Cell-based PK assays a useful additional tool for large molecule … · 2018-06-13 · Cell-based...
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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