update from the ebf liquidupdate from the ebf liquid ...li idliqu id m iliicrosamp ling. bi l...

Update from the EBF LiquidUpdate from the EBF Liquid Microsampling Consortium

Presenter: Stephen Whiteon behalf of EBF

thEBF 8th Open Symposium19th Nov 2015

BarcelonaBarcelona

http://www.europeanbioanalysisforum.eu

Consortium MembershipConsortium Membership

1 AstraZeneca1. AstraZeneca2. Charles River Laboratories3. Covance

A consortium is an association of two or more individuals, companies, organizations or governments (or3. Covance

4. Envigo5. GlaxoSmithKline

organizations or governments (or any combination of these entities) with the objective of participating in a common activity or pooling their

6. H Lundbeck A/S7. Janssen R&D8 LGC

a common activity or pooling their resources for achieving a common goal.

8. LGC9. PRA 10 QPS Netherlands B V

Consortium is a Latin word, meaning "partnership", "association" 10. QPS Netherlands B.V

11. Sanofi12. TNO

or "society" and derives from consors 'partner', itself from con-'together' and sors 'fate', meaning g , gowner of means or comrade.

http://www.europeanbioanalysisforum.eu 2

EBF & Liquid Microsamplingq p gDBS Focus

Workshop @ EBF pOS (2013)

EBF LMS Consortium d fi i t t ddefining strategy and

designing experiments

Perform experiments

2014 2015 2016

2 Publications from EBF

Oct‐2013: Start of Liquid Positioning or

LMS Consortium

Oct 2013: Start of Liquid Micro sampling (LMS)

consortium

Positioning or recommendation paper

from EBF on LMS


EBF LMS Consortium Discussion

3 main points identified for focus and further3 main points identified for focus and further discussion:

Impact on assay validation, additional experiments may be required to represent samples and alleviate concerns Matrix stability in small volumes / capillaries Matrix stability of diluted samples Whole blood stability in small volumes / capillaries

Sample manipulation – to investigatep p g Sample homogeneity – to investigate


Recent Achievments & ActivitiesRecent Achievments & Activities

2013 C ti W k h 2013 Consortium Workshop

European Bioanalysis Forum continued plans to support li id i li Bi l i (2014) V l 6 N 14liquid microsampling. Bioanalysis (2014) Vol. 6, No. 14, Pages 1897-1900.

E Bi l i F R fl ti bi l ti l European Bioanalysis Forum - Reflection on bioanalyticalassay requirements used to support Liquid Microsampling.Bioanalysis (2014) Vol. 6, No. 19, Pages 2581-2586y ( ) , , g

Two experimental protocols finalised

“Manipulation of Small Sample Volumes”– “Manipulation of Small Sample Volumes”

– “Homogeneity of Small Sample Volumes”

– Execution of these work plans is in progress


Highlights from 2014 “philosophical” paperS l M i l tiSample Manipulation

C t EBF LMS ti thi kiCurrent EBF LMS consortium thinking:

Sample integrity throughout its lifetime (collection, storage and extraction) should be supported by experiments performed duringextraction) should be supported by experiments performed during assay development / validation

Therefore its not crucial whether diluent added on collection or Therefore its not crucial whether diluent added on collection or analysis

Recommend against introducing new semantics such as primary g g p yand secondary sample

Ensure experimental evidence validates your approach

But also think about what is practical

Consortium will perform experiments to aid understanding Consortium will perform experiments to aid understanding

European Bioanalysis Forum - Reflection on bioanalytical assay requirements used to support Liquid Mi li Bi l i (2014) V l 6 N 19 P 2581 2586


Microsampling. Bioanalysis (2014) Vol. 6, No. 19, Pages 2581-2586

Highlights from 2014 “philosophical” paperg g p p p pSample Homogeneity

C t EBF LMS ti thi kiCurrent EBF LMS consortium thinking: It is not yet known if homogeneity is a real or perceived

concernconcern

Targeted experiments will give us a better insight on this topictopic

Consortium to generate experimental data

However: Experimental evidence should validate your approach

QCs prepared in same volume and handled in the same way l ill hi hli ht ias samples will highlight issues

European Bioanalysis Forum - Reflection on bioanalytical assay requirements used to support Liquid Mi li Bi l i (2014) V l 6 N 19 P 2581 2586


Microsampling. Bioanalysis (2014) Vol. 6, No. 19, Pages 2581-2586

Experimental ProtocolsExperimental Protocols


Small Volume Handling ProtocolSmall Volume Handling ProtocolPipettes CapillariesPipettes

Evaluation of positive/air displacement pipettes and fixed /variable pipettes are

Capillaries

Evaluation of end-to-end capillaries from 2 different manufacturers arepipettes and fixed /variable pipettes are

to be performed, with each company investigating 2 pipettes.

from 2 different manufacturers are to be performed by each company.

Volumes to be evaluated are 1, 2, 4Volumes evaluated will be 1, 2, 4 & 8 µL (n=6 replicates per pipette for each volume).

Volumes to be evaluated are 1, 2, 4 & 8 µL for Drummond and for Vitrex capillaries (n=6 replicates per capillary type for each volume).) p y yp )

All sites will perform the experiments using water (control) and plasma.p p g ( ) p

The experiments are to be performed by 2 operators per site (an experienced daily pipette/capillary user and a trained, but infrequent user).


Small Volume Handling ProtocolEmerging Data (lab 1)Emerging Data (lab 1)

Lab 1 Accuracy Data ‐Water Lab 1 Accuracy Data ‐ Plasma

105 0110.0115.0120.0125.0

(%)

105 0110.0115.0120.0125.0

(%)

80 085.090.095.0100.0105.0

Accuracy

80 085.090.095.0100.0105.0

Accuracy

75.080.0

7 uL (pipette)8 uL (capillary)

4 uL 2 uL 1 uL75.080.0


4 uL 2 uL 1 uL

Lab 1 Precision Data ‐Water Lab 1 Precision Data ‐ Plasma

15.0%

20.0%

Lab 1 Precision Data Water

15.0%

20.0%

Lab 1 Precision Data Plasma

5.0%

10.0%

CV (%

)

5.0%

10.0%CV

(%)

10

0.0%7 uL (pipette)8 uL (capillary)

4 uL 2 uL 1 uL0.0%


4 uL 2 uL 1 uL

Small Volume Handling ProtocolEmerging Data (lab 2)Emerging Data (lab 2)

Lab 2 Accuracy Data ‐Water Lab 2 Accuracy Data ‐ Plasma

105 0

115.0

125.0

(%)

115.0

125.0

%)

85.0

95.0

105.0

Accuracy (

85.0

95.0

105.0

Accuracy (%

75.08 uL 4 uL 2 uL 1 uL

75.08 uL 4 uL 2 uL 1 uL

20.0%

Lab 2 Precision Data ‐Water20.0%

Lab 2 Precision Data ‐ Plasma

15.0%

20.0%

)

15.0%

%)

5.0%

10.0%

CV (%

)

5.0%

10.0%CV

(%

11

0.0%8 uL 4 uL 2 uL 1 uL

0.0%8 uL 4 uL 2 uL 1 uL

Homogeneity ProtocolThis experiment has been designed to understand how samples derived in thisunderstand how samples derived in this manner using two commonly employed approaches (Vitrex micro hematocritapproaches (Vitrex micro hematocrittube and Drummond plasma capillaries) may differ from those obtained bymay differ from those obtained by conventional processes (controls).

Specifically, the experiment will p y pdemonstrate whether plasma derived by centrifugation of a capillary is homogenous and therefore, whether sub-aliquots taken from the sample are


equivalent.

Homogeneity ProtocolPlasma transferred to 2 x 4 µL capillaries for

analysis

Capillary broken to give plasma portion

(10 – 15 µL) y( µ )

Plasma Homogeneity Tests

Vitrex 32 µLVitrex 32 µL micro‐hematocrit

tubeCompare

Plasma separated

Compare concentration data

Plasma (25‐35 µL) pushed out of capillary into separate tube

Plasma aliquots (5 µL) pipetted for analysis

Plasma Homogeneity Tests

Drummond 70 µL .....

Plasma

µplasma capillary

Compare concentration data


Plasma separated

concentration data

Homogeneity ProtocolVitrex CapillariesVitrex Capillaries Spike blood with compound

Fill 18 x Vitrex caps (32 uL)

6 caps frozencaps (32 uL)

with blood, plug, centrifuge

& break

12 x 8 µL caps filled with plasma

6 caps frozen 6 caps diluted before freezing & break

2 x 4 µL cap filled with plasma from

Cap1

6 caps frozen

with plasma from each of 6 Vitrex caps

26 caps frozen

Cap2 Above is performed per concentration and per analyte. Total of 72 blood capillaries, 48 x 8 µL plasma capillaries and 48 x 4 µL plasma capillaries


µL plasma capillaries and 48 x 4 µL plasma capillaries.

Homogeneity ProtocolDrummond CapillariesDrummond Capillaries

Below is performed per concentration (2), per analyte (2). Total 12 Drummond devices – 3 per analyte and per concentration

d

Plasma collected into 500 L Micre t be and fro enFill 3 Drummond

caps (70 uL) with blood & centrifuge

µL Micrewtube and frozen Plasma collected into 1100 µL Micronic and frozeng

(per concentration, per analyte)

3 collections of ca 35 µL

µL Micronic and frozen Plasma collected into one tube and diluted 10 foldof ca 35 µL

plasma tube and diluted 10‐fold

For each of the above take 6 replicates of 5 µL for analysis


Caps= capillaries

Test Compounds for Homogeneity ProtocolLC-MS (21) LBA(3) ICP-MS (3)

AtenololpKa= 9 43 MW=266 336

AtropinelogP = 1 8

BuprenorphineMW=467 64

Rituximab CarboplatinpKa 9.43 , MW 266.336, PPB~6-16%, logP=0.0965

logP 1.8pKa = 9.4MW = 289

MW 467.64, pKa=8.31/9.37, LogD=-0.27protein binding 96%

CabazitaxelMW=835 938 LogD=3 3

Clobazam DiazepamlogