EP4673750A2 - Cartographie peptidique de perfusion à haut débit - Google Patents

Cartographie peptidique de perfusion à haut débit

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Publication number
EP4673750A2
EP4673750A2 EP24764594.8A EP24764594A EP4673750A2 EP 4673750 A2 EP4673750 A2 EP 4673750A2 EP 24764594 A EP24764594 A EP 24764594A EP 4673750 A2 EP4673750 A2 EP 4673750A2
Authority
EP
European Patent Office
Prior art keywords
polypeptides
polypeptide
solution
generating
mass
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
Application number
EP24764594.8A
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German (de)
English (en)
Inventor
Alexander HEBERT
Joshua Coon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wisconsin Alumni Research Foundation
Original Assignee
Wisconsin Alumni Research Foundation
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Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Publication of EP4673750A2 publication Critical patent/EP4673750A2/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • Biotherapeutic proteins are vital medicines for a wide range of diseases with extracellular targets.
  • these protein-based drugs acquire deleterious modifications during production and storage that can impact safety and efficacy.
  • Production, formulation, and storage conditions must be optimized for stability of these drugs to ensure molecular fidelity.
  • Numerous assays are implemented to monitor their quality, such as capillary isoelectric focusing for charge variants, top-down or middle-down mass spectrometry (MS) for oxidation analysis, glycan analysis to check for humanization, and peptide mapping for site specific modification levels. Of these, only peptide mapping generates site specific modification levels used to identify critical quality attributes (CQAs).
  • CQAs critical quality attributes
  • Peptide mapping is especially powerful for the analysis of monoclonal antibodies (mAbs) because their sequences typically only differ in specific regions. Modification sites that do not have deleterious effects in one mAb can be de-prioritized in other mAbs.
  • mAbs monoclonal antibodies
  • Peptide mapping entails liquid chromatography (LC) coupled with MS (LC-MS) analysis of enzymatically produced peptides from the target protein.
  • peptides composing the complete sequence and common chemical and biological modifications e.g., oxidation, deamidation, isomerization, glycosylation, clipping, etc.
  • the signals for the modified and non-modified (native) peptides are compared to generate and estimate of the modification stoichiometry. These measurements can be correlated with bioactivity/binding analyses to inform strategies for production, formulation, and storage of biotherapeutic proteins to specifically protect the critical amino acids.
  • the system is able to analyze polypeptides and other molecules to generate MS data without the need for a chromatography step prior to injection of the analytes into the mass spectrometer while retaining the accuracy required by end users, such as pharmaceutical companies and those monitoring the quality and efficacy of biotherapeutics.
  • the present invention provides an alternative to LC-MS based protein mapping assays.
  • the alternative methods and systems described herein have the potential to significantly increase peptide analysis throughput while providing accuracy and sequence coverage comparable to conventional protein mapping methods.
  • the present invention provides a method for preparing and analyzing multiple polypeptides via direct infusion mass spectrometry (MS).
  • MS direct infusion mass spectrometry
  • Direct infusion also known as “direct injection” refers to the direct analysis of a sample by mass spectrometry with little to no separation or purification steps being performed on analytes within the sample prior to the sample being inserted into the MS device. Most often this is accomplished by use of electrospray ionization where a liquid sample is infused into an electrospray emitter directly. The sample is then ionized by electrospray ionization and the ions are then subsequently measured by mass spectrometry.
  • the sample is a complex mixture of polypeptides resulting from a proteolytic digest of a single protein or simple protein mixture, including but not limited to a monoclonal antibody (mAb) or antibody drug conjugate.
  • mAb monoclonal antibody
  • This mixture may contain several hundred polypeptides and, following direct infusion, m/z peaks in the resulting mass spectrum are detected for many, if not the majority, of the polypeptides in the mixture.
  • the removal of conventional chromatography and other separation steps can significantly improve the time and costs requirements of such analyses.
  • One embodiment of the invention provides a method for analyzing a plurality of polypeptides in a sample comprising the steps of: a) simultaneously injecting the plurality of polypeptides into an ion source of a mass spectrometer (MS) device and generating a plurality of precursor ions from the polypeptides; and b) performing one or more MS scans on each precursor ion generated from the plurality of polypeptides, thereby generating spectral data for each precursor ion.
  • MS mass spectrometer
  • the MS scans may be a full mass range scan (which provides a full mass spectrum of each analyte), a narrow mass range scan (which provides a mass spectrum over a specified m/z range), a segmented mass range scan, or combinations thereof.
  • a segmented mass range scan the mass range of interest is broken into two or more segments, each segment is scanned in succession, and the resulting scans are combined together. This increases the spectral dynamic range allowing smaller ion intensities to be seen.
  • the sample is a liquid sample that is directly injected into an electrospray emitter of an MS device, including but not limited to an Orbitrap MS device.
  • the MS device provides a resolving power equal to or greater than 100,000, has a scan rate of at least 0.5 scans per second (0.5 Hz), and the multiple scans are performed over a time period of at least 10 seconds.
  • the method preferably does not comprise an additional liquid or gas chromatography step and/or salt removal step.
