The importance of clearly defined bioanalytical data

EBF – Focus Workshop, Lisbon, June 21-22, 2017

Roland F. StaackpRED Pharmaceutical Sciences , DMPK and Bioanalytical R&D, Roche Innovation Center Munich

Target-bindingcompetent Drug

Cata-/Metabolism?„Aggregation“?

Chemical Conversion?(„hot spots“)

Active Drug

Soluble (neutralizing) binding partners?

What is the problem? Why isn’t there “one PK”?Factors to be considered for defining a BA strategy

Total Drug

2

Ø Clear Communication* ® harmonized nomenclature**Ø Appropriate BA methods which provide clearly defined data!

*Dudal, Staack, Stoellner, Fjording , Vieser, Pascual, Brudny-Kloeppel, Golob . Bioanalysis. 2014 May;6(10):1339-48.* Lee, Kelley, King, Yang, Salimi-Moosavi, Tang, Lu, Kamerud, Ahene, Myler, Rogers.AAPS J. 2011 Mar;13(1):99-110.**Heinrich, Staack, Stubenrauch, Papadimitriou, Bioanalysis. 2015 Dec;7(24):3057-62

Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Drug

What defines the BA data/result?

ADAsol.

target

Technology?

LC-MSLBA

?

mAb<I.D.>Assay

Reagents?

Assay Procedure?

Incubation/Wash Incubation/Wash Incubation/Wash

Assay Format?

„Platform Assays“Stubenrauch K, et alJ Pharm Biomed Anal. 2013 Jan;72:208-15. J Pharm Biomed Anal. 2009 May 1;49(4):1003-8. mAb<human IgG > mAb<human IgG >

„Total“ Drug?

(pre-clinic)

„Target Capture Assays“

Target

„Active“ Drug?

3Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

What does this mean for bioanalytical scientists?

Ø Clarity what „analyte“ information is required!

Ø Question defines the technology/assay!

Ø BA method needs to be characterized!

* https://www.bioanalysis-zone.com/2016/03/11/large-molecule-analysis-by-lc-ms-survey-infographic

Do you characterize your LC-MS orhybrid LBA/LC-MS method with

regard to influences of soluble binding partner e.g. soluble target,

anti-drug antibodies (ADAs)?

Bioanalysis-Zone Survey*:

4Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 1: The role of assay format & reagents® Deamidation

Ø Analyte: therapeutic mAb with deamidation hotspots in CDR!

Ø BA assay panel to support mouse SDPK study

Cal

c. S

erum

Con

cent

ratio

n[µ

g/m

L]

0 96 192 288 384 480

0.1

1

10

100

Id2-binding-comp.Id1-binding-comp.

TotalTarget-binding-comp.

PK assay, target-binding competent/active drug

PK assay, total drug

PK assay, ID1-binding competent drug

PK assay, ID2-competent drug

5Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 1: Reagent characterization“Binding competent” – “total” – “binding incompetent”

6Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 2: The role of assay reagents® „parent drug“ vs. „active drug“

ELISA 1

ELISA 2

0.001

0.010

0.100

1.000

10.000

0 20 40 60 80 100

Plasmaconcen

tration(µg/mL)

Time(h)

ELISA1

ELISA2

PDMarker

Correlationw

ithPD

P

û

0.01

0.10

1.00

10.00

Cholesterolefflux

1:100(µ

g/mL)

„First Hint“Æ different detectability of

„chymase digest products“ by the two mAbs

mAb 1mAb 2

Difference between thetwo capture mAbs?

Catabolism?

7Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 2: Reagent characterization® by IAE-LC-HRMS

HRMS Analysis

Structure Elucidationof „active catabolites“

ImmunoaffinityExtraction

Predominant catabolite: TN-ApoA-I minus 168 Da

ØRapid loss of dipeptidealanine-proline (AP) at N-terminus

Catabolite specific PK by intact mass MS combined with ELISA data

0.1

1

10

100

1000

0 10 20 30 40 50 60 70 80

Conc

entr

atio

n(µ

g/m

l)

Time (h)

Elisa 1TN-ApoA1Catabolite 1 (-AP)Catabolite 2

Indication that„ELISA 2“ detects

TN-ApoA1via N-Terminus

PD due toactive catabolite(s)

Zell, Husser, Staack, Jordan, Richter, Schadt, Pähler, Anal Chem. 2016 Dec 6;88(23):11670-11677 8Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 3: The role of assay format & procedure® Does the assay format define the result?

Target capture FormatØ Active drug assay!?

