Toward generic methods for monoclonal antibody quantification by LCMS/MS

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Toward generic methods for monoclonal antibody quantification by LC-MS/MS Olivier Heudi and Samuel Barteau DMPK-Bioanalytics Novartis Basel DMBA

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2 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Outline Introduction Monoclonal antibodies (mAbs) as therapeutic proteins Rationale of LC-MS/MS Methods for Therapeutic Strategies for LC-MS/MS assay development Methods development and validation for mAb quantification Protein cleavage with labeled peptide as ISTD Protein cleavage with analog protein as ISTD Protein cleavage with analog labeled protein as ISTD Assay Development Time and Throughput Conclusions

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4 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Rationale of LC-MS/MS method for mAbs quantification Pros High Sensitivity High throughput Measures active mAb Established and accepted by regulatory Cons Time consuming and expensive in assay development (>2months)* Narrow linear dynamic range Potential matrix interferences Compound and matrix specific Pros Rapid assay development (2+ weeks) Good sensitivity (Nano-molar) High selectivity (unique peptide is measured) Good throughput Wide dynamic range (> 103) Generic approach Cons No measure of mAb activity Unknown acceptance by regulatory ELISA LC-MS/MS *Target-interaction assays, currently under investigation may reduce the method development time

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5 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Strategies for LCMS/MS mAbs analysis Trypsin Digestion 1. Specificity 2. Sensitivity 3. AA composition 4. Chromatography In silico prediction of the most unique peptides LC-MS/MS peptide quantification

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6 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only In-silico prediction of the most unique peptide 39 AAAAAAAAAAAAAAAAAAAAA 59 99 AAAAAAAAAAAAAAAAAAAAAAAA 122 25 AAAAAAAAAAAAAAA 39 50 AAAAAAAAAAAA 61 An isotopic variant of one of the unique peptide was synthesized In the absolute quantification study of the anti-ILß mAb‘s PK, this peptide will be used as internal standard. Identity A bio-informatics tool was constructed based on sequence alignment with germline and mAb.

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7 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Sample preparation strategies Tryptic Digestion LC-MS/MS Albumin Depletion IEF Fractionation Others

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Assay 1 Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry Hagman C et al. Anal.Chem, (2008); 80, 1290-1296

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9 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Method workflow Digest pH 3.5 37?C, O/N Albumin serum depletion Tryptic digestion Sample clean-up Digest 37?C, O/N µLC-MS/MS LC-MS/MS 30 min ISTD addition Denaturation Reduction/Alkylation

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10 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only SDS-PAGE of albumin depleted serum

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11 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Proteins bound to albumin depletion columns

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12 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Albumin depletion and trypsin digestion Blank 2 mg/mL mAb

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13 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Preliminary validation data Good S:N (LLOQ 2 µg/mL), no background in monkey serum Linear from 2-1000 µg/mL, R2 =0.998

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Yang et al. Anal. Chem, (2007); 79, 9294 -9301 LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup Assay 2

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15 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Method workflow mAb

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16 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Protein Internal Standard Bovine fetuin Protein size: 38 KD Many tryptic peptides are unique from serums of common study species Spiked into samples at the beginning of the assay Monitor whole process of the assay, including protein digest step

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17 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only 2D-SPE Cleanup First dimension of SPE: Reversed phase 1D-SPE cleanup Second dimension of SPE: strong cation exchange 1 µg/mL somatropin in plasma 2D-SPE cleanup

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18 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only LC-MS/MS assay selectivity mAb MS/MS transition 786.71 / 886.94 IS MS/MS transition 738.09 / 879.50 Peptide ion of IS (bovine fetuin): TPIVGQPSIPGGPVR and mAb mAb Blank serum IS RT: 6.00 - 10.00 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100

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19 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Calibration and precision data Assay calibration curve Accuracy and Precision Linear range: 0.5-1000 µg/mL R2: 0.9943 Normalized by Protein

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Heudi et al. Anal Chem; (2008), 80 4200-4207 Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using stable isotopically labeled antibody as internal standard and protein cleavage isotope dilution mass spectrometry Assay 3

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22 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Method workflow

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23 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only T – G – P – F – D – T – W – G – Q – G – T – L – V – T – V – S – S – A – S – T – K 13C4, 15N-Threonine Strategies for IS labeling

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24 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only ESI-MS TOF spectra of light and heavy chains

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27 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Intra- and inter- day precision and accuracy

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28 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only mAb concentration (µg/mL) obtained by LC-MS/MS Administration route: subcutaneous Dose: 50 mg/kg Species: marmoset N/A not available

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29 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only mAb concentration (µg/mL) obtained by LC-MS/MS NA: Not Available Administration route: subcutaneous Dose: 150 mg/kg Species: marmoset

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30 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Where de we lose our mAb

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31 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Time (h) mAb concentration (µg/mL) PK study results: LC-MS/MS vs ELISA Administration : subcutaneous Dose: 150 mg/kg Species: marmoset

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32 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Serum sample IS and trypsin addition Trypsin Digest Assay development Peptide selection LC-MS/MS optimization Sample cleanup optimization Preliminary validation Work Flow --- 96-well plate format 1 week 1 week Day 1 Over night Development time 2 weeks Analysis time 1 batch 2 days Assay development time and throughput +

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33 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Conclusions The LC-MS/MS bio-assay provides a simple and feasible approach to the bioanalysis of therapeutic proteins for early R&D Demonstrated better accuracy and precision Quicker assay development Comparable throughput to small molecule 96-well plate format sample preparation Short LC-MS/MS run Towards universal LC-MS/MS approach Applicability to several species Labeled or analog protein as internal standard Sensitivity needs to be improved Validation data on several mAbs are needed

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34 | PBA_2008 | Olivier Heudi| 09.06.2008 | | Business Use Only Acknowledgements Axel Römer (A&M Bergheim, Germany)

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	olivier.heudi@novartis.com 4 Years ago.



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