Development of a hybrid assay for the quantification of a mab drug in human serum at the low ng/ml levels

Transcription

1 Development of a hybrid assay for the quantification of a mab drug in human serum at the low ng/ml levels EBF Open Symposium November 2018 marco.michi@aptuit.com

2 Presentation Topics Current scenario and sponsor requirements Process to identify and test signature peptide Interference issue and investigation Immunocapture-Mass Spectrometry Hybrid assay development Ion mobility HRMS vs triple quad Conclusions PAGE 1

3 The Current Scenario and Requirements Monoclinal antiblody under clinical investigation in a Phase I trial Currently used ELISA assay has an LLQ of 40 ng/ml ELISA method sensitivity is impaired by inter-subjects specificity issue The main requirement is to develop an assay with an LLQ of 10 ng/ml in human serum PAGE 2

4 Process to identify and test signature peptide Current scenario and sponsor requirements Process to identify and test signature peptide Interference issue and investigation Immunocapture-Mass Spectrometry Hybrid assay development Ion mobility HRMS vs Triple quad Conclusion PAGE 3

5 In-Silico Digestion Software used: Skyline and UNIFI Background proteome: Homo Sapiens uniprot-proteome Only three peptides from heavy chain and two from light chain were identified as suitable for quantification The following rules have been applied for the peptide selection - No Methionine or Histidine susceptible to oxidation - No Cysteine can form S-S - No N terminal Glutamic or C terminal Glutamine cyclization - No Asparagine-Glycine or Aspartic-Glycine susceptible to deamidation - No glycosylation site - No ragged ends (RR, KK, RK, KR) - 5 to 25 AA PAGE 4

6 In-Vitro Digestion Candidate peptide selection process Chromatography transfer and optimization Peptide mapping (MSe) Candidate Peptide Identification Optimization for: Chromatography Mass spec conditions Working solution and spiked plasma samples Digestion protocol PAGE 5

7 Transfer to triple quad Only one signature peptide (LLI) demonstrate an adequate sensitivity even if about 4 times less than ALPAPIEK (general signature peptide for human mab) All the other candidate peptides showed a much lower sensitivity even after thorough method optimization PAGE 6

8 Interference issue and investigation Current scenario and sponsor requirements Process to identify and test signature peptide Interference issue and investigation Immunocapture-Mass Spectrometry Hybrid assay development Ion mobility HRMS vs Triple quad Conclusion PAGE 7

9 Interference Issue Human Serum Analysis Interference observe in all matrix lots of plasma and serum High sensitivity drop when moving from working solution to plasma samples Retention time for LLI Blank 5000 ng/ml PAGE 8

10 Interference investigation SPE and pellet digestion have been tested to remove ion suppression and interference Sensitivity was generally increased using pellet digestion SPE didn t improve the sensibility The interference pattern didn t improve Direct digest (reference) Pellet digestion 3 x sensitivity gain Pellet digestion + SPE 3 x sensitivity gain PAGE 9

11 Phospholipids and Albumin removal Signal monitored: Albumin abundance was monitored following peptides AEFAEVSK, QTALVELVK and LVNEVTEFAK Phospholipids monitored : Phosphatidylcholine (m/z = 496, m/z = 524) and Lysophosphatidylcholine (m/z = 758, m/z = 786) SPE MCX used to purify digested samples Results: Monitored Phospholipids and Albumin peptides were not eluted at the mab signature peptide retention time Pellet digestion using IPA and TCA did reduce the albumin content of more than 70% (non linearity detector region) Phospholipids depletion using mixed mode strong cation exchange SPE was between 50% to 80% PAGE 10

12 Interference in-silico investigation LLI peptide is a doubly charged peptide Modified peptides with a Dmass ~ 0.7x2 amu may interfere on Q1 - Same consideration for Q3 (for triple quad) Interfering modified peptides should have similar retention time (similar Hydrophobicity Index) Interfering modified peptides must belong to human proteome or to trypsin - As no interference were observed in PBS digested samples, trypsin interference was not considered Skyline - SRM collider tool, was used to investigate potential interference PAGE 11

13 Interference in-silico investigation Q1 LLI potential interfering peptides n=472 PAGE 12

14 Interference in-silico investigation Q1 Q3 LLI potential interfering peptides n=52 In particular 3 peptides have only 3 different AAs when compared to LLI. - This may explain why 3 interference peaks were observed PAGE 13

15 Interference Investigation using IMS-qTof LLI peptide PBS QC HDMSe experiment to identify drift time for MEN1112 signature peptide HSMRM method using observed drift was applied to: - QC prepared in PBS - QC prepared in human serum PBS Serum QC - Control human serum NO IMPROVEMENTS have been observed Serum Blank PAGE 14

16 How to fix the Issue? BLAST search on the identified potential interfering peptides has been performed Peptides belong to Immunoglobulin Kappa Variable chain This make impossible their isolation by immunoprecipitation using protein A, G or L Alternative signature peptides are not available Drug Anti-id immunoprecipitation is considered the only possibility Anti-id drug PAGE 15

17 Hybrid assay development Current scenario and sponsor requirements Process to identify and test signature peptide Interference issue and investigation Immunocapture-Mass Spectrometry Hybrid assay development Ion mobility HRMS vs Triple quad Conclusion PAGE 16

18 Immunoprecipitation Two different capturing agent have been tested - Natural antigen - Drug anti-id antibody Magnetic beads immune-captured samples from PBS, human serum and diluted human serum (50%) for: - Blanks - LLQ - HLQ Samples have been monitored for two LLI peptide transitions: IgG generic peptides have also been monitored for: - ALPAPIEK - GPSVFPLAPSSK PAGE 17

19 Immuno-precipitated Samples PBS / Human Serum 50% data The sensitivity in matrix and PBS samples is the same Immuno 7.5ug PBS 2uL Immuno 7.5ug HUSE/PBS 2uL PAGE 18

20 Immuno-precipitated Samples LLQ vs Blank Interference issue has been fixed using hybrid assay 10 ng/ml Human Serum Blank Human Serum PAGE 19

21 Ion mobility HRMS vs Triple quad Current scenario and sponsor requirements Process to identify and test signature peptide Interference issue and investigation Immunocapture-Mass Spectrometry Hybrid assay development Ion mobility HRMS vs Triple quad Conclusion PAGE 20

22 VION Scheme PAGE 21

23 Tof-Pseudo MRM vs. Tof-MS Scan Settings: Same scan time for both channel MS scan Same extraction tolerance (12ppm) centred on observed m/z Results Pseudo MRM mode signal is about 8 times higher than MS scan Pseudo MRM mode noise is similar to MS mode noise Pseudo MRM PAGE 22

24 Ion Mobility Tof-MS Scan vs. Tof-MS Scan Settings: Same scan time for both HD MS scan Same extraction tolerance (12ppm) centred on observed m/z Results Ion mobility MS scan signal is about 40% higher than MS scan Ion mobility MS scan noise is 50% less of the MS scan mode noise MS scan PAGE 23

25 Ion Mobility Tof-Pseudo MRM vs. Tof- Pseudo MRM Settings: Same scan time for both HS PSEUDO MRM Same extraction tolerance (12ppm) centred on observed m/z Data derived from the same injection Results Ion mobility Pseudo MRM signal is more than double than Pseudo MRM Ion mobility Pseudo MRM noise is 50% less of the Pseudo MRM noise PSEUDO MRM PAGE 24

26 Ion Mobility Tof MRM vs. Tof MRM Settings: Q1 high resolution HS MRM Low Ion Mobility resolution Same extraction tolerance (12ppm) Results Ion mobility MRM signal is higher than MRM signal Both noises are very low (few counts) Average HSMRM noise is about 40% lower then MRM noise MRM PAGE 25

27 Ion Mobility Tof Pseudo MRM vs. Ion Mobility Tof MRM Settings: Q1 high resolution HS MRM Low Ion Mobility resolution Same extraction tolerance (12ppm) Results Ion mobility Pseudo MRM signal is more than three times higher the ion mobility MRM signal Ion mobility Pseudo MRM noise is about three times higher the ion mobility MRM noise HS Pseudo MRM The two S/N are similar but Pseudo MRM has an higher intensity PAGE 26

28 What is the best IMS-QToF acquisition mode? Results discussion: Ion mobility MS scan S/N is about 80% higher than MS scan S/N Pseudo MRM S/N is more than 8 times higher than MS scan S/N Ion mobility pseudo MRM S/N is about 5 times higher than pseudo MRM S/N Ion mobility pseudo MRM S/N is similar to Ion mobility MRM S/N - Ion mobility pseudo MRM signal 3 times the ion mobility MRM signal S/N gain factor HS MRM MRM MS Scan HS Pseudo MRM PAGE 27

29 Vion vs Xevo TQS Comparison of ion mobility pseudo MRM vs triple quad MRM - LLQ Sample at 10ng/mL - 10µL injection volume - LLQ CV% = 9.7% without the use of an internal standard IMS-qTof HS Pseudo MRM S/N=16 Triple quad MRM S/N=18 PAGE 28

30 Conclusions Interference issue has been fixed by using an hybrid assay with anti-id immuno-capture step The final sensitivity allow the quantification at concentrations below the 10ng/mL level required CV% obtained without the use of a labelled mab is already good (<10%) The developed Hybrid assay has a sensitivity much greater than the ELISA assay Ion mobility did improve significantly the IMS-qToF sensitivity in all acquisition modes Vion S/N was similar to that of Xevo TQS (very high performance triple quad) Labelled mab is under production and will be used in the method validation PAGE 29

31 Acknowledgement Menarini Team Giuseppe Raucci Rossana Bugianesi Paolo Mazzei Angela Capriati Andrea Pellacani PAGE 30

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