Inactive/neutralizedforms

9Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Challenge 2:

„Active“ drug quantification in theabsence of a „drug-target binding“ interaction as a mimicry for activity

Use of „cell-based assay“ forappropriate method characterization

Presentation by Martin Schäfer14:00 -14:20

Challenge 1:

Impact of assay procedureon correctactive drug quantification

Use of „free analyte QCs“ forappropriate assay validation

Presentation by Eginhard Schick15:40 -16:00

Inactive/neutralizedforms

Case 3: The role of assay format & procedure® „active“ & „total intact“ drug using the same reagentsTarget capture Format

Ø Active drug assay!?

Total-intact drug

Acid Dissociation

Target capture FormatØActive Total drug assay!

10Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 4: The role of an appropriate BA strategy® „Total“ drug did not provide the crucial information

0 5 10 15 20 251

10

100

1000

Time [h]

T ot altest c

ompo

und

c oncen

tration[ ng/mL]

day 28

Ø Analyte: Therapeutic Peptide

Ø BA Method: “Total” drug by LC-MS

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

LC-MS

day 267 ADA +

Increased „total“ drug exposure!

…but what is the active exposure?

Presentation by Martin Schäfer14:00 -14:20

Cell-based assaysto provide the required info!

11Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 5: The role of the „capture step“® Impact of capture step on „active“ drug quantification

Drug DenaturationReductionAlkylation

Tryptic

digestion

Hybrid L

C-M

S

LC-MS/MSDetection

Signature peptide

Drug

FluorescenceDetection

LB

A (G

yros)

12Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 5: Assay procedure – „capture step“® Free & Total QCs for method evaluations

Total QCs

–50% Total –80% Total

Active/Free QCs

identical active/free drug conc.

Hybrid LCMSIncubation times: Ø „overnight“ Ø 10 min

Jordan, Ohnami, StaackManuscript in preparation

Detailed Data:

Abstract submitted to10th EBF open MeetingGregor Jordan

13Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Case 5: Assay procedure – „capture step“® Timing is critical – total QCs misleading!

Dru

g co

nc. (µg

/mL)

TotalQC1 TotalQC2

Tota

l (on

)P

Tota

l (10

min

.)P

Tota

l (on

)P

Tota

l (10

min

.)P

Free

(10

min

.)P

Free

(10

min

.)P

Free

(on)û

Free

(on)û®

shift

to to

tal

FreeQC(-50%total)

FreeQC(-80%total)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

Jordan, Ohnami, StaackManuscript in preparation

Detailed Data:

Abstract submitted to10th EBF open MeetingGregor Jordan

14Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

Ø mAb-based / heterogeneous

Case 6: The role of clearly defined methods fortechnologies comparison® „Total“ drug by LBA & LC-MS

„Total“ ELISATN-ApoA1-specificØ colorimetric assay

15Roland Staack, EBF Focus Workshop, Lisbon, June 21-21, 2017

CobasâTina-Quant ApoA1 assay

Ø Immunoturbidimetric assay

Ø pAb-based/ homogeneous

„Total“ LC-MSTN-ApoA1-specific

„clearly defined“ methods® no „technology“ bias!

„Total“ LBA – „Total“ LC-MS Comparison

„Total“ ELISATN-ApoA1-specificØ colorimetric assay

Ø mAb-based / heterogeneous

Summary & Conclusion

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Project Team

ØClarity what information is required to enable decision makingØ„harmonized“ nomenclature helpful for common understanding within the team

Bioanalytical Functions

ØBioanalytical method(s) which provide clearly defined data!

ØDetailed characterization „beyond“ routine package might be necessaryØreagent selection/characterization ® biochemical characterization

Ø„orthogonal“ methods ® LBA, LC-MS, cell-based

Ø„alternative QC concepts“ ® „free analyte QCs“

Prerequisites for an optimal bioanalytical project support:

Importance will be increasing with increasing complexity of our molecules! (® multi-domain biologics)

Acknowledgement

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Case 1Thomas EmrichAntje WalzGennady SamokhinHerbert BirnböckHubert KettenbergerJasmin Sydow

Case 2Manfred Zell Gregor JordanChristophe HusserWolfgang RichterSimone SchadtAlex PählerNicole JusitiesJürgen FingerleJean Luc MarySilke Mohl

Case 3Gregor JordanEginhard SchickUwe DahlMartin SchäferMaria Viert

Case 4

Martin Schäfer Steven ChallandEginhard SchickKatja HeinigSabine WildonDominik HainzlLutz Müller

Case 5Gregor JordanIchio Ohnami

Case 6

Nicole JustiesGregor JordanBerthold LauseckerWolfgang RichterFranziska RegennassKean Luc MaryJürgen FingerleSilke Mohl

Julia Heinrich, Apollon Papadimitriou, Lisa Benincosa, Thomas Singer

Doing now what patients need next

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