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WO2025166281A1 - Plateforme d'analyse par spectrométrie de masse à détection de charge d'aavs - Google Patents

Plateforme d'analyse par spectrométrie de masse à détection de charge d'aavs

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Publication number
WO2025166281A1
WO2025166281A1 PCT/US2025/014183 US2025014183W WO2025166281A1 WO 2025166281 A1 WO2025166281 A1 WO 2025166281A1 US 2025014183 W US2025014183 W US 2025014183W WO 2025166281 A1 WO2025166281 A1 WO 2025166281A1
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WIPO (PCT)
Prior art keywords
antibody
aav
flow rate
minute
viral vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/014183
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English (en)
Inventor
Chen DU
Victoria COTHAM
Shunhai WANG
Ning Li
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Publication of WO2025166281A1 publication Critical patent/WO2025166281A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/728—Intermediate storage of effluent, including condensation on surface
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray

Definitions

  • Gene therapy biopharmaceuticals mediate therapeutic effects by transcription and/or translation of transferred genetic material, such as integrating genetic material into the host genome, and are used to treat, prevent or cure a disease.
  • gene therapy is one of the most investigated therapeutic modalities in preclinical and clinical settings.
  • concerns have been raised about the safety of gene therapy and the critical need for safer gene delivery vectors.
  • a better understanding of gene delivery vectors and advancing the manufacture of safe and effective vectors is necessary to mitigate safety risks.
  • Gene delivery vectors are important to ensure efficient gene delivery to the target tissue and cells.
  • the ideal gene delivery system should have high gene transfer efficiency, low toxicity to the cell, and single cell specificity to the intended target.
  • vectors can be divided into non-viral vectors and viral vectors. Due to the high gene transfer efficiency of viral vectors, they have been widely used in clinical trials.
  • Adeno-associated virus is the most widely used viral vector for in vivo gene therapy applications. AAVs have low immunogenicity and they can enable long-term, stable gene expression. The use of AAVs for gene therapy has created the 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.
  • Biopharmaceutical products should meet very high standards of purity. Thus, it is important to monitor any impurities in such biopharmaceutical products at different stages of drug development, production, storage and handling. Residual impurities should be at an acceptable low level prior to conducting clinical studies. Residual impurities are also a concern for biopharmaceutical products intended for end-users. For example, an inherent characteristic of the AAV manufacturing process is the production of empty capsids, a capsid which lacks any packaged transgene. The presence of empty capsids in drug products should be monitored and can be unacceptable above a certain threshold. Sometimes, even empty capsids could result in an unwanted immune response and may reduce the potency of the gene therapy drug.
  • CDMS Charge-detection mass spectrometry
  • This disclosure sets forth newly developed size-exclusion chromatography (SEC)-based flow injection (FI) systems and methods to enable online desalting and long- lasting infusion for native MS and CDMS measurement of AAVs and AAV-antibody conjugates.
  • SEC size-exclusion chromatography
  • FI flow injection
  • the systems can comprise a liquid chromatography system, comprising a first pump coupled to a size exclusion chromatography column, wherein said column is further coupled to a sample loop, and a second pump coupled to said sample loop; and a mass spectrometry system, comprising a microflow nanospray ion source, wherein said mass spectrometry system is coupled to said sample loop, and said mass spectrometry system is configured to perform charge detection mass spectrometry analysis of a polypeptide or viral vector.
  • said first pump is configured for a flow rate of about 100 pL/minute to about 300 pL/minute. In another aspect, said first pump is configured for a flow rate of about 200 pL/minute. In a further aspect, said first pump is configured for a flow rate of about 10 pL/minute to about 300 pL/minute.
  • said first pump is configured for a flow rate of about 10 pL/minute, about 20 pL/minute, 50 pL/minute, about 75 pL/minute, about 100 pL/minute, about 125 pL/minute, about 150 pL/minute, about 175 pL/minute, about 200 pL/minute, about 225 pL/minute, about 250 pL/minute, about 275 pL/minute, or about 300 pL/minute.
  • said first pump is configured for a flow rate of about 20 pL/minute.
  • said first pump is configured for a flow rate of about 200 pL/minute.
  • said size exclusion chromatography column has an internal diameter of about 4 mm to about 5 mm. In another aspect, said size exclusion chromatography column has an internal diameter of about 4.6 mm. In a further aspect, said size exclusion chromatography column has an internal diameter of about 0.5 mm to about 1.5 mm, about 1 mm to about 2 mm, or about 1 mm to about 5 mm. In yet another aspect, said size exclusion chromatography column has an internal diameter of about 1 mm or about 1.5 mm.
  • said size exclusion chromatography column has an internal diameter less than about 5 mm, about 4.5 mm, about 4 mm, about 3.5 mm, about 3 mm, about 2.5 mm, about 2 mm, about 1.5 mm, or about 1 mm.
  • said size exclusion chromatography column has a length of about 100 mm to about 200 mm. In another aspect, said size exclusion chromatography column has a length of about 150 mm. In a further aspect, said size exclusion chromatography column has a length of about 40 mm to about 60 mm or about 40 mm to about 200 mm. In yet another aspect, said size exclusion chromatography column has a length of about 50 mm, about 75 mm, about 100 mm, about 125 mm, about 150 mm, about 175 mm, or about 200 mm.
  • said sample loop has a volume of about 100 pL to about 300 pL. In another aspect, said sample loop has a volume of about 200 pL. In a further aspect, said sample loop has a volume of about 50 pL to about 100 pL, about 50 pL to about 300 pL, or about 100 pL to about 300 pL.
  • said sample loop has a volume of about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 110 pL, about 120 pL, about 130 pL, about, 140 pL, about 150 pL, about 160 pL, about 170 pL, about 180 pL, about 190 pL, or about 200 pL. In yet another aspect, said sample loop has a volume of about 60 pL.
  • said second pump is configured for a flow rate of about 0.5 pL/minute to about 2 pL/minute. In another aspect, said second pump is configured for a flow rate of about 0.5 pL/minute, about 1 pL/minute, about 1.5 pL/minute, about 2 pL/minute, or about 2.5 pL/minute. In another aspect, said second pump is configured for a flow rate of about 2 pL/minute.
  • the system further comprises a gas input to the microflow nanospray ion source configured at a flow rate of about 0 L/min to about 2 L/min, about 0 L/min to about 1 L/min, or about 1 L/min to about 2 L/min.
  • the gas is a sheath gas.
  • said first pump is configured for a flow rate that is greater than the flow rate for which said second pump is configured.
  • said sample loop comprises tubing connecting an output of said size exclusion chromatography column and an input of said microflow nanospray ion source.
  • said polypeptide or viral vector of interest is an adeno-associated viral vector.
  • said adeno-associated viral vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 rhlO, rh39, rh43, rh74, Avian AAV, Sea Lion AAV, Bearded Dragon AAV, and variations thereof, and combinations thereof.
  • this disclosure further provides methods for identifying, characterizing, and/or quantitating a polypeptide or viral vector of interest.
  • the methods can comprise (a) subjecting a sample including a polypeptide or viral vector of interest to size exclusion chromatography separation at a first flow rate to form a desalted sample; (b) injecting said desalted sample into an electrospray ion source at a second flow rate to form ions; and (c) subjecting said ions to charge detection mass spectrometry analysis to identify, characterize, and/or quantitate said polypeptide or viral vector of interest.
  • said polypeptide or viral vector of interest is an adeno-associated viral vector.
  • said adeno-associated viral vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rhlO, rh39, rh43, rh74, Avian AAV, Sea Lion AAV, Bearded Dragon AAV, variations thereof, and combinations thereof.
  • said viral vector of interest is an antibody- viral vector conjugate.
  • said characterizing comprises quantitating a number of antibodies in said antibody- viral vector conjugate.
  • the viral vector can comprise a first cognate member of a specific binding pair covalently bound together, and the antibody is linked to a second cognate member of the specific binding pair covalently bound together.
  • the first member and second cognate member can be a system selected from the group consisting of Spy Tag:Spy Catcher, Spy Tag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, SnoopTag:SnoopCatcher, Isopeptag:Pilin-C, Isopeptag:Pilin-N, SnoopTagJr:SnoopCatcher, DogTag:DogCatcher, SdyTg:SdyCatcher, Jo:In, 3kptTag: 3kptCatcher, 4oqlTaq/4oqlCatcher, NGTag/NGCatcher, Rumtrunk/Mooncake, Snoop ligase, GalacTag, Cpe, Ececo, and Corio.
