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LBA and LC-MS: Advantages of Incorporating Both Platforms in Large Molecule Drug Development

Surinder Kaur, Ph. D. Director, ADC Programs & MS BioAnalytical Sciences, Genentech S. San Francisco, California

EBF Focus-Workshop

Bioanalytical Strategies for Large Molecules in Modern Drug Development: LBA and LC-MS United

21st June, 2017

Overview

IQ Consortium Large Molecule LCMS Working Group

Ligand binding, LC-MS/MS and hybrid IA-LC-MS/MS strategies

Advantages of having diverse large molecule platforms

Case study: Unusual clinical PK investigation

Summary and acknowledgements

S. Kaur, EBF Workshop, Lisbon, 6.21.17 2

IQ Consortium Large Molecule LCMS Working Group

•  Kickoff 2016 •  Mission: Best use of LC-MS for biotherapeutic bioanalysis and biotransformations •  Upcoming: Joint IQ/AAPS LCMS/LBA Workshop at AAPS, San Diego, 11/2017

AbbVie:  Jens  Sydor    Amgen:  Hongyan  Li  Bayer:  Erik  Haaf    BMS:  Jianing  Zeng  Celgene:  Ma=hew  Myers  Daiichi  Sankyo:  Joseph  Pav  Eli  Lilly:  Mike  Berna  Genentech:  Surinder  Kaur  GSK:  Jack  Kellie  Merck:  Kevin  Bateman  NovarJs:  Jim  Glick  NovarJs:  Carsten  Krantz  Pfizer:  Hendrik  Neubert  

Pierre  Fabre:  Stéphane  Couffin  Roche:  Faye  Vazvaei  Sanofi:  Olivier  Pasquier  Takeda:  Dhiman  Ghosh  Vertex:  Zhenmin  Liang  BI:  Lin-­‐zhi  Chen  Sea=le  GeneJcs:  Shawna  Hengel  UCB:  Mark  Jairaj  UCB:  Ludovicus  Staelens  

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New Product formats for Biotherapeutics: Drive Need for Complex Bioanalysis

Cyclic Peptides

Antibody-drug conjugates

Bispecific antibodies

Fusion proteins and novel macromolecule conjugates

PEGylated molecules

Small protein formats and novel protein scaffolds

More complicated biotherapeutics

Analytes are mixtures and have various catabolic products

Different in vivo PK properties

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A Role of Bioanalysis in Drug Discovery & Development: Pharmacokinetic Assays for Exposure Assessment

Exploratory PK Assays: Exposure in efficacy and Tox species to understand Therapeutic Window

Preclinical Development

Clinical Development Filing/Approval Discovery

Molecule Selection

IND Enabling Studies

Phase I Phase II Phase III Post Marketing

Validated PK Assays: Allow exposure assessment in Tox species to determine first in human dose

* IND

Validated PK Assays: Allow exposure response understanding to determine optimum dose and dose regimen

Quantitative PK assays play a key role in drug discovery & development

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Monoclonal Antibody

Growth FactorDigoxin

Analysis of Large and Small Molecule Drugs: Past Trends Molecular Structure was a Major Driver

6

Ligand Binding Assays: Fundamental Characteristics

Ligand Binding Assays: E.g. ELISA

Antigen or Anti-Hu IgG

Anti-Hu IgG HRP

Therapeutic Antibody

Protein Calibration Curve

unknown

Reagents •  One or two high quality binding reagents required

for performance

Sensitivity

•  New micro-fluidic platforms for super sensitivity:

Singulex, Quanterix

Selectivity

•  Binding specificity (2 epitopes) •  Total or free format •  Potential for interference •  Sample dilution typical •  “Free” or “total” format

