Characterization of Monoclonal Antibodies Using UHPLC-HRAM MS The power of the Q Exactive BioPharma for online intact, subunit analysis, middle down, and peptide mapping Jonathan L. Josephs and Aaron O. Bailey. Life Sciences Mass Spectrometry The world leader in serving science
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Two Types of Orbitrap Measurements for Intact Proteins Average mass resolution High Mass Range (HMR) mode Works for all proteins (mabs) Lowest resolving power Isotopic resolution Protein mode Small / medium-sized proteins (mab subunits) Highest resolving power 9 7 3 1 Average mass 9 7 3 1 Monoisotopic mass ReSpect deconcolution algorithm Xtract deconvolution algorithm 3
Intact Protein Analysis in BioPharma Finder Workflow software for intact protein mass determination Supports all Orbitrap mass spectrometers Includes 2 deconvolution algorithms: Xtract for isotopically resolved proteins ReSpect TM for isotopically unresolved proteins (e.g. IgG) Batch processing/automation 4
Sliding Window Deconvolution is the smart choice for chromatography [ [ [ [ [ t t t I t i t I t i t I t i t n s y e n v i e l a R e n s y e n v i e l a R e n s y e n v i e l a R e mab 8. 8.2 8.4 8.6 8.8 RT(min) 9. 9.2 9.4 9.6 9.8 8. 8.2 8.4 8.6 8.8 RT(min) 9. 9.2 9.4 9.6 9.8 8. 8.2 8.4 8.6 8.8 RT(min) 9. 9.2 9.4 9.6 9.8 mab + 2 drugs mab + 4 drugs Components integrated 5
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Subunit Analysis in Protein Mode on Q Exactive Plus LC-MS analysis of IdeS-digested, reduced Trastuzumab 9 7 3 1 Fc LC Fd 1 2 3 4 5 6 7 8 Time (min) Fc * 28+ * 92. 92.5 1 R=1, Sliding Window Xtract TM deconvolution LC.2 ppm 23428.519 23 LC * Fc A2GF 1.9 ppm 252.415 mass * 978 1 Fd.1 ppm 25367.515 Fc A2G1F 1.4 ppm 25382.551 24+ 26+ Fd * * 977 1 7
Subunit Analysis in Protein Mode on Q Exactive HF LC-MS analysis of reduced Trastuzumab Light Chain R = 1, Full MS * 24+ * 977.5 978. 978.5 LC HC 4 5 6 7 8 9 1 11 12 Time (min) Heavy Chain R = 15, Full MS R = 2, SIM MS 46+ 1.7 1.9 111.1 111.3 l a t i v e I n t e n s i t y R e 9 7 3 1 7 9 1 1 13 1 1 1 LC.8 ppm 23428.542 23425 23475 Mass Xtract TM deconvolution for monoisotopic masses 1 1 1 1 ReSpect TM deconvolution for average masses l a t i v e I n t e n s i t y R e HC A2GF 2.8ppm 1.26 Mass HC A2G1F 1.2ppm 763.44 8
Denaturing LC-MS in HMR mode Sensitive intact analysis of Trastuzumab antibody * 47+ 3 3 31 31.1 ng ( pg).5 ng 1. ng 1. ng. ng. ng Q Exactive Plus in HMR Mode with MAbPac RP 2.1 x mm, 2 µl/min.9 ppm 1456.34 1.2 ppm 148218.17 2.2 ppm 1483.16 pg injection 147 1 148 148 148 Mass ReSpect TM deconvolution 1. ppm 1456.6.4 ppm 148218.28 2.6 ppm 1483.9 1 148 Mass ng injection 9
Navigating Intact Protein complexity with High Mass Range (HMR) mode * 49+ MAbPac RP 5 1 Time (min) 3 7 R=17, 3 325 33 335 Denaturing MS is compatible with reverse phase HPLC separation. MAbPac SEC-1 5 1 Time (min) 25+ 3 7 * R=35, 59 593 59 59 Native MS is best for co-eluting high complexity samples like ADCs HMR mode intact analysis of Trastuzumab shows same answer in denaturing or native conditions 1
Two reasons why Native MS intact analysis is a powerful addition to a comprehensive ADC characterization stategy Cysteine-linked Lysine-linked (1) Preservation of structural non-covalent interactions (2) Increased separation in high mass range denatured native 3 7 11
Native LC-MS of Brentuximab vedotin, cysteine-linked ADC 2 4 6 8 1 Time (min) Size Exclusion LC-MS Intact Brentuximab vedotin 3 7 DAR2 DAR4 DAR6 Challenges: Optimizing desalting (Size Exclusion LC) Optimizing desolvation (MS) Can achieve balance of clean spectra and preservation of non-covalent forms Average Drug-to-Antibody Ratio (DAR) 4.7 Relative Intensity 147 12 DAR 2 x GF 1486.53 2 x GF 17.6 (-762.25 Da) 2 x GF 153355., 2 x GF 155987.81 DAR8 2 x GF 158632. 1 149 1 1 15 153 15 15 1 157 1 159 1 Mass Previously reported DAR 3.9 4.3 static nanospray Dabaene et al., Anal Chem. 14 Nov 4;86(21):1674-83