  • Spy Tag002:SpyCatcher002 and SpyTag003:SpyCatcher003 are different iterations of Spy Tag:Spy Catcher.
  • said size exclusion chromatography separation and said mass spectrometry analysis are performed under native conditions.
  • a mobile phase for said size exclusion chromatography is ammonium acetate.
  • a concentration of said ammonium acetate is about 100 mM to about 200 mM.
  • a concentration of said ammonium acetate is about 150 mM.
  • said first flow rate is about 100 pL/minute to about 300 pL/minute. In another aspect, said first flow rate is about 200 pL/minute. In a further aspect, said first pump is configured for a flow rate of about 10 pL/minute to about 300 pL/minute.
  • said first pump is configured for a flow rate of about 10 pL/minute, about 20 pL/minute, 50 pL/minute, about 75 pL/minute, about 100 pL/minute, about 125 pL/minute, about 150 pL/minute, about 175 pL/minute, about 200 pL/minute, about 225 pL/minute, about 250 pL/minute, about 275 pL/minute, or about 300 pL/minute.
  • said first pump is configured for a flow rate of about 20 pL/minute.
  • said first pump is configured for a flow rate of about 200 pL/minute.
  • said first flow rate is controlled by a first pump.
  • said desalted sample is contained in a sample loop.
  • said sample loop comprises tubing connecting said size exclusion chromatography column and said electrospray ion source.
  • a volume of said sample loop is about 100 pL to about 300 pL.
  • a volume of said sample loop is about 200 pL.
  • said sample loop has a volume of about 50 pL to about 100 pL, about 50 pL to about 300 pL, or about 100 pL to about 300 pL.
  • said sample loop has a volume of about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, about 110 pL, about 120 pL, about 130 pL, about, 140 pL, about 150 pL, about 160 pL, about 170 pL, about 180 pL, about 190 pL, or about 200 pL. In yet another aspect, said sample loop has a volume of about 60 pL.
  • said second flow rate is controlled by a second pump. In another aspect, said first flow rate is greater than said second flow rate.
  • said electrospray ion source is a microflow nanospray ion source.
  • said injecting is performed for from 30 minutes to 240 minutes. In another aspect, said injecting is performed for about 30 minutes, about 60 minutes, about 180 minutes, or about 240 minutes.
  • the method further comprises subjecting the results of said charge detection mass spectrometry analysis to frequency correction.
  • the method further comprises injecting said desalted sample into said electrospray ion source at said first flow rate to form ions, and subjecting said ions to mass spectrometry analysis to further identify, characterize, and/or quantitate said polypeptide or viral vector of interest.
  • FIG. 1A is a schematic diagram of an Orbitrap analyzer for charge-detection mass spectrometry (CDMS) analysis, according to an exemplary aspect.
  • CDMS charge-detection mass spectrometry
  • FIG. IB depicts a calculation of ion mass using CDMS analysis, according to an exemplary aspect.
  • FIG. 1C depicts a comparison of a protein mass spectrum to a single ion CDMS, according to an exemplary aspect.
  • FIG. ID depicts an example of highly glycosylated spike proteins that can be resolved using CDMS, according to an exemplary aspect.
  • FIG. IE depicts an example of large AAV vectors that can be resolved using CDMS, according to an exemplary aspect.
  • FIG. 2A depicts single ion data acquisition for STORI analysis of Orbitrapbased CDMS, according to an exemplary aspect.
  • FIG. 2B depicts analysis of ion data using STORI analysis, according to an exemplary aspect.
  • FIG. 2C depicts data processing of STORI analysis, according to an exemplary aspect.
  • FIG. 3A depicts a static infusion system for an ultra-high mass range (UHMR) mass spectrometry system, according to an exemplary aspect.
  • UHMR ultra-high mass range
  • FIG. 3B depicts a total ion chromatogram (TIC) trace of static infusion of an AAV sample, according to an exemplary aspect.
  • TIC total ion chromatogram
  • FIG. 4A depicts a high flow configuration of a size exclusion chromatographyflow injection (SEC-FI) MS system, according to an exemplary aspect.
  • SEC-FI size exclusion chromatographyflow injection
  • FIG. 4B depicts a low flow configuration of a SEC-FI-CDMS system, according to an exemplary aspect.
  • FIG. 5A depicts efficient online desalting of an AAV8 sample using high-flow SEC-FI (native MS), according to an exemplary aspect.
  • FIG. 5B depicts efficient stable infusion of an AAV8 sample using low-flow SEC-FI-CDMS (single ion CMDS), according to an exemplary aspect.
  • FIG. 6A depicts optimized parameters for SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 6B depicts an optimized low flow configuration SEC-FI-CDMS system, according to an exemplary aspect.
  • FIG. 6C depicts a comparison of a non-optimized (“Standard”) SEC-FI-MS analysis and an optimized (“AAV”) SEC-FI-MS analysis, according to an exemplary aspect.
  • FIG. 6D depicts a comparison of a non-optimized (“Standard”) SEC-FI-CDMS analysis and an optimized (“AAV”) SEC-FI-CDMS analysis, according to an exemplary aspect.
  • FIG. 7A depicts mass measurements of empty and full AAV8 using Orbitrapbased SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 7B depicts a comparison of SEC-FI-CDMS analysis with and without sheath gas, according to an exemplary aspect.
  • FIG. 8A depicts a comparison of SEC-FI-MS analysis of empty AAV8 with and without STORI frequency correction, according to an exemplary aspect.
  • FIG. 8B depicts a comparison of SEC-FI-MS analysis of full AAV8 with and without STORI frequency correction, according to an exemplary aspect.
  • FIG. 9A depicts native MS analysis of VP3-only AAV using static infusion, according to an exemplary aspect.
  • FIG. 9B depicts SEC-FI-CDMS analysis of VP3-only AAV, according to an exemplary aspect.
  • FIG. 10A depicts SEC-FI-CDMS analysis of VP3-only AAV at varying mixing ratios, according to an exemplary aspect.
  • FIG. 10B depicts a comparison of calculated and measured VP3-only AAV at varying mixing ratios, according to an exemplary aspect.
  • FIG. 11A depicts total ion chromatograms from high-flow SEC-FI analysis of a sample of mixed empty and full AAV vectors, according to an exemplary aspect.
  • FIG. 11B depicts SEC-FI-CDMS analysis of full and empty AAV at varying mixing ratios, according to an exemplary aspect.
  • FIG. 11C depicts a comparison of predicted and measured masses of full and empty AAV, according to an exemplary aspect.
  • FIG. 11D depicts a comparison of SEC-FI-CDMS measurements of full and empty AAV masses across replicates, according to an exemplary aspect.
  • FIG. 12A depicts native SEC-FI-MS analysis of full and empty AAV at varying mixing ratios, according to an exemplary aspect.
  • FIG. 12B depicts SEC-FI-CDMS analysis of full and empty AAV at varying mixing ratios, according to an exemplary aspect.
  • FIG. 12C depicts a comparison of calculated and measured full AAV at varying mixing ratios, according to an exemplary aspect.
  • FIG. 12D depicts a comparison of full AAV measured at varying mixing ratios using AUC or native SEC-FI-MS, according to an exemplary aspect.
  • FIG. 12E depicts a comparison of full AAV measured at varying mixing ratios using AUC or SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 13A depicts native SEC-FI-MS analysis of empty AAV8 capsid at varying injection amounts, according to an exemplary aspect.
  • FIG. 13B depicts SEC-FI-CDMS analysis of empty AAV8 capsid at varying injection amounts, according to an exemplary aspect.
  • FIG. 14A depicts a measurement of empty, partially filled, and full AAV vectors using low-flow SEC-FI analysis for a first AAV8 lot, according to an exemplary aspect.
  • FIG. 14B depicts a measurement of empty, partially filled, and full AAV vectors using low-flow SEC-FI analysis for a second AAV8 lot, according to an exemplary aspect.
  • FIG. 14C depicts a measurement of empty, partially filled, and full AAV vectors using low-flow SEC-FI analysis for a third AAV8 lot, according to an exemplary aspect.
  • FIG. 14D depicts a measurement of empty, partially filled, and full AAV vectors using low-flow SEC-FI analysis for a fourth AAV8 lot, according to an exemplary aspect.
  • FIG. 14E depicts a comparison of measurements of empty, partially filled, and full AAV vectors using low-flow SEC-FI analysis across four AAV8 lots, according to an exemplary aspect.
