HEAVY PEPTIDE APPROACH TO ACCURATELY MEASURE UNPROCESSED C-TERMINAL LYSINE IN ANTIBODIES

Abstract

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-MS.sup.2) run.

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-MS.sup.2).

7. The method of claim 4, wherein the unprocessed C-terminal K is analyzed and quantified in a single LC-MS.sup.2 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.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0024] 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.

[0025] FIGS. 1A and 1B. FIG. 1A is a schematic of the assay of the invention showing a peptide representing the C-terminus of an antibody fully processed “clipped” (.i.e., lacking a C-terminal lysine (K)) mixed with four (4) other peptides representing the C-terminus of an antibody that is unprocessed “unclipped” (i.e., having a C-terminal lysine (K)) to form a response curve peptide mix. This response curve peptide mix is then mixed with a sample representing a potential antibody manufacturing sample (mAb Digest), sufficient to accurately quantitate the amount of C-terminal lysine (K) present. FIG. 1B represents the analytical peaks observed for each of the species when subjected to Liquid Chromatography Mass Spectroscopy (M/Z).

[0026] FIGS. 2A-2E show the structures of five heavy peptide standards. FIGS. 2A-2D show four SLSLSLGK (SEQ ID NO:2) “unclipped” heavy peptides standards containing .sup.13C and .sup.15N isotopes (indicated by •), Δ4, Δ8, Δ12, and Δ16 K peptides, respectively. FIG. 2E shows the heavy isotopic SLSLSLG (SEQ ID NO:1) standard, Δ4 des-K. containing .sup.13C and .sup.15N isotopes (indicated by •).

[0027] FIG. 3 shows a calibration curve (CC) exhibiting a proportional relationship between “clipped” and “unclipped” peptides.

[0028] FIG. 4 shows an exemplary response curve using heavy chain (HC) C-terminal peptides in accordance with an embodiment of the present invention.

[0029] FIGS. 5A and 5B. FIG. 5A shows a UV chromatogram of an equimolar mixture of “unclipped” SLSLPGK (SEQ ID NO:4) and “clipped” SLSLSPG (SEQ ID NO:3). FIG. 5B shows an extracted ion chromatogram (XIC) of equimolar amounts of the SLSLPGK (SEQ ID NO:4) and SLSLSPG (SEQ ID NO:3) reagent set in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0030] 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.

[0031] 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).

[0032] 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).

[0033] 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.

[0034] 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, .sup.13C and .sup.15N isotopes.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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) Analysis

[0041] The 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.

[0042] Typically, the assay of the invention is structured as shown in FIGS. 1A and 1B where five (5) heavy peptides are co-incubated in the presence of an antibody digest to produce a detectable signal. The detectable signal can indicate an accurate measure of the “clipped” and “unclipped” C-terminal lysine (K).

[0043] 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 Kits

[0044] The 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).

[0045] 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.

[0046] 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 Invention

[0047] It 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.

[0048] For example, the invention allows for perfecting the manufacture and safeguarding of a number of antibody therapies.

[0049] Such antibody therapies include:

[0050] 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.

[0051] 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.

EXAMPLES

[0052] The 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 Methods

[0053] The 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”, 3.sup.rd ed. 2001; Ausubel et al. “Short Protocols in Molecular Biology”, 5.sup.th 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”, 4.sup.th 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 (4.sup.th Ed. 2012) and “Culture of Animal Cells” by Freshney (7.sup.th Ed., 2015), as well as current electronic versions.

[0054] 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.

[0055] 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-MS.sup.2 run. Unprocessed C-terminal K may be quantified in a single LC-MS.sup.2 peptide mapping experiment.

[0056] 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.

[0057] The following working examples demonstrate exemplary methods for identifying and quantifying PTMs after antibody synthesis.

Example 1

Assay Design and Methods for Calibration

[0058] This example shows the experimental design of the assay of the invention for calibrating the understanding of antibody C-terminus lysine (K) structure.

[0059] 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.

[0060] 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).