Multiplexing

•  Some multiplexing possible

Assay Training

•  Established industry expertize •  Trained analyst pool available

Outsourcing •  Ready CRO capacity •  Low capital investment •  Assay maintenance

requirements S. Kaur, EBF Workshop, Lisbon, 6.21.17 7

Direct Digestion LC-MS/MS Assays: Fundamental Characteristics

Enzymatic digestion

Peptide Digest

Direct Digestion LC-MS/MS Assays

Protein Calibration Curve

Protein Concentration

Pept

ide

/ IS

ratio

unknown

MRM

LC-MS/MS

Stable isotope-labeled IS

Serum/Plasma Proteome

Reagents •  No binding reagents required •  Stable labeled peptide/protein

Sensitivity/ Identity

•  Limited sensitivity with direct digestion (1 µg/ml for mAb)

•  Catabolite ID information

Selectivity

•  Based on (i) retention time, (ii) peptide mass, (iii) peptide fragmentation

•  Low potential for interference •  No dilution (except Cmax) •  Total Format

Multiplexing

•  Highly multiplexed

Assay Training

•  New industry expertise •  Limited trained analyst pool

Outsourcing •  Few expert CROs •  Building CRO capacity a slow

process •  Initial capital investment •  Robust assay transfer & facile

assay maintenance S. Kaur, EBF Workshop, Lisbon, 6.21.17 8

Enzymatic digestion

Peptide Digest

Immunoaffinity-LC-MS/MS Assays

Protein Calibration Curve

Protein Concentration

Pept

ide

/ IS

ratio

unknown

Immuno-Enrichment

MRM

LC-MS/MS

Magnet

Protein A, Anti-Hu IgG or Antigen Capture Stable isotope-labeled IS

Combining Ligand Binding Approach with LC-MS/MS: Hybrid Immunoaffinity (IA)-LC-MS/MS

Bio$n-­‐An$  Id  or  (ECD)  

Magnet  

   

Streptavidin  Coated    Paramagne$c  Bead  

Generic  Capture  

Specific  Capture  

Therapeu$c      mAb  

Magnet  

Protein  A,  G  or  (xhuIgG)  Bead  

Endogenous  Ab  

Therapeu$c    mAb  

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Platform Reagents Sensitivity/ Identity

Selectivity/ Multiplexing

Training/Outsourcing

LBA

•  One or two high quality binding reagents

for performance

•  New micro-fluidic platforms for super sensitivity:

Singulex, Quanterix

•  Binding specificity •  Total or free format •  Potential for interference •  Sample dilution typical •  Multiplexed possible

•  Established analysts Ready CRO capacity

•  Low capital investment

•  Assay maintenance requirements

Direct Digest LC-MS/MS

•  No binding reagents required

•  Stable labeled peptide/protein

•  Limited sensitivity (1 µg/mL for mAb) •  Catabolite ID

•  Based on (i) retention time, (ii) peptide mass, (iii) peptide fragmentation

•  Low potential for interference

•  No dilution (except Cmax) •  Total Format •  Highly multiplexed

•  Few expert CROs •  Building CRO

capacity a slow process

•  Initial capital investment

•  Robust assay transfer

Hybrid IA-LC-MS/MS

•  One reagent used to enrich analyte

•  Otherwise, as above

•  pg/mL sensitivity with anti-peptide reagents

•  Otherwise, as above

•  Binding specificity •  Total or free format •  Otherwise, as above

•  New industry expertise

•  Limited analyst pool •  Otherwise, as above

Differences and Similarities for LBA, Direct Digestion LC-MS/MS and Immunoaffinity LC-MS/MS

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Considerations for choice of assay platform: LBA, LC-MS/MS, Hybrid IA-LC-MS/MS

“Free/Total” or “Generic/Specific” measurement required?

Assay sensitivity requirements

What binding reagents are available?

Exploratory or validated assay?

Is there a need to compare to historical platform data?

Resources/time available for assay development

Will the assay be outsourced?