  • FIG. 15A depicts a measurement of empty, partially filled, and full AAV 1 and AAV5 vectors using SEC-FI-CDMS analysis, according to an exemplary aspect.
  • FIG. 15B depicts a comparison of expected and measured empty, partially filled, and full AAV1 and AAV5 vectors, according to an exemplary aspect.
  • FIG. 15C depicts a comparison of measured empty, partially filled, and full AAV1 and AAV5 vectors using AUC or SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 16A illustrates an antibody-AAV conjugate, according to an exemplary aspect.
  • FIG. 16B illustrates a legend for FIGs. 16C-16H, according to an exemplary aspect.
  • FIG. 16C depicts a mass spectrum of a sample with a 1:0 ratio of AAV9- SpyT:SpyC-mAb obtained using low-flow SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 16D depicts a mass spectrum of a sample with a 1:4 ratio of AAV9- SpyT:SpyC-mAb obtained using low-flow SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 16E depicts a mass spectrum of a sample with a 1:8 ratio of AAV9- SpyT:SpyC-mAb obtained using low-flow SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 16F depicts a mass spectrum of a sample with a 1:12 ratio of AAV9- SpyT:SpyC-mAb obtained using low-flow SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 16G depicts a mass spectrum of a sample with a 1:16 ratio of AAV9- SpyT:SpyC-mAb obtained using low-flow SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 16H depicts a mass spectrum of a sample with a 1:100 ratio of AAV9- SpyT:SpyC-mAb obtained using low-flow SEC-FI-CDMS, according to an exemplary aspect.
  • FIG. 17A depicts measured masses of AAV-antibody conjugates using SEC-FI- CDMS, according to an exemplary aspect.
  • FIG. 17B depicts a relative abundance of AAV-antibody conjugate species, according to an exemplary aspect.
  • FIG. 18A depicts a SEC-FI-CDMS analysis of AAV surface charge as a function of anti- AAV antibody binding at a ratio of 1:0 of AAV9-SpyT:SpyC-mAb, according to an exemplary aspect.
  • FIG. 18B depicts a SEC-FI-CDMS analysis of AAV surface charge as a function of anti- AAV antibody binding at a ratio of 1:4 of AAV9-SpyT:SpyC-mAb, according to an exemplary aspect.
  • FIG. 18C depicts a SEC-FI-CDMS analysis of AAV surface charge as a function of anti- AAV antibody binding at a ratio of 1:8 of AAV9-SpyT:SpyC-mAb, according to an exemplary aspect.
  • FIG. 18D depicts a SEC-FI-CDMS analysis of AAV surface charge as a function of anti- AAV antibody binding at a ratio of 1:12 of AAV9-SpyT:SpyC-mAb, according to an exemplary aspect.
  • FIG. 18E depicts a SEC-FI-CDMS analysis of AAV surface charge as a function of anti- AAV antibody binding at a ratio of 1:16 of AAV9-SpyT:SpyC-mAb, according to an exemplary aspect.
  • FIG. 18F depicts a SEC-FI-CDMS analysis of AAV surface charge as a function of anti- AAV antibody binding at a ratio of 1:100 of AAV9-SpyT:SpyC-mAb, according to an exemplary aspect.
  • FIG. 19A depicts TIC traces from SEC-FI-CDMS charge calibration using various protein standards, according to an exemplary aspect.
  • FIG. 19B depicts charge calibration coefficients across four protein standards using the SEC-FI-CDMS platform, according to an exemplary aspect.
  • FIG. 20A depicts a measurement of empty, partially full, and full AAV 1 vectors comprising a 3’ template (top) or a 5’ template (bottom) using SEC-FI-CDMS analysis, according to an exemplary aspect.
  • FIG. 20B depicts a comparison of measured empty, partially filled, and full AAV1 vectors comprising a 3’ template or a 5’ template, according to an exemplary aspect.
  • FIG. 21A depicts SEC-FI-CDMS analysis of full and empty AAV 1 comprising a 3’ template and AAV1 comprising a 5’ template at varying mixing ratios, according to an exemplary aspect.
  • FIG. 21B depicts a comparison of measured full AAV1 comprising a 3’ template and AAV1 comprising a 5’ template at varying mixing ratios, according to an exemplary aspect.
  • FIG. 22A depicts SEC-CDMS analysis of empty and full AAV 1 comprising a 3’ template or a 5’ template, according to an exemplary aspect.
  • FIG. 22B depicts SEC-CDMS analysis of empty and full AAV 1 comprising a 3’ template or a 5’ template, according to an exemplary aspect.
  • FIG. 22C depicts SEC-CDMS analysis of empty and full AAV 1 comprising a 3’ template or a 5’ template, according to an exemplary aspect.
  • FIG. 22D depicts SEC-CDMS analysis of empty and full AAV 1 comprising a 3’ template or a 5’ template, according to an exemplary aspect.
  • FIG. 22E depicts SEC-CDMS analysis of empty and full AAV 1 comprising a 3’ template or a 5’ template, according to an exemplary aspect.
  • FIG. 23A depicts a comparison of measured empty, partially filled, and full AAV1 comprising a 3’ template, a 5’ template, or a sample comprising both an AAV 1-3’ template and an AAV1-5’ template at either time 0 (TO) or after incubated at 37°C for 14 days (37°C 14d), according to an exemplary aspect.
  • FIG. 23B depicts a comparison of the percentage of empty, partially filled, and full AAV1 based on the conditions as shown in FIG. 23A.
  • FIG. 24A depicts a comparison of measured empty, partially filled, full, and heavy molecular weight (HMW) species of AAV1 comprising a 3’ template, a 5’ template, or a sample comprising both an AAV 1-3’ template and an AAV 1-5’ template at either time 0 (TO) or after incubated at 37°C for 14 days (37°C 14d), according to an exemplary aspect.
  • HMW heavy molecular weight
  • FIG. 24B depicts SEC-CDMS analysis of empty, partially filled, full, and heavy molecular weight (HMW) species of AAV1 comprising a 3’ template, a 5’ template, or a sample comprising both an AAV 1-3’ template and an AAV 1-5’ template at either time 0 (TO) or after incubated at 37°C for 14 days (37°C 14d), according to an exemplary aspect.
  • HMW heavy molecular weight
  • FIG. 25 depicts mass v. charge heat maps of empty, partially filled, full, and heavy molecular weight (HMW) species of AAV1 comprising a 3’ template, a 5’ template, or a sample comprising both an AAV1-3’ template and an AAV1-5’ template before (e.g., time 0 (TO)) and after thermally stressed (e.g., 37°C for 14 days (37°C 14d)), according to an exemplary aspect.
  • HMW heavy molecular weight
  • FIG. 26A depicts comparison of measurement of AAV1- 3’ template before and after thermal stress, according to an exemplary aspect.
  • FIG. 26B depicts high molecular weight (HMW) and low molecular weight (LMW) species of AAV1 3’ template after thermally stressed, according to an exemplary aspect.
  • FIG. 26C depicts mass v. charge heat map of empty, partially filled, full, and heavy molecular weight (HMW) and low molecular weight (LMW) species of AAV 1 comprising a 3’ template, according to an exemplary aspect.
  • Adeno-associated viruses have been widely used as gene delivery vectors to deliver genetic material, such as delivering nucleic acids for gene therapy.
  • AAVs provide the advantages of non-pathogenicity and low immunogenicity.
  • AAVs are nonpathogenic members of the Parvoviridae family under Dependovirus genus and require helpers, such as Adenovirus or Herpesvirus, for infection (Venkatakrishnan el 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, pages 4974- 4984).
  • AAV encapsulates a single-stranded DNA genome of about 4.7 kilobases (kb) in an icosahedral capsid which is made of a shell of capsid viral proteins.
  • Recombinant AAV genomes are nonpathogenic and do not integrate into a host’s genome, but exist as stable episomes that provide long-term expression.
  • AAV serotypes make a very useful system for preferentially transducing specific cell types.
  • AAV-based therapy has the advantages of being non-pathogenic and non-toxic, having cell type-specific infection, and offering different serotypes with varying cell transduction efficiencies.
  • a disadvantage is that AAV production, purification, and characterization are more complex compared to, for example, antibody therapies.
  • the purity of AAVs is defined by several product-related impurities, including empty capsids, capsids containing partial or incorrect genomes, and aggregated or degraded capsid, as well as residual host cell proteins (HCP).
  • HCP residual host cell proteins
  • a characteristic of the AAV manufacturing process is the production of empty capsids, namely, capsids lacking any packaged transgene.
