Robert E. Murphy, Arvind Kinhikar, Joselyn Del Rosario, Ryan Preston, Mike Shields, and Nancy Levin

Transcription

1 Combined use of Immunoassay and Two-Dimensional Liquid Chromatography Mass Spectrometry for the Detection and Identification of Metabolites from Biotherapeutic Pharmacokinetic Samples Robert E. Murphy, Arvind Kinhikar, Joselyn Del Rosario, Ryan Preston, Mike Shields, and Nancy Levin CovX, Pfizer BioTherapeutics Research and Development September 15, 211

2 Agenda CovX Technology Anti-Idiotype Capture Reagent Immunoassays Developed and PK Results Two-dimensional Liquid Chromatography/MS method 2DLC/MS PK Results and Intact Protein Metabolite Identification Immunoassay and 2DLC/MS correlation Summary 1

3 CovX Technology Pharmacophore fusion onto catalytic antibody ACTIVE MOLECULE SPONTANEOUS COVALENT ASSEMBLY IN SOLUTION ANTIBODY HOMOGENEOUS FUSION PROTEIN 2

4 A closer look at the fusion reaction Intact SEC/MS characterization T (minutes after addition) Mid-reaction time point Final product (2 peptides fused) Deconvoluted Mass Spectra (149 to 155 amu) 3

5 CovX-bodies Evolution Peptides to proteins to bi-functional bioconjugates 4 Mono-functional peptide CovX-Bodies Substitute protein for peptides: protein CovX-Bodies Bi-functional peptide CovX-Bodies

6 Transforming Peptides into Drugs Antibody and peptide are both key CovX-Body design elements Peptide and linker design dramatically impacts CovX-Body properties 5 5

7 GLP-1 Model 6 GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR DPP-4 enzyme -HA EGTFTSDVSSYLEGQAAKEFIAWLVKGR Exendin-4 HGEGTFTSDLSKQMEEEAVR LFIEWLKNGGPSSGAPPPSG EGTFTSDLSKQMEEEAVRLF IEWLKNGGPSSGAPPPS

8 Anti-Idiotype Development: Specific and Universal Capture Reagent 7 Anti-Idiotype MAb Variable Constant CovX Body Fc Domain Peptide Fab Anti-Idiotype Antibody that binds to the variable domains of another antibody Develop non-blocking private anti-idiotype that is specific for CovX body Utility Generic capture or detection reagent for all CovX Bodies More sensitive assays; lower backgrounds Single format for preclinical and clinical assays Vendor independence

9 PK Immunoassays Developed 8 Total Active TMB Color TMB Color HRP 3) HRP-Antihuman IgG SA-HRP 3) Anti-peptide (N-term Specific) 2) CovX body 2) CovX body 1) Anti-Id 1) Anti-Id Block Block Block Block Block Block Block

10 Conc (ug/ml) Conc (ug/ml) Conc (ug/ml) Mouse PK Immunoassay Results GLP-1 CovX-body 5. Exendin-4 CovX-body Time (hrs) 6. Blue Total Green Active Time (hrs) Modified Exendin-4 CovX-body Time (hrs) 9

11 1 Anti-Idiotype Column Preparation -38 umoles of resin + 12 nmoles of anti-id in ph 9. buffer -react for 2 hrs, then quench with 1M tris -wash with PBS and 1M NaCl -pack 2 x 5 mm column with 15uL of resin

12 Two-Dimensional Liquid Chromatography with UV and MS Detection Step 1: Anti-Id Analysis and condition RPLC Step 2: Collect fraction minutes Step 3: RPLC/MS analysis Auto Pump 1 Pump 1 Waste Anti-Id UV UV V:P1 2uL loop RPLC Pump 2 V:P2 V:P2 Pump 2 V:P1 QTOF QTOF QTOF 11 Pump 1 UV Pump 2 11

13 First Dimension Anti-ID Chromatography with UV Detection CovX Body Std in mouse serum, UV at 28 nm Anti-Id column Protein A column 12 12

14 Conc (ug/ml) First Dimension Anti-ID Chromatography with UV Detection 7 Mouse PK Curves of Three related CovX Bodies using Anti-ID Column and UV Detection at 28 nm Time (hrs) 13 GLP-1 Exendin-4 Modified Exendin-4 13

15 Two-Dimensional Liquid Chromatography with MS Detection GLP-1 CovX-body PK samples PK at 6 hrs PK at 1 hr PK at 5 min Dosing solution 14 14

16 Two-Dimensional Liquid Chromatography with MS Detection Conc (ug/ml) Ab+GLP-1-21 Da GLP-1 CovX-body PK samples PK at 6 hrs Immunoassay Results 35. PK at 1 hr GLP-1 Total GLP-1 Active PK at 5 min Time (hrs) Ab+GLP-1 Dosing solution HAEGTFTSDVSSYLEGQA AK EFIAWLVKGR 15 15

17 Peptide Stability in DPP-4 by RPLC/MS and MS/MS CVX1227 with DPP enzyme, day June7a (5.18) Cm (246:264) Incubated with DPP-4 overnight 1.16e GLP-1 peptide -28 Da (-HA) June7a (4.973) Cm (247:261) 1 GLP-1 peptide HAEGTFTSDVSSYLEGQAAK EFIAWLVKGR m/z 16 16

