.

[0016] In one aspect, enriched HCP is subjected to denaturing agents, alkylating agents, and reducing agents to produce denatured, reduced, and alkylated samples.

[0017] In one respect, the sample contains an AAV vector. In a specific respect, the AAV vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, their variants and combinations thereof.

[0018] In one aspect, the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL. In a specific aspect, the amount of the sample is about 20 µL, about 25 µL, about 30 µL, about 40 µL, about 50 µL, about 60 µL, about 70 µL, about 80 µL, about 90 µL, or about 100 µL, including any and all values ​​in between.

[0019] In one respect, the pH of the solution in which the interacting peptide ligands bind is in the range of about pH 6.0 to about pH 8.0. In a specific respect, the pH is about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

[0020] In one aspect, the volume of the interacting peptide ligand is from about 0.4 µL to about 15 µL. In a specific aspect, the volume of the interacting peptide ligand is about 0.4 µL, about 1 µL, about 1.5 µL, about 2 µL, about 2.5 µL, about 3 µL, about 4 µL, about 5 µL, about 6 µL, about 7 µL, about 8 µL, about 9 µL, about 10 µL, about 11 µL, about 12 µL, about 13 µL, about 14 µL, about 15 µL, or about 20 µL, including any and all values ​​in between.

[0021] In one aspect, the digestive enzyme includes trypsin. In a specific aspect, the enzymatic digestion conditions include contacting the denatured, reduced, and alkylated sample with at least one digestive enzyme, optionally wherein the at least one digestive enzyme includes trypsin.

[0022] In one respect, the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer.

[0023] In one respect, the mass spectrometer is coupled to a liquid chromatography system.

[0024] This disclosure further provides a method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising: (a) contacting a sample containing at least one HCP impurity with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby generating a slurry; (b) washing the slurry containing at least one HCP impurity to remove unbound material; and (c) eluting bound HCP to generate enriched HCP. (d) subjecting the enriched HCP sample to a denaturing agent to produce a denatured sample; (e) subjecting the denatured sample to a reducing agent to produce a denatured and reduced sample; (f) subjecting the denatured and reduced sample to an alkylating agent to produce a denatured, reduced and alkylated sample; (g) subjecting the denatured, reduced and alkylated sample to enzymatic digestion conditions to produce a peptide digest; and (h) subjecting the peptide digest to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to identify and / or characterize the at least one HCP impurity.

[0025] In one respect, the sample contains an AAV vector. In a specific respect, the AAV vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, their variants and combinations thereof.

[0026] In one aspect, the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL. In a specific aspect, the amount of the sample is about 20 µL, about 25 µL, about 30 µL, about 40 µL, about 50 µL, about 60 µL, about 70 µL, about 80 µL, about 90 µL, or about 100 µL, including any and all values ​​in between.

[0027] In one aspect, the volume of the interacting peptide ligand is from about 0.4 µL to about 15 µL. In a specific aspect, the volume of the interacting peptide ligand is about 0.4 µL, about 1 µL, about 1.5 µL, about 2 µL, about 2.5 µL, about 3 µL, about 4 µL, about 5 µL, about 6 µL, about 7 µL, about 8 µL, about 9 µL, about 10 µL, about 11 µL, about 12 µL, about 13 µL, about 14 µL, about 15 µL, or about 20 µL, including any and all values ​​in between.

[0028] In one aspect, enriched HCP is subjected to a denaturing agent to produce a denatured sample. In a specific aspect, the denaturing agent includes heat, high pH, ​​low pH, reducing agents, or ionizing agents.

[0029] In one aspect, the enriched HCP is subjected to an alkylating agent to produce an alkylated sample. In a specific aspect, the alkylating agent includes iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.

[0030] In one aspect, the enriched HCP is subjected to a reducing agent to produce a reduced sample. In one specific aspect, the reducing agent includes dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), or combinations thereof.

[0031] In one aspect, the digestive enzyme includes trypsin. In a specific aspect, the enzymatic digestion conditions include contacting the denatured, reduced, and alkylated sample with at least one digestive enzyme, optionally wherein the at least one digestive enzyme includes trypsin.

[0032] In one respect, the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer.

[0033] In one respect, the mass spectrometer is coupled to a liquid chromatography system. Attached Figure Description

[0034] The invention and the following detailed description will be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of these methods are shown in the drawings to illustrate the disclosed methods; however, these methods are not limited to the specific embodiments disclosed. In the drawings:

[0035] Figure 1A The diagram illustrates the average HCP detected using different concentrations of ProteoMiner™ beads via the enrichment method of this disclosure, compared to direct digestion, according to an exemplary embodiment.

[0036] Figure 1B The diagram illustrates the number of unique peptides detected using different concentrations of ProteoMiner™ beads via the enrichment method of this disclosure, compared to direct digestion, according to an exemplary embodiment.

[0037] Figure 1C The number of HCPs identified using varying sample volumes, according to an exemplary embodiment, is shown.

[0038] Figure 1D The number of unique peptides identified using varying sample volumes, according to an exemplary embodiment, is shown.

[0039] Figure 1EThe diagram illustrates the number of HCPs identified at different pH values ​​using the enrichment method of this disclosure, compared to direct digestion, according to an exemplary embodiment.

[0040] Figure 1F The diagram illustrates the number of HCPs of several AAV serotypes identified at different pH values ​​using the enrichment method of this disclosure, according to an exemplary embodiment.

[0041] Figure 2A The diagram illustrates the number of HCPs identified from an intact AAV8 capsid sample by the enrichment method of this disclosure, compared to direct digestion, according to an exemplary embodiment.

[0042] Figure 2B The diagram illustrates the number of HCPs identified from an intact AAV8 capsid sample in three replicates using the enrichment method of this disclosure, according to an exemplary embodiment.

[0043] Figure 3A The diagram illustrates the number of HCPs identified from several empty capsid AAV subtypes using the enrichment method of this disclosure, compared to direct digestion, according to an exemplary embodiment.

[0044] Figure 3B A Venn diagram is shown of HCPs identified from several empty capsid AAV subtypes using the enrichment method of this disclosure, according to an exemplary embodiment, compared to direct digestion.

[0045] Figure 4 The illustration shows a comparison of the percentage of previously reported HCPs using ProteoMiner™ with the percentage of HCPs detected by the enrichment methods of this disclosure, according to an exemplary embodiment.

[0046] Figure 5A The quantification of two different peptides, VFEHIGK (SEQ ID NO: 1) and NVAVDELSR (SEQ ID NO: 2), before and after the enrichment method of this disclosure, is shown according to an exemplary embodiment.

[0047] Figure 5B MS2 mass spectra of two low-abundance peptides, EIEIDIEPTDKVER (SEQ ID NO: 3) and TAFQEALDAAGDK (SEQ ID NO: 4), are shown according to an exemplary embodiment and using the enrichment method of this disclosure.

[0048] Figure 6 A comparison of the TIC chromatograms of a sample prepared by direct digestion and an AAV product prepared by digestion via a filter, according to an exemplary embodiment, is shown.

[0049] Figure 7A The TIC chromatogram of the sample prepared by direct digestion is shown.

[0050] Figure 7B It is prepared by direct digestion of the sample. Figure 7A The corresponding MS mass spectra were collected at retention times from 50 to 81 min.

[0051] Figure 7C The TIC chromatogram of the sample prepared by the ProteoMiner™ enrichment method is shown.

[0052] Figure 7D The sample was prepared using the ProteoMiner™ enrichment method. Figure 7C The corresponding MS mass spectra were obtained. MS mass spectra were collected from retention times of 50 to 90 min.

[0053] Figure 8 This is a schematic diagram of a method of the present disclosure according to one embodiment. Detailed Implementation

[0054] The disclosed methods can be more readily understood by referring to the following detailed descriptions in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed methods.

[0055] Unless otherwise expressly stated, any description of possible mechanisms or modes of action or reasons for improvement is intended to be illustrative only, and the disclosed methods should not be limited by the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0056] When a numerical range is described or established herein, the range includes its endpoints and all individual integers and fractions within that range, and also includes each of the narrower ranges formed by all the various possible combinations of those endpoints and internal integers and fractions to form a subgroup of larger values ​​within that range, to the same extent as each of those narrower ranges is explicitly described. When a numerical range is stated herein as being greater than the stated value, the range remains finite and is limited at its upper end by values ​​operable in the context of the disclosure herein. When a numerical range is stated herein as being less than the stated value, the range remains limited at its lower end by non-zero values. It is not intended that the range of a method be limited to the specific values ​​stated when the range is defined. All ranges are inclusive and composable.

[0057] When a value is expressed as an approximation using the antecedent “about,” it should be understood that the specific value forms another embodiment. Unless the context explicitly states otherwise, a reference to a specific numerical value includes at least that specific value.

[0058] It should be understood that, for clarity, certain features of the disclosed methods described herein in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed methods described above and below a single embodiment may also be provided individually or in any sub-combination.

[0059] This disclosure provides a method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising: (a) contacting a sample containing at least one HCP impurity with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby generating a slurry; (b) washing the slurry containing at least one HCP impurity to remove unbound material; (c) eluting bound HCP to generate an enriched HCP sample; (d) subjecting the enriched HCP to enzymatic digestion conditions to generate a peptide digest; and (e) subjecting the peptide digest to tandem mass spectrometry (MS / MS) analysis to enrich, identify, and / or characterize the at least one HCP impurity.

[0060] This disclosure provides a method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising: (a) contacting a sample containing at least one HCP impurity with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby generating a slurry; (b) washing the slurry containing at least one HCP impurity to remove unbound material; and (c) eluting bound HCP to generate an enriched HCP sample. (d) subjecting the enriched HCP sample to a denaturing agent to produce a denatured sample; (e) subjecting the denatured sample to a reducing agent to produce a denatured and reduced sample; (f) subjecting the denatured and reduced sample to an alkylating agent to produce a denatured, reduced, and alkylated sample; (g) subjecting the denatured, reduced, and alkylated sample to enzymatic digestion conditions to produce a peptide digest; and (h) subjecting the peptide digest to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to identify and / or characterize the at least one HCP impurity.

[0061] In some embodiments, the enriched HCP is subjected to a denaturing agent to produce a denatured sample.

[0062] In some embodiments, the denaturing agent includes heat, high pH, ​​low pH, reducing agent, or liquid-dissolving agent.

[0063] In some embodiments, the enriched HCP is subjected to an alkylating agent to produce an alkylated sample.

[0064] In some embodiments, the alkylating agent includes iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), 4-vinylpyridine, or combinations thereof.

[0065] In some embodiments, the enriched HCP is subjected to a reducing agent to produce a reduced sample.

[0066] In some embodiments, the reducing agent includes dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or a combination thereof.

[0067] In some embodiments, the enriched HCP is subjected to denaturing agents, alkylating agents, and reducing agents to produce denatured, reduced, and alkylated samples.

[0068] In some embodiments, the viral vector is an AAV vector.

[0069] In some embodiments, the AAV vector comprises a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof.

[0070] In some embodiments, the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL.

[0071] In some embodiments, the peptide ligand library comprises a binding solution with a pH of about pH 6.0 to about pH 8.0, optionally wherein the pH is about 7.0.

[0072] In some embodiments, the volume of the peptide ligand library is from about 0.4 µL to about 15 µL.

[0073] In some embodiments, the enzymatic digestion conditions include contacting the denatured, reduced, and alkylated sample with at least one digestive enzyme.

[0074] In some embodiments, the at least one digestive enzyme includes trypsin.

[0075] In some embodiments, the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer. In some embodiments, the mass spectrometry system is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or an orbital trap-based mass spectrometer. In some embodiments, the mass spectrometer is coupled to a liquid chromatography system.

[0076] As used in this article, the singular forms “a / an” and “the” contain the plural.

[0077] Throughout the specification and claims, various terms are used in connection with different aspects of the specification. Unless otherwise stated, such terms shall have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0078] The term “comprising / including” is intended to include instances covered by the terms “substantially consisting of” and “consisting of”; similarly, the term “substantially consisting of” is intended to include instances covered by the term “consisting of”.

