HEAVY PEPTIDE APPROACH TO ACCURATELY MEASURE UNPROCESSED C-TERMINAL LYSINE IN ANTIBODIES
The present disclosure provides a method for measuring post-translational modifications in proteins such as antibodies. In particular, the method may be used to quantify C-terminal truncation in antibodies that incorporates heavy isotopic standards for both the unprocessed C-terminal K peptide and the truncated C-terminal K peptide to build a calibration curve and quantify this PTM using mass spectrometry. Quantification of post-translational modifications may occur in a single liquid chromatography tandem mass spectrometry (LC-MS2) run.
This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63/041,015, filed Jun. 18, 2020
SEQUENCE LISTINGAn official copy of the sequence listing is submitted concurrently with the specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “10760US01_SEQ_LIST_ST25.txt” created on Jun. 14, 2021 and having a size of 1 KB. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTIONAspects of the invention are generally directed to methods for characterizing antibody sequence fidelity. More specifically, the present disclosure provides methods for identifying and quantifying a post-translational modification (PTM) in the Fc region after antibody synthesis, such as C-terminal lysine (K) truncation (clipping), using heavy isotopic standards to establish a calibration curve and quantify the PTM in a single liquid chromatography tandem mass spectrometry (LC-MS2) run.
BACKGROUND OF THE INVENTIONTherapeutic monoclonal antibodies (mAbs) and bispecific antibodies (bsAbs) play a key role in treating many disorders. The advantages of this class of drugs, including high specificity and affinity to an expansive variety of molecular targets, warrant their continued development and have led to approvals for treatment of health conditions like asthma, rheumatoid arthritis, and elevated low density lipoprotein cholesterol, among many others. While the commercial and scientific success of therapeutic antibodies is unprecedented, their inherent benefits are tempered by their large size, complexity, and chemical heterogeneity, necessitating that a host of methods be used to evaluate their safety and efficacy.
A significant fraction of these methods is devoted to evaluating PTMs, a product quality attribute (PQA) and major source of mass and charge heterogeneity. The PTM complement of a single antibody is diverse, but common modifications are shared among almost all mAbs and bsAbs, such as C-terminal lysine truncation, glycosylation, N-terminal pyro-Glu formation, oxidation, amidation, deamidation, succinimide intermediate formation, glycation, isomerization, cysteinylation, and trisulfide bonding.
Careful monitoring of these PTM levels enables their control through predefined acceptance criteria and has become a common strategy for two distinct reasons: (1) numerous reports have shown that PTMs, especially when located in a complementarity determining region (CDR), can affect the stability and bioactivity of an antibody, and (2) variability in PTM levels could indicate a lack of process control.
Post-translational modifications are assayed at the global level with chromatographic and electrophoretic techniques, including methods like size exclusion chromatography multi angle laser light scanning (SEC-MALLS), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), imaged capillary isoelectric focusing (cIEF), and cation exchange chromatography (CEX). Such methods have enjoyed wide acceptance but typically identify only the most abundant modifications without determining their specific locations within the amino acid sequence.
For example, the acidic species in a CEX chromatogram will most likely contain PTMs like deamidation, glycation, and cysteinylation, and the basic species will be comprised of modifications like unprocessed C-terminal K, oxidation, and isomerization. However, as the amino acid locations of these PTMs are indeterminable in global analyses, it is challenging to determine if they are located in a CDR and at what abundance.
The integration of highly sensitive mass spectrometer detectors with an ever-increasing number of liquid chromatography column chemistries and enzymatic treatment conditions has resulted in a mature suite of PTM characterization methods. Intact mass analysis of an antibody via liquid chromatography mass spectrometry (LC-MS) does not yield site-specific PTM data, but it requires minimal sample preparation and can provide an analysis of larger PTMs with the additional benefit of mass identification.
Disulfide bond reduction and/or limited digestion with enzymes like IdeS, papain, GingisKHAN®, and FabALACTICA® marginally increase sample preparation time but enable subunit level resolution of PTM localization that can be further increased by fragmenting each subunit using electron transfer dissociation (ETD) or another tandem mass spectrometry (MS2) approach. However, site-specific localization and quantification of PTMs across a wide dynamic range are most commonly performed from the “bottom-up” using a technique called peptide mapping.