18 Two-Dimensional Liquid Chromatography with MS Detection Exendin-4 CovX-body PK samples CVX1844_21 3ug/mL water 5. 31Aug9a (64.25) M1 [Ev-64554,It33] (Gs,2.,2817:4,1.,L33,R33); Cm (1211:1479) PK at 48hrs e Aug9a (64.13) M1 [Ev-6599,It33] (Gs,2.,2817:4,1.,L33,R33); Cm (1212:1483) 1 PK at 31 hrs e Aug9a (64.393) M1 [Ev-6479,It32] (Gs,2.,2817:4,1.,L33,R33); Cm (128:1477) PK at 24 hrs e Aug9a (7.742) M1 [Ev-6586,It36] (Gs,2.,2817:4,1.,L33,R33); Cm (1212:148) 1 Dosing solution e mass

19 Conc (ug/ml) 5. Ab+2xpeptides -2 and 4 Da CVX1844_21 3ug/mL water 31Aug9a (64.25) M1 [Ev-64554,It33] (Gs,2.,2817:4,1.,L33,R33); Cm (1211:1479) Two-Dimensional Liquid Chromatography with MS Detection Aug9a (64.13) M1 [Ev-6599,It33] (Gs,2.,2817:4,1.,L33,R33); Cm (1212:1483) Aug9a (64.393) M1 [Ev-6479,It32] (Gs,2.,2817:4,1.,L33,R33); Cm (128:1477) PK at 48hrs PK at 31 hrs PK at 24 hrs Aug9a (7.742) M1 [Ev-6586,It36] (Gs,2.,2817:4,1.,L33,R33); Cm (1212:148) Dosing solution -HG Exendin-4 CovX-body PK samples 9.7e ; IEWLKNGGPSSGAPPPS mass Ab+2xpeptides Immunoassay Results e4 2.29e Time (hrs) Total Active HGEGTFTSDLSKQMEEEAVR 1.1e5 LFIEWLKNGGPSSGAPPPSG EGTFTSDLSKQMEEEAVRLF 18

20 CVX225_21 PK at 6hr 5. 31Aug9a (64.126) M1 [Ev-63974,It31] (Gs,2.,2781:45,1.,L33,R33); Cm (1212:1463) PK at 48 hrs Two-Dimensional Liquid Chromatography with MS Detection Modified Exendin-4 CovX-body e Aug9a (64.38) M1 [Ev-8195,It32] (Gs,2.,2571:4,1.,L33,R33); Cm (121:1461) PK at 31 hrs ; e Aug9a (64.392) M1 [Ev-7484,It33] (Gs,2.,2667:4,1.,L33,R33); Cm (1213:1462) PK at 24 hrs ; e Aug9a (64.326) M1 [Ev-7654,It4] (Gs,2.,2667:4,1.,L33,R33); Cm (1212:1463) 1 19 PK at 6 hrs e mass

21 Two-Dimensional Liquid Chromatography with MS Detection Conc (ug/ml) CVX225_21 PK at 6hr 5. 31Aug9a (64.126) M1 [Ev-63974,It31] (Gs,2.,2781:45,1.,L33,R33); Cm (1212:1463) 1 Ab+2xpeptides -68 Da PK at 48 hrs Modified Exendin-4 CovX-body e4 Immunoassay Results 31Aug9a (64.38) M1 [Ev-8195,It32] (Gs,2.,2571:4,1.,L33,R33); Cm (121:1461) 1 PK at 31 hrs e4 Total Active Aug9a (64.392) M1 [Ev-7484,It33] (Gs,2.,2667:4,1.,L33,R33); Cm (1213:1462) 1 1 PK at 24 hrs Aug9a (64.326) M1 [Ev-7654,It4] (Gs,2.,2667:4,1.,L33,R33); Cm (1212:1463) PK at 6 hrs e4 Ab+2xpeptides e5 Time (hrs) mass

22 Conc (ug/ml) Immunoassay Results using C-terminal Specific Ab Using C-Term Specific Ab TMB Color 6. Modified Exendin-4 CovX-body Immunoassay Results SA-HRP Total Active Exendin C-term 3) Anti-peptide (C-term Specific) 2) CovX body 1) Anti-Id Time (hrs) Block Block Block Block 21 21

23 Conc (ug/ml) Conc (ug/ml) Time (hrs) Modified Exendin-4 CovX-Body -Excellent PK and half-life -Some degradation (not active) -Immunoassay active and 2 peptide additions by MS correlate Peptide Optimization Results Immunoassay and 2DLC/MS Comparison IA Total IA Active MS 1PA-21 MS 1PA GLP-1 CovX-Body 14. -Poor 12.PK and half-life 1. -Degraded quickly 8. -Immunoassay 6. (IA) active and 1 peptide 4. addition (PA) by MS correlate IA Total IA Active MS 2PA-68 MS 2PA Time (hrs)

24 Summary 23 Anti-Idiotype reagent greatly reduces background interference in immunoassay and 2DLC/MS and is a universal reagent for CovX-Body capture 2DLC/MS is a powerful technique for the automated analysis of intact proteins from serum 2DLC/MS can be used to confirm immunoassay results when using the same reagents to capture CovX-Bodies Mass spectrometry of intact proteins is useful for the identification of in-vivo metabolites Analysis of PK samples for intact biotherapeutics and in-vivo metabolites is a powerful research tool for improving drug development optimization

25 Questions/Discussion Acknowledgements: James Kerr Gary Ward Rodney Lappe CHAPTER 5: INSTRUMENTATION FOR COMPREHENSIVE MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY CHAPTER 6: METHOD DEVELOPMENT IN COMPREHENSIVE MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY CHAPTER 18 THE ANALYSIS OF SURFACTANTS BY MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY 24