[0079] To manufacture biopharmaceutical products, it is crucial to obtain products with high purity, as residual hematoxylin and eosin (HCP) can compromise the safety and stability of the product. Several methods have been developed for characterizing and identifying HCPs in mAbs, including non-mass spectrometry (MS) techniques such as enzyme-linked immunosorbent assay (ELISA) and gel-based analyses, as well as MS-based methods. The use of mass spectrometry (MS) for HCP analysis in mAb drugs has experienced rapid development and is increasingly used for HCP characterization. This is primarily due to the high sensitivity and ability to identify individual HCPs, which greatly aids risk assessment efforts. A key aspect of developing sensitive MS methods for HCP analysis is reducing the dynamic range between mAb peptides and HCP peptides. Various methods have been utilized to achieve this goal. These methods include using protein A affinity columns to deplete mAbs (protein A depletion), precipitating mAbs by inadequately digested antibodies (restriction digestion), enriching HCPs using anti-HCP antibodies (ELISA-immunocapture coupled with LC-MS / MS), enriching HCPs by removing mAbs using molecular weight cutoff filters (filtration) or size exclusion chromatography (SEC), and enriching HCPs using ProteoMiner™ beads with or without restriction digestion (PMLD or PM).

[0080] ProteoMiner™ beads are a library of bead-based peptide ligands that can bind to different types of proteins.

[0081] Adeno-associated viruses (AAVs) have been widely used as gene delivery vectors to deliver genetic material, such as nucleic acids for gene therapy. AAVs offer the advantages of non-pathogenicity and low immunogenicity. AAVs are non-pathogenic members of the Parvoviridae family within the genus Dependovirus and require helper viruses (such as adenoviruses or herpesviruses) for infection (Venkatakrishnan et al., Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1 - Unique N-Terminal Domain and Its Role in Capsid Trafficking, Journal of Virology, May 2013, Vol. 87, No. 9, pp. 4974-4984). AAVs encapsulate approximately 4.8 kilobases (kb) of single-stranded DNA genome within an icosahedral capsid composed of capsid viral protein shells. The recombinant AAV genome is non-pathogenic and does not integrate into the host genome, but rather exists as a stable appendage providing long-term expression. AAV serotyping is a very useful system for preferential transduction of specific cell types.

[0082] Overall, AAV-based therapies offer advantages such as non-pathogenicity and non-toxicity, cell type-specific infection, and the availability of different serotypes with varying cell transduction efficiencies. The disadvantage is that AAV production, purification, and characterization are more complex compared to, for example, antibody therapies. Fully packaged AAV consists of an icosahedral capsid containing approximately 4.8 kb of single-stranded genome. An empty capsid has a molecular weight of approximately 3750 kDa, while a complete capsid with approximately 4.7 kb of single-stranded genome has a molecular weight of approximately 5100 kDa. The purity of AAV is defined by several product-related impurities, including empty capsids, capsids containing partial or incorrect genomes, aggregated or degraded capsids, and residual HCP.

[0083] For the manufacture of biopharmaceutical products, high purity is crucial, as residual HCPs can compromise the safety and stability of the product. Characterization of HCPs in viral vector production presents numerous challenges. For example, the location of HCPs must be considered. HCPs may be located inside the vector, associated with the target genome, and / or as part of the AAV capsid. Furthermore, AAV serotypes can have a profound impact on the identified HCPs.

[0084] Huang et al. (“A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies,” Anal. Chem. 2017 May 16; 89 (10):5436-5444) describe a sample preparation method using trypsin digestion for shotgun proteomics characterization of HCP impurities in antibody samples. Huang’s sample preparation method preserves the antibody almost intact while HCPs are digested. Compared to conventional trypsin digestion sample preparation, Huang’s method can reduce the dynamic range of HCP detection using mass spectrometry by one to two orders of magnitude. As demonstrated in HCP incorporation experiments, Huang’s method can detect HCPs with molecular weights greater than 60 kDa at 0.5 ppm, such as rPLBL2. For example, using Huang's method, sixty mouse HCP impurities were detected in RM 8670 (NIST mAb, a standard of NIST monoclonal antibodies expressed in mouse cell lines obtained from the National Institute of Standards and Technology, Gaithersburg, MD).

[0085] Doneanu et al. (“Enhanced Detection of Low‐Abundance HostCell Protein Impurities in High‐Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography,” Anal. Chem. 20 Oct 20, 2015; 87(20):10283-10291) reported the use of liquid chromatography-mass spectrometry (LC-MS) to detect low-abundance HCP impurities down to 1 ppm in antibody samples. Doneanu's method involved using a novel charge-surface modified C18 stationary phase to alleviate column saturation challenges, incorporating traveling-wave ion mobility separation of co-eluted peptide precursors, and improving the cleavage efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor cleavage with mobility drift time. HCP impurities ranging from 10 to 50 ppm could be identified using 2D-HPLC combined with ion mobility mass spectrometry. However, the cycle times for 2D-LC or 2D-HPLC can be very long. Furthermore, these methods may not be sensitive enough for analyzing low levels of HCP, such as less than 10 ppm. Other methods for identifying HCP impurities include preparing samples to enrich HCP by removing antibodies from the sample, such as using affinity purification or limited digestion. Additionally, capturing HCP using polyclonal antibodies is another common method.

[0086] Due to the extremely high sample complexity, the analytical techniques required for identifying HCP impurities face the challenge of processing matrix molecules that are approximately one million times larger than the analyte, such as HCP or HCP peptides. Enriching HCPs to detection-compatible levels is difficult because HCP impurities are typically present in protein biopharmaceuticals at low levels, such as 1–100 ppm. Developing universal sample preparation procedures to enrich HCPs (or HCP peptides) or remove matrix background without understanding the properties and characteristics of HCPs can be very challenging (Doneanu et al.).

[0087] Chen et al. (“Improved host cell protein analysis in monoclonal antibody products through ProteoMiner,” Anal. Biochem. 2020 Dec 1; 610:113972) describe a method for enriching HCPs using interacting peptide ligands, specifically ProteoMiner™ beads. The method disclosed herein improves upon previously described ProteoMiner™ methods for HCP enrichment, identification, and quantification.

[0088] This application provides a method for enriching hexapeptides (HCPs) using interacting peptide ligands, such as combinatorial ligand libraries. In some exemplary embodiments, ProteoMiner™ beads (Bio-Rad Laboratories, Inc., Hercules, CA) are combinatorial hexapeptide libraries immobilized on beads for HCP enrichment. When peptide-ligand-conjugated beads are applied to a sample containing various protein species, each protein species can bind to its interacting peptide ligand. HCPs bind to their interacting peptide ligands primarily through hydrophobic interactions, combined with weak interactions such as ionic interactions and hydrogen bonds.

[0089] High-abundance protein species can saturate their interacting peptide ligands due to their excess presence, as the number of interacting peptide ligands corresponding to each protein species in a combinatorial ligand library is finite. In the presence of excess high-abundance proteins, the limited number of corresponding interacting peptide ligands can easily saturate. Excess high-abundance proteins that cannot bind to interacting peptide ligands can be washed away from the beads. Since the number of low-abundance proteins in the sample is relatively low compared to high-abundance proteins, low-abundance proteins may not saturate their corresponding interacting peptide ligands. Therefore, low-abundance proteins can be relatively enriched compared to high-abundance proteins. After the enrichment process, a wide dynamic range of protein concentration can be significantly reduced to allow for the detection of low-abundance proteins.

[0090] Several methods have been employed to identify HCPs, such as gel electrophoresis and / or digestion coupled with liquid chromatography-mass spectrometry (LC-MS), or digestion followed by enrichment with liquid chromatography (LC) and then LC-MS. Additionally, direct digestion, immunoprecipitation, natural digestion, and molecular weight cutoff filtration techniques have been implemented. However, the dynamic concentration and location of HCPs in AAV, as well as the limitations of sample volume and AAV concentration, are major challenges for monitoring and removing HCP impurities. To overcome this problem, methods for enriching, identifying, and characterizing HCPs have been developed.

[0091] This application provides a method for the enrichment, identification, and characterization of HCPs using ProteoMiner™ enrichment beads for gene therapy products, particularly AAV products. In some exemplary embodiments, this method improves the dynamic range of HCP detection by one to two orders of magnitude compared to direct digestion and allows for a 5- to 10-fold increase in the number of HCPs detected from AAV materials. Furthermore, it was found that detergents are completely removed without interfering with the detection of HCP peptides. This method can be applied to other gene therapy products and can be used for troubleshooting and risk assessment to identify potentially problematic HCPs.

[0092] This disclosure provides a method for meeting the above requirements by providing a method for identifying HCPs in biopharmaceutical products to mitigate safety risks. The exemplary embodiments disclosed herein satisfy the above requirements and long-standing needs.

[0093] Unless otherwise stated, 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 disclosure pertains. Although methods and materials similar to or equivalent to any methods and materials described herein may be used in practice or testing, specific methods and materials are now described.

[0094] The term “a” should be understood to mean “at least one”; and the terms “about” and “approximately” should be understood to allow for variations in the standard, as would be understood by one of ordinary skill in the art; and where the scope is provided, the endpoints are included. As used herein, the terms “include,” “includes,” and “including” are meant to be non-restrictive and are understood to mean “comprise,” “comprises,” and “comprising,” respectively.

[0095] As used herein, the term "protein" or "protein of interest" can encompass any amino acid polymer having covalently linked amide bonds. A protein comprises one or more amino acid polymer chains, commonly referred to in the art as a "peptide." A "peptide" is a polymer consisting of amino acid residues, associated naturally occurring structural variants, and their synthetic, non-natural analogs linked by peptide bonds. A "synthetic peptide or polypeptide" refers to a non-natural peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated peptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. Proteins can include one or more polypeptides to form a single functional biomolecule.

[0096] As used herein, the term "therapeutic protein" includes any of the following: protein, recombinant protein for research or therapy, trap protein and other chimeric receptor Fc fusion proteins, chimeric protein, antibody, monoclonal antibody, polyclonal antibody, human antibody and bispecific antibody.

[0097] In another exemplary aspect, the protein may comprise antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. The protein of interest may comprise any of the following: biotherapeutic proteins, recombinant proteins for research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. The protein may be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives, such as CHO-K1 cells). For a recent review of biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non‐human sialylation” (Darius Ghaderi et al., 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147–176 (2012), the entire contents of which are incorporated herein by reference). In some exemplary embodiments, the protein includes 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, FLAGtag, maltose-binding protein (MBP), chitin-binding protein (CBP), glutathione S-transferase (GST) myc epitopes, fluorescent labels, and other dyes. Proteins can be classified based on their composition and solubility, and thus can include simple proteins, such as globular and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucins, pigment proteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary and secondary derived proteins.

[0098] As used herein, the term "recombinant protein" refers to a protein resulting from the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In some exemplary embodiments, the recombinant protein may be an antibody, such as a chimeric antibody, a humanized antibody, or a fully human antibody. In some exemplary embodiments, the recombinant protein may be an isotype antibody selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In some exemplary embodiments, the antibody molecule is a full-length antibody (e.g., IgG1), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0099] As used herein, the term "antibody" includes immunoglobulin molecules and their multimers (e.g., IgM) comprising four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In various embodiments of this disclosure, the FR of the anti-ET-1 antibody (or its antigen-binding portion) may be identical to the human germline sequence or may be natural or artificially modified. The common amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes the antigen-binding fragment of the complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., encompass any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody may be derived from the complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. This DNA is known and / or readily available from, for example, commercial sources, DNA libraries (containing, for example, phage-antibody libraries), or may be synthetic. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable conformation, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0100] As used herein, an "antibody fragment" comprises a portion of a complete antibody, such as the antigen-binding region or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv bifunctional antibodies, dAb fragments, Fd' fragments, Fd fragments, and separated complementarity-determining region (CDR) regions, as well as trifunctional antibodies, tetrafunctional antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of variable regions of the immunoglobulin heavy and light chains, and a scFv protein is a recombinant single-chain polypeptide molecule in which the immunoglobulin light and heavy chain variable regions are linked by peptide linkers. In some exemplary embodiments, the antibody fragment comprises a sufficient amino acid sequence of a parent antibody that is a fragment that binds the same antigen as the parent antibody; in some exemplary embodiments, the fragment binds the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for antigen binding. Antibody fragments can be generated in any manner. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of a complete antibody and / or by recombination of a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments can be produced entirely or partially synthesized. Antibody fragments may optionally comprise single-chain antibody fragments. Alternatively or additionally, antibody fragments may comprise, for example, multiple chains linked together by disulfide bonds. Antibody fragments may optionally comprise multi-molecular complexes. Functional antibody fragments typically comprise at least about 50 amino acids, and more typically comprise at least about 200 amino acids.