Peptide mapping methods require enzymatic digestion of the antibody, yielding a peptide mixture that is separated by liquid chromatography and detected by ultraviolet/visible (UV/Vis) absorbance before being ionized and infused into a mass spectrometer. Full MS spectra are acquired, and peptides are selected and fragmented to produce MS2 spectra that are used to validate a peptide's identity or localize a PTM on a peptide containing more than one potential modification site. While peptide mapping can potentially induce preparation-related artifacts onto the antibody sequence and significantly increases the time and complexity of an experiment, it is the most sensitive PTM characterization method and is site specific.
Quantification of each modification can be performed using UV or extracted ion chromatograms (XICs), but UV quantification is obfuscated by co-eluting peptides and is inherently less sensitive than modern mass spectrometers. Because of this, XIC-based quantification is routinely performed, and an MS-based peptide mapping assay allows for identification, localization, and quantification of all relevant PTMs with a detection limit of less than 0.1% under optimal conditions.
The advantages of PTM quantification by XIC are accompanied by some unique disadvantages that affect the method's accuracy and precision. Many of these issues are related to differences in the ionization of an unmodified peptide versus the modified form due to: (1) ion suppression of one or both peptide forms from co-eluting peptide peaks, (2) the difference in solvent environment between the two peptide forms eluting at separate retention times, (3) the disparity in ionization efficiency between the modified peptide relative to the unmodified, and (4) variability among mass spectrometers. Peptide mapping quantification of all PTMs is influenced by these factors, but the C-terminal K truncation (des-K) value is particularly impacted due to differences in ionization efficiencies and a reduction in the peptide's predominant charge state to 1+ compared to the unprocessed form (K, z=2+).
This PTM readily occurs because of carboxypeptidase activity during production from mammalian tissue culture cells, and the resulting predominant form in a mAb or bsAb is des-K. Therefore, the percent relative abundance of unprocessed C-terminal K is typically calculated in relation to the sum of K and des-K. Unprocessed C-terminal K is not thought to be an efficacy or safety concern in antibodies since it is not in a CDR and has been shown to be rapidly lost upon injection with a half-life of roughly one hour, but careful monitoring of this PTM demonstrates process control, and it has been reported that antibodies with more basic pl values may also have increased tissue uptake and blood clearance.
For these reasons, unprocessed C-terminal K measurement is still critical, and previous efforts found that the percentage of K is overestimated during peptide map quantification as the additional K on the C-terminus of the peptide sequence increases the ionization efficiency relative to the des-K peptide. Some attempts to minimize this error include using only the most abundant charge state to calculate the XIC area under the curve (AUC) for each peptide or using a correction factor determined by injecting equal molar amounts of each peptide onto the LC column and gauging the mass spectrometer response.
However, while the first method decreases the magnitude of the unprocessed C-terminal K value, it does so with no empirical knowledge of how much this value should be decreased by, and the second assumes that the correction factor remains static across the possible concentration range of unprocessed C-terminal K, in the presence of potentially co-eluting peptides, and among different mass spectrometers.
Accordingly, it would be desirable to provide a simple method for accurate and precise quantification of a PTM, such as quantification of an unprocessed C-terminal lysine (K), in antibodies and other proteins.
Therefore, an object of the invention is to provide an improved method for the precise quantification of a PTM, such as quantification of an unprocessed C-terminal lysine (K), in antibodies and other proteins.
SUMMARY OF THE INVENTIONThe present invention provides methods useful for the accurate and precise characterization of PTMs in proteins, such as antibodies, using a heavy peptide approach. More specifically, the present disclosure provides quantification of unprocessed C-terminal lysine (K) in antibodies. Methods of quantifying C-terminal K using a heavy peptide approach are provided.
In one exemplary embodiment of the present invention, a method of quantifying unprocessed C-terminal K involves mixing a set of heavy C-terminal peptide standards to a peptide digest, generating a calibration curve of the unprocessed C-terminal K peptide's signal response relative to that of the des-K peptide, and analyzing and quantifying the mass of the unprocessed C-terminal K. In one embodiment, the mass of the unprocessed C-terminal lysine (K) is analyzed and quantified in a single LC-MS2 peptide mapping experiment.