  • Empty capsids are impurities that may result in an unwanted immune response and may reduce potency of the gene therapy drug.
  • An empty capsid has a molecular weight of about 3750 kDa, while a full capsid with an about 4.7 kb single-stranded genome has a molecular weight of about 5100 kDa.
  • a heterogeneous population of partially filled capsids, containing process-related impurities or a truncated transgene may also be produced during manufacturing.
  • full capsids Aggregated or degraded capsids, which may present as low or high molecular weight species in a sample, also must be monitored, as they may reduce viral titer and efficacy while increasing the load of potentially immunogenic viral protein.
  • the ratio of empty-full capsids, empty-partially filled-full capsids, the presence of degraded or aggregated capsids, and the presence of low molecular weight species or high molecular weight species may be critical quality attributes (CQA) for a viral vector gene therapy product.
  • CQA critical quality attributes
  • CDMS charge detection mass spectrometry
  • FIG. 1A depicts an example schematic diagram of an Orbitrap analyzer for charge-detection mass spectrometry (CDMS) analysis.
  • CDMS charge-detection mass spectrometry
  • CDMS has proven particularly powerful for evaluating the mass of large and/or highly heterogeneous samples, such as AAV vectors, e.g., full, partial filled, or empty AAV capsids, where individual charge states cannot be resolved, thus rendering conventional MS based techniques incapable of mass interpretation, as illustrated in FIG. ID and FIG. IE.
  • image current detection improves the efficiency of single ion collection, multiplexing single ion detection to one ion at a defined frequency per acquisition. Long time-domain signals are subject to ion decay, resulting in decreased signal amplitude, which can lead to underestimation of z and mass.
  • Selective Temporal Overview of Resonant Ions (STORI) analysis can be used to determine z using the rate of signal accumulation instead of its total amount.
  • STORI Selective Temporal Overview of Resonant Ions
  • Orbitrap-based CDMS may be achieved through Direct Mass Technology (DMT) (Kafader et al., 2020, Nat. Methods, 17(4):391 -394) using STORI analysis.
  • DMT Direct Mass Technology
  • each ion is analyzed using a STORI plot, as shown in FIG. 2B.
  • the induced current signal is integrated and plotted over time.
  • the STORI slope of an individual ion is proportional to z.
  • a charge calibration function is used to calibrate STORI slopes.
  • the data is processed as shown in FIG. 2C.
  • the mass domain spectrum is calculated, with the mass of each ion determined by multiplying m/z and z.
  • the masses are binned into a histogram (CDMS spectrum).
  • this disclosure sets forth a SEC-FI platform that streamlines and automates the sample introduction method.
  • the developed platform uses a dual high-flow/low-flow configuration that enables fast online size exclusionbased desalting and buffer exchange under high flow conditions, as well as stable and long- lasting sample infusion with limited dilution under low flow conditions. Additionally, this dual flow configuration was coupled with a MnESI source equipped with a multinozzle M3 emitter for stable, robust, and sensitive nano-infusion. These systems and methods are particularly powerful for Orbitrap-based CDMS analysis of AAV, as shown in the Examples below.
  • protein or “protein of interest” can include any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more amino acid polymer chains, generally known in the art as “polypeptides.” “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. As used herein, the term polypeptide includes proteins, variants thereof, fragments thereof, and peptides, whether synthetic, naturally occurring, or derived from a larger polypeptide, for example through digestion or truncation. “Synthetic peptide or polypeptide” refers to a non-naturally occurring peptide or polypeptide.
  • Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • Various solid phase peptide synthesis methods are known to those of skill in the art.
  • a protein may comprise one or multiple polypeptides to form a single functioning biomolecule.
  • a protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like.
  • Proteins of interest or polypeptides of interest can include any of bio-therapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells).
  • yeast systems e.g., Pichia sp.
  • mammalian systems e.g., CHO cells and CHO derivatives like CHO-K1 cells.
  • proteins comprise modifications, adducts, and other covalently linked moieties.
  • 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-epitope, fluorescent labels and other dyes, and the like.
  • avidin streptavidin
  • biotin glycans
  • glycans e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other
  • Proteins can be classified on the basis of compositions and solubility and can thus include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.
  • the term “recombinant protein” refers to a protein produced as the result of the transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell.
  • the recombinant protein can be an antibody, for example, a chimeric, humanized, or fully human antibody.
  • the recombinant protein can be an antibody of an isotype selected from group consisting of: IgG, IgM, IgAl, IgA2, IgD, or IgE.
  • the antibody molecule is a full-length antibody (e.g., an IgGl) or alternatively the antibody can be a fragment (e.g., an Fc fragment or a Fab fragment).
  • antibody as used herein includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CHI, 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).
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigenbinding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, for example, commercial sources, DNA libraries (including, e.g., phageantibody libraries), or can be synthesized.
  • the DNA may 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 configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antibody fragments include, but are not limited to, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fc fragment, a Fc/2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd’ fragment, a Fd fragment, and an isolated complementarity determining region (CDR) region, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multi specific antibodies formed from antibody fragments.
  • CDR complementarity determining region
  • Fv fragments are the combination of the variable regions of the immunoglobulin heavy and light chains, and ScFv proteins are recombinant single chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by a peptide linker.
  • an antibody fragment comprises a sufficient amino acid sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some aspects, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen.
  • An antibody fragment may be produced by any means.
  • an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence.
  • an antibody fragment may be wholly or partially synthetically produced.
  • An antibody fragment may optionally comprise a single chain antibody fragment.
  • an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide linkages.
  • An antibody fragment may optionally comprise a multi-molecular complex.
  • a functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids.
  • bispecific antibody includes an antibody capable of selectively binding two or more epitopes.
  • Bispecific antibodies generally comprise two different heavy chains with each heavy chain specifically binding a different epitope — either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or 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 on the same or a different target (e.g., on the same or a different protein).
  • Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen.
  • nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
  • a typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.
  • BsAbs can be divided into two major classes, those bearing an Fc region (IgG-like) and those lacking an Fc region, the latter normally being smaller than the IgG and IgG-like bispecific molecules comprising an Fc.
  • the IgG-like bsAbs can have different formats such as, but not limited to, triomab, knobs into holes IgG (kih IgG), crossMab, orth-Fab IgG, Dual-variable domains Ig (DVD-Ig), two-in-one or dual action Fab (DAF), IgG-single-chain Fv (IgG-scFv), or Kk-bodics.
  • the non-IgG-like different formats include tandem scFvs, diabody format, single-chain diabody, tandem diabodies (TandAbs), Dual-affinity retargeting molecule (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the entire teachings of which are herein incorporated).
  • the methods of producing bsAbs are not limited to quadroma technology based on the somatic fusion of two different hybridoma cell lines, chemical conjugation, which involves chemical cross -linkers, and genetic approaches utilizing recombinant DNA technology.
  • multispecific antibody refers to an antibody with binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (z.e., bispecific antibodies, bsAbs), antibodies with additional specificities such as trispecific antibody and KIH Trispecific can also be addressed by the systems and methods disclosed herein.
  • monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology.
  • a monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.
  • Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • a “sample” can be obtained from any step of a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in the downstream processing, final concentrated pool (FCP), drug substance (DS), or a drug product (DP) comprising the final formulated product.
  • CCF cell culture fluid
  • HCCF harvested cell culture fluid
  • FCP final concentrated pool
  • DS drug substance
  • DP drug product
  • the sample can be selected from any step of the downstream process of clarification, chromatographic production, or filtration.
  • the drug product can be selected from manufactured drug product in the clinic, shipping, storage, or handling.
  • the sample is a biological sample.
  • biological sample refers to a sample taken from a living organism, for example a human or non-human mammal.
  • a biological sample may comprise or consist of, for example, whole blood, plasma, serum, saliva, tears, semen, cheek tissue, organ tissue, urine, feces, skin, or hair.
  • a sample may be taken from a patient, for example, a clinical sample.
  • a sample may be taken from a non-human animal, for example, a preclinical sample.
  • a sample may be taken from a non-human animal subjected to gene therapy in order to produce at least one protein of interest or polypeptide of interest that may be included in the sample.
  • a sample is a further processed form of any of the aforementioned examples of samples.
  • the protein of interest or polypeptide of interest can be produced from mammalian cells.
  • the mammalian cells can be of human origin or non- human origin can include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells), established cell lines and their strains (e.g., HEK293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13,
  • IgG is a preferred class, and includes subclasses IgGl (including IgGIX and IgGlK), IgG2, IgG3, and IgG4.
  • the protein of interest or polypeptide of interest is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antibody fragment, an antigen-binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, a fusion protein, a receptor fusion protein, an antibody-derived protein, or combinations thereof.
  • the antibody is an IgGl antibody. In one aspect, the antibody is an IgG2 antibody. In one aspect, the antibody is an IgG4 antibody. In one aspect, the antibody is a chimeric IgG2/IgG4 antibody. In one aspect, the antibody is a chimeric IgG2/IgGl antibody. In one aspect, the antibody is a chimeric IgG2/IgGl/IgG4 antibody. Derivatives, components, domains, chains, and fragments of the above are also included.
  • the antibody is selected from the group consisting of an antiProgrammed Cell Death 1 antibody (e.g. an anti-PDl antibody as described in U.S. Pat. App. Pub. No. US2015/0203579A1), an anti-Programmed Cell Death Ligand-1 antibody (e.g. an anti-PD-Ll antibody as described in in U.S. Pat. App. Pub. No. US2015/0203580A1), an anti-DII4 antibody, an anti-Angiopoietin-2 antibody (e.g. an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), an anti-Angiopoietin-Like 3 antibody (e.g.
  • an antiProgrammed Cell Death 1 antibody e.g. an anti-PDl antibody as described in U.S. Pat. App. Pub. No. US2015/0203580A1
  • an anti-DII4 antibody e.g. an anti-Angiopoietin-2 antibody
  • an anti- AngPtl3 antibody as described in U.S. Pat. No. 9,018,356 an anti-platelet derived growth factor receptor antibody (e.g. an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827), an anti-Erb3 antibody, an anti-Prolactin Receptor antibody (e.g. anti-PRLR antibody as described in U.S. Pat. No. 9,302,015), an anti-Complement 5 antibody (e.g. an anti-C5 antibody as described in U.S. Pat. App. Pub. No US2015/0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g. an anti-EGFR antibody as described in U.S.
  • an anti-GCGR antibody as described in U.S. Pat. App. Pub. Nos. US2015/0337045A1 or US2016/0075778A1
  • an anti-VEGF antibody an anti-ILlR antibody
  • an interleukin 4 receptor antibody e.g., an anti-IL4R antibody as described in U.S. Pat. App. Pub. No. US2014/0271681A1 or U.S. Pat. Nos. 8,735,095 or 8,945,559
  • an anti-interleukin 6 receptor antibody e.g. an anti-IL6R antibody as described in U.S. Pat. Nos.
  • an anti-ILl antibody an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti- IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g. anti- IL33 antibody as described in U.S. Pat. App. Pub. Nos. US2014/0271658A1 or US2014/0271642A1), an antiCluster of differentiation 3 antibody (e.g. an anti-CD3 antibody, as described in U.S. Pat. App. Pub. Nos. US2014/0088295A1 and US20150266966A1, and in U.S. Application No.
  • an anti-Cluster of differentiation 20 antibody e.g. an anti-CD20 antibody as described in U.S. Pat. App. Pub. Nos. US2014/0088295A1 and US20150266966A1, and in U.S. Pat. No. 7,879,984
  • an anti-CD19 antibody e.g. an anti-CD20 antibody as described in U.S. Pat. App. Pub. Nos. US2014/0088295A1 and US20150266966A1, and in U.S. Pat. No. 7,879,984
  • an anti-CD19 antibody e.g. an anti-CD28 antibody
  • an anti-Cluster of Differentiation-48 antibody e.g. anti-CD48 antibody as described in U.S. Pat. No.
  • an anti-Fel dl antibody e.g. as described in U.S. Pat. No. 9,079,948
  • an antiinfluenza virus antibody e.g. anti-RSV antibody as described in U.S. Pat. App. Pub. No. US2014/0271653A1
  • an anti-Middle East Respiratory Syndrome virus antibody e.g. an anti-MERS-CoV antibody as described in U.S. Pat. App. Pub. No. US2015/0337029A1
  • an anti-Ebola virus antibody e.g. as described in U.S. Pat. App. Pub. No.
  • an anti-Zika virus antibody an anti-Severe Acute Respiratory Syndrome (SARS) antibody (e.g., an anti-SARS-CoV antibody), an anti-CO VID- 19 antibody (e.g., an anti-SARS-CoV-2 antibody), an anti-Lymphocyte Activation Gene 3 antibody (e.g. an anti-LAG3 antibody, or an anti-CD223 antibody), an anti-Nerve Growth Factor antibody (e.g. an anti-NGF antibody as described in U.S. Pat. App. Pub. No. US2016/0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176) and an anti-Activin A antibody.
  • SARS severe Acute Respiratory Syndrome
  • an anti-SARS-CoV antibody an anti-CO VID- 19 antibody
  • an anti-Lymphocyte Activation Gene 3 antibody e.g. an anti-LAG3 antibody, or an anti-CD223 antibody
  • an anti-Nerve Growth Factor antibody
  • the bispecific antibody is selected from the group consisting of an anti-CD3xanti-CD20 bispecific antibody (as described in U.S. Pat. App. Pub. Nos. US2014/0088295A1 and US20150266966A1), an anti-CD3xanti-Mucin 16 bispecific antibody (e.g., an anti-CD3xanti-Mucl6 bispecific antibody), an anti-CD3xBCMA bispecific antibody, and an anti-CD3xanti-Prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3xanti-PSMA bispecific antibody). See also U.S. Patent Publication No. US 2019/0285580 Al.
  • a MetxMet antibody an agonist antibody to NPR1, an LEPR agonist antibody, a MUC16xCD28 antibody, a GITR antibody, an IL-2Rg antibody, an EGFRxCD28 antibody, a Factor XI antibody, antibodies against SARS-CoV-2 variants, a Fel d 1 multi-antibody therapy, and a Bet v 1 multi-antibody therapy.
  • Derivatives, components, domains, chains and fragments of the above also are included.
  • the protein of interest or polypeptide of interest comprises a combination of any of the foregoing.
  • the protein of interest or polypeptide of interest is selected from the group consisting of Alirocumab, Atoltivimab, Maftivimab, Odesivimab, Odesivimab- ebgn, Casirivimab, Imdevimab, Cemplimab and Cemplimab-rwlc (human IgG4 monoclonal antibody that binds to PD-1), Sarilumab, Fasinumab, Nesvacumab, Dupilumab (human monoclonal antibody of the IgG4 subclass that binds to the IE-4R alpha (a) subunit and thereby inhibits Interleukin 4 (IE-4) and Interleukin 13 (IE- 13) signaling), Trevogrumab, Evinacumab, Evinacumab-dgnb, Fianlimab, Gareto
  • Additional exemplary antibodies include Ravulizumab-cwvz, Abciximab, Adalimumab, Adalimumab-atto, Ado-trastuzumab, Alemtuzumab, Atezolizumab, Avelumab, Basiliximab, Belimumab, Benralizumab, Bevacizumab, Bezlotoxumab, Blinatumomab, Brentuximab vedotin, Brodalumab, Canakinumab, Capromab pendetide, Certolizumab pegol, Cetuximab, Denosumab, Dinutuximab, Durvalumab, Eculizumab, Elotuzumab, Emicizumab- kxwh, Emtansine alirocumab, Evolocumab, Golimumab, Guselkumab, Ibritumomab t
  • Biosimilars are described in various ways depending on the jurisdiction, but share a common feature of comparison to a previously approved biological product in that jurisdiction, usually referred to as a “reference product.”
  • a biosimilar is a biotherapeutic product similar to an already licensed reference biotherapeutic product in terms of quality, safety and efficacy, and is followed in many countries, such as the Philippines.
  • a biosimilar in the U.S. is currently described as (A) a biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; and (B) there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.
  • an interchangeable biosimilar or product may be substituted for the previous product without the intervention of the health care provider who prescribed the previous product.
  • a biosimilar is a biological medicine highly similar to another biological medicine already approved in the EU (called “reference medicine”) and includes consideration of structure, biological activity, efficacy, and safety, among other things, and these guidelines are followed by Russia.
  • a biosimilar product currently refers to biologies that contain active substances similar to the original biologic drug and is similar to the original drug in terms of quality, safety, and effectiveness, with no clinically significant differences.
  • a biosimilar currently is a product that has bioequivalent/quality-equivalent quality, safety, and efficacy to a reference product already approved in Japan.
  • biosimilars currently are referred to as “similar biologies,” and refer to a similar biologic product which is similar in terms of quality, safety, and efficacy to an approved reference biological product based on comparability.
  • a biosimilar medicine currently is a highly similar version of a reference biological medicine.
  • a biosimilar currently is a biotherapeutic product that is similar in terms of quality, safety, and efficacy to an already licensed reference product.
  • a biosimilar currently is derived from an original product (a comparator) with which it has common features.
  • a biosimilar currently is a biological therapeutic product that is similar to an existing biological product registered in Singapore in terms of physicochemical characteristics, biological activity, safety and efficacy.
  • a biosimilar currently is a new biological medicinal product developed to be similar in terms of quality, safety and efficacy to an already registered, well established medicinal product.
  • a biosimilar currently is a biologic drug that is highly similar to a biologic drug that was already authorized for sale.
  • South Africa a biosimilar currently is a biological medicine developed to be similar to a biological medicine already approved for human use. Production of biosimilars and its synonyms under these and any revised definitions can be undertaken according to the inventions.
  • the protein of interest or polypeptide of interest is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein).
  • an Fc-fusion protein is a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety.
  • the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG.
  • the receptor Fc-fusion protein contains two or more distinct receptor chains that bind to either a single ligand or multiple ligands.
  • an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the IL1R1 extracellular region fused to Fc of hlgGl; see U.S. Pat. No. 6,927,004, which is herein incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl fused to Fc of hlgGl; see U.S. Pat. Nos.
  • IL-1 trap e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the IL1R1 extracellular region fused to Fc of hlgGl
  • a VEGF trap e
  • an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one or more of one or more antigen-binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.
  • the protein of interest comprises a combination of any of the foregoing.
  • vector refers to a recombinant plasmid or virus (“viral vector”) that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.
  • vectors derived from AAV are particularly attractive for delivering genetic material because (i) they are able to infect (transduce) a wide variety of non-dividing and dividing cell types including muscle fibers and neurons; (ii) they are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, for example, interferon- mediated responses; (iii) wild type AAVs have never been associated with any pathology in humans; (iv) in contrast to wild type AAVs, which are capable of integrating into the host cell genome, replication-deficient AAV vectors generally persist as episomes, thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (v) in contrast to other vector systems, AAV vectors do not trigger a significant
  • a “recombinant AAV vector” refers to a polynucleotide vector including one or more heterologous sequences (z.e., nucleic acid sequence not of AAV origin) that may be flanked by at least one, e.g., two, AAV inverted terminal repeat sequences (ITRs).
  • rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
  • a “capsid” is the protein shell of a virus, which encloses the genetic material.
  • a full capsid contains genetic material and is required to provide therapeutic benefit.
  • An empty capsid lacks the genome and therefore lacks the ability to provide therapeutic benefit to the patient.
  • a “viral particle” refers to a particle composed of at least one viral capsid and an encapsulated viral genome. While AAV is described in this disclosure as a model virus or viral particle, it is contemplated that the disclosed methods can be applied to profile a variety of viruses, e.g., the viral families, subfamilies, and genera. In some aspects, the viral capsid, virus, or viral particle belongs to a viral family selected from the group consisting of Adenoviridae, Parvoviridae, Retroviridae, Baculoviridae, and Herpesviridae.
  • the viral capsid, virus, or viral particle belongs to a viral genus selected from the group consisting of Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus, Tetraparvovirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus, Utovirus, Mardivirus, Simplexvirus, Varicellovirus, Cytomegalovirus, Muromegalovirus,
  • Proboscivirus Roseolovirus, Lymphocryptovirus, Macavirus, Percavirus, and Rhadinovirus.
  • a sample can be prepared prior to or following enrichment steps, separation steps, and/or analysis steps.
  • Preparation steps can include alkylation, reduction, denaturation, digestion, derivatization, and/or deglycosylation.
  • protein alkylating agent refers to an agent used for alkylating certain free amino acid residues in a protein.
  • Non-limiting examples of protein alkylating agents are iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N- ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.
  • protein denaturing can refer to a process in which the three- dimensional shape of a molecule is changed from its native state. Protein denaturation can be carried out using a protein denaturing agent.
  • a protein denaturing agent include heat, high or low pH, reducing agents like DTT (see below) or exposure to chaotropic agents.
  • reducing agents like DTT see below
  • chaotropic agents can be used as protein denaturing agents. Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.
  • Non-limiting examples for chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroylsarcosine, urea, and salts thereof.
  • protein reducing agent refers to the agent used for reduction of disulfide bridges in a protein.
  • protein reducing agents used to reduce a protein are dithiothreitol (DTT), B-mercaptoethanol, Ellman’s reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1), or combinations thereof.
  • DTT dithiothreitol
  • B-mercaptoethanol Ellman’s reagent
  • hydroxylamine hydrochloride sodium cyanoborohydride
  • TCEP-HC1 tris(2-carboxyethyl)phosphine hydrochloride
  • a conventional method of protein analysis, reduced peptide mapping involves protein reduction prior to LC-MS analysis.
  • non-reduced peptide mapping omits the sample preparation step of reduction in order to preserve endogenous disulfide bonds.
  • non-reduced preparation may be used, for example, in order to preserve an endogenous disulfide bond between Fab arms of an antibody or antibody-derived protein.
  • partially-reduced preparation may be used, for example, in order to reduce the disulfide bond between Fab arms of an antibody or antibody-derived protein without fully reducing the protein.
  • the term “digestion” refers to hydrolysis of one or more peptide bonds of a protein or polypeptide.
  • hydrolysis There are several approaches to carrying out digestion of a protein in a sample using an appropriate hydrolyzing agent, for example, enzymatic digestion or non-enzymatic digestion.
  • the term “digestive enzyme” refers to any of a large number of different agents that can perform digestion of a protein or polypeptide.
  • hydrolyzing agents that can carry out enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), outer membrane protein T (OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), IdeZ, IgdE, glyseria
  • IdeS or a variant thereof is used to cleave an antibody below the hinge region, producing an Fc fragment and a Fab2 fragment.
  • Digestion of an analyte may be advantageous because size reduction may increase the sensitivity and specificity of characterization and detection of the analyte using LC-MS.
  • digestion that separates out an Fc fragment and keeps a Fab2 fragment for analysis may be preferred. This is because variable regions of interest, such as the complementaritydetermining region (CDR) of an antibody, are contained in the Fab2 fragment, while the Fc fragment may be relatively uniform between antibodies and thus provide less relevant information.
  • CDR complementaritydetermining region
  • IdeS digestion has a high efficiency, allowing for high recovery of an analyte.
  • the digestion and elution process may be performed under native conditions, allowing for simple coupling to a native LC-MS system.
  • IdeS or variants thereof are commercially available and may be marketed as, for example, FabRICATOR® or FabRICATOR Z®.
  • liquid chromatography refers to a process in which a biological/chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase.
  • liquid chromatography include reversed phase (RP) liquid chromatography, ion-exchange (IEX) chromatography, size exclusion chromatography (SEC), affinity chromatography, hydrophobic interaction chromatography (HIC), hydrophilic interaction chromatography (HILIC), or mixed-mode chromatography (MMC).
  • RP reversed phase
  • IEX ion-exchange
  • SEC size exclusion chromatography
  • HIC hydrophobic interaction chromatography
  • HILIC hydrophilic interaction chromatography
  • MMC mixed-mode chromatography
  • a sample can be subjected to any one of the aforementioned chromatographic methods or a combination thereof.
  • Analytes separated using chromatography will feature distinctive retention times, reflecting the speed at which an analyte moves through the chromatographic column.
  • Analytes may be compared using a chromatogram, which plots retention time on one axis and measured signal on another axis, where the measured signal may be produced from, for example, UV detection or fluorescence detection.
  • the methods and systems of the present invention include the use of size exclusion chromatography.
  • Size exclusion chromatography or gel filtration relies on the separation of components as a function of their molecular size. Separation depends on the amount of time that the substances spend in the porous stationary phase as compared to time in the fluid. The probability that a molecule will reside in a pore depends on the size of the molecule and the pore.
  • the ability of a substance to permeate into pores is determined by the diffusion mobility of macromolecules which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all; and, for very small macromolecules the probability of penetration is close to unity. While components of larger molecular size move more quickly past the stationary phase, components of small molecular size have a longer path length through the pores of the stationary phase and are thus retained longer in the stationary phase.
  • the chromatographic material can comprise a size exclusion material wherein the size exclusion material is a resin or membrane.
  • the matrix used for size exclusion is preferably an inert gel medium which can be a composite of cross-linked polysaccharides, for example, cross-linked agarose and/or dextran in the form of spherical beads. The degree of cross-linking determines the size of pores that are present in the swollen gel beads.
  • Molecules greater than a certain size do not enter the gel beads and thus move through the chromatographic bed the fastest. Smaller molecules, such as detergent, protein, DNA and the like, which enter the gel beads to varying extent depending on their size and shape, are retarded in their passage through the bed. Molecules are thus generally eluted in the order of decreasing molecular size.
  • the mobile phase used to obtain an eluate from size exclusion chromatography can comprise a volatile salt.
  • the mobile phase can comprise ammonium acetate, ammonium bicarbonate, or ammonium formate, or combinations thereof.
  • nSEC-MS native SEC-MS
  • mass analyzer includes a device that can separate species, that is, atoms, molecules, or clusters, according to their mass.
  • species that is, atoms, molecules, or clusters, according to their mass.
  • mass analyzers that could be employed are time-of-flight (TOF), magnetic electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and also the technique of accelerator mass spectrometry (AMS).
  • TOF time-of-flight
  • Q quadrupole mass filter
  • QIT quadrupole ion trap
  • FTICR Fourier transform ion cyclotron resonance
  • AMS accelerator mass spectrometry
  • the mass spectrometer can work on nanoelectro spray or nanospray.
  • nanoelectrospray or “nanospray” as used herein refers to electrospray ionization at a very low solvent flow rate, typically hundreds of nanoliters per minute of sample solution or lower, often without the use of an external solvent delivery.
  • the electrospray infusion setup forming a nanoelectro spray can use a static nanoelectro spray emitter or a dynamic nanoelectro spray emitter.
  • a static nanoelectro spray emitter performs a continuous analysis of small sample (analyte) solution volumes over an extended period of time.
  • a dynamic nanoelectro spray emitter uses a capillary column and a solvent delivery system to perform chromatographic separations on mixtures prior to analysis by the mass spectrometer.
  • the mass spectrometer may use a microflow nanospray ion source, for example Newomics® MnESI (microflow nanospray electrospray ionization) ion source, with M3 emitter.
  • MnESI microflow nanospray electrospray ionization
  • the ion source has multiple nozzles working together to split a single microflow stream evenly into multiple nanoflows.
  • the MnESI source can be linked to a high-flow LC system using a T-splitter. This configuration allows a microflow to enter the MnESI, while the remaining analytical flow is directed towards an additional detector or system.
  • the mass spectrometer can be coupled to a liquid chromatography-multiple reaction monitoring system. More generally, a mass spectrometer may be capable of analysis by selected reaction monitoring (SRM), including consecutive reaction monitoring (CRM) and parallel reaction monitoring (PRM).
  • SRM selected reaction monitoring
  • CCM consecutive reaction monitoring
  • PRM parallel reaction monitoring
  • MRM multiple reaction monitoring
  • MRM can be typically performed with triple quadrupole mass spectrometers wherein a precursor ion corresponding to the selected small molecules/peptides is selected in the first quadrupole and a fragment ion of the precursor ion is selected for monitoring in the third quadrupole (Yong Seok Choi et al., Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimers disease biomarker candidates, 930 JOURNAL OF CHROMATOGRAPHY B 129-135 (2013)).
  • LC-MS can be performed under native conditions.
  • native conditions can include performing mass spectrometry under conditions that preserve non-covalent interactions in an analyte.
  • Native mass spectrometry is an approach to study intact biomolecular structure in the native or near-native state.
  • the term “native” refers to the biological status of the analyte in solution prior to subjecting to the ionization.
  • Several parameters, such as pH and ionic strength, of the solution containing the biological analytes can be controlled to maintain the native folded state of the biological analytes in solution.
  • native mass spectrometry is based on electrospray ionization, wherein the biological analytes are sprayed from a nondenaturing solvent.
  • native MS allows for better spatial resolution compared to non-native MS.
  • Elisabetta Boeri Erba & Carlo Pe-tosa The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE pp. 1176— 1192 (2015).
  • a Thermo Q Exactive UHMR (Thermo Fisher Scientific, Bremen, Germany) equipped with a microflow-nanospray electrospray ionization (MnESI) source and a microfabricated monolithic multinozzle (M3) emitter (Newomics, Berkley, CA) was used for native MS and CDMS analysis.
  • MnESI microflow-nanospray electrospray ionization
  • M3 emitter Newomics, Berkley, CA
  • a sample loop was added to trap the desalted analytes followed by nano-ESI spray achieved at a low flow rate (1 pL/min) using a secondary pump.
  • Online SEC-CDMS analysis of the protein standards was performed in a similar configuration using an Acquity BEH200 SEC guard column (4.6x150 mm, 1.7pm, 200 A) and 200 pL sample loop, or an optimized column (1.0x50 mm) and 60 pL sample loop.
  • CDMS spectra were acquired using Direct Mass Technology (DMT) and processed using Proteinaceous STORIBoard with frequency correction function developed by Thermo Fisher Scientific.
  • Example 1 Size exclusion chromatography-flow injection for charge-detection mass spectrometry injection
  • Charge-detection mass spectrometry typically involves hundreds to thousands of single ion measurements to generate statistically significant spectra, which in turn requires stable sample infusion for 30 minutes to several hours.
  • CDMS Charge-detection mass spectrometry
  • static infusion of an AAV sample AAV signal was detected, but unstable.
  • Static infusion involves an expert operator, lacks robustness, can be subject to frequent tip clogging, and involves offline buffer exchange into MS-compatible buffers prior to analysis, which likely leads to AAV samples precipitating and aggregating in solution.
  • FIG. 3A An example of a static infusion system is shown in FIG. 3A, and a corresponding total ion chromatogram (TIC) trace is shown in FIG. 3B.
  • Two desirable features for improving a sample introduction platform for CDMS analysis of AAVs include a platform with online size-exclusion-based desalting and buffer exchange, and stable and long-lasting sample infusion with limited dilution.
  • size exclusion chromatography-flow injection (SEC-FI) methods and systems were developed, as illustrated in FIG. 4A and FIG. 4B.
  • the system includes a microflow nanospray ion source equipped with a multinozzle M3 emitter for stable, robust, and sensitive nano-infusion. Both a high-flow configuration and a low-flow configuration are possible with the developed system.
  • the high-flow configuration allows for efficient online desalting, with no buffer exchange required. Size exclusion-based online desalting and native MS measurement can be used to quickly assess the full/empty ratio of AAV vectors.
  • the loading pump is coupled to the SEC column.
  • the desalted sample can be trapped in an extended sample loop.
  • a survey run is used to establish the timing of the valve switch to trap desalted sample in the extended loop.
  • the effectiveness of online desalting of an AAV sample using the high-flow SEC-FI-MS system is shown in a native mass spectrum (MS) in FIG. 5A.
  • the low-flow configuration allows for stable and long-lasting sample infusion in the range of about 30 minutes to several hours with minimal sample dilution, thus allowing Orbitrap-based CDMS analysis.
  • the nano pump is directly coupled to the sample loop and facilitates stable spray at flow rates as low as 1 pL/minute. As many as 200 minutes of infusion time are possible.
  • the stability of injection of an AAV sample using the low-flow SEC-FI-MS system is shown in a CDMS in FIG. 5B.
  • AAV Adapted SEC-FI a new SEC column with a smaller volume and a shorter sample loop were used (“AAV Adapted SEC-FI”), as shown in FIG. 6A and FIG. 6B.
  • AAV Adapted SEC-FI a new SEC column with a smaller volume and a shorter sample loop were used (“AAV Adapted SEC-FI”), as shown in FIG. 6A and FIG. 6B.
  • AAV Adapted SEC-FI a similar desalting result was achieved while the signal intensity of AAVs was increased in both native MS and single ion CDMS, as shown in FIG. 6C and FIG. 6D.
  • the effective ion counts were doubled compared to the previous configuration.
  • the frequency correction function was applied to the SEC-FI-MS AAV analysis, allowing for accurate AAV mass calculations. Additionally, more ions were retained and peaks were better resolved after the corrective signal processing, as shown in FIG. 8A and FIG. 8B.
  • AAVs containing only VP3 protein were evaluated using the SEC-FI-CDMS platform.
  • AAVs containing only VP3 are relatively homogeneous, so charge states can be readily resolved using native MS spectra.
  • the mass determined by SEC- FI-CDMS was higher than those from native MS and CDMS using static infusion, suggesting that there are higher amounts of solvation, as shown in FIG. 9A and FIG. 9B.
  • the method was performed using lot material of a full AAV8 vector, spiked into an AAV8 control lot with only empty capsid, as shown in FIG. 11A. In this manner, a variety of mixtures of empty and full capsids were produced, with the full capsid percentage ranging from 0% to 89%.
  • a native MS survey scan was performed for each sample using high flow SEC-FI. Samples were quantified by directly integrating native MS spectra at m/z 26-3 Ik for empty capsids and 33-48k for full capsids.
  • the platform was additionally evaluated for the ability to detect and analyze AAVs even at low concentrations. This was achieved by varying the injection amount of empty AAV8 capsids. Both native MS and CDMS responses showed a strong linear relationship to the injection amount, as shown in FIG. 13A and FIG. 13B. Masses for empty AAV8 capsids could be detected and accurately assigned even at a low titer of 3.0E11 capsids/mL. Therefore, the platform demonstrated high sensitivity suitable for application to regular AAV programs.
  • Example 4 Application of SEC-FI-CDMS for AAV analysis
  • Orbitrap-based SEC-FI-CDMS analysis with frequency correction was applied for the analysis of AAV vectors.
  • Empty, partially-filled, and full vectors were separated, and the average mass of each was measured, as shown in FIG. 14E.
  • the developed platform was further applied to analyze other AAV serotypes with a higher amount of partial capsid contamination, for example AAV1 and AAV5.
  • the partial capsids cannot be easily distinguished in conventional native MS spectra.
  • FIG. 15A The masses of empty, partial, and full capsids measured by SEC-FI-CDMS aligned well with expected values, as shown in FIG. 15B, and with those obtained from an alternative CDMS method.
  • the relative abundance of empty, partial, and full capsids showed a similar trend across three analytical methods: AUC, an alternative CDMS method, and the developed SEC-FI-CDMS platform, as shown in FIG. 15C.
  • the discrepancies in abundance are likely due to different ages of the samples. It should be noted that the abundance data from CDMS are estimated using mass range, and careful peak fitting is necessary to calculate accurate abundance. The average masses of empty, partially-filled and full vectors of each serotype is shown in Table 2. Overall, the developed platform was capable of detecting and quantifying partial capsids, and may be applied to enhance quality and purity assessment of manufactured A A Vs.
  • SEC-FI-CDMS was additionally shown to be capable of resolving variations of AAV-mAb conjugation as a function of increasing anti- AAV mAb, as illustrated in FIG. 16A.
  • Anti- AAV mAb can bind to 5-fold axis of capsids with 12 theoretical binding sites.
  • the degree of SpyC-mAb decoration of AAV9-SpyT with increasing ratios of SpyC-mAb to AAV9-SpyT as measured by the developed Orbitrap-based SEC-FI-CDMS method was assessed, as shown in FIGs. 16C-16H, see FIG. 16B for legend of FIGs. 16C-16H.
  • the results from the developed SEC-FI-CDMS method were comparable to an alternative CDMS method. Compared to a naked AAV9 control, a mass shift to higher mass range was observed in the CDMS spectra of AAV conjugates by a stepwise addition of antibody mass.
  • the SEC-FI-CDMS platform was further applied to reveal changes in AAV surface charge as a function of anti- AAV mAb binding.
  • Two distributions of charge states were observed for AAV-antibody conjugate samples in the charge-mass heatmap, which correlated with naked AAVs and antibody-conjugated AAVs, respectively, as shown in FIGs. 18A-FIG. 18F. This result suggests a surface charge change upon antibody conjugation, demonstrating the value of measuring charge and mass simultaneously using the CDMS approach.
  • SEC-FI-CDMS platform was also applied to establish CDMS charge calibration.
  • SEC-FI enables the CDMS charge calibration function to be established using various protein standards.
  • CDMS acquisition time of protein standards is over 120 minutes to ensure a reliable fitting of the charge calibration coefficient, requiring a long and stable injection time.
  • Charge calibration coefficients across four protein standards using the SEC-FI-CDMS platform was within a 2% difference, as shown in FIG. 19A and FIG. 19B.
  • the SEC-FI platform set forth in the present disclosure streamlines the sample introduction process through a dual high-flow/low-flow setup.
  • the high-flow configuration allows for fast online desalting and native MS screening, which can be applied for quick assessment of the full/empty ratio of AAV vectors.
  • Full/empty ratios were determined by the relative abundance of capsids from online SEC-MS spectra, which were in good agreement with those calculated by AUC.
  • the low-flow configuration facilitates stable, sensitive, and robust sample infusion and long acquisition times for CDMS.
  • the low-flow configuration enables Orbitrap-based CDMS analysis for approximately 30 minutes to several hours with minimal sample dilution.
  • the masses of empty, partial, and full AAVs from three AAV serotype samples were accurately determined by SEC-FI-CDMS, which showed good agreement with predicted values.
  • the optimized SEC-FI-CDMS platform enabled efficient and robust mass analysis for AAVs.
  • the masses and relative abundance of different AAV components determined using SEC-FI-CDMS are consistent with those from calculation or from orthogonal methods.
  • SEC-FI-CDMS resolves and quantifies the distribution of antibody in AAV-mAb non-covalent conjugates, which could be too complex to resolve using other methods.
  • the unique charge detection capability of CDMS also provides valuable insights into the surface charge of AAV-mAb conjugates.
  • Dual-vector AAVs offer a novel approach for delivering therapeutic transgenes that exceed the packaging capacity of AAV. This is achieved through complementary gene fragments that are independently packaged and recombine in vivo. Despite the promise of this approach, traditional characterization techniques lack the resolution to accurately quantify capsids in these complex mixtures. The characterization and quantification of dual vector AAVs is crucial for understanding vector reconstitution efficiencies but challenging due to the inherent heterogeneity of capsids and the complexity of transgenes. To address this challenge, a method utilizing a size-exclusion chromatography (SEC)-based online desalting coupling to native charge detection mass spectrometry (CDMS) was used to characterize dual vector AAVs samples.
  • SEC size-exclusion chromatography
  • CDMS native charge detection mass spectrometry
  • SEC-CDMS was used to resolve vectors comprising 3’ genomes or 5’ genomes based on payload mass.
  • the observed mass difference between AAV1-4 (3’) and AAV1-3 (5’) is consistent with mass difference expected for the 3’ vs. 5’ payloads (-130 kDa) (FIG. 20A and 20B).
  • the percentage of full capsids determined by SEC-FI-CDMS corresponds closely with those from mass photometry (VPC), indicating that 50% and 65% of the capsids are full, respectively.
  • SEC-CDMS was used to resolve dual-vector AAVs in a mixture. Desalting SEC was directly coupled to CDMS using an extended sample loop for online analysis of AAV. This approach enables fast online buffer exchange and extended CDMS acquisition times for sensitive and robust mass analysis, and precise quantification of empty, partial, and full capsids.
  • This technique was applied to characterize dual- vector AAVs by preparing a series of mixed samples with known genome titers of individual vectors, with expected ratios ranging from approximately 0:100 to 100:0. The individual vector components were well resolved from the mixtures based on a payload mass difference of -130 kDa (FIGs. 21 A and 21B). Quantification of each component using Gaussian peak fitting showed excellent agreement with expected values, with deviations within 4% (FIGs. 21A and 21B).
  • FIG. 26C depicts mass v. charge heat map of empty, partially filled, full, and heavy molecular weight (HMW) and low molecular weight (LMW) species of AAV1 comprising a 3’ template, according to an exemplary aspect.
  • HMW heavy molecular weight
  • LMW low molecular weight
  • Mass versus Charge heat maps show stress stability of AAV vectors comprising a 5’ genome, 3’ genome, or both (FIG. 25).
  • the empty and filled capsids of thermal stressed samples show broader charge distributions compared to the control samples.
  • the HMW (heavy molecular weight) particles presumably consist of aggregates with the released ssDNA, show higher average charge states than empty/full capsids.

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Abstract

La présente invention concerne des systèmes et des procédés pour identifier, caractériser et quantifier des vecteurs viraux. Des échantillons de vecteurs viraux peuvent être soumis à un dessalage basé sur une chromatographie d'exclusion de taille utilisant un flux élevé et ensuite injectés dans une source d'ions à électronébulisation utilisant un faible flux pour une analyse par spectrométrie de masse à détection de charge.
PCT/US2025/014183 2024-02-01 2025-01-31 Plateforme d'analyse par spectrométrie de masse à détection de charge d'aavs Pending WO2025166281A1 (fr)

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