[0061] FIG. 1A is a schematic of the assay of the invention showing a peptide representing the C-terminus of an antibody fully processed “clipped” (.i.e., lacking a C-terminal lysine (K)) mixed with four (4) other peptides representing the C-terminus of an antibody that is unprocessed “unclipped” (i.e., having a C-terminal lysine (K)) to form a response curve peptide mix. This response curve peptide mix is then mixed with a sample representing a potential antibody manufacturing sample (mAb Digest), sufficient to accurately quantitate the amount of C-terminal lysine (K) present. FIG. 1B represents the analytical peaks observed for each of the species when subjected to Liquid Chromatography Mass Spectroscopy (M/Z).

[0062] FIGS. 2A-2E show heavy isotopic standards. .sup.13C and .sup.15N are indicated by •. FIGS. 2A-2D show heavy isotopic SLSLSLGK (SEQ ID NO:2) Δ4, Δ8, Δ12, and Δ16 K peptides, respectively. FIG. 2E shows the heavy isotopic SLSLSLG (SEQ ID NO:1) standard, Δ4 des-K. The peptide standards were dissolved in 10% ACN, 0.1% TFA and combined into two calibration curve sets according to the C-terminal sequence (LGK or PGK). Each set contained equimolar concentrations of Δ4 des-K and K peptide as well as Δ8, Δ12, and Δ16 K peptides at molar ratios of 1:10, 1:100, and 1:1000 K to des-K, respectively. The mixture was analyzed by XIC as shown in FIG. 3.

[0063] An equimolar mixture of SLSLSPGK (SEQ ID NO:4) and SLSLSPG (SEQ ID NO:3) was quantified by UV chromatography, as shown in FIG. 4A. Corresponding K AUC/des-K AUC values for PGK peptides were 1.08. Similarly, K AUC/des-K AUC values for LGK peptides were 1.07. Equimolar amounts of the SLSLSPGK (SEQ ID NO:4) and SLSLSPG (SEQ ID NO:3) reagent set were quantified by XIC, as shown in FIG. 4B. Heavy AUC/light AUC values for PGK and LGK peptides are shown in Table 1.

TABLE-US-00001 TABLE 1 Heavy/light Heavy AUC/ Peptide isotope light AUC SLSLSPGK Δ4/Δ0 0.98 Δ8/Δ0 1.00 Δ12/Δ0  0.99 Δ16/Δ0  1.00 SLSLSPG Δ4/Δ0 0.99 SLSLSLGK Δ4/Δ0 1.02 Δ8/Δ0 1.04 Δ12/Δ0  1.06 Δ16/Δ0  1.03 SLSLSLG Δ4/Δ0 1.00

[0064] As can be seen, the values of the heavy peptides were approximately equal to the corresponding light peptides.

[0065] 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.

[0066] As shown in Table 2, the calibration curve corrected values were closely aligned with the expected % lysine.

TABLE-US-00002 TABLE 2 SLSLSLGK (SEQ ID NO: 2) SLSLSPGK (SEQ ID NO: 4) CC CC Expected Corrected % Corrected % % K % K Difference % K Difference 50.0 49.6 0.8 50.8 1.5 9.1 8.9 2.0 9.3 2.5 1.0 0.9 8.9 1.0 7.1 0.1 0.1 2.0 0.1 3.5

Example 2

[0067] Unprocessed C-Terminal Lysine Quantification of mAbs

[0068] This example shows the experimental design of the assay of the invention for understanding the antibody C-terminus lysine (K) structure.

[0069] 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-MS.sup.2 run.

Antibody Digestion

[0070] Equal 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-MS.SUP.2 .Parameters

[0071] Aliquots 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.

[0072] 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.

[0073] Experiments requiring MS.sup.2 identification by data dependent acquisition (DDA), a single dd-MS.sup.2 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.

[0074] 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.

[0075] Orbitrap Fusion Lumos parameters for MS.sup.1 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 MS.sup.2 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).

[0076] Relevant LC-MS.sup.2 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.

[0077] 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.