  • Injecting the plurality of polypeptides into the ion source of the MS device optionally includes preparing the polypeptides comprising the steps of: a) denaturing the plurality of polypeptides thereby generating a plurality of denatured polypeptides; b) treating the plurality of denatured polypeptides with a reducing agent, thereby generating a plurality of reduced polypeptides; c) treating the plurality of reduced polypeptide with a first solution causing the plurality of reduced polypeptide to precipitate out of the first solution, thereby generating a polypeptide precipitate; and e) dissolving the polypeptide precipitate thereby generating a plurality of dissolved polypeptides, followed by injecting the plurality of dissolved polypeptides into the ion source to generate precursor ions.
  • mass spectrometry data is obtained on each of the plurality of dissolved or digested polypeptides.
  • the mass spectrometry data may include mass-to-charge ratios obtained from MS1 data, MS2, or both.
  • the spectral data is generated by averaging spectral data obtained from multiple scans for each precursor ion. Site specific mutations, post translational modifications, or combinations thereof, are detected and/or measured in the polypeptides.
  • the biological activity of each of the polypeptides is measured and a link between the biological activity and the site specific mutations, post translational modifications, or combinations thereof, are recorded for each polypeptide.
  • the polypeptides are generated from a protein of interest that is chemically or enzymatically digested.
  • the plurality of polypeptides are biotherapeutics (or portions thereof), including but not limited to antibodies and/or therapeutic proteins.
  • a specific sequence or region of a control polypeptide or polypeptide standard is known, such as a region of an antibody that affects activity or stability of the antibody, or a region that is susceptible to degradation or modification.
  • a further embodiment of the invention comprises comparing one or more specific regions of each of the plurality of polypeptides with a corresponding region of a control polypeptide and determining if a chemical modification is present in one or more of the plurality of polypeptides.
  • the spectral data generated for each precursor ion is compared to calculated mass-to-charge values for predicted polypeptides and modified polypeptides able to be generated from the protein. It is then determined whether the precursor ions correspond to one or more of the predicted polypeptides or modified polypeptides of the protein.
  • Sequence coverage is used herein to refer to the amount of a protein amino acid sequence that corresponds to precursor ions detected and analyzed by the MS device.
  • the present method provides a sequence coverage of at least 90%, at least 92%, at least 95%, or 100% of the of the protein’s amino acid sequence.
  • At least a portion of the plurality of precursor ions are fragmented, thereby generating product ions (also referred to as fragment ions).
  • the mass- to-charge ratios of the product ions are measured to generate product ion mass spectrometry data, and the generated product ion mass spectrometry data is compared with predicted fragment ion data of the protein. Comparing the generated product ion mass spectrometry data to the predicted fragment ion data of the protein can confirm the spectral data for the precursor ions and can confirm the sequence order of the polypeptides that generate the precursor ions, which can provide sequence confirmation.
  • the direct infusion method of the present invention also enables relative quantification of the polypeptides in the sample.
  • the peak intensity of two or more detected precursor ions in MS1 events or their product ions in MS/MS events are compared to each other and the relative abundance in the sample of each polypeptide generating the precursor ions is then calculated from the compared peak intensities.
  • the polypeptides generating the two or more detected precursor ions comprise a first polypeptide and a variant of the first polypeptide having a modified amino acid sequence, a modified structure, or a modified chemical formula. As a result, the relative abundance of the first polypeptide to the modified polypeptide in the sample is able to be calculated.
  • Such modifications to the polypeptide include, but are not limited to, oxidation, deamidation, isomerization, glycosylation, substitutions of amino acids, and clipping.
  • modifications to the polypeptide include, but are not limited to, oxidation, deamidation, isomerization, glycosylation, substitutions of amino acids, and clipping.
  • specific regions a manufactured antibody or therapeutic protein may be compared to a control protein or antibody to determine if the manufactured antibody or therapeutic protein has undergone a modification that would affect the stability or activity of the molecule.
  • the MS device provides a resolving power equal to or greater than 100,000, or equal to or greater than 200,000, or equal to or greater than 480,000, or equal to or greater than 960,000.
  • the MS device preferably has a scan rate of at least one scan per second (1 Hz), at least two scans per second (2 Hz), at least five scans per second (5 Hz), or at least ten scans per second (10 Hz).
  • the MS device has a time period of approximately 6-10 seconds to scan the analytes eluting from the chromatography column before the analytes are moved to make room for the next batch of analytes.
  • using the direct infusion method of the present invention allows the MS device to perform multiple scans over a time period of at least 5 seconds, over a time period of at least 10 seconds, over a time period of at least 20 seconds, over a time period of at least 30 seconds, over a time period of at least 45 seconds, over a time period of at least 60 seconds, over a time period of at least 90 seconds, or over a time period of at least 120 seconds.
  • An additional embodiment of the invention provides methods for preparing a polypeptide or a plurality of polypeptides using a method that enables rapid MS analysis compatible with direct infusion and without requiring additional purification or separation steps. This method is also beneficial in that it does not remove small polypeptides that are often removed during chromatography or other separation steps, thereby allowing MS detection and analysis of these polypeptides.
  • the present invention provides a method for analyzing a polypeptide comprising the steps of: a) denaturing the polypeptide thereby generating a denatured polypeptide; b) treating the denatured polypeptide with a reducing agent, thereby generating a reduced polypeptide; c) treating the reduced polypeptide with a first solution causing the reduced polypeptide to precipitate out of the first solution, thereby generating a polypeptide precipitate; d) optionally, washing the polypeptide precipitate with a second solution; and e) dissolving the polypeptide precipitate with a third solution thereby generating a dissolved polypeptide.
  • the dissolved polypeptide is then preferably digested, thereby generating a digested polypeptide; and the dissolved polypeptide or digested polypeptide is directly injected into an ion source of a mass spectrometer device.
  • the method further comprises obtaining mass spectrometry data on the digested polypeptide and measuring site specific mutations, post translational modifications, or combinations thereof, in the polypeptide.
  • the mass spectrometry data may include MS1 data, MS2, or both.
  • the method does not comprise an additional salt removal step (other than what is achieved by step d), an additional liquid chromatography step, an additional gas chromatography step, and/or other additional separation steps.
  • the present invention provides a method for analyzing one or more polypeptides comprising the steps of: a) depositing the one or more polypeptides in a container or carousel having one or more separated wells or chambers, wherein the one or more polypeptides are deposited in separate wells or chambers in the container; b) denaturing the one or more polypeptides thereby generating one or more denatured polypeptides; c) treating the one or more denatured polypeptides with a reducing agent, thereby generating one or more reduced polypeptides; and d) treating the one or more reduced polypeptides with a first solution causing the one or more reduced polypeptide to precipitate out of solution, thereby generating one or more polypeptide precipitates.
  • the container or carousel is a multi-well plate, including but not limited to filter plates, and the one or more polypeptides are deposited in separate wells in the multi-well plate.
  • the one or more polypeptides are a plurality of polypeptides.
  • the one or more polypeptide precipitates are then optionally washed with a second solution one or more times.
  • the method further comprises dissolving the one or more polypeptide precipitates, either washed or unwashed, with a third solution thereby generating one or more dissolved polypeptides, and, optionally, digesting the one or more dissolved polypeptides or polypeptide precipitates, thereby generating one or more digested polypeptides.
  • Each of the one or more dissolved polypeptides or digested polypeptides is injected from the container into an ion source of a mass spectrometer device able to generate mass spectrometry data on the plurality of polypeptides.
  • the contents of each well or container is sequentially injected into the ion source.
  • the one or more polypeptides are a plurality of polypeptides.
  • the denaturing step comprises treating the one or more polypeptides with a chemical agent able to denature proteins and polypeptides as is known in the art.
  • the denaturing step comprises contacting each of the one or more polypeptides with one or more of guanidinium chloride, urea, a detergent, or solutions thereof.
  • the one or more polypeptides are contacted with a solution containing of guanidinium chloride or urea.
  • the reducing agent may be any chemical agent known in the art able to reduce the denatured polypeptides, including but not limited to dithiolthreitol (DTT) and solutions thereof.
  • the one or more denatured polypeptides or reduced polypeptides are additionally treated with an alkylating agent.
  • the alkylating agent may be any alkylating agent known in the art suitable for use with polypeptides including, but not limited to, iodoacetate, iodoacetic acid, iodoacetamide (all referenced herein as IAA) and solutions thereof.
  • the first solution may be any chemical agent able to cause polypeptides, particularly the reduced polypeptides, to precipitate out of solution, including acids, organic solvents, salts, and metals as is known in the art.
  • the first solution comprises an organic solvent, such as methanol (MeOH).
  • the second solution is optionally used to wash the one or more polypeptide precipitates in order to reduce or remove salts and other unwanted contaminants.
  • the second solution comprises water, MeOH, and combinations thereof.
  • the second solution comprises 70%-95% MeOH, such as a 20/80 or 10/90 water to MeOH mixture.
  • the third solution is used to dissolve the one or more polypeptide precipitates back into solution, and may be any solution able to dissolve the polypeptide precipitates, including but not limited to ammonium acetate, ammonium bicarbonate, and other buffer solutions.
  • the digesting step comprises treating the one or more dissolved polypeptides with an enzyme or a chemical agent able to digest proteins and polypeptides as is known in the art.
  • the denaturing step comprises contacting each of the dissolved polypeptides with a proteolytic enzyme, including but not limited to trypsin, pepsin, chymotrypsin, papain, calpain, serrapeptase, thermolysin, carboxypeptidase, and combinations thereof.
  • an acid quenching step is performed on the resulting digested polypeptides by adding an acidic solution, including but not limited to a formic acid solution, to the digested polypeptides.
  • Methods described herein are advantageous in that they can prepare the one or more polypeptides for MS analysis in a sufficiently purified form so as to allow the dissolved polypeptides or digested polypeptides to be injected into a MS device without requiring additional salt removal steps and/or chromatography steps.
  • the method does not comprise an additional salt removal step other than what is achieved by washing the polypeptide precipitates with the second solution.
  • the method does not comprise a liquid chromatography step and/or or other additional separation step.
  • the biological activity of each of the analyzed polypeptides is compared with the biological activity of a control peptide.
  • the polypeptides are antibodies or other biotherapeutics
  • the biological activity of the analyzed polypeptides are compared with the desired or expected activity of the corresponding normal active antibody or biotherapeutic to determine if the analyze polypeptides have altered activity and to determine if the altered activity corresponds to a chemical or structural modification.
  • therapeutic or commercially produced antibodies can be compared to the normal active antibody to determine if chemical modifications affecting activity have arisen during production or storage. Examples of chemical modifications that may alter the biological activity of the polypeptides include, but are not limited to, oxidation, deamidation, isomerization, glycosylation, substitutions of amino acids, and clipping.
  • each sample is analyzed within 24 hours using the above methods on a single mass spectrometry device, preferably at least 100 samples within 24 hours, preferably at least 150 samples within 24 hours, preferably at least 200 samples within 24 hours, preferably at least 250 samples within 24 hours, preferably at least 300 samples within 24 hours, preferably at least 400 samples within 24 hours, preferably at least 500 samples within 24 hours, or more preferably at least 600 samples within 24 hours.
  • each sample is analyzed at an average time of ⁇ 5.0 min per sample, preferably ⁇ 4.5 min per sample, preferably ⁇ 4.0 min per sample, preferably or ⁇ 3.0 min per sample.
  • each sample is an antibody or therapeutic protein that is digested to generate a plurality of polypeptides.
  • the sample being analyzed comprises a mixture of at least 50 different polypeptides, at least 100 different polypeptides, at least 150 different polypeptides, at least 200 different polypeptides, or at least 300 different polypeptides.
  • the generated spectral data preferably includes spectral data from precursor or product ions generated from polypeptides having ten amino acids or less, eight amino acids or less, six amino acids or less, or four amino acids or less.
  • mass spectrometry data is obtained for over 50% of the amino acids of the analyzed polypeptides, preferably over 60% of the amino acids of the analyzed polypeptides, preferably over 70% of the amino acids of the analyzed polypeptides, preferably over 80% of the amino acids of the analyzed polypeptides, preferably over 85% of the amino acids of the analyzed polypeptides, preferably over 90% of the amino acids of the analyzed polypeptides, or preferably over 95% of the amino acids of the analyzed polypeptides.
  • obtaining mass spectrometry data includes detecting and/or measuring specific mutations, post translational modifications, or combinations thereof, of the amino acids.
  • the one or more polypeptides are treated so as to induce a chemical modification in the polypeptides.
  • the one or more polypeptides are treated with a chemical reagent, including but not limited to H2O2, or heat, time, light, etc. to generate susceptibility to chemical modifications in the polypeptides.
  • the one or more peptides are a plurality of polypeptides deposited in a multi- well plate, wherein the plurality of polypeptides are deposited in separate wells, and varying concentrations of the chemical reagent (e.g., H 2 O 2 ) or varying temperatures are added to different wells.
  • the chemical reagent e.g., H 2 O 2
  • the present invention provides a system for analyzing a plurality of polypeptides comprising: a) a series of containers or a multi-well plate able to contain a plurality of polypeptides in separate containers or wells; b) a reagent kit comprising, separately from one another, a denaturing solution, a reducing agent solution, a first solution able to cause reduced polypeptides to precipitate, a second solution wherein the second solution is a wash solution, a third solution able to dissolve polypeptide precipitates, and optionally, a solution comprising an enzyme able to digest the plurality of polypeptides; and c) an automated system for injecting the plurality of peptides in each of the separate containers or wells into a mass spectrometer or other molecular analyzer.
  • the automated system is able to inject the plurality of peptides directly into the mass spectrometer or other molecular analyzer without going through a separation system, such as a chromatography columns or system.
  • the series of containers may be one or more, preferably two or more, five or more, ten or more, or twenty or more, containers, including but not limited to test tubes.
  • the reagent kit additionally comprises an alkylating agent solution and an acid quench solution.
  • the reagent kit comprises guanidinium chloride, urea, or a detergent as part of a denaturing solution, dithiolthreitol (DTT) as part of the reducing agent solution, iodoacetate, iodoacetic acid, and/or iodoacetamide (IAA) as part of the alkylating agent solution, methanol (MeOH) as the first solution, a MeOH/water mixture as the second solution, ammonium acetate as the third solution, and trypsin as part of the digesting enzyme solution.
  • DTT dithiolthreitol
  • IAA iodoacetamide
  • the multi-well plate is a filter plater and the system optionally further comprises an automated fluid delivery system for adding each solution and/or reagent to each well and draining each well of any liquid after each step.
  • the reagent kit comprises an additional chemical reagent solution able to induce a chemical modification in the polypeptides, including but not limited to oxidation.
  • the reagent kit may comprise one or more H 2 O 2 solutions.
  • the reagent kit comprises solutions of the chemical reagent in varying concentrations, wherein the solutions of varying concentrations may be added to different wells.
  • FIG. 1 shows different regions and modifications of an antibody that may be analyzed using peptide mapping with a mass spectrometer.
  • FIG. 2 illustrates a sample preparation step performed in a filter plate in an embodiment of the present invention.
  • FIG. 3 shows a mass spectrum collected from an antibody sample analyzed using a method in an embodiment of the present invention.
  • FIG. 4A shows a representative mass spectrum where oxidation of a peptide results in stereoisomers and split peaks in conventional LC-MS peptide mapping.
  • FIG. 4B shows a representative mass spectrum using the direct infusion method of the present invention where all isomers have the same mass and coalesce into a single peak that additionally has a distinguishable mass shift.
  • FIGs. 4G and 4D show peptide mapping results of a digested NIST mAb for both LC-MS analysis and direct infusion MS (inf-MS) analysis of the present invention (DTLMISR in Fig. 4C - SEQ ID NO:1 ; FNWYVDGVEVHNAK in Fig. 4D - SEQ ID NO:2).
  • FIG. 4E provides a graph showing untreated and H2O2 treated NIST mAb, Sigma mAb, infliximab, rituximab, and cetuximab samples prepared and analyzed by both conventional LC-MS peptide mapping and inf-MS peptide mapping of an embodiment of the present invention.
  • FIG. 4F shows that the methods are able to provide consistent results across the different antibodies for untreated and H 2 O 2 treated samples.
  • FIG. 5A shows a zoomed-in m/z region from an MS/MS spectrum of a peptide (GFYPSDIAVEWESNGQPENNYK - SEQ ID NO:3) from a mAb sample analyzed by an inf-MS method of an embodiment of the present invention.
  • FIGs. 5B-5C provide graphs showing that the inf-MS method of an embodiment of the present invention generates comparable results to convention peptide mapping for calculating the ratios of deamidated peptides to native peptides in mAb samples.
  • FIGs. 6A-6B shows MS data comparing the ability of an inf-MS method of an embodiment of the present invention to generate accurate and comparable glycan occupation data for mAb samples.
  • FIG. 7 shows a mass spectrum resulting from direct injection of a tryptic digest of the therapeutic antibody adalimumab according to an embodiment of the present invention.
  • FIG. 8 shows the sequence of the adalimumab heavy chain (SEQ ID NO: 4) and light chain (SEQ ID NO:5) annotated with detected polypeptides.
  • FIG. 9 shows MS/MS analysis and sequence confirmation of polypeptide #94 from the adalimumab digestion (SEQ ID NO: 6).
  • FIG. 10 is a plot showing the percentage of theoretically calculated m/z peaks that could occur from a direct injection of the tryptic peptides of adalimumab that can be resolved from one another at various mass analyzer resolving powers.
  • the x-axis is resolving power calculated at m/z 200. At a resolving power of about 100,000, approximately 80 percent of possible m/z peaks were resolved.
  • FIG. 11 is a graphic showing whether various glycosylated and modified variants of the peptide EEQYNSTYR (SEQ ID NO:7) can be detected (lighter colored boxes) in the MS1 signal by accurate mass. Note for the peptides that exist at higher abundances (> 5%) the signals are easily observed in the MS1 scan. For those with relative abundances lower than ⁇ 3.5%, reduction of the m/z range down as low as 2 m/z can allow for detection.
  • FIG. 12 shows a MS/MS scan confirming the amino acid sequence of MS1 identified m/z peaks for (GFYPSDIAVEWESNGQPENNYK - SEQ ID NO:3) and allowing for detection and quantification of deamidation.
  • a key to detecting deamidation in peptides is the use of very high resolving powers to distinguish the deamidated peptide product ions from those generated by the 13 C isotope containing non-modified version.
  • precursor ion is used herein to refer to an ion which is produced during ionization stage of mass spectrometry analysis, including the MS1 ionization stage of MS/MS analysis.
  • product ion is used to refer to an ion which is produced during a fragmentation process of a precursor ion, including the MS2 ionization stage of MS/MS analysis.
  • mass spectrometry refers to an analytical technique for the determination of the elemental composition of an analyte, including but not limited to polypeptides.
  • the mass spectrometry principle consists of ionizing analytes to generate charged species (i.e. , precursor ions) or species fragments (i.e., product ions) and measurement of their mass-to-charge ratios.
  • Mass spectrometry data corresponding to analyte ion and analyte ion fragments is presented in mass-to-charge (m/z) units representing the mass-to-charge ratios of the analyte precursor ions and/or analyte product ions.
  • mass-to-charge (m/z) units representing the mass-to-charge ratios of the analyte precursor ions and/or analyte product ions.
  • samples containing a mixture of polypeptides can be ionized and the resulting precursor ions separated according to their mass-to-charge ratio. Selected precursor ions can then be fragmented and further analyzed according to the mass-to-charge ratio of the product ions.
  • mass spectrometer refers to a device which creates ions from a sample, separates the ions according to mass to charge ratios, and measures the abundance of each detected m/z peak. Because charge (z) can be calculated from the spectra, mass can be determined.
  • Mass spectrometers include multistage mass spectrometers which fragment the mass-separated precursor ions and separate the product ions by mass-to-charge ratio one or more times. Multistage mass spectrometers include tandem mass spectrometers which fragment the mass-separated precursor ions and separate the product ions by mass-to-charge ratio once.
  • ion source refers to a component of a mass spectrometer which produces ions from a sample.
  • ion sources include, but are not limited to, electrospray ionization (ESI) sources, matrix assisted laser desorption/ionization (MALDI) sources, atmospheric pressure chemical Ionization (APCI) sources.
  • ESI electrospray ionization
  • MALDI matrix assisted laser desorption/ionization
  • APCI atmospheric pressure chemical Ionization
  • signal-to-noise ratio refers to the level of a desired signal to the level of background noise.
  • the term “mass-to-charge ratio” refers to the ratio of the mass of a species (i.e., a precursor ion or product ion) or to the charge state of a species.
  • the term “m/z unit” refers to a measure of the mass to charge ratio.
  • the Thomson unit (abbreviated as Th) is an example of an m/z unit and is defined as the absolute value of the ratio of the mass of an ion (in Daltons) to the charge of the ion (with respect to the elemental charge).
  • Mass spectrometer resolving power is a quantitative measure of how well m/z peaks in a mass spectrum are separated (i.e., resolved). There are a variety of conventions to calculate resolving power.
  • “Scan rate” refers to the number of scans per second performed by the mass spectrometer device. For example a scan rate of 10 Hz means the acquisition of 10 spectra per second.
  • “Mass spectrometer dynamic range” can be defined as the ratio between the minimal and the maximal concentration of compounds that can be detected simultaneously in a given sample. For example, mass spectrometers suitable for the present invention typically have a dynamic range of at least 3 orders of magnitude, at least 4 orders of magnitude, at least 5 orders of magnitude, or at least 6 orders of magnitude.
  • protein refers to a class of compounds comprising one or more polypeptide chains and/or modified polypeptide chains. Proteins can be modified by naturally occurring processes such as post-translational modifications or co- translational modifications. Exemplary post-translational modifications or co- translational modifications include, but are not limited to, phosphorylation, glycosylation, deamidation, lipidation, prenylation, sulfonation, hydroxylation, acetylation, methylation, methionine oxidation, the addition of cofactors, proteolysis, and assembly of proteins into macromolecular complexes. Modification of proteins can also include non-naturally occurring derivatives, analogues and functional mimetics generated by chemical synthesis. Exemplary derivatives include chemical modifications such as alkylation, acylation, carbamylation, iodination or any modification that derivatizes the protein.
  • peptide and “polypeptide” are used synonymously in the present description, and refer to a class of compounds composed of amino acid residues chemically bonded together by amide bonds (or peptide bonds). Peptides and polypeptides are polymeric compounds comprising at least two amino acid residues or modified amino acid residues. Modifications can be naturally occurring or non-naturally occurring, such as modifications generated by chemical synthesis.
  • Modifications to amino acids in peptides include, but are not limited to, phosphorylation, glycosylation, deamidation, lipidation, prenylation, sulfonation, hydroxylation, acetylation, methylation, methionine oxidation, alkylation, acylation, carbamylation, iodination and the addition of cofactors.
  • Peptides and polypeptides can be generated by substantially complete digestion or by partial digestion of proteins.
  • Peptide mapping is a vital tool for biotherapeutic drug development.
  • Demand for this technique is increasing due to demand within pharmaceutical companies to use site-specific information to inform therapeutic strategies, as well as from groups, such as the FDA, to ensure that these therapies are safely produced and administered.
  • a typical pharmaceutical company is likely to conduct thousands of peptide mapping assays each year.
  • the costs associated with existing techniques is substantial given the time required ( ⁇ 60 minutes per sample), the requisite equipment (advanced LC-MS systems), and the need for highly skilled technicians.
  • biotherapeutic proteins such as antibodies
  • acquire deleterious modifications during production and storage Figure 1.
  • Peptide mapping assays such as liquid chromatography-mass spectrometry (LC-MS) analyses of tryptic digests of these molecules, can identify and measure site-specific changes that ultimately reduce bioactivity. The results from these assays can also be used to make improvements and changes to production and formulation strategies with the goal of protecting critical amino acids.
  • LC-MS liquid chromatography-mass spectrometry
  • the present invention provides a high throughput peptide mapping platform by combining a plate-based sample preparation method with direct infusion MS analysis. Protein samples are reduced, alkylated, digested, and acid quenched in a filter plate (a type of multi-well plate). Samples are then each infused sequentially using a NanoMate® or other automated infusion system directly to a mass spectrometer (i.e., a high resolution Orbitrap).
  • the result is an effective assay for identifying site specific modifications in therapeutic antibodies, with their initial results showing high fidelity to LC based methods (>90% sequence coverage) for methionine and tryptophan oxidation, asparagine glycosylation, protein n-terminal pyrolysis, clipping, amino acid sequence variants, and succinimide intermediates of asparagine deamidation and aspartic acid isomerization.
  • Adding targeted ultrahigh resolution MS2 scans (500,000 resolving power at 200 m/z) into the scan sequence further enabled the ability to measure site-specific deamidation.
  • the high-throughput direct infusion method in an embodiment of the present invention can provide similar information to conventional LC-MS methods but taking only approximately 0.1 to 3 minutes, preferably approximately 0.1 to 2 minutes, or approximately 0.1 to 1 minute per sample, which could translate to a 20X increase in throughput.
  • the sequence coverage provided by the present invention compares favorably to conventional methods despite faster analysis time and simpler preparation.
  • the infusion method of the present invention performs similarly to conventional LC-MS peptide mapping in comparing methionine and tryptophan oxidation levels of NIST monoclonal antibody treated with increasing amounts of hydrogen peroxide (>90% sequence coverage). Additional comparisons for antibody deamidation and glycosylation have also been completed with similar results.
  • Example 1 - This example describes a peptide mapping method for high throughput sample preparation and analysis by performing direct infusion mass spectrometry analyses on tryptic peptide mixes without chromatography.
  • a plate-based sample preparation has been developed that is compatible with direct injection MS analysis. This method is capable of rapidly preparing and analyzing 96 samples completely within 8 hours (4 hours of sample preparation, and 4 hours of MS analysis). Protein samples are cleaned, reduced, alkylated, digested and acid quenched in a filter plate. Samples are then filtered and infused sequentially using a NanoMate® system directly to the mass spectrometer. With the unbiased sample preparation method and high resolution Orbitrap survey scans, >95% sequence coverage are observed through exact mass comparison to theoretical peptide masses.
  • Plate Method Developing a plate-based sample preparation that is compatible to infusion MS was key to matching the throughput of the sample preparation with that of the data collection.
  • studies have reported plate-based peptide mapping methods. However, these have been coupled to chromatographic separation which removes the salt from the rest of sample during the loading step. These methods are unlikely to be compatible with direct infusion without additional desalting steps.
  • These examples describe a method (see generally Figure 2) where all sample preparation steps are performed in a filter plate. In this example, the samples are first added to 8 M guanidine chloride in an individual filter well, followed by reduction and alkylation with dithiolthreitol (DTT) and iodoacetate (IAA), respectively.
  • DTT dithiolthreitol
  • IAA iodoacetate
  • the protein is then precipitated with methanol and washed repeatedly to desalt the sample, all within the same well.
  • the protein is then resuspended in ammonium acetate and digested with trypsin (still in the well). The digestion is then quenched with formic acid and the peptides are eluted from the filter to a second 96-well plate and are ready for analysis.
  • any preparation method must be easily compatible with varied formulations.
  • This plate method is compatible with antibody samples with concentrations greater than 2 mg/mL; less concentrated samples may need to be pre-concentrated prior to analysis.
  • Sample matrices will commonly have salts and detergents, which are not compatible with infusion-MS and must be removed prior to analysis.
  • the precipitation step removes detergents better than gel permeation chromatography or molecular weight cutoff filters.
  • the samples can be infused into a mass spectrometer using electrospray ionization.
  • An Advion Nanomate® system is typically used which directly accepts the 96 well plate of samples and automates the infusion and ionization step.
  • the resultant ionized tryptic peptides can be analyzed with any interfaced mass spectrometer - such as Orbitrap hybrid systems capable of high-resolution MS and MS n experiments. Shown in Figure 3 is a partial mass spectrum that has been collected from an antibody sample using the methods described herein. All annotated m/z peaks are identified peptides resulting from the mAb. Sequence coverage is generally above 95%.
  • Oxidation analysis Protein therapeutics are susceptible to oxidative damage prior to being administered to the patient. Primarily, methionine and tryptophan residues acquire deleterious oxidation modifications. When these modifications occur in the complementarity determining region (CDR) or fragment crystallizable (FC) region, this chemical modification can decrease bioactivity.
  • CDR complementarity determining region
  • FC fragment crystallizable
  • Conventional peptide mapping experiments separate out the oxidized and native versions of a given peptide using chromatography and mass spectrometry. The oxidation level is then estimated by dividing the signal from the oxidized peptide by the total signal from the peptide of interest. Oxidation is a chemical process and thus leads to stereoisomers in methionine and positional isomers in tryptophan.
  • Deamidation analysis converts asparagine residues into aspartic acid and iso-aspartic acid. This modification can alter both the charge state and structure.
  • deamidated peptides are difficult to separate from the unmodified versions by reversed-phase chromatography and the extra modified species further complicates data analysis.
  • Deamidation adds 1 Da mass shift to the peptide; however, this mass shift is 19 mDa different from the first 13 C isotope of the native peptide. These species are indistinguishable under most mass spectrometry conditions.
  • Figure 5A presents a zoom-in of this m/z region from an MS/MS spectrum of the GFYPSDIAVEWESNGQPENNYK (SEQ ID NO:1) (commonly referred to as “PENNYK”) peptide from a mAb sample analyzed by inf-MS.
  • PENNYK GFYPSDIAVEWESNGQPENNYK
  • Figures 5B and 5C provide evidence that inf-MS can generate comparable results for calculating the ratios of deamidated to native peptides in mAb samples.
  • FIG. 6A and 6B provide data comparing the ability of inf-MS to generate accurate and comparable glycan occupation data for mAb samples. These data can be obtained from either the MS m/z signals (as used here) of the various peptides or through MS/MS spectra.
  • Example 2 - This example describes an additional general filter plate preparation method suitable for the present invention.
  • a polypeptide such as an antibody
  • GnHCI guanidinium chloride
  • Different amounts of the polypeptide may be used depending on the polypeptide to be analyzed.
  • the polypeptide is reduced and/or alkylated, precipitated out of solution using MeOH, and then optionally washed.
  • the precipitate is dissolved in approximately 25 mM ammonium acetate and digested using trypsin or other known digestive enzyme or chemical agent.
  • An acid quenching step is optionally performed and then the polypeptide fragments are injected into a mass spectrometer or other analyzing device, without the need for a chromatography step or salt removing step.
  • Example 3 - Figure 7 presents a spectrum resulting from direct infusion of polypeptides that result from the trypsin digestion of a therapeutic antibody (adalimumab, i.e., Humira®), prepared as described herein, into an Orbitrap hybrid mass spectrometer.
  • the spectrum contains hundreds of discrete m/z peaks ( ⁇ 500 isotopic clusters) that result from the various tryptic peptides of the digested mAb.
  • in silico peptide masses are generated from trypsin digestion of the known protein sequence, and each peptide’s theoretical m/z values are calculated in the expected charge states.
  • Those calculated values are then matched to the measured m/z values using a m/z tolerance.
  • This tolerance is typically ranges from 1 to 20 parts per million (5 to 10 ppm being common, and 5 ppm being the typical choice in this experiment) and results in accurate mapping of the measured m/z values to theoretical m/z values calculated from the expected peptide sequences.
  • This process i.e., direct injection, is particularly effective because the mixture is not very complex relative to other biological mixtures (such as cell lysates), with the vast majority of the detected m/z peaks resulting from the various tryptic peptides of the antibody.
  • Figure 7 the most abundant m/z peaks are annotated for each peptide sequence.
  • Polypeptides are detected spanning the entire light and heavy chain ( Figure 8) allowing confirmation of the expected sequence of the mAb.
  • the peak intensities were used. For example, the intensity of the modified and unmodified versions of a given peptide m/z signal can be compared to calculate the relative amounts of each.
  • cleavage site Occasionally a protease cleavage site is missed and the resulting longer peptide, contains two or more of the expected tryptic peptides (see peptide 55, for example, in Figure 8). Such missed cleavage peptides may be used to confirm the sequence and to also inform on the relative abundances of any modifications/variants.
  • MS/MS tandem MS experiments
  • DDA data dependent acquisition mode
  • DIA data independent mode
  • the product ions in these MS/MS spectra can be used to confirm the accurate mass MS1 peak assignments.
  • MS1 scan only the intact mass of the polypeptide is known. That mass indicates if the polypeptide’s mass is consistent with the expected amino acids but does not indicate if the amino acids are in the correct sequence order.
  • the mass accuracy of the instrument is not always high enough to guarantee that the detected polypeptide is not composed of a different combination of amino acids (and possibly modifications) which happen to sum to a similar m/z.
  • the mass accuracy of the instrument is not always high enough to guarantee that the detected polypeptide is not composed of a different combination of amino acids (and possibly modifications) which happen to sum to a similar m/z.
  • the resulting MS/MS spectrum allows the order of the amino acids and site-specific location of any modifications to be determined.
  • a modification or sequence variant can then be localized, or the stoichiometry of a particular modification can be determined (i.e., deamidation).
  • Figure 9 presents an example of MS/MS analysis of peptide #94 from adalimumab.
  • Example 4 This example describes the sequence coverage and sequence confirmation of an analyzed protein. Sequence coverage is generally defined here as the amount of the protein sequence being analyzed that has been detected. For example, if the protein analyte in question contains 100 amino acids and peptides containing 90 of those amino acids are observed, then it is reported that 90% of the sequence has been covered.
  • Detection of a polypeptide is done in two ways. First, and as described above, the exact mass of the observed m/z signals are compared with the predicted m/z values for all possible polypeptides. If a match is made within a specific mass tolerance window (typically 1 to 10 ppm), this peptide is reported and part of the sequence is indicated as being detected. A further level of confirmation can be obtained by collecting MS/MS ( Figure 9). In this way, predicted product ions of the matched sequence are compared to those observed in the experimental MS/MS spectrum. Established algorithms and tools for this comparison are known in the art and are used to calculate a confidence score and ultimately to confirm the detection of the matched peptide sequence.
  • a specific mass tolerance window typically 1 to 10 ppm
  • Example 5 There are several key figures of merit that are required in the MS system to achieve the results described in the examples above.
  • the mass analyzer used must have the capability to resolve these, often closely spaced, peptide m/z signals.
  • Theoretical analysis shows that resolving powers in excess of approximately 100,000 (@ m/z 200) provide the best performance (Figure 10). That is, at this resolving power approximately 80% of the possible m/z peaks can be resolved.
  • the MS analyzer should have the largest dynamic range possible.
  • the system should allow the detection of a peptide that is up to 1 ,000 times, preferably up to 10,000 times, more abundant than another polypeptide, i.e., up to four orders of magnitude. Such capability would allow for the detection of modified and variant peptides as low as 0.1% relative abundance.
  • Signal to noise ratios, and detection limits can also be increased by narrowing the m/z range of ions that are allowed to be injected and polypeptides have been observed with as low as 0.02 % relative abundance with this approach.
  • Figure 11 shows how detection sensitivity is improved for low level modified peptides as the injection m/z range is narrowed.
  • the MS system ideally can perform tandem MS experiments. While not necessary, this capability allows for the confirmation of sequences as described above.
  • the MS/MS analysis requires high resolution analysis so that the deamidation modification can be distinguished from the isotopic cluster of the non-modified peptides (see Figure 12).

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Abstract

L'invention concerne une plateforme de cartographie peptidique à haut débit combinant un procédé de préparation d'échantillon basé sur une plaque avec une analyse par MS de perfusion directe. Les échantillons de protéines sont nettoyés, réduits, alkylés, digérés et trempés à l'acide dans une plaque filtrante. Les échantillons sont ensuite chacun perfusés séquentiellement directement à un dispositif de spectromètre de masse sans étapes supplémentaires de chromatographie ou de purification classiques. Le résultat est un dosage efficace d'identification des modifications spécifiques à un site présents chez des anticorps thérapeutiques, leurs résultats initiaux présentant une fidélité élevée à des procédés basés sur la chromatographie en phase liquide (> 90 % de couverture de séquence).
EP24764594.8A 2023-03-01 2024-02-29 Cartographie peptidique de perfusion à haut débit Pending EP4673750A2 (fr)

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