In another embodiment, the resulting response curve may span a concentration ratio range of 1:1000-1:1 K to des-K peptide. In other embodiments, the resulting response curve has an error of less than 10%. In one embodiment, the peptide is an antibody. For example, the method is useful for analyzing and quantifying the mass of the unprocessed C-terminal lysine (K) in monoclonal antibodies (mAbs).
In another embodiment, the disclosed method is useful for analyzing and quantifying the mass of the unprocessed C-terminal lysine (K) in bispecific antibodies (bsAbs).
Advantages of the invention include but are not limited to: a robust and highly accurate assay using analytical chemistry designed for the detection of C-terminal lysines (K) at the end of an antibody using heavy peptides in a single comparative step; a robust and highly accurate assay using analytical chemistry for high speed and accurate manufacture of therapeutic antibodies; therapeutic antibodies produced to a higher level of confidence as a result of the above as part of manufacturing train; wide application for perfecting the manufacture of antibodies in clinical development and in commercial use, and kits containing heavy peptides, comparative standards, and instructions for use in carrying out the assays of the invention.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosure and together with the description, serve to explain the principles of the disclosure.
The term “C terminal lysine (K)” or “K peptide” refers to an amino acid lysine residue or “K” residue that can be present or absent on the end of the heavy chain of an antibody.
The term “truncated peptide” or “(des-K)” refers to a representative portion of a protein having the C-terminal amino acid sequence of an antibody missing a C-terminal lysine (K).
The term “analyzing and quantifying the percentage of K peptide” refers to comparing the difference between a first and second assay signal sufficient to ascertain the difference between an antibody, or representative peptide thereof, which shows the presence of absence of a C-terminal lysine (K).
The term “analytical chemistry or chemistries” refers to quantitative analysis of molecules for the purpose of carrying out the invention, and in particular liquid chromatography mass spectrometry.
The term “heavy peptides” refers to any peptide of the invention, or equivalents thereof, wherein at least one or more carbon or nitrogen atoms of the peptide is a heavy isotope thereof, for example, 13C and 15N isotopes.
The term “peptide digest” refers to peptide mix resultant from exposing an antibody, as described herein, when incubated with one or more enzymes capable of digesting an antibody protein sequence such that a polypeptide sequence representative of the C-terminus of the antibody is released.
The term “unclipped” refers to an antibody C-terminal sequence or a representative polypeptide sequence thereof, wherein the C-terminal sequence is has a terminal lysine (K) amino acid residue.
The term “clipped” refers to an antibody C-terminal sequence or a representative polypeptide sequence thereof, wherein the C-terminal sequence is missing a terminal lysine (K) amino acid residue.
The term “antibody” refers to a therapeutic immunobinder, e.g., a monoclonal antibody, bi- or multi-specific antibody, that is suitable for introducing into a subject for modulating a disease or disorder, for example, an immune or oncological disorder. A “drug antibody” can be, for example, a bispecific antibody that can bind to two (2) targets.
The term “antibody” is to be construed broadly as describing monoclonal antibodies, bispecific antibodies, antibody compositions with multi-specificity, as well as antibody fragments (e.g., Fab, F(ab′)2, scFv and Fv), antibody derivatives, variants, and analogs.
Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used.
2. Improved Assays for Antibody C-Terminal Lysine (K) AnalysisThe invention provides a peptide-based for assay for accurately quantitating the undesirable amount of antibody C-terminal lysine (K). The assays of the invention are essential quality control tools for evaluating an antibody candidate, for example, in clinical trials or in commercial use.
Typically, the assay of the invention is structured as shown in
The assay of the invention, using a novel set of heavy peptides and analytical chemistries (e.g., liquid chromatography and mass spectrophotometry) can be calibrated to provide highly accurate measurements. This assay fidelity is key for the manufacture of complex protein molecules, in particular, therapeutic antibodies designed to be introduced into human patients.
3. Assay KitsThe invention also provides kits for carrying out the assay of the invention. A key step in the assay for determining accurate and true measures of the presence of C-terminal lysines (K) is the use of one or more heavy peptides of sufficient plurality, that when admixed with appropriate standards and a sample, provide a readable signal. The signal is typically measured using analytical chemistries, for example, Liquid Chromatography Mass Spectroscopy (LCMS).
Accordingly, exemplary components of the kit consist of:
1. standard peptides “clipped”
2. standard peptides “unclipped”
3. standard heavy peptides (“clipped” and “unclipped”) including one or more of the following exemplary peptides disclosed herein; and
4. instructions for use, including instructions for calibration, data extraction, analysis, and interpretation.
Accordingly, the invention provides for a convenient test kit and instructions for perfecting an important antibody manufacturing chemistry, manufacturing, and controls (CMC) endpoint.
5. Wide Application of the InventionIt should be appreciated that current invention provides for the accurate determination of the fine structure and exact amino acid sequence of a therapeutic antibody. Accordingly, the invention compliments and improves the CMC (Chemistry, Manufacturing, and Controls) of any commercially produced therapeutic antibody.
For example, the invention allows for perfecting the manufacture and safeguarding of a number of antibody therapies.
Such antibody therapies include:
abciximab, adalimumab, adalimumab-atto, ado-trastuzumab emtansine, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cetuximab, daclizumab (Zenapax), daclizumab (Zinbryta), daratumumab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, evolocumab, golimumab, golimumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab ixekizumab, mepolizumab, natalizumab, necitumumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rituximab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, sarilumab, rituximab and hyaluronidaseguselkumab, inotuzumab ozogamicin, adalimumab-adbm, gemtuzumab ozogamicin, bevacizumab-awwb, benralizumab, and emicizumab-kxwh. trastuzumab-dkst, infliximab-qbtx, ibalizumab-uiyk, tildrakizumab-asmn, burosumab-twza, and erenumab-aooe.
Other therapeutic antibodies of interest for various indications subject to the invention include: aflibercept, for treating eye disorders; rilonacept for treating blindness and metastatic colorectal cancer; alirocumab for treating familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease (ASCVD); dupilumab for treating atopic dermatitis; sarilumab for treating rheumatoid arthritis and COVID-19; cemiplimab for treating PD-1 related disease; and antibodies for treating Ebola.
EXAMPLESThe examples below are provided for illustrative purposes and should not be construed as limiting the invention which is defined by the appended claims. All references and patents recited within the present application are included herein by reference.
Materials and MethodsThe present invention, when practiced by the person skilled in the art, may make use of conventional techniques in the field of pharmaceutical chemistry, immunology, molecular biology, cell biology, recombinant DNA technology, and assay techniques, as described in, for example, Sambrook et al. “Molecular Cloning: A Laboratory Manual”, 3rd ed. 2001; Ausubel et al. “Short Protocols in Molecular Biology”, 5th ed. 1995; “Methods in Enzymology”, Academic Press, Inc.; MacPherson, Hames and Taylor (eds.). “PCR 2: A practical approach”, 1995; “Harlow and Lane (eds.) “Antibodies, a Laboratory Manual” 1988; Freshney (ed.) “Culture of Animal Cells”, 4th ed. 2000; “Methods in Molecular Biology” vol. 149 (“The ELISA Guidebook” by John Crowther) Humana Press 2001, and later editions of these treatises (e.g., “Molecular Cloning” by Michael Green (4th Ed. 2012) and “Culture of Animal Cells” by Freshney (7th Ed., 2015), as well as current electronic versions.
Methods useful for quantifying and analyzing PTMs in proteins are provided within the disclosure. More specifically, the present disclosure provides methods for quantifying and analyzing C-terminal lysine (K) in proteins, for example, antibodies. The methods include applying a set of heavy C-terminal peptide standards to a digested protein. The protein may be digested by proteases such as trypsin and other suitable enzymes.
The method may involve spiking calibration curves into antibody digests and injecting approximately equimolar amounts of heavy des-K peptide to digested des-K peptide onto a column in each LC-MS2 run. Unprocessed C-terminal K may be quantified in a single LC-MS2 peptide mapping experiment.
The method may involve generating a calibration curve spanning a ratio range of 1:1000-1:1 K to des-K peptide. The calibration curve may have an error of less than 10%, less than 9%, or less than 8%. Mass spectra may be quantified using various spectrometers, such Thermo Q-Exactive Plus 3, Q-Exactive Plus 4 or Orbitrap Fusion Lumos mass spectrometers.
The following working examples demonstrate exemplary methods for identifying and quantifying PTMs after antibody synthesis.
Example 1 Assay Design and Methods for CalibrationThis example shows the experimental design of the assay of the invention for calibrating the understanding of antibody C-terminus lysine (K) structure.
All light and heavy isotopic peptide standards were purchased from New England Peptide (Gardner, Mass.). Trifluoroacetic acid (TFA), formic acid (FA), tris [2-carboxylethyl] phosphine hydrochloride (TCEP-HCl), and Optima LC/MS grade acetonitrile (ACN) were obtained from Thermo Fisher Scientific (Rockford, Ill.) while glacial acetic acid and iodoacetamide (IAM) were procured from Sigma-Aldrich (St. Louis, Mo.). Sequencing grade modified trypsin, ultrapure urea, and ultrapure 1 M Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) were purchased from Promega (Madison, Wis.), Alfa Aesar (Haverhill, Mass.), and Invitrogen (Carlsbad, Calif.), respectively. Milli-Q water was purified by a Millipore Milli-Q Advantage Δ10 Water Purification System.
Isotopic HC C-terminal peptide standards were used to normalize the mass spectrometer response between corresponding light unprocessed and truncated peptides for quantification of percent lysine. Peptide standards included SLSLSLG (SEQ ID NO:1), SLSLSLGK (SEQ ID NO:2), SLSLSPG (SEQ ID NO:3) and SLSLSPGK (SEQ ID NO:4).
An equimolar mixture of SLSLSPGK (SEQ ID NO:4) and SLSLSPG (SEQ ID NO:3) was quantified by UV chromatography, as shown in
As can be seen, the values of the heavy peptides were approximately equal to the corresponding light peptides.
To determine the accuracy of the method, known quantities of light des-K and K were spiked into the reagent sets across the 1:10-1:1000 K to des-K peptide ratio range and measured using the calibration curve corrected method.
As shown in Table 2, the calibration curve corrected values were closely aligned with the expected % lysine.
Unprocessed C-Terminal Lysine Quantification of mAbs
This example shows the experimental design of the assay of the invention for understanding the antibody C-terminus lysine (K) structure.
For antibody analysis, the calibration curves were spiked into antibody digests so that an approximately equimolar amount of heavy des-K peptide to digested des-K peptide was injected onto the column in each LC-MS2 run.
Antibody DigestionEqual weights of five IgG4 mAb samples were buffer exchanged into 5 mM acetic acid and 5 mM TCEP-HCl before denaturation and reduction at 80° C. for ten minutes. The samples were further denatured in 4 M urea/0.1 M Tris-HCl, pH 7.5 and alkylated with 5 mM IAM at room temperature in the dark for 30 minutes. Urea concentration was lowered to 1 M by adding 0.1 M Tris-HCl, pH 7.4, and the antibodies were digested at a 1:20 antibody to trypsin ratio at 37° C. for 4 hours. Enzymatic activity was quenched by acidifying the samples in 0.2% TFA.
LC-MS and LC-MS2 ParametersAliquots of 5 μg of antibody digest was injected onto a 2.1 mm×150 mm Waters Acquity Ultra Performance Liquid Chromatography (UPLC) Charged Surface Hybrid (CSH) C18 column with 1.7 μm particles. Peptides were separated on this column with a Waters Acquity I-Class UPLC set to a flow rate of 250 μL/min and column temperature of 40° C. The gradient consisted of a 0.1-35% increase of organic mobile phase (ACN and 0.1% FA) relative to water and 0.1% FA over 95 minutes.
Mass data was acquired using a Thermo Q-Exactive Plus using QE Plus 3 and 4 systems and/or Orbitrap Fusion Lumos mass spectrometer. Full mass scans were performed on the Q-Exactive Plus acquired an m/z range of 300-2000 at 140,000 resolution (m/z 200) for an ion population limited by an automatic gain control (AGC) target set to 1×106 or a maximum ion injection time (max IT) of 50 ms.
Experiments requiring MS2 identification by data dependent acquisition (DDA), a single dd-MS2 loop began by isolating and fragmenting each of the five most intense peptide ions with a 1.5 Th window using higher energy collisional dissociation (HCD) at a normalized collision energy of 30 was used.
Fragment ion population data was collected using an AGC target of 1×105 or a max IT of 100 ms and then scanned at 17,500 resolution, at which point the sampled precursor was placed on an exclusion list for 10 seconds to ensure the analysis of less intense ions.
Orbitrap Fusion Lumos parameters for MS1 acquisition were the same as for the QE-Plus, with the exceptions being resolution set to 120,000 (m/z 200) and ACG target to 5×105. Differences in MS2 settings were: (1) limiting DDA by a cycle time of one second instead of by number of precursors, (2) setting AGC target to 2×104, (3) controlling max IT with 50 ms but allowing for continued injection if parallelizable time was available, and (4) scanning at 15,000 resolution (m/z 200).
Relevant LC-MS2 raw files were analyzed with Byonic 3.0 using custom fasta files for each antibody according to the following parameters: (1) Cleavage Sites—R, K, (2) Cleavage Side—C-terminal, (3) Digestion Specificity—Fully Specific, (4) Precursor Mass Tolerance—10 ppm, (5) Fragmentation Type—QTOF/HCD, Fragment Mass Tolerance—20 ppm, (6) Fixed and Variable Modifications—Fixed C Carbamidomethyl, Variable M Oxidation, Variable E/Q to pE, and Variable C-term K Loss, and (7) Glycan Modifications—50 common biantennary N-glycans. Ion chromatograms for the 1+ and 2+ charge states of light and heavy C-terminal peptides were extracted in Thermo Xcalibur 3.1 by the Genesis algorithm set to a 10 ppm m/z tolerance. Quantitative AUC measurements were exported to Microsoft Excel, where calibration curves ranging from 1:1000-1:1 K to des-K were constructed to calculate the percentage of unprocessed C-terminal K in each sample.
Table 3 shows the results obtained using the calibration curve correction method compared to normal, uncorrected peptide mapping. As shown in Table 3, the percentage of C-terminal lysine is overestimated during peptide quantification using uncorrected peptide mapping in comparison to the CC corrected method of the present disclosure.
This example shows the experimental design of the assay of the invention for assaying bispecific antibodies (BsAbs).
Multiple IgG4-based bsAbs (7 seven) (containing both SLSLSLGK (SEQ ID NO:2) and SLSLSPGK (SEQ ID NO:4) C-terminal sequences) were digested as described above. Calibration curves were spiked into the antibody digests and approximately equimolar amount of heavy des-K peptide to digested des-K peptide was injected onto the column in each LC-MS2 run. Corresponding bsAb digests were subjected to traditional, uncorrected peptide mapping.
Table 4 shows the results obtained using the calibration curve correction method compared to normal, uncorrected peptide mapping of the PGK C-terminal sequences. As shown in Table 4, the percentage of C-terminal lysine is overestimated during peptide quantification using uncorrected peptide mapping in comparison to the CC corrected method of the present disclosure.
Table 5 shows the results obtained using the calibration curve correction method compared to normal, uncorrected peptide mapping of the LGK C-terminal sequences. As shown in Table 5, the percentage of C-terminal lysine is overestimated during peptide quantification using uncorrected peptide mapping in comparison to the CC corrected method of the present disclosure.
Five IgG4 mAbs and one IgG1 mAb were digested as described above. Calibration curves were spiked into the antibody digests and approximately equimolar amount of heavy des-K peptide to digested des-K peptide was injected onto the column in each LC-MS2 run. Corresponding mAb digests were subjected to traditional, uncorrected peptide mapping. Mass data were acquired using a Thermo Q-Exactive Plus and an Orbitrap Fusion Lumos mass spectrometer.
As shown in Table 6, when using the CC corrected method, there was zero to little difference in percent lysine when quantified using either the QE-Plus or Fusion mass spectrometer. However, greater variability of percent lysine was seen across instruments when uncorrected peptide mapping was used.
Multiple (7) IgG4-based bsAbs (containing both SLSLSLGK (SEQ ID NO:2) and SLSLSPGK (SEQ ID NOA4) C-terminal sequences) were digested as described above. Calibration curves were spiked into the antibody digests and approximately equimolar amount of heavy des-K peptide to digested des-K peptide was injected onto the column in each LC-MS2 run. Corresponding bsAb digests were subjected to traditional, uncorrected peptide mapping.
Mass data were acquired using a Thermo Q-Exactive Plus and an Orbitrap Fusion Lumos mass spectrometer. As shown in Table 7, when using the CC corrected method, there was zero to little difference in percent lysine when quantified using either the QE-Plus or Fusion mass spectrometer. However, greater variability of percent lysine was seen across instruments when uncorrected peptide mapping was used.
While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain details described herein can be varied without departing from the basic principles of the invention.
Claims
1. A method for quantifying unprocessed C-terminal lysine in a peptide (K peptide), comprising:
- mixing a set of heavy C-terminal peptide standards with a peptide digest;
- generating a calibration curve of a peptide signal of the unprocessed C-terminal K response relative to that of a truncated (des-K) peptide; and
- analyzing and quantifying the percentage of K peptide using liquid chromatography mass spectrometry.
2. The method according to claim 1, wherein the set of heavy C-terminal peptide standards comprises equimolar concentrations of Δ4 K peptide:Δ4 des-K peptide, Δ8 K peptide:Δ4 des-K peptide, Δ12 K peptide:Δ4 des-K peptide, and Δ16 K peptide:Δ4 des-K peptide.
3. The method of claim 2, wherein the equimolar concentrations are at molar ratios of 1:1, 1:10, 1:100, and 1:1000 K peptide to des-K peptide.
4. The method of claim 3, wherein the K peptide standard is SEQ ID NO:2 and the des-K peptide standard is SEQ ID NO:1.
5. The method of claim 3, wherein the K peptide standard is SEQ ID NO:4 and the des-K peptide standard is SEQ ID NO:3.
6. The method of claim 1, wherein the percentage of K peptide is analyzed using liquid chromatography tandem mass spectrometry (LC-MS2).
7. The method of claim 4, wherein the unprocessed C-terminal K is analyzed and quantified in a single LC-MS2 peptide mapping run.
8. The method of claim 1, wherein the K peptide is an antibody.
9. The method of claim 8, wherein the antibody is a monoclonal or bispecific antibody.
10. The method of claim 1, wherein an error of the calibration curve is less than 10%.
11. A method for quantifying unprocessed C-terminal lysine in a peptide (K peptide), comprising:
- digesting a protein with a protease to produce a peptide digest;
- mixing the peptide digest with a set of heavy C-terminal peptide standards, wherein the set of heavy C-terminal peptide standards comprises equimolar concentrations of Δ4 K peptide:Δ4 des-K peptide, Δ8 K peptide:Δ4 des-K peptide, Δ12 K peptide:Δ4 des-K peptide, and Δ16 K peptide:Δ4 des-K peptide;
- generating a calibration curve of a peptide signal of the unprocessed C-terminal K response relative to that of a truncated (des-K) peptide; and
- analyzing and quantifying the percentage of K peptide using liquid chromatography mass spectrometry.
12. The method of claim 11, wherein the K peptide standard:des-K peptide standard is SEQ ID NO:2:SEQ ID NO:1 or SEQ ID NO:4:SEQ ID NO:3.
13. A kit for quantifying unprocessed C-terminal lysine in a peptide (K peptide), comprising:
- des-K peptide standards;
- K peptide standards;
- heavy des-K-peptide standards;
- heavy K-peptide standards; and
- instructions for use.
14. The kit of claim 13, further comprising instructions for calibration, data extraction, analysis, and interpretation.
15. The kit of claim 13, wherein the des-K peptide standards and heavy des-K peptide standards are SEQ ID NO:1 and the K peptide standards and heavy K peptide standards are SEQ ID NO:2.
16. The kit of claim 13, wherein the des-K peptide standards and heavy des-K peptide standards are SEQ ID NO:3 and the K peptide standards and heavy K peptide standards are SEQ ID NO:4.
Type: Application
Filed: Jun 18, 2021
Publication Date: Jan 13, 2022
Inventors: Tyler Greer (Elmsford, NY), Milos Cejkov (Ridgewood, NJ), Reid O'Brien Johnson (Hartsdale, NY), Xiaojing Zheng (Croton-on-Hudson, NY), Ning Li (New Canaan, CT)
Application Number: 17/351,896