[0101] The term "bispecific antibody" (bsAb) refers to an antibody capable of selectively binding to two or more epitopes. Bispecific antibodies typically comprise two distinct heavy chains, each specifically binding to a different epitope on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). If a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), then the affinity of the first heavy chain for the first epitope is typically at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be located on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be prepared, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, a nucleic acid sequence encoding a variable sequence of a heavy chain that recognizes different epitopes of the same antigen can be fused with a nucleic acid sequence encoding a constant region of a different heavy chain, and such sequences can be expressed in cells expressing immunoglobulin light chains.

[0102] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain. The immunoglobulin light chain does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and bind to one or more epitopes bound by the heavy chain antigen-binding region, or can associate with each heavy chain such that one or both heavy chains can bind to one or two epitopes. bsAbs can be divided into two main categories: those with an Fc region (IgG-like) and those lacking an Fc region, the latter typically smaller than IgG and IgG-like bispecific molecules that include an Fc region. IgG-like bsAbs can take various forms, such as, but not limited to, trifunctional antibodies, knob-in-pore IgG (kih IgG), crossMab, orthogonal Fab IgG, dual variable domain Ig (DVD-Ig), dual-acting or dual-functional Fab (DAF), IgG-single-chain Fv (IgG-scFv), or κλ bodies. Non-IgG-like forms include tandem scFv, biantibody forms, single-chain bifunctional antibodies, tandem bifunctional antibodies (TandAb), dual-affinity retargeting molecules (DART), DART-Fc, nanobodies, or antibodies generated via docking lock (DNL) methods (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Müller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265–310 (2014), the entire contents of which are incorporated herein by reference). Methods for generating bsAbs are not limited to tetrahybridoma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation involving chemical cross-linking agents, and gene methods utilizing recombinant DNA technology.

[0103] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity to at least two different antigens. While these molecules will typically bind to only two antigens (e.g., bispecific antibodies, bsAb), antibodies with additional specificity, such as trispecific antibodies and KIH trispecific antibodies, are also considered.

[0104] 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 in any manner available or known in the art, including any eukaryotic, prokaryotic, or phage clone. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display technologies, or combinations thereof.

[0105] As used herein, "protein drug product" or "biopharmaceutical product" comprises an active ingredient that may be wholly or partially biological in nature. In one aspect, a protein drug product may include peptides, proteins, fusion proteins, antibodies, antigens, vaccines, peptide-drug conjugates, antibody-drug conjugates, protein-drug conjugates, cells, tissues, or combinations thereof. In another aspect, a protein drug product may include recombinant, engineered, modified, mutated, or truncated versions of peptides, proteins, fusion proteins, antibodies, antigens, vaccines, peptide-drug conjugates, antibody-drug conjugates, protein-drug conjugates, cells, tissues, or combinations thereof.

[0106] As used herein, a “sample” refers to a molecular mixture containing at least viral particles (such as AAV particles) or empty viral capsids, manipulated according to the methods of this disclosure, including, for example, separation, analysis, extraction, concentration, and so on. The sample can be obtained from any step of a biological process, such as cell culture medium (CCF), harvested cell culture medium (HCCF), any step in a downstream process, active pharmaceutical ingredient (DS), or pharmaceutical product (DP) containing the final formulation. In some specific exemplary embodiments, the sample can be selected from any step of a downstream process, such as clarification, chromatographic generation, or filtration. In some specific exemplary embodiments, the pharmaceutical product can be selected from pharmaceutical products manufactured in a clinical, transport, storage, or processing manner.

[0107] As used herein, the term "impurity" can include any undesirable protein present in a protein sample or protein biopharmaceutical product. Impurities can include process- and product-related impurities. Impurities can include non-protein molecules, including but not limited to chemical and biochemical processing agents, inorganic salts, solvents, carriers, and other leachables. Impurities can further be of known structure, partially characterized, or unidentified. Process-related impurities can originate from the manufacturing process and can fall into three main 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, carriers, or total DNA) derived from the host organism. Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other mediator components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing agents (e.g., cyanogen bromide, guanidine, oxidants, and reductants), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.

[0108] As used herein, the term "host cell protein" (HCP) includes proteins derived from host cells. Host cell proteins can be process-related impurities, which can originate from the manufacturing process and can comprise three main categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from the host organism and nucleic acids (host cell genome, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antibiotics, serum, and other mediator components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidants, and reductants), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachable substances. In some exemplary embodiments, the types of HCP process-related impurities in the composition can be at least two.

[0109] The presence of host cell proteins in biotherapeutic products can be considered as a higher or lower risk based on a number of measurable factors. One such factor is the concentration or abundance (quantity) of HCP impurities in the biotherapeutic product. HCPs at sufficiently low abundances may have no perceptible effect, as measured by methods such as ELISA or mass spectrometry. The level at which an HCP poses a considerable risk can depend on the specific identity of the HCP, and this level can be considered unacceptable in the product and can be monitored as a critical quality attribute (CQA). A particular HCP may be known to pose a specific level of risk, for example, based on the level of its enzymatic activity as an enzyme.

[0110] Relatedly, the criticality of the presence of HCPs may depend on their function, particularly their function related to the components of the biotherapeutic product. For example, HCP lipases that may or are known to degrade polysorbate present in the biotherapeutic product of interest can be closely monitored, and may have a low threshold for how many HCP impurities are permissible in the biotherapeutic product. Other particularly relevant HCPs may be, for example, proteases that may or are known to degrade the protein of interest in the biotherapeutic product, or immunogenic HCPs that may or are known to elicit an immune response when administered to a subject. Using the methods of this disclosure, those skilled in the art can assess the abundance, distribution, and / or identity of HCP impurities in the context of the biotherapeutic product of interest to determine whether an HCP impurity is relevant, and based on said determination, the impurity can be removed using chromatographic separation methods or other separation methods during the production of the biotherapeutic product.

[0111] 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 1,000 times, about 10,000 times, about 100,000 times, or about 1,000,000 times the concentration of the at least one HCP. Another way to express relative concentration is, for example, in parts per million (ppm). It should be understood that when ppm is used to describe the concentration of low-abundance proteins or peptides (such as HCPs) in a sample containing high-abundance proteins or peptides (such as therapeutic proteins), 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 1,000 ppm, less than about 10 ppm, or less than about 1 ppm.

[0112] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to polymers of amino acids and / or amino acid analogs linked by peptide bonds or peptide bond mimics. The twenty naturally occurring amino acids and their single-letter and three-letter names are as follows: Alanine (Ala); Cysteine ​​(Cys); Aspartic acid (Asp); Glutamic acid (Glu); Phenylalanine (Phe); Glycine (Gly); Histidine (H His); Isoleucine (He); Lysine (Lys); Leucine (L Leu); Methionine (M Met); Asparagine (Asn); Proline (Pro); Glutamine (Gln); Arginine (Arg); Serine (S Ser); Threonine (T Thr); Valine (V Val); Tryptophan (W Trp); and Tyrosine (Y Tyr).

[0113] As used herein, “vector” refers to a recombinant plasmid or virus (“viral vector”) containing nucleic acid to be delivered to host cells in vitro or in vivo. Vectors derived from AAVs are particularly attractive for delivering genetic material because (i) they are capable of infecting (transducing) a variety of non-dividing and dividing cell types, including myofibrils and neurons; (ii) they lack viral structural genes, thereby eliminating the natural host cell response to viral infection, such as an interferon-mediated response; (iii) wild-type AAVs have never been associated with any pathology in humans; (iv) unlike wild-type AAVs, which can integrate into the host cell genome, replication-deficient AAV vectors typically persist as episomes, thus limiting the risk of insertional mutagenesis or oncogene activation; and (v) unlike other vector systems, AAV vectors do not trigger a significant immune response (see ii), thus allowing for the long-term expression of therapeutic transgenes (provided their gene products are not rejected).

[0114] "Recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterologous sequences (e.g., nucleic acid sequences from non-viral sources).

[0115] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (e.g., nucleic acid sequences not derived from AAV), which may be side-joined with at least one (e.g., two) AAV inverted terminal repeat (ITR) sequences. When present in host cells that are already infected with a suitable helper virus (or are expressing a suitable helper function) and are expressing AAV rep and cap gene products (e.g., AAV rep and Cap proteins), such rAAV vectors can be replicated and packaged into infectious viral particles.

[0116] "Virus particle" refers to a particle composed of at least one viral capsid protein and an encapsulated viral genome. Although AAVs are described in this disclosure as model viruses or virus particles, the disclosed methods are intended to be applicable to the analysis of a variety of viruses, such as viridae, subfamilies, and genera. In some aspects, the viral capsid, virus, or virus particle belongs to the viridae family selected from the groups consisting of adenoviridae, parvoviridae, retroviridae, baculoviridae, and herpesviridae. In some aspects, the viral capsid, virus, or virus particle belongs to the genera selected from the groups consisting of: adenovirus, avian adenovirus, fish adenovirus, mammalian adenovirus, sialidovirus, bifidus, short-nucleovirus, hepatic nucleovirus, isovirus, invertebrate shrimp nucleovirus, Aleutian parvovirus, avian parvovirus, Boca parvovirus, coronavirus parvovirus, parvovirus-dependent, red parvovirus, proparvovirus, tetraparvovirus, alpha retrovirus. Genus: β-retrovirus, δ-retrovirus, ε-retrovirus, γ-retrovirus, lentivirus, foam virus, baculovirus A, baculovirus B, baculovirus D, baculovirus C, infectious laryngotracheitis virus, Marek's virus, herpes simplex virus, varicella virus, cytomegalovirus, murine cytomegalovirus, proboscis virus, roseola virus, latent lymphovirus, Macavirus, Percavirus, and spider monkey virus.

[0117] The "capsid" is the protein shell of a virus that encloses its genetic material. Three viral capsid proteins, VP1, VP3, form a 60-subunit icosahedral capsid in a 1:1:10 ratio. A complete capsid contains genetic material and is essential for providing therapeutic benefits. An empty capsid lacks a genome and therefore lacks the ability to provide therapeutic benefits to patients.

[0118] As used herein, the term "gene therapy" refers to a method of treating genetic diseases by modifying or manipulating genes of interest. A key step in gene therapy is the efficient delivery of a vector to appropriate tissues or cells. Non-limiting examples of gene therapy products may include plasmid DNA, viral vectors, non-viral vectors, bacterial vectors, human gene editing technologies, and patient-derived cell gene therapy products.

[0119] As used herein, the term "solid support" can include any surface capable of binding proteins or peptides. Non-limiting examples of solid supports may include affinity resins, beads, and coated plates or microplates. Solid supports may be attached to molecules capable of binding to proteins or peptides, including affinity reagents, antigen-binding molecules, or interacting peptide ligands. In some exemplary embodiments, the solid support comprises beads linked to interacting peptide ligands. In some exemplary embodiments, the solid support comprises ProteoMiner™ beads.

[0120] In some exemplary embodiments, samples may be prepared prior to LC-MS analysis. Preparation steps may include reduction, denaturation, alkylation, dilution, digestion, and separation (e.g., centrifugation).

[0121] As used herein, “protein denaturation” or “denaturation” can refer to the process by which the three-dimensional shape of a molecule changes from its native state. Protein denaturation can be performed using protein denaturing agents. Non-limiting examples of protein denaturing agents include heat, high or low pH, reducing agents such as DTT, or exposure to a dissociating agent. Several dissociating agents can be used as protein denaturing agents. Dissociating solutes increase the entropy of a system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic interactions. Non-limiting examples of dissociating agents include butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroyl sarcosine, urea, and their salts.

