Cell-based PK assays a useful additional "tool" for large molecule bioanalysis

Martin SchäferRoche Pharma Research and Early Development, Pharmaceutical Sciences, DMPK & Bioanalytical R&D, Roche Innovation Center Munich, Germany

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisThe need to understand active exposure

Drug-Treatment

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 2

“The validity of a nonclinical safety study relies upon the demonstration of active drug exposure throughout the dosing phase of the study.”1

à Is true for clinical studies, too.

Exposure Safety

Structural mod.Drug

Ligand-Drug Complex

ADA-Drug Complex

Drug

Exposure

1 Ponce et al., Regul.Toxicol.Pharmacol. 54(2), 164-182 (2009)Active ?

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisThe technical options to assess active exposure

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 3

Ligand-binding based LC-MS Cell based

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisThe technical options to assess active exposure

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 4

Ligand-binding based LC-MS Cell based

Current gold standard foractive exposure assessment

Limitations for multidomaincompounds

Standard for total exposureassessment

Hybrid LC-MS is underdiscussion for active exposureassessment

Might gain more importancewith multidomain biologicsinvolving multiple cellularfunctions

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisCell-based PK assay when ligand binding assay is not available

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 5

Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum2

2 Wei et al., Journal of Pharmaceutical and Biomedical Analysis, 43 (2) 2007

• Proliferation of 32D cells in response to therapeutic components with erythropoietin stimulating properties

• Cell based PK assay was fully validated according to guidelines including robustness, linearity, accuracy, precision, limit of quantitation (LOQ) and specificity

• Assay was successfully implemented in a rat study to compare PK properties of two different therapeuticcomponents

• Important Factors influencing the Performance:

• Cell density, subculturing frequency and influence of bleeding frequency on plasma quality

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisCell-based PK assay as a complementary tool to characterize a ligand binding assay3

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 6

3 Staack, Jordan, Viert, Schäfer, Papadimitriou, Heinrich. Bioanalysis; 2015 (24):3097-106

TherapeuticProtein

Toxindomaine

Targetingdomaine

• Since no defined binding target as mimickry for the toxin activity is available, there is no simple surrogate measure of drug activity that might be monitored by a LBA

• A cell based PK assay that quantifies cell viability and can, therefore, be described as a potency assay was used as the true mimicry of activity.

Cell-basedPK Assay

LBA-basedPK Assay

SubstrateHPPA

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisCell-based PK assay as a complementary tool to characterize a ligand-binding assay3

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 7

• Effect of human ADA-positive samples on recovery

• Cell-based PK Assay was needed to confirm correctness of LBA PK assay to quantify active drug, especially as in this case where an activity surrogate is lacking (eg. toxin)

• Challenges of the cell-based PK Assay:

- throughput

- variabilty

- short incubation times (= reduced risk to overestimate active drug)

3 Staack, Jordan, Viert, Schäfer, Papadimitriou, Heinrich. Bioanalysis; 2015 (24):3097-106

Multiple R-squared: 0.87

F-statistic: 186.6 on 1 and 28 DF, p-value: 6.605e-14

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Recovery Cell-Based PK Assay

Rec

over

y LB

A-B

ased

PK

Ass

ay

-2 -1 0 1 2

-0.1

0-0

.05

0.00

0.05

0.10

Normal Q-Q Plot

Theoretical Quantiles

Sam

ple

Qua

ntile

s

LBA-basedPK Assay

Cell-basedPK Assay

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisCell-based Ex-vivo Potency Assay to semi-quantitatively assess active drug exposure

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 8

• Compound: Peptide therapeutic that leads to bodyweight loss

• Individual Cynomolgus Monkey Plasma PK Profile Following 0.05 mg/kg s.c. Administration

• At day 267 drug clearance tends to be decreased & a higher initial starting concentration is observed

• Does this increase in apparent exposure of drug translate into higher drug activity - efficacy/PD

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisCell-based Ex-vivo Potency Assay to semi-quantitatively assess active drug exposure

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 9

• Was required in a pre-clinical study to complement total drug quantification by LC-MS/MS and an unsteady PD marker to assess active drug exposure2

Post-dose=131 ng/mL

Pre-dose+ 131 ng/mL test compound

2. Dilution to 2 nM1. Spiking of pre-dose 3. Cell-based potency assay 4. Quantification of potency

GS Gi

AC

AMP cAMPATP

340 nm 620 nm 665 nm

cAMP

cAMP

cAMP

cAMPFRET

+

+ -

2 Schäfer, Challand, Schick, Bader, Hainzl, Heinig, Müller, Papadimitriou, Heinrich.Bioanalysis; 2015 (24):3063-72

Example

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisCell-based Ex-vivo Potency Assay to semi-quantitatively assess active drug exposure

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 10

• Despite an apparent increased total drug (LC-MS/MS) level and reduced clearance, a PD marker (Loss of Bodyweight=BW) indicated a loss of efficacy that could be confirmed by a measured loss of cellular drug activity (cAMP) in the cell-based Ex vivo Potency Assay

• Quantification of total drug levels by LC-MS/MS may provide only limited information on drug activity in vivo

• The Ex vivo Potency Assay enables relative quantification of drug activity

LC-MS/MSPD & cellular activity

day 267 = ADA +ve cAMP

Bodyweight

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisWhere cell-based PK assays offer more information

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 11

Effect of

ADA-Formation

Immune-cellTumor

Kill

Bispec. Biologic

ADA 1

ADA 2

Immune-cellTumor

Kill

Lysis

Mode of Action

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisWhere cell-based PK assays offer more information

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 12

Ligand binding PK assay

Effect of ADA-Formation

Cell based PK assay(s)

ADA 1

ADA 2

ADA 1&2

Read-out: Tumor cell lysis Immune-cell act.

Loss of Assay Signal

Loss of Assay Signal

Loss of Assay Signal

No tumor cell lysis

No tumor cell lysyis

No tumor cell lysis

Immune-cell act.

ADA 2

ADA 1

Red. Immune-cell act.

No Immune-cell act.

No ADAs Assay Signal Tumor cell lysis No Immune-cell act.

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisBenefits and challenges of cell based PK assays

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 13

Benefits

• Assays that are based on a cellular response offer the advantage to mimic the desired effect of the therapeutic protein more closely than any other in vitro method

Challenges

• Effect by non-therapeutic protein components ofthe study sample on the cellular response (eg. chemotherapeutics effect on cellular proliferation)

• Effect by cell-cycle

• Limited dynamic range, Assay throughput andsensitivity

• Incubation time

• Operator skills/expertise

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisConclusion

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 14

Ulrich Brinkmann and Roland E. Kontermann. mAbs Vol. 9 , Iss. 2,2017

• So far ligand-binding based PK assays were seen as the gold standard for assessing exposure

• In many cases cell based PK assays have been proven as useful tools to significantly support and/or extend the data package based on Ligand-binding or LC-MS/MS-based compound quantification

• More complex multidomain biologics will increase the need for cell based PK assays as details of MoA (especially in context of enzymatic activity) that drive efficacy will get nearly impossible to be reflected in classical ligand binding based PK assays

Cell-based PK assays a useful additional "tool" for large molecule bioanalysisAcknowledgement

pRED Pharmaceutical Sciences

All colleagues in pRED LMBARoland StaackKay StubenrauchHerbert BirnboeckJulia HeinrichApollon PapadimitriouLisa BenincosaThomas Singer

EBF - Focus Workshop 21-22 June 2017, Lisbon

Cell-based PK assays a useful additional "tool" for large molecule bioanalysis – Martin Schaefer Roche 15

Doing now what patients need next