Overall implementation and Regulatory strategy

Mix of technologies, collaboration, cross-training important

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K. Peng*, K. Xu* (*contributed equally), L. Liu, R. Hendricks, R. Delarosa, R. Erickson, N. Buda, M. Leabman, A. Song, S. Kaur, and S.K. Fischer, “Critical role of bioanalytical strategies in investigation of clinical pharmacokinetics, a Phase I case study”, MAbs 6 (6) 1500-08 (2014). doi: 10.4161/mabs.36208

Case Study: Investigating Unusual Phase I Clinical PK using ELISA and Hybrid LC-MS/MS

•  Fully human RG7652 mAb targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) for treatment of hypercholesterolemia

•  Target binding ELISA designed to quantify total RG7652

•  Phase I showed unexpected nonlinear dose normalized exposure

•  Is this an assay artifact or biology?

•  Nonlinear PK results confirmed using hybrid LC-MS/MS

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ELISA Showed Unexpected Nonlinear Phase I PK

SA-coated plate

biotin-target

Mu anti-drug mAb

HRP anti-mu IgG

Clinical  ELISA  format:  target-­‐binding  

Assay sensitivity: 0.1 ug/mL •  Non-dose proportional increase in AUC

•  Could PK non-linearity be due to assay artifacts?

Therapeutic mAb A

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Rapidly Develop Orthogonal LC-MS/MS Assay for Quantification of mAb RG7652 in Human Serum

•  Signature Peptides selected from the CDRs or variable domains

L. Liu, K. Xu, S. Kaur

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Assay Performance Using Affinity Enrichment

Protein  A  Capture   An$-­‐Id  Capture  0.05 ug/mL

Human serum background

2.0 3.0 4.0 5.0 Time, min

0

2000

4000

6000

8000

9500

1.0

Human serum background

1.5 ug/mL

1.0 2.0 3.0 4.0 5.0 0.0

5000.0

1.0e4

1.5e4

2.0e4

2.4e4

Time, min

0.05 – 125 ug/mL 1.5 – 125 ug/mL

Calibration range

Direct  Diges$on  

Inte

nsity

1.0 2.0 3.0 4.0 5.0 Time, min

0.0

5000

1.0e4

1.5e4

1.9e4

3.0 ug/mL

Human serum background

3.0 – 100 ug/mL

Trypsin

All forms of mAb

Protein  A    AnJ-­‐Id  mAb    

Affinity Enrichment

Signature peptide

FNWYVDGVEVHNAK

y9

Trypsin

Increasing Sensitivity of Total LC-MS/MS PK Assay by Affinity Enrichment of Signature Peptide

Other immunoglobulins

+ Rb anti-peptide pAb

Signature peptide

L. Liu, K. Xu, S. Kaur

•  Direct serum digestion ensures measuring the total Ab conc. •  Immuno-enrichment by anti-peptide antibody enhances assay sensitivity

LC-MS/MS with Anti-ID Capture Allows Detection of Total Concentrations of mAb RG7652 in Presence of Soluble Antigen at Clinically Relevant Levels

Endogenous antigen levels in patients range from 0.13 ug/mL to 0.59 ug/mL

Good Correlation between LC-MS/MS and ELISA PK Data Rapidly Confirmed Unusual Nonlinear PK

Summary: Based on LC-MS/MS & ELISA comparable data unusual nonlinear clinical PK results were readily confirmed

Summary

•  LBA, micro-fluidic LBA, LC-MS/MS and and IA-LC-MS/MS provide complimentary methods for protein quantification

•  Unusual large molecule PK results can be rapidly investigated using a combination of diverse platforms

•  Multiple platforms provide advantages during all stages of discovery and drug development

BioAnalytical Sciences/ ADC Group Mass Spectrometry: Keyang Xu, Luna Liu, Carl Ng, Dian Su, Jintang He Mass Spectrometry: Ola Saad, Neelima Koppada, Violet Lee, Suk-Joon Hyung Immunoassays: Randy Dere Montse Carrasco, Helen Davis, Connie Mahood, Kyu Hong, Collaborator Groups and Teams Administrative Assistant Cassie Duenas Dev Sci Management An Song, Patty Siguenza, Sara Kenkare

Acknowledgements

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