[0122] Proteins may exhibit unique sensitivities to denaturation. For example, viral proteins assembled into the viral capsid may have reduced denaturation susceptibility compared to monomeric or smaller multimeric proteins (such as host cell proteins). This difference in denaturation susceptibility can be utilized to preferentially denature a specific protein or class of proteins while leaving another protein or class of proteins in a substantially natively folded state. In some aspects, denaturation can be performed at temperatures selected to substantially denature a protein or class of proteins (e.g., non-viral proteins, such as host cell proteins) while leaving the proteins in the viral capsid substantially folded. This can be termed, for example, mild denaturation, limited denaturation, partial denaturation, or differential denaturation. The partially denatured sample can then be subjected to a digestion step to produce peptide digests. Because denatured proteins are more readily digested by digestive enzymes, the peptide digests will preferentially include peptides from more denatured proteins (e.g., non-viral proteins, such as HCP) compared to peptides from more natively folded proteins (e.g., viral capsid proteins). This can be termed, for example, partial digestion, differential digestion, or restrictive digestion. Compared to the original sample, the peptide digest will enrich peptides of non-viral proteins, such as HCP. This enrichment can be used for subsequent analyses, such as liquid chromatography-mass spectrometry, to sensitively and accurately identify, characterize, and quantify non-viral proteins.

[0123] As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds in a protein. Several methods exist for digesting proteins in a sample using appropriate hydrolytic agents, such as enzymatic digestion or non-enzymatic digestion. Digesting a protein into its constituent peptides produces a "peptide digest," which can be further analyzed using peptide mapping.

[0124] As used herein, the term "digestive enzyme" refers to any of a large number of different agents capable of digesting proteins. Non-limiting examples of hydrolysants capable of enzymatic digestion include proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, Aspergillus pepsin I, LysN protease (Lys-N), LysC endonuclease (Lys-C), asp-N endonuclease (Asp-N), Arg-C endonuclease (Arg-C), Glu-C endonuclease (Glu-C), or outer membrane protein T (OmpT), immunoglobulin degrading enzyme (IdeS) from Streptococcus pyogenes, thermophilic proteases, papain, streptomycin, V8 protease, or bioactivated fragments or homologs thereof, or combinations thereof. For a recent review of the available techniques for protein digestion, see Switzar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar, Martin Giera & Wilfried MA Niessen, 12 JOURNAL OF PROTEOME RESEARCH 1067–1077 (2013)).

[0125] Conventional methods use digestive enzymes at conditions and concentrations sufficient to completely digest all proteins in a sample prior to LC-MS analysis. This disclosure surprisingly reveals that the identification and quantification of low-abundance proteins (such as HCPs) can be improved through restrictive digestion, meaning that selective denaturation and digestion conditions prevent complete digestion of proteins in the sample. In some exemplary embodiments, the proteins undergo mild or partial denaturation prior to digestion, such that more denatured specific proteins or classes of proteins are preferentially digested.

[0126] As used herein, the term "protein reducing agent" or "reducing agent" refers to a reagent used to reduce disulfide bonds in proteins. Non-limiting examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), or combinations thereof. The reduction step can be performed sequentially or simultaneously with other sample preparation steps. For example, the reduction and denaturation steps can be performed simultaneously (by adding the reducing agent while incubating the sample at high temperature), allowing the reducing agent to access cysteine ​​residues exposed to the solvent through denaturation.

[0127] As used herein, the term "protein alkylating agent" or "alkylating agent" refers to an agent used to alkylate certain 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.

[0128] As used herein, the term "liquid chromatography" refers to a process in which a biological and / or chemical mixture carried by a liquid may separate into components due to the differential distribution of components as it flows through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some aspects, the sample or eluent may be subjected to any one or a combination of the aforementioned chromatographic methods.

[0129] As used herein, the term "mass spectrometer" encompasses a device capable of identifying specific molecular species and measuring their accurate mass. The term also means any molecular detector containing peptides or polypeptides that can be characterized. A mass spectrometer can comprise three main components: an ion source, a mass analyzer, and a detector. The role of the ion source is to generate gaseous ions. Analyte atoms, molecules, or clusters can be transferred into the gas phase and ionized simultaneously (e.g., by electrospray ionization) or by a separate process. The choice of ion source depends on the application.

[0130] Mass spectrometers can be coupled to liquid chromatography-multiple reaction monitoring systems. More typically, mass spectrometers can be used for analysis via selected reaction monitoring (SRM), including continuous reaction monitoring (CRM) and parallel reaction monitoring (PRM).

[0131] As used in this article, “multiple reaction monitoring” or “MRM” refers to a mass spectrometry-based technique with high sensitivity, specificity and wide dynamic range that can accurately quantify small molecules, peptides and proteins in a complex matrix (Paola Picotti and Ruedi Aebersold, Selected reaction monitoring–based proteomics: workflows, potential, pitfalls and future directions, 9 Nature Methods 555–566 (2012)). MRM can typically be performed using a triple quadrupole mass spectrometer, where the precursor ion corresponding to the selected small molecule / peptide is selected in the first quadrupole, and the fragment ion of the precursor ion for monitoring is selected in the third quadrupole (Yong Seok Choi et al., Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimers disease biomarker candidates, 930 JOURNALOF CHROMATOGRAPHY B 129–135 (2013)).

[0132] SRM / MRM / Selected Ion Monitoring (SIM) is a method used in tandem mass spectrometry where ions of a specific mass are selected in the first stage of the tandem mass spectrometer, and the ion products of the fragmentation reaction of the precursor ions are selected in the second stage of the second mass spectrometer for detection. Examples of triple quadrupole mass spectrometers (TQMS) capable of performing MRM / SRM / SIM include, but are not limited to, the QTRAP® 6500 system (Sciex), the QTRAP® 5500 system (Sciex), the Triple QTriple Quad 6500 system (Sciex), the Agilent 6400 series triple quadrupole LC / MS system, and the Thermo Scientific™ TSQ™ triple quadrupole system.

[0133] In addition to MRM, peptide selection can also be quantified using parallel reaction monitoring (PRM). PRM is an application of SRM, which uses a high-resolution mass spectrometer to detect all transitions in parallel in a single analysis. PRM offers high selectivity, high sensitivity, and high throughput for quantifying selected peptides (Q1), and thus proteins. Multiple peptides can be selected specifically for each protein. The PRM methodology uses a quadrupole mass spectrometer to separate target precursor ions, fragments the target precursor ions in a collision cell, and then detects the resulting product ions in an orbital trap mass analyzer. PRM can be used for peptide and / or protein identification using quadrupole time-of-flight (QTOF) or hybrid quadrupole-orbit trap (QOrbitrap) mass spectrometers. Examples of QTOF include, but are not limited to, the TripleTOF® 6600 system (Sciex), the TripleTOF® 5600 system (Sciex), the X500R QTOF system (Sciex), the 6500 series Precision Mass Quadrupole Time-of-Flight (Q-TOF) (Agilent), and the Xevo G2-XS QT quadrupole time-of-flight mass spectrometer (Waters). Examples of QObitrap include, but are not limited to, the Q Exactive™ Hybrid Quadrupole-Orbittrap Mass Spectrometer (Thermo Scientific) and the Orbitrap Fusion™ Tribrid™ (ThermoScientific).

[0134] Non-limiting advantages of PRM include: eliminating most interferences; providing higher accuracy and attomole-level limits of detection and quantitation; enabling reliable identification of peptides via mass spectrometry library matching; reducing assay development time because no pre-selected target conversion is required; and ensuring UHPLC-compatible data acquisition speeds through spectral multiplexing and advanced signal processing.

[0135] The mass spectrometer in the method or system of this application can be, for example, an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, wherein the mass spectrometer can be coupled to a liquid chromatography system, and wherein the mass spectrometer is capable of performing LC-MS (liquid chromatography-mass spectrometry) or LC-PRM-MS (liquid chromatography-parallel reaction monitoring-mass spectrometry) analysis. In some exemplary embodiments, PRM-MS is used for peptide identification.

[0136] In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" encompasses a technique for obtaining structural information about sample molecules through the use of multi-stage mass selection and mass separation. A prerequisite is that the sample molecules are converted to the gas phase and ionized, such that fragmentation occurs in a predictable and controllable manner after a first mass selection step. MS / MS or MS2 can be performed by first selecting and separating precursor ions (MS1) and fragmenting them to obtain meaningful information. Tandem MS has been successfully performed with various analyzer combinations. Which analyzer to combine for a particular application can be determined by many different factors, such as sensitivity, selectivity, and speed, as well as size, cost, and availability. The two main categories of tandem MS methods are spatial tandem and temporal tandem, but hybrid cases also exist where a temporal tandem analyzer is spatially coupled or coupled to a spatial tandem analyzer. A spatial tandem mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-capture mass analyzers. Specific m / z separation functions can be designed to select ions in one segment of the instrument, dissociate them in an intermediate region, and then transfer the product ions to another analyzer for m / z separation and data acquisition. In time-tandem, mass spectrometer ions generated in the ion source can be captured, isolated, fragmented, and m / z separated in the same physical device.

[0137] Peptides identified by mass spectrometry can be used as alternative representatives of intact proteins and their post-translational modifications. These peptides can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter derived from possible peptides in protein sequence databases. Characterization includes, but is not limited to, protein identification, amino acid sequencing of protein fragments, protein sequencing determination, protein quantification, localization of post-translational modifications, identification of post-translational modifications, or comparability analysis, or combinations thereof.

[0138] In some exemplary aspects, the mass spectrometer may use nanoelectrospray or nanospray ionization. As used herein, the term "nanoelectrospray" or "nanospray" refers to the electrospray ionization of a sample solution, typically hundreds of nanoliters per minute or less, at very low solvent flow rates, without the use of external solvent delivery. Electrospray delivery settings that form nanosprays may use static or dynamic nanoelectrospray emitters. Static nanoelectrospray emitters perform continuous analysis of small sample (analyte) solution volumes over extended time periods. Dynamic nanoelectrospray emitters use capillary columns and solvent delivery systems to perform chromatographic separation of mixtures prior to analysis by the mass spectrometer.

[0139] As used herein, the term "database" refers to a collection of protein sequences that may exist in a sample (e.g., in the form of a file in FASTA format). The relevant protein sequences may originate from the cDNA sequences of the species being studied. Public databases that can be used to search for relevant protein sequences include databases hosted by, for example, Uniprot or Swiss-prot. These databases can be searched using what is referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search for uninterpreted MS / MS spectra against all possible sequences in the database and provide interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools include Mascot (matrixscience.com), Spectrum Mill (chem.agilent.com), PLGS (waters.com), PEAKS (bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / proteinpilot), Phenyx (phenyx‐ms.com), Sorcerer (sagenresearch.com), OMSSA (pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (proteinmetrics.com / products / byonic), or Sequest (fields.scripps.edu / sequest).

[0140] An alternative embodiment of this disclosure includes a method for enriching, identifying, and / or characterizing at least one host cell protein impurity in a sample containing at least one mAb or protein, the method comprising contacting a sample containing at least one HCP impurity with a solid support, wherein the solid support is linked to an interacting peptide ligand capable of interacting with the at least one HCP impurity to generate a slurry; washing the slurry containing beads containing at least one HCP impurity to remove unbound material; eluting bound HCP to generate an enriched HCP sample; subjecting the enriched HCP to enzymatic digestion conditions to generate a peptide digest; and subjecting the peptide digest to tandem mass spectrometry (MS / MS) analysis to enrich, identify, and / or characterize the at least one HCP impurity.

[0141] It should be understood that this disclosure is not limited to any of the aforementioned proteins, therapeutic proteins, antibodies, recombinant proteins, host cell proteins, protein drug products, samples, AAVs, viruses, serotypes, vectors, protein alkylating agents, protein denaturants, protein reducing agents, digestive enzymes, chromatographic methods, mass spectrometers, databases, bioinformatics tools, pH ranges or values, temperatures or concentrations, and any protein, therapeutic protein, antibody, recombinant protein, host cell protein, protein drug product, sample, AAV, virus, serotype, vector, protein alkylating agent, protein denaturant, protein reducing agent, digestive enzyme, chromatographic method, mass spectrometer, database, bioinformatics tools, pH, temperature or concentration can be selected in any suitable manner.

[0142] This disclosure will be more fully understood by referring to the following examples. However, the following examples should not be construed as limiting the scope of this disclosure.