TABLE-US-00003 TABLE 3 CC Corrected C-term Lys % Uncorrected C-term Lys % Standard Standard Antibody Mean Deviation Mean Deviation mAb 1 5.7 0.2 10.4 0.4 mAb 2 6.9 0.7 11.5 0.7 mAb 3 6.3 0.2 12.0 0.3 mAb 4 9.8 0.4 15.8 0.2 mAb 5 11.8 0.4 19.1 1.1

Example 3

Unprocessed C-Terminal Lysine Quantification of Bispecific Antibodies (BsAbs)

[0078] This example shows the experimental design of the assay of the invention for assaying bispecific antibodies (BsAbs).

[0079] 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-MS.sup.2 run. Corresponding bsAb digests were subjected to traditional, uncorrected peptide mapping.

[0080] 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-US-00004 TABLE 4 CC Corrected C-term Lys % Uncorrected C-term Lys % Antibody Standard Standard (PGK) Mean Deviation Mean Deviation bsAb 1 14.3 0.1 23.9 0.3 bsAb 2 15.3 0.0 23.5 0.6 bsAb 3 15.8 0.0 27.3 1.0 bsAb 4 16.4 0.2 27.0 0.5 bsAb 5 16.9 0.2 25.7 1.2 bsAb 6 20.0 0.1 30.1 0.6 bsAb 7 26.4 0.3 37.3 0.3

[0081] 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.

TABLE-US-00005 TABLE 5 CC Corrected C-term Lys % Uncorrected C-term Lys % Antibody Standard Standard (LGK) Mean Deviation Mean Deviation bsAb 1 2.0 0.1 3.5 0.1 bsAb 2 2.5 0.1 4.1 0.1 bsAb 3 2.2 0.1 3.7 0.2 bsAb 4 2.5 0.2 4.2 0.1 bsAb 5 2.7 0.0 4.7 0.1 bsAb 6 3.4 0.1 5.7 0.1 bsAb 7 5.1 0.1 8.4 0.2

[0082] 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.

[0083] 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.

TABLE-US-00006 TABLE 6 Antibody CO Corrected C-term Lys % Uncorrected C-term Lys % (C-term) QE-Plus Fusion % RSD QE-Plus Fusion % RSD IgG4 5.5 5.5 0.4 10.0 9.1 6.5 mAb 1 IgG4 6.5 6.5 0.2 10.9 9.8 7.5 mAb 2 IgG4 6.6 6.5 0.7 12.2 10.0 14.0 mAb 3 IgG4 9.7 9.6 0.7 16.0 14.8 5.5 mAb 4 IgG4 12.0 11.6 2.6 20.0 17.7 8.8 mAb 5 IgG1 0.7 0.6 8.6 1.1 0.8 19.2 mAb 1 (PGK)

[0084] 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.

[0085] 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.

TABLE-US-00007 TABLE 7 CC Corrected Uncorrected C-term Lys % C-term Lys % C- QE- % QE- % Antibody terminal Plus Fusion RSD Plus Fusion RSD bsAb 1 PGK 14.2 14.8 2.7 24.1 18.9 17.0 LGK 2.1 2.1 2.6 3.6 3.2 9.2 bsAb 2 PGK 15.3 15.5 1.2 24.2 19.2 16.3 LGK 2.6 2.5 2.5 4.0 4.2 3.3 bsAb 3 PGK 15.8 16.3 2.2 28.1 20.8 20.9 LGK 2.3 2.2 3.8 3.7 3.3 7.7 bsAb 4 PGK 16.3 16.9 2.2 28.1 20.8 20.9 LGK 2.6 2.5 2.1 4.3 3.9 6.2 bsAb 5 PGK 16.7 17.3 2.2 24.8 21.1 11.4 LGK 2.7 2.7 1.2 4.6 4.3 4.2 bsAb 6 PGK 20.1 20.4 1.3 30.6 25.1 14.0 LGK 3.5 3.4 2.6 5.8 5.3 6.0 bsAb 7 PGK 26.1 26.9 2.0 37.6 31.9 11.8 LGK 5.2 5.1 0.9 8.6 8.0 5.0

[0086] 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.