Intact analysis of Trastuzumab Emtansine lysine-linked ADC Denaturing vs. Native conditions Denatured R=17, Native R=35, 3 3 5 6 7 7 53+ 52+ 51+ + 49+ 48+ 29 29 3 3 3 26+ 25+ 59 6 61 Native MS allows greater separation of co-eluting species charge states 13
Native LC-MS in High Mass Range (HMR) mode Intact ADC Analysis of Trastuzumab Emtansine 25+ 26+ 24+ R=7, 6 6 6 zoom Size Exclusion Chromatography ADC Buffer salts can be diverted to waste 2 4 6 8 1 Time (min) 59 6 61 6 9 7 3 1 1 149 1 1 15 153 15 15 1 Mass GF/G1F DAR Mass Accuracy (ppm) Relative Abundance D 6.49 9.19 D1 21.69 34.26 D2.5 59.3 D3 6.81. D4 5.17 91.16 D5 6.69 67.42 D6 15..46 D7 6.28 24.28 D8 3.78 3.84 Trastuzumab Emtansine Lysine-linked ADC Average Drug-to-Antibody Ratio (DAR) 3.71 14
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Workflow for a middle down experiment 1gG (1kDa) IdeS Reduction Sample Prep LC (25kDa) Fd (25kDa) Fc/2 (25kDa) LC-MS Fc/2 4.43 4.77 5.26 LC 6.25 4 5 6 7 8 LC 838.28 938.79 977.78 Time (min) 166.53 1117.32 7.29 Fd 1234.88 1379.92 8.19 8.49 782.46 1466.17 1563.85 757.38 1678.26 1 1 1 1 Type of fragmentation available: Q Exactive Plus/HF: HCD Orbitrap Tribrid Fusion/Lumos: HCD, CID, ETD and EThcD (UVPD - not commercial) 16
Workflow for a middle down experiment 1gG (1kDa) IdeS Reduction Sample Prep LC (25kDa) Fd (25kDa) Fc/2 (25kDa) LC-MS Fc/2 4.43 4.77 5.26 LC 6.25 4 5 6 7 8 LC 838.28 938.79 977.78 Time (min) 166.53 1117.32 7.29 Fd 1234.88 1379.92 8.19 8.49 782.46 1466.17 1563.85 757.38 1678.26 1 1 1 1 MS/MS Due to the complexity of the spectra in top-down analysis, high resolution is required 17
Sequence coverage with HCD fragmentation on an Q Exactive Plus (Trastuzumab) 9 7 3 1 HCD MS/MS R=1, z=26 z=25 z=7 z=6 z=6 z=8 z=6 z=6 z=7 z=4 z=4 z=6 7 9 1 1 13 1 Xtract TM deconvolution Light Chain 49% residue cleavages Fd 38% residue cleavages 1 1 2 mass Fc 39% residue cleavages 18
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Trastuzumab peptide mapping Trastuzumab Accurate peptide IDs are based on high fidelity MS/MS spectra visualized in a color coded display by BioPharma Finder software.
Top 15 most abundant glycoforms measured by peptide mapping Protein Residue # Modification Category Sequence Confidence Automatic detection by BioPharma Finder Average % Abundance % Abundance Run 1 % Abundance Run 2 % Abundance Run 3 Heavy Chain 3 N3+A2GF Glycoform EEQYNSTYR 35.22 34.7 34.7 36.24 Heavy Chain 3 N3+A2G1F Glycoform EEQYNSTYR 31.77 31.22 31.53 32.55 Heavy Chain 3 N3+A1GF Glycoform EEQYNSTYR 9.31 1.83 9.53 7.59 Heavy Chain 3 N3+A2G2F Glycoform EEQYNSTYR 5.54 5. 5.56 5.58 Heavy Chain 3 N3+A2G Glycoform EEQYNSTYR 4.79 4.66 4.85 4.85 Heavy Chain 3 N3+M5 Glycoform EEQYNSTYR 4.72 4.78 4.83 4.53 Heavy Chain 3 N3+A1G1F Glycoform EEQYNSTYR 3.53 4.6 3.65 2.87 Heavy Chain 3 N3+A1G Glycoform EEQYNSTYR 2.35 2.54 2.45 2.5 Heavy Chain 3 N3+A2G1 Glycoform EEQYNSTYR 2.32 2.25 2.39 2.31 Heavy Chain 3 N3+Unglycosylated Glycoform EEQYNSTYR 1.97 1.94 2. 1.97 Heavy Chain 3 N3+A2S1G1F Glycoform EEQYNSTYR.82.76.82.88 Heavy Chain 3 N3+A1G1 Glycoform EEQYNSTYR.59.61.64.54 Heavy Chain 3 N3+M6 Glycoform EEQYNSTYR.59.56.. Heavy Chain 3 N3+A2S1GF Glycoform EEQYNSTYR..39.41.41 Heavy Chain 3 N3+M4 Glycoform EEQYNSTYR.39.38.43.35 21
Top 15 most abundant glycoforms measured by peptide mapping Automatic detection by BioPharma Finder R e l a t i v e A b u n d a n c e 9 7 3 1 9 7 3 1 1194.441 938.4489 1121.1 1275.76 1356.968 Predicted MS/MS 625.9684 366.1395 836.79 1645.6963 1937.7946 99.8472 2375.2 b 2 23.1499 (GnM) 366.1392 b 4 483.3 b 5 612.3422 b 6 741.3969 Y1-F² + 937.9435 M1² + 1121.128 M2F² + 1275.6 M3² + 1283.641 Acquired MS/MS A1G² + 1384.28 1857.8456 Y1 21.9683 1 1 1 1 2 22
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