[0143] sequence list

[0144]

[0145] Example

[0146] The following list of examples is intended to supplement, rather than replace or supersede, the preceding description.

[0147] Example 1. A method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising:

[0148] (a) A sample containing at least one HCP impurity is brought into contact with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby producing a slurry;

[0149] (b) Wash the slurry containing at least one HCP impurity to remove unbound material;

[0150] (c) Elution of bound HCP to produce an enriched HCP sample;

[0151] (d) subjecting the enriched HCP to enzymatic digestion conditions to produce peptide digests; and

[0152] (e) subject the peptide digest to tandem mass spectrometry (MS / MS) analysis to enrich, identify and / or characterize the at least one HCP impurity.

[0153] Example 2. The method according to Example 1, wherein the enriched HCP is subjected to a denaturing agent to produce a denatured sample.

[0154] Example 3. The method according to Example 2, wherein the denaturant includes heat, high pH, ​​low pH, reducing agent or liquid-dissolving agent.

[0155] Example 4. The method according to any one of Examples 1 to 3, wherein the enriched HCP is subjected to an alkylating agent to produce an alkylated sample.

[0156] Example 5. The method according to Example 4, wherein the alkylating agent comprises iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), 4-vinylpyridine, or a combination thereof.

[0157] Example 6. The method according to any one of Examples 1 to 5, wherein the enriched HCP is subjected to a reducing agent to produce a reduced sample.

[0158] Example 7. The method according to Example 6, wherein the reducing agent comprises dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), or a combination thereof.

[0159] Example 8. The method according to any one of Examples 1 to 7, wherein the enriched HCP is subjected to a denaturing agent, an alkylating agent and a reducing agent to produce a denatured, reduced and alkylated sample.

[0160] Example 9. The method according to any one of Examples 1 to 8, wherein the viral vector is an AAV vector.

[0161] Example 10. The method according to Example 9, wherein the AAV vector comprises a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof.

[0162] Example 11. The method according to any one of Examples 1 to 10, wherein the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL.

[0163] Example 12. The method according to any one of Examples 1 to 11, wherein the peptide ligand library comprises a binding solution with a pH of about pH 6.0 to about pH 8.0, optionally wherein the pH is about 7.0.

[0164] Example 13. The method according to any one of Examples 1 to 12, wherein the volume of the peptide ligand library is about 0.4 µL to about 15 µL.

[0165] Example 14. The method according to any one of Examples 1 to 13, wherein the enzymatic digestion conditions include contacting the denatured, reduced and alkylated sample with at least one digestive enzyme.

[0166] Example 15. The method according to Example 14, wherein the at least one digestive enzyme comprises trypsin.

[0167] Example 16. The method according to any one of Examples 1 to 15, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer.

[0168] Example 17. The method according to any one of Examples 1 to 16, wherein the mass spectrometer is coupled to a liquid chromatography system.

[0169] Example 18. A method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising:

[0170] (a) A sample containing at least one HCP impurity is brought into contact with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby producing a slurry;

[0171] (b) Wash the slurry containing at least one HCP impurity to remove unbound material;

[0172] (c) Elution of bound HCP to produce an enriched HCP sample;

[0173] (d) subject the enriched HCP sample to a denaturing agent to produce a denatured sample;

[0174] (e) subjecting the denatured sample to a reducing agent to produce a denatured and reduced sample;

[0175] (f) subjecting the denatured and reduced sample to an alkylating agent to produce a denatured, reduced and alkylated sample;

[0176] (g) subjecting the denatured, reduced, and alkylated sample to enzymatic digestion conditions to produce peptide digests; and

[0177] (h) The peptide digest is subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to identify and / or characterize the at least one HCP impurity.

[0178] Example 19. The method according to Example 18, wherein the viral vector is an AAV vector.

[0179] Example 20. The method according to Example 19, wherein the AAV vector comprises a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof.

[0180] Example 21. The method according to Example 20, wherein the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL.

[0181] Example 22. The method according to any one of Examples 18 to 21, wherein the volume of the interacting peptide ligand is about 0.4 µL to about 15 µL.

[0182] Example 23. The method according to any one of Examples 18 to 22, wherein the peptide ligand library comprises a binding solution with a pH of about pH 6.0 to about pH 8.0, optionally wherein the pH is about 7.0.

[0183] Example 24. The method according to any one of Examples 18 to 23, wherein the denaturant includes heat, high pH, ​​low pH, reducing agent, or liquid-dissolving agent.

[0184] Example 25. The method according to any one of Examples 18 to 24, wherein the alkylating agent comprises iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), 4-vinylpyridine, or a combination thereof.

[0185] Example 26. The method according to any one of Examples 18 to 25, wherein the reducing agent comprises dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), or a combination thereof.

[0186] Example 27. The method according to any one of Examples 18 to 26, wherein the enzymatic digestion conditions include contacting the denatured, reduced and alkylated sample with at least one digestive enzyme, optionally wherein the at least one digestive enzyme includes trypsin.

[0187] Example 28. The method according to any one of Examples 18 to 27, wherein the mass spectrometry system is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or an orbital trap-based mass spectrometer.

[0188] Example 29. The method according to any one of Examples 18 to 28, wherein the mass spectrometer is coupled to the liquid chromatography system.

[0189] Example

[0190] The following examples are provided to further describe some of the embodiments disclosed herein. These examples are intended to illustrate, and not limit, the disclosed embodiments.

[0191] Material

[0192] An exemplary solid support with a peptide ligand library attached thereto is the ProteoMiner™ bead. The ProteoMiner™ Protein Enrichment Small Volume Kit was obtained from Bio-Rad (Hercules, CA). The chromatographic solvent was LC-MS grade, from Fisher Scientific (Waltham, MA). AAV reference materials, including empty capsids of AAV1, AAV5, AAV8, and AAV9, and an intact capsid of AAV8, were obtained from Charles River. Sodium deoxycholate (SDC), sodium lauroyl sarcosinate (SLS), 2-chloroacetamide (CAA), tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP), and the Proteomics Dynamic Range Standards Kit UPS2 were obtained from Sigma-Aldrich (St. Louis, MO). Iodoacetamide (IAM), dithiothreitol (DTT), trifluoroacetic acid (TFA), and UltraPure 1 M Tris-HCl pH 8.0 were obtained from Thermo Fisher. AAV8 and AAV9 empty capsids with UPS2 incorporation were prepared by diluting UPS2 (10.6 µg / ampoule protein) in 32 µL of water and adding 2 µL of diluted UPS2 to 500 µL of AAV reference material with empty capsids.

[0193] Using ProteoMiner TM Sample preparation for protein enrichment kit

[0194] The ProteoMiner™ Protein Enrichment Kit was used to enrich proteins in samples. HCPs from AAV reference materials were enriched using ProteoMiner™ enrichment beads following a similar protocol described by Chen et al. with some modifications. The AAV reference materials were stored in PBS buffer containing 0.01% Pluronic F-68 (pH 7). The viral particle titers of the AAV reference materials with empty capsids ranged from 1.20 x 10⁻⁶. 12 VP / mL to 2.30 x 10 12The viral genome titer of the AAV8 reference material with intact capsids was 7.97 x 10⁻⁶ VP / mL. 11 GC / mL. The pH of the empty capsid of the AAV reference material was adjusted by adding 50 mM acetic acid (pH 6) or 50 mM Tris-HCl, pH 8.0 (pH 8). The ProteoMiner™ beads were washed three times with 200 µL of wash buffer provided in the enrichment kit, followed by one wash with water. The beads were resuspended in water, and 10 µL of the bead slurry was added to the AAV reference material and incubated by rotation at room temperature for 2.5 h. The slurry beads with enriched proteins were loaded onto a homemade pipette tip and centrifuged at 200 × g for 5 min until dry. The beads were then washed three times with 100 µL of wash buffer, followed by one wash with water. Finally, the enriched proteins were eluted three times with 10 µL of PTS buffer containing 12 mM SDC, 12 mM SLS, 10 mM TCEP, and 40 mM CAA. The eluent was then denatured, reduced, and alkylated in PTS buffer at 95 °C for 5 min, cooled to room temperature, diluted with 120 µL of 0.1 M Tris-HCl to pH 8.0, and digested overnight at 37 °C with 10 ng of trypsin. The digested peptides were acidified by adding 10 µL of 10% TFA, centrifuged at 14,000 × g for 20 min, and the peptide-containing supernatant was collected for sequential desalting and nanoliter LC-MS / MS analysis.

[0195] Sample preparation using direct digestion

[0196] Direct digestion was performed by adding 10 µL of denaturing buffer containing 36 mM SDC / SLS, 30 mM TCEP, and 120 mM CAA to 20 µL of AAV reference material and heating at 95 °C for 5 min. The denatured, reduced, and alkylated AAV reference material was then diluted with 120 µL of 0.1 M Tris-HCl (pH 8.0) and digested overnight at 37 °C with 100 ng trypsin. The digested peptides were acidified by adding 10 µL of 10% TFA, centrifuged at 14,000 × g for 20 min, and the peptide-containing supernatant was collected for sequential desalting and nanoliter LC-MS / MS analysis.

[0197] Nanoliter LC-MS / MS Analysis

[0198] The peptide mixture was resuspended in 12 µL of 0.1% formic acid (FA) aqueous solution, and 10 µL was analyzed using an UltiMate 3000 RSLC nanoliter LC system interfaced with an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). The peptides were loaded at 5 µL / min onto a trapping column (20 cm × 0.075 mm Acclaim PepMap 100 C18) and sequentially onto an analytical column (30 cm × 0.075 mm, 1.7 µm, 100 Å, CoAnn Technologies) at 0.25 µL / min for desalting and separation. Mobile phase A was an aqueous solution of 0.1% FA, and mobile phase B was an acetonitrile solution of 0.1% FA. The gradient was set as follows: 4% mobile phase B for 6 min, reaching a high of 20% B during the 84 min time period, increasing to 36% B within 35 min, and then rapidly increasing to 95% B within 9 min, before maintaining 95% B for 9 min. OrbitrapExploris operated in data-dependent mode (DDA) with a full MS scan at 60,000 resolution in the m / z range of 380–1600, and a predicted automatic gain control (AGC) value of 3e. 6 The maximum injection time is 20 ms, and MS / MS events are performed at a resolution of 15,000 at m / z 200-2000, with a predicted AGC of 1e. 5 The maximum injection time is 60 ms.

[0199] Data Analysis

[0200] Raw mass spectrometry data were searched using the Sequest HT and Mascot search engines with Proteome Discoverer software (version 2.4) compared to UniProt Homo Sapiens (version 2022). Mass spectra were searched with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da. Trypsin digestion was performed, with a maximum of two missed cleavage sites. Ureylmethylation of cysteine ​​(+57.0214 Da) was included as a static modification, and oxidation of methionine (+15.9949 Da) and deamidation of asparagine (+0.984 Da) were included as variable modifications. The false discovery rate for proteins and peptides was set to 0.01, and HCP was identified as positive when at least two peptides were found.

[0201] Example 1. Optimization of enrichment methods using the ProteoMiner™ Protein Enrichment Kit

[0202] The effectiveness of enriching HCP using different amounts of ProteoMiner™ beads was evaluated using pH-adjusted AAV8 reference material incorporating UPS2 with empty capsids. ProteoMiner™ is an exemplary library of peptide ligands. UPS2 is a commercially available proteomics standard comprising 48 human proteins spanning a wide concentration dynamic range across many orders of magnitude. The pH of the AAV8 reference material was adjusted to pH 6.0 by adding 2.5 µL of 1M acetic acid, and then added to 500 µL according to a previous ProteoMiner™ enrichment protocol. The limit of detection was evaluated by incorporating 1 / 16 of UPS2 (0.66 µg protein) into 500 µL of pH-adjusted empty AAV8 capsids. This method was performed using varying amounts of ProteoMiner™ beads, including 1 / 2 ProteoMiner™ kit (10 µL beads), 1 / 5 ProteoMiner™ kit (4 µL beads), 1 / 20 ProteoMiner™ kit (1 µL beads), and 1 / 50 ProteoMiner™ kit (0.4 µL beads). The beads were incubated with 100 µL of pH-adjusted AAV8 material incorporated into UPS2, and the number of identified HCPs was compared to alternative direct digestion methods. An average of 167 HCPs were detected after ProteoMiner™ enrichment, while only 45 HCPs were detected by direct digestion. Figure 1A As shown in Table 1, after applying different amounts of enrichment beads, all UPS2 standards with concentrations higher than 6.25 fmol were detected. In contrast, Table 1 also shows that not all UPS2 standards with concentrations less than 4 ng / mL can be detected using the direct digestion method. When different amounts of beads were applied, the number of HCPs identified (…) Figure 1A ) or the number of unique peptides identified from 6.25 fmol of UPS2 standards ( Figure 1B No discernible changes were observed. This indicates that, in terms of detection limit and sensitivity, enrichment efficiency remains unaffected by the number of beads used. Therefore, all subsequent experiments used 1 µL of enrichment beads to minimize variations in bead quantity and conserve experimental materials.

[0203] Table 1. Detection limits of HCP in UPS2 standards from empty-capsulated AAV8 samples incorporating UPS2, comparing the enrichment method of this disclosure using different concentrations of ProteoMiner™ beads with direct digestion.

[0204]

[0205] Example 2. Parameter optimization using the ProteoMiner™ Protein Enrichment Kit

[0206] The method of this disclosure was further optimized by comparing a range of sample volumes. Due to the low titers during drug production, the amount of AAV material is often limited. This method was performed for HCP analysis using AAV8 reference material incorporating UPS2 and AAV9 reference material adjusted to pH 6.0. Different volumes of reference material (20 µL, 50 µL, and 100 µL) were compared to determine the minimum sample volume required for HCP analytical detection. Figure 1C As shown, the number of HCPs identified was consistent for both 50 µL and 100 µL sample volumes. Compared to 20 µL, the number of HCPs increased by 60% and 46%, respectively, in the AAV8 and AAV9 reference materials when using 50 µL or 100 µL samples. However, only 5 out of 8 UPS2s (6.25 fmol) were detected from the 50 µL sample, compared to 8 out of 8 UPS2s (6.25 fmol) detected from the 100 µL sample. Figure 1D As shown.

[0207] Furthermore, it was found that the identity of HCPs enriched from 50 µL AAV reference material overlapped by approximately 60% with that enriched from 100 µL, such as... Figure 1D As shown in the figure. The results indicate that the detection limit can be achieved at 0.5 ng / mL when using 100 µL of sample, and the detection limit increases as the amount of sample used for enrichment decreases. 100 µL of AAV reference material was used for the remaining experimental procedures.

[0208] It has been reported that the binding efficiency of enrichment beads to antibody drugs is affected by pH. Similarly, the binding efficiency of enrichment beads to HCPs in AAV products may differ from that in antibody drugs. Therefore, the method disclosed herein has been further optimized to evaluate the pH of the binding solution for AAV products. AAV reference material was initially stored in PBS buffer containing 0.01% Pluronic F-68 (pH 7.0). The pH was adjusted to 6.0 by adding 50 mM acetic acid and to 8 by adding 50 mM Tris-HCl (pH 8.0). Enrichment was performed by incubating 1 µL of ProteoMiner™ beads with 100 µL of AAV reference material. For AAV9 reference material with an empty capsid, 191 HCPs were detected under acidic conditions, 262 under neutral conditions, and 109 under alkaline conditions, as shown below. Figure 1E As shown.

[0209] To assess whether neutral pH is the optimal incubation condition for HCP enrichment of all AAV serotypes, AAV1, AAV5, and AAV8 reference materials were compared with empty capsids at pH 6.0 and pH 7.0 using the methods disclosed herein. Figure 1F As shown, compared with acidic pH conditions, the number of HCPs detected from different AAV serotypes increased by 15.6% to 50.6% when AAV reference materials and enrichment beads were incubated together under neutral pH conditions. Therefore, neutral pH was chosen for enriching HCPs from AAV samples.

[0210] The optimized conditions are used to evaluate the reproducibility of the method disclosed herein. For example... Figure 2A As shown, approximately 146 HCPs were identified from AAV8 reference material with an intact capsid using the enrichment method of this disclosure, which is 4.3 times the number of HCPs identified using direct digestion. In the three preparations, 131 common HCPs were identified, representing 80.9% of all proteins, such as... Figure 2B As shown. High reproducibility indicates a high confidence level in protein identification, which is crucial for HCP analysis.

[0211] Example 3. Case study using the ProteoMiner™ Protein Enrichment Kit

[0212] The optimized method described in Example 2 was applied to several different AAV serotypes and compared with a direct digestion method. A significant increase in the number of HCPs identified from AAV1, AAV5, AAV8, and AAV9 reference materials with empty capsids was observed, such as... Figure 3A As shown, this enhancement was observed in all AAV serotypes. Figure 3A The enrichment method of this disclosure was used to detect over 80% of newly identified HCPs, revealing less than 1.2% of missed proteins compared to direct digestion methods. These findings demonstrate that the method of this disclosure is comprehensive and capable of detecting large numbers of HCPs present in low abundance that would otherwise be undetectable by direct digestion.

[0213] A total of 860 HCPs were identified from the AAV1, AAV5, AAV8, and AAV9 reference materials by ProteoMiner™ enrichment coupled with nano-liquid chromatography-tandem mass spectrometry (LC-MS / MS). Figure 4The results of the analysis are presented, showing that of the 860 HCPs identified, 267 were previously reported in the literature to originate from AAV products produced by the human cell line HEK293. These proteins include frequently reported HCPs such as nucleophosphorin, SET protein, nucleolin, and RNA-binding proteins such as small nucleoribonucleoproteins SmD2 and D3, heterogeneous nucleoribonucleoproteins A / B, and PABP. Additionally, some HCPs commonly found in monoclonal antibodies (mAbs), such as peroxidase 1, ubiquitin, actin, and Hsc70, which potentially regulate cellular function, were also detected in the AAV products. Furthermore, the enrichment method disclosed herein identified 593 previously unreported HCPs.

[0214] The optimized method was used to evaluate the reduction in dynamic range in AAV9 reference materials. 0.264 µg of UPS2 protein was incorporated into empty AAV9 capsid reference materials, and the protein detection range was determined. Seven UPS2 standards ranging from 0.1 ng / mL to 414.7 ng / mL were selected for comparison. The concentration of each UPS2-incorporated standard was calculated by comparing the relative abundance of UPS2 standard peptides to capsid protein peptides in samples prepared using the optimized enrichment method and the direct digestion method. The enrichment factor was calculated by comparing the calculated concentration with the incorporated concentration, as shown in Table 2. The enrichment factor of the UPS2 standard prepared by direct digestion was close to 1, as no enrichment occurred. Figure 4 As shown. For direct digestion, some standards (such as serum albumin and peroxidase) showed slightly higher enrichment factors than 1, which may be due to the pre-existing HCP in the AAV reference material. The enrichment factors of samples treated with ProteoMiner™ ranged from 1.5 to 25, as shown. Figure 4 As shown in the image.

[0215] Table 2. Enrichment factors of UPS2 standards detected from AAV9 empty caps containing UPS2 using direct digestion and ProteoMiner™ enrichment.

[0216]

[0217] 1. The enrichment factor is calculated using Formula 1:

[0218]

[0219] 2. The dynamic range of direct digestion is calculated using Formula 2:

[0220]

[0221] 3. PM / Direct Digestion Ratio is calculated by dividing the PM enrichment factor by the direct digestion enrichment factor.

[0222] For UPS2 incorporation concentrations higher than 1.6 ng / mL, the dynamic range between the UPS2 peptide and the AAV capsid protein peptide, as determined by the enrichment method, is within 1.4E. -03 With 3.0E -01 Between, and for direct digestion, at 2.5E -04 With 3.0E -02 This indicates that enrichment increased the concentration by 3.6 to 34.1 times. Figure 5A Demonstrated catalase peptide VFEHIGK 2+ (SEQ ID NO: 1) and ribosyl dihydronicotinamide dehydrogenase [quinone] [quinone oxidoreductase 2] [NQO2] peptide NVAVDELSR 2+ Signal changes at (SEQ ID NO: 2). For 0.6 ng / mL neddylin and 0.1 ng / mL thioredoxin, the dynamic range between the enriched UPS2 peptide and AAV capsid peptide was 3.5E. -04 and 7.2E -04 Furthermore, it can be predicted that the dynamic range between these two UPS2 peptides and capsid protein peptides will be in the E... -05 Within the range. Due to the potentially low range of neddyline and thioredoxin, and likely other host cell proteins at low ng / mL or below 1 ng / mL, they are undetectable by direct digestion preparation. Figure 5B The enriched 0.6 ng / mL neddylin peptide EIEIDIEPTDKVER was shown. 3+ (SEQ ID NO: 3) and 0.1 ng / mL of thioredoxin peptide TAFQEALDAAGDK 2+ MS2 mass spectrum of (SEQ ID NO: 4). By applying this enrichment method, a significant improvement in the sensitivity for detecting these low-abundance HCPs (< 1 ng / mL) was observed.

[0223] Example 4. Complete detergent removal after enrichment bead treatment

[0224] ProteoMiner™ protein enrichment technology is used to enrich low-abundance proteins, so only proteins (such as antibodies, AAV capsid proteins, and HCPs) bind to the beads. Detergents commonly used in mAb pharmaceutical products and / or AAV products (polysorbate and Pluronic F-68) do not bind to the enrichment beads and are therefore removed from the protein mixture after enrichment. Figure 6The total ion chromatograms (TICs) comparing direct digestion (top panel) with AAV products prepared by direct digestion using a filter (bottom panel) are shown. Molecular weight cutoff filters encounter difficulties in removing these detergents, thus filtration is useless for reducing detergent interference. Furthermore, significant loss of HCP was observed during buffer exchange due to HCP adhesion to the filter membrane. In contrast, Figure 7A and 7C The TIC and corresponding MS signals of the direct digestion method are shown, where the signal of Pluronic F-68 is more than 20 times stronger than that of viral protein peptides. This significant difference in signal levels poses a major challenge to the detection of HCP peptides, as the abundance of detectable HCP peptides is as low as 1 / 100 to 1 / 10,000 compared to viral protein peptides. Figure 7B and Figure 7D The TIC and corresponding MS mass spectra of the peptide mixture after enrichment using the methods disclosed herein are shown. The detergent peaks were completely eliminated by the enrichment beads, and certain HCP peptides at levels similar to viral protein peptides were observed in the TIC spectra.

[0225] Example 5. Analysis of host cell proteins in AAV products using ProteoMiner™ protein enrichment technology

[0226] Information on hepatocellular carcinoma (HCP) in gene therapy products is limited and has not yet been integrated into host cell engineering or purification processes. In this study, ProteoMiner™ beads efficiently enriched HCP in adeno-associated virus (AAV) products while simultaneously removing the detergent Pluronic F-68 without loss of low-abundance HCP. HCP enrichment increased by up to 34-fold compared to direct digestion. The detection limit was significantly reduced, enabling the detection of HCP at levels as low as 0.1 ng / mL after ProteoMiner™ treatment. The findings of this study provide insights into HCP in AAV products and may facilitate process development and host cell line optimization. The high sensitivity of this method also contributes to the detection of low-abundance HCP, thereby improving the safety and quality of AAV-based gene therapy products.

[0227] introduce

[0228] Host cell proteins (HCPs) are proteins produced by the host organism and retained in pharmaceutical products (DPs). After undergoing multiple rigorous purification steps during processing, the concentration of HCPs in DPs is typically low. However, despite their low abundance, HCPs are still considered important quality properties because they pose potential risks including immunogenicity, protein cleavage or glycosylation, degradation, and excipient instability.

[0229] A major challenge in HCP analysis in gene therapy is the limited product quality. HCP enrichment methods for monoclonal antibody (mAb) drug formulations typically require at least 1 mg mAb to accumulate sufficient HCP for MS detection. However, the quantity of gene therapy products available for HCP analysis (such as AAV materials) is usually limited to 10 µg or less. Therefore, methods such as molecular weight cutoff cannot be applied to separate HCP from AAV capsid proteins, as most low-abundance HCPs are adsorbed by the filter and rarely flow through the membrane. Pluronic F-68, a commonly used detergent in AAV products, is also difficult to remove by filtration, and the detergent's MS signal severely interferes with the detection of HCP peptides. Anti-HCP polyclonal antibodies used for immunocapturing HCP from mAb drugs have been found to be insufficient to recognize all HCP from AAV products. Furthermore, since capsid proteins do not contain disulfide bonds, limiting the amount of trypsin used does not prevent capsid protein digestion; therefore, HCP analysis in AAV materials cannot be improved by restrictive digestion.

[0230] ProteoMiner™ beads are a library of bead-based peptide ligands that bind to different protein types. In this study, a method for HCP analysis of AAV products was developed using ProteoMiner™ enriched beads. Compared to direct digestion, this method improves the dynamic range of HCP detection by one to two orders of magnitude and achieves a 5- to 10-fold increase in the number of HCPs detected from AAV materials. Detergent is completely removed, thus avoiding interference with HCP peptide detection. This method could be applied to other gene therapy products and used for troubleshooting and identifying potentially problematic HCPs for risk assessment.

[0231] Materials and methods

[0232] The ProteoMiner™ Protein Enrichment Small Volume Kit was purchased from Bio-Rad™ (Hercules, CA). LC-MS grade solvents were purchased from Fisher Scientific (Waltham, MA). AAV reference materials, including empty capsids of AAV1, AAV5, AAV8, and AAV9, and intact capsids of AAV8 (74.8% intact) and AAV9 (82.3% intact), were purchased from Charles River Laboratory and expressed in HEK293 cells. Sodium deoxycholate (SDC), sodium lauroyl sarcosinate (SLS), 2-chloroacetamide (CAA), tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP), and the Proteomics Dynamic Range Standards Kit (UPS2) were purchased from Sigma-Aldrich (St. Louis, MO). Iodoacetamide, dithiothreitol, trifluoroacetic acid (TFA), and UltraPure 1 M Tris-HCl, pH 8.0, were purchased from Thermo Fisher. Empty capsids of AAV 8 incorporating UPS2 were prepared by diluting UPS2 (10.6 µg / ampoule protein) in 32 µL of water; subsequently, 2 µL of diluted UPS2 was added to 500 µL of AAV reference material with empty capsids. UPS2 is a dynamic range standard used in proteomics, comprising a mixture of six of eight proteins spanning five orders of magnitude. This allows it to be used to assess enrichment factors for recombinant proteins present in UPS2 standards.

[0233] Sample preparation using enriched beads

[0234] HCPs from AAV reference materials were enriched using ProteoMiner™ enrichment beads, similar to and modified by the protocol described by Chen et al. (A Highly Sensitive LC-MS / MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics; J Pharm Sci 2021, 110, 3811-3818.). The AAV reference materials were stored in PBS buffer at pH 7 containing 0.01% Pluronic F-68. The viral particle titers of the AAV reference materials with empty capsids ranged from 1.20 × 10⁻⁶. 12 VP / mL to 2.30 × 10 12 The viral genome titer of the AAV8 reference material with intact capsids was 7.97 × 10⁻⁶ VP / mL. 11GC / mL. The pH of the empty capsid of the AAV reference material was adjusted to pH 8.0 by adding 50 mM acetic acid (pH 6) or 50 mM Tris-HCl. The enriched beads were washed three times with 200 µL wash buffer from the ProteoMiner™ Enrichment Kit and once with water, then resuspended in water. Subsequently, 10 µL of the resuspended enriched beads were added to the AAV reference material and incubated by rotation at room temperature for 2.5 h. The slurry containing the protein-enriched beads was loaded onto a homemade pipette tip, centrifuged at 200 × g for 5 min until dry, washed three times with 100 µL wash buffer and once with water, and eluted three times with 10 µL PTS buffer containing 12 mM SDC, 12 mM MSLS, 10 mM TCEP, and 40 mM CAA. The eluent was then denatured, reduced, and alkylated in PTS buffer at 95 °C for 5 min, cooled to room temperature, diluted with 120 µL of 0.1 M Tris-HCl, pH 8.0, and digested overnight at 37 °C with 10 ng of trypsin. The digested peptides were acidified by adding 10 µL of 10% TFA, centrifuged at 14,000 × g for 20 min, and the peptide-containing supernatant was collected for sequential desalting and nanoliter LC-MS / MS analysis.

[0235] Sample preparation using direct digestion

[0236] Direct digestion was performed by adding 10 µL of denaturation buffer containing 36 mM SDC / SLS, 30 mM TCEP, and 120 mM CAA to 20 µL of AAV reference material and heating at 95 °C for 5 min. The denatured, reduced, and alkylated AAV reference material was then diluted with 120 µL of 0.1 M Tris-HCl, pH 8.0, and digested overnight at 37 °C with 100 ng trypsin. The digested peptides were acidified by adding 10 µL of 10% TFA and centrifuged at 14,000 × g for 20 min. The peptide-containing supernatant was collected for sequential desalting and nanoliter LC-MS / MS analysis.

[0237] Nanoliter LC-MS / MS Analysis

[0238] The peptide mixture was resuspended in 12 µL of 0.1% formic acid (FA) aqueous solution, and 10 µL was loaded onto an UltiMate 3000 RSLC nanoliter LC system interfaced with an OrbitrapExploris 480 mass spectrometer (Thermo Fisher Scientific). The peptides were sequentially loaded at 5 µL / min onto a trapping column (20 cm × 0.075 mm Acclaim PepMap100 C18) at 40 °C and at 0.25 µL / min onto an analytical column (30 cm × 0.075 mm, 1.7 µm, 100 Å, CoAnn Technologies) at 40 °C for desalting and separation. Mobile phase A was an aqueous solution of 0.1% FA, and mobile phase B was an acetonitrile solution of 0.1% FA. The gradient started at 4% mobile phase B and persisted for 6 min, increasing to 20% B within 84 min, and further increasing to 36% B within 35 min, before rapidly increasing to 95% B within 9 min and holding for 9 min. OrbitrapExploris was run in data-dependent mode, performing a full MS scan at 60,000 resolution in the m / z range of 380–1600, with a predicted automatic gain control value of 3 × 10⁻⁶. 6 The maximum injection time is 20 ms, and MS / MS events are performed at a resolution of 15,000 at m / z 200-2000, with predictive automatic gain control of 1 × 10⁻⁶. 5 The maximum injection time is 60 ms.

[0239] Data Analysis

[0240] Raw MS data were searched using the Sequest HT and Mascot search engines and Proteome Discoverer software (version 2.4) against the UniProt Homo sapiens database (version 2022). Mass spectra were searched with a mass tolerance of 10 ppm for precursor ions and 0.02 Da for fragment ions. Trypsin digestion was performed, with a maximum of two missed cleavage sites. Ureylmethylation of cysteine ​​(+57.0214 Da) was included as a static modification, and oxidation of methionine (+15.9949 Da) and deamidation of asparagine (+0.984 Da) were included as variable modifications. The false discovery rate for proteins and peptides was set to 0.01, and HCP was considered positive when at least two unique peptides were found.

[0241] Results and discussion

[0242] Optimization of bead quantity in enrichment methods

[0243] The effectiveness of enriching HCPs using different bead amounts was evaluated using UPS2-infused, pH-adjusted AAV8 reference material with empty capsids. Following the ProteoMiner™ enrichment protocol, 2.5 µL of 1 M acetic acid was added to 500 µL of AAV8 product to adjust the pH to approximately 6. Subsequently, 1 / 16 of UPS2 (0.66 µg protein) was incorporated into 500 µL of pH-adjusted AAV8 empty capsids to assess the limit of detection. 1 / 2, 1 / 5, 1 / 20, or 1 / 50 of the ProteoMiner™ kit (10 µL beads), 1 / 5, 4 µL, 1 / 20, or 1 / 50 (0.4 µL beads) of the ProteoMiner™ kit were incubated with 100 µL of pH-adjusted AAV8 material incorporating UPS2. An average of 167 HCPs were detected after ProteoMiner™ enrichment, while only 45 HCPs were detected by direct digestion. Figure 1A After applying enrichment beads, all UPS2 standards above 6.25 fmol were detected, while one-quarter of these lower concentration (≤4 ng / mL) standards could not be detected by direct digestion (Table 1). The number of HCPs (…) was also observed when different amounts of beads were applied. Figure 1A ) or the number of unique peptides identified from 6.25 fmol of UPS2 standards ( Figure 1B No discernible changes were identified. These results indicate that, in terms of detection limit and sensitivity, enrichment efficiency is not affected by the number of beads used. All subsequent experiments used 1 µL of enrichment beads.

[0244] Evaluation of the amount of sample used in the experiment

[0245] Due to the low titers during drug production, the quantity of AAV materials is often limited. Therefore, to evaluate the HCP analysis and detection limit, we adjusted 20 µL, 50 µL, and 100 µL of AAV8 and AAV9 reference materials to pH 6 and then incubated them with 1 µL of ProteoMiner™ beads. The aim was to determine the minimum sample volume to evaluate the detection limit for HCP analysis. The number of HCPs detected was compared to the detection limit using AAV8 reference material incorporating UPS2 and pH-adjusted AAV9 reference material. The number of HCPs in the AAV8 and AAV9 reference materials increased by 60% when using 50 µL or 100 µL of sample, and by 46% when using 20 µL. Surprisingly, the number of HCPs detected remained the same when enriched with 100 µL of sample as when using 50 µL. Figure 1CHowever, only five out of eight UPS2 at 6.25 fmol were detected from a 50 µL sample, compared to eight out of eight UPS2 at 6.25 fmol from a 100 µL sample. Figure 1D When 100 µL of sample was applied, a detection limit of 0.5 ng / mL was observed, and the detection limit increased as the amount of sample used for enrichment decreased. 100 µL of LAAV reference material was used for all subsequent experimental procedures.

[0246] Optimization of solution pH in enrichment methods

[0247] The pH of the binding solution for the AAV product was evaluated. The AAV reference material was initially stored in PBS buffer containing 0.01% Pluronic F-68, pH 7. The material was adjusted to pH 6 by adding 50 mM acetic acid, or to pH 8 by adding 50 mM Tris-HCl, pH 8.0. Enrichment was performed by incubating 1 µL of ProteoMiner™ beads with 100 µL of AAV reference material. For the AAV9 reference material with an empty capsid, 191 HCPs were detected under acidic conditions, 262 HCPs were detected under neutral conditions, and 109 HCPs were detected under alkaline conditions. Figure 1E To evaluate the neutral incubation conditions for AAV serotypes used for HCP enrichment, additional experiments were performed on AAV1, AAV5, and AAV8 reference materials with empty capsids at pH 6 and pH 7. Compared to acidic conditions, the number of HCPs detected from different AAV serotypes increased by 15.6%–50.6% when the AAV reference materials were incubated with enrichment beads under neutral conditions. Figure 1F Therefore, a neutral pH was chosen for enriching HCP from AAV samples.

[0248] Method reproducibility

[0249] The reproducibility of the method was evaluated in triplicate under the experimental conditions, which included incubating 1 µL of RotooMiner™ beads with 100 µL of AAV reference material in PBS buffer. Approximately 146 HCPs were identified from the AAV8 reference material with intact capsids using enrichment methods, a number 4.3 times that identified by direct digestion. In three runs, 131 common HCPs were identified, representing 80.9% of all proteins. Figure 2B High reproducibility indicates a high level of confidence in protein identification—a property of HCP analytical methods.

[0250] The application of enrichment beads increased the identification of HCP in all AAV serotypes by 5 to 10 times.

[0251] The number of HCPs identified using the enrichment method in this study from AAV1, AAV5, AAV8, and AAV9 reference materials with empty capsids was 5 to 10 times that of direct digestion. Figure 3B This enhancement was observed in all AAV serotypes. Over 80% of newly identified HCPs were detected using enrichment methods, thus revealing less than 1.2% of missed proteins compared to direct digestion methods. These findings suggest that this method can comprehensively detect many HCPs present in low abundance that would otherwise be undetectable by direct digestion.

[0252] Assessment of the reduction in dynamic range of AAV9 reference material containing UPS2-doped standards

[0253] To assess the reduction in the dynamic range of the enrichment method, 1 / 40 UPS2 (0.264 µg protein) was incorporated into 200 µL of empty LAAV9 capsid. Seven UPS2 standards ranging from 0.1 ng / mL to 414.7 ng / mL were selected for comparison. The concentration of each UPS2-incorporated standard was calculated by comparing the relative abundance of the UPS2 standard peptide to the capsid protein peptide in samples prepared by the enrichment method and the direct digestion method. The enrichment factor was calculated by comparing the calculated concentration with the incorporation concentration (Table 3). The enrichment factor of the UPS2 standards prepared by direct digestion was close to 1 because no enrichment occurred. Some standards (such as serum albumin and peroxidase) had enrichment factors slightly greater than 1, which may be due to endogenous HCP in the AAV reference material. The enrichment factors of the samples treated with ProteoMiner™ ranged from 1.5 to 25. For UPS2 concentrations above 1.6 ng / mL, the dynamic range between the UPS2 peptide and the AAV capsid protein peptide was 3.5 × 10⁻⁶ for the enrichment method. -4 With 3.0 × 10 -1 Between, and for direct digestion, the dynamic range between UPS2 peptide and AAV capsid protein peptide is 2.5 × 10⁻⁶. -4 With 3.0 × 10 -2 The concentrations ranged from 3.6 to 34.1 times after enrichment, indicating an increase of 3.6 to 34.1 times. (VFEHIGK, a catalase peptide) 2+ Ribosyl dihydronicotinamide dehydrogenase [quinone] [quinone oxidoreductase 2] [NQO2] peptide NVAVDELSR 2+ The signal changes are shown in Figure 5A In the enriched samples, UPS2 and AAV capsid peptides showed a dynamic range of 3.5 × 10⁻⁶ ng / mL for neddylin and thioredoxin at 0.6 ng / mL. -4 and 7.2 × 10 -4In the case of direct digestion, the predicted dynamic range between these two UPS2 peptides and the capsid protein peptide is approximately 10. -5 Therefore, neddylin and thioredoxin, as well as other host cell proteins that may be present at low ng / mL or even lower concentrations, are undetectable in direct digestion preparation. Enriched to 0.6 ng / mL, the neddylin peptide EIEIDIEPTDKVER... 3+ and 0.1 ng / mL of thioredoxin peptide TAFQEALDAAGDK 2+ MS2 mass spectrum ( Figure 5B The results show that the sensitivity of detecting these low-abundance HCPs (< 1 ng / mL) using enrichment methods is significantly improved. The enrichment factors of the remaining incorporated HCPs are reported in Table 3.

[0254] Table 3. Summary of enrichment factors and enrichment factor comparisons for HCP incorporated into AAV reference materials using ProteoMiner™ enrichment method and direct digestion.

[0255]

[0256] Complete detergent removal after enrichment bead treatment

[0257] ProteoMiner™ technology is used to enrich low-abundance proteins in pharmaceutical products using bead-based peptide ligands. Only proteins (such as antibodies, AAV capsid proteins, and host cell proteins) bind to the beads. Detergents (polysorbate or Pluronic F-68) do not bind to the enrichment beads and are therefore removed from the protein mixture after enrichment. Molecular weight cutoff filters cannot easily remove these detergents, and therefore filtration does not reduce detergent interference. Figure 6 Additionally, we observed significant loss of HCP during buffer exchange, likely due to their adhesion to the filter membrane. In the case of direct digestion, the TIC and MS signals of the Pluronic F-68 were more than 20 times that of the viral protein peptides. Figures 7A to 7B This significant difference in signal levels poses a major challenge to the detection of HCP peptides, as the abundance of detectable HCP peptides is only 1 / 100 to 1 / 10,000 of that of viral protein peptides. TIC chromatography and corresponding MS mass spectra of the enriched peptide mixture are shown in (…). Figures 7C to 7D In the TIC spectrum, the detergent peak was completely eliminated by the enrichment beads, and certain HCP peptides at levels similar to viral protein peptides were observed.

[0258] in conclusion

[0259] To date, the inability to apply MS-based HCP analysis methods to gene therapy products in this field has been hampered by several challenges. These include limited sample quantities, interference from detergents used during viral vector production, and incomplete coverage by anti-HCP antibodies.

[0260] In this study, the disclosed method was demonstrated to successfully detect HCPs in gene therapy viral vector products. Comprehensive optimizations were performed to develop a sensitive HCP enrichment method for AAV products. The method disclosed herein increases the number of detected HCPs by 5 to 10-fold and improves the dynamic range by up to two orders of magnitude compared to direct digestion. The method achieves a reproducibility of 80.9% and can detect HCPs as low as 0.1 ng / mL.

[0261] By providing comprehensive and orthogonal information about AAV HCPs, this approach can serve as a valuable tool for troubleshooting and identifying potentially problematic HCPs. Additionally, this approach can help assess the risks associated with AAV products.

[0262] In this study, a total of 847 host cell proteins (HCPs) from adeno-associated virus (AAV) types 1, 5, 8, and 9 were identified. This identification was achieved by combining ProteoMiner™ enrichment with nanoliter LC-MS / MS. These HCPs can be classified according to their biological functions. For example, structural proteins such as desmosome plaque protein, filamentin-A, myosin-9, reticulin, tubulin, actin, vimentin, adhesion plaque protein, and ezrin were identified. Transport proteins such as serum proteins, α-2-macroglobulin-like proteins, apolipoproteins, and haptoglobins were also discovered. Enzymes, including protein-glutamine, caspase-14, inosine-5'-monophosphate, fructose-bisphosphate, gamma-glutamyl cyclotransferase, cathepsins, glyceraldehyde-3-phosphate, arginase-1, triose phosphate, peroxidase-2, carboxypeptidase, and transaldolase, were also identified. Binding proteins, such as RNA-binding proteins, annexins, leucine-rich proteins, polypyrimidines, and calcine-binding proteins, were also identified. Additionally, transcriptional proteins, heat shock proteins, immunoglobulins, glycoproteins, and ribonucleoproteins were identified in HCPs (Table 4). HCPs that can regulate cellular function, including but not limited to peroxidase 1, ubiquitin, actin, and Hsc70, were also detected in AAV products. Figure 4 The ProteoMiner™ enrichment method identified 584 previously unreported HCPs. The presence of HCP impurities can affect drug efficacy, and some HCPs, such as cathepsins, can lead to viral protein degradation. Therefore, the identification of these low-abundance HCPs, which may not be easily detected using other methods, can be used for troubleshooting and optimizing cell line production and purification.

[0263] Table 4. Summary of HCP gene names identified from all serotypes of AAV reference materials prepared using all different enrichment parameters and digestion conditions (Column 1). HCPs identified from UPS2 standards using ProteoMiner™ enrichment as a negative control (Column 2). HCPs identified from AAV standards without UPS2 standards (Column 3). HCPs identified from all serotypes of AAV reference materials prepared using all different enrichment parameters and digestion conditions and reported in the referenced paper (Column 4).

[0264]

[0265] Those skilled in the art will understand that various changes and modifications can be made to the preferred embodiments disclosed herein, and such changes and modifications can be made without departing from the spirit of the invention. Therefore, the following claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.

[0266] The disclosure of each patent, patent application, and publication cited or described herein is hereby incorporated in its entirety by reference.

Claims

1. A method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising: (a) A sample containing at least one HCP impurity is brought into contact with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby producing a slurry; (b) Wash the slurry containing at least one HCP impurity to remove unbound material; (c) Elution of bound HCP to produce an enriched HCP sample; (d) subject the enriched HCP to enzymatic digestion conditions to produce peptide digests; as well as (e) subject the peptide digest to tandem mass spectrometry (MS / MS) analysis to enrich, identify and / or characterize the at least one HCP impurity.

2. The method of claim 1, wherein the enriched HCP is subjected to a denaturing agent to produce a denatured sample.

3. The method according to claim 2, wherein the denaturing agent comprises heat, high pH, ​​low pH, reducing agent, or liquid-dissolving agent.

4. The method according to any one of claims 1 to 3, wherein the enriched HCP is subjected to an alkylating agent to produce an alkylated sample.

5. The method according to claim 4, wherein the alkylating agent comprises iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), 4-vinylpyridine, or a combination thereof.

6. The method according to any one of claims 1 to 5, wherein the enriched HCP is subjected to a reducing agent to produce a reduced sample.

7. The method according to claim 6, wherein the reducing agent comprises dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or a combination thereof.

8. The method according to any one of claims 1 to 7, wherein the enriched HCP is subjected to a denaturing agent, an alkylating agent, and a reducing agent to produce a denatured, reduced, and alkylated sample.

9. The method according to any one of claims 1 to 8, wherein the viral vector is an AAV vector.

10. The method of claim 9, wherein the AAV vector comprises a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof.

11. The method according to any one of claims 1 to 10, wherein the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL.

12. The method according to any one of claims 1 to 11, wherein the peptide ligand library comprises a binding solution with a pH of about pH 6.0 to about pH 8.0, optionally wherein the pH is about 7.

0.

13. The method according to any one of claims 1 to 12, wherein the volume of the peptide ligand library is about 0.4 µL to about 15 µL.

14. The method according to any one of claims 1 to 13, wherein the enzymatic digestion conditions comprise contacting the denatured, reduced, and alkylated sample with at least one digestive enzyme.

15. The method of claim 14, wherein the at least one digestive enzyme comprises trypsin.

16. The method according to any one of claims 1 to 15, wherein the mass spectrometer is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer.

17. The method according to any one of claims 1 to 16, wherein the mass spectrometer is coupled to a liquid chromatography system.

18. A method for enriching, identifying, and / or characterizing at least one host cell protein (HCP) impurity in a sample containing at least one viral vector, the method comprising: (a) A sample containing at least one HCP impurity is brought into contact with a solid support having a library of peptide ligands attached thereto capable of interacting with the at least one HCP impurity, thereby producing a slurry; (b) Wash the slurry containing at least one HCP impurity to remove unbound material; (c) Elution of bound HCP to produce an enriched HCP sample; (d) subject the enriched HCP sample to a denaturing agent to produce a denatured sample; (e) subjecting the denatured sample to a reducing agent to produce a denatured and reduced sample; (f) subjecting the denatured and reduced sample to an alkylating agent to produce a denatured, reduced and alkylated sample; (g) subjecting the denatured, reduced and alkylated sample to enzymatic digestion conditions to produce peptide digests; as well as (h) The peptide digest is subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis to identify and / or characterize the at least one HCP impurity.

19. The method according to claim 18, wherein the viral vector is an AAV vector.

20. The method of claim 19, wherein the AAV vector comprises a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV2 / 8, AAV9, AAV10, AAV11, AAV12, variants thereof, and combinations thereof.

21. The method of claim 20, wherein the amount of the sample containing at least one host cell protein impurity is from about 20 µL to about 100 µL.

22. The method according to any one of claims 18 to 21, wherein the volume of the interacting peptide ligand is from about 0.4 µL to about 15 µL.

23. The method according to any one of claims 18 to 22, wherein the peptide ligand library comprises a binding solution with a pH of about pH 6.0 to about pH 8.0, optionally wherein the pH is about 7.

0.

24. The method according to any one of claims 18 to 23, wherein the denaturing agent comprises heat, high pH, ​​low pH, reducing agent, or liquid-dissolving agent.

25. The method according to any one of claims 18 to 24, wherein the alkylating agent comprises iodoacetamide (IOA / IAA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), 4-vinylpyridine, or a combination thereof.

26. The method according to any one of claims 18 to 25, wherein the reducing agent comprises dithiothreitol (DTT), β-mercaptoethanol, Elman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or a combination thereof.

27. The method according to any one of claims 18 to 26, wherein the enzymatic digestion conditions comprise contacting the denatured, reduced, and alkylated sample with at least one digestive enzyme, optionally wherein the at least one digestive enzyme comprises trypsin.

28. The method according to any one of claims 18 to 27, wherein the mass spectrometry system is an electrospray ionization mass spectrometer, a nanoelectrospray ionization mass spectrometer, or an orbital trap-based mass spectrometer.

29. The method according to any one of claims 18 to 28, wherein the mass spectrometer is coupled to the liquid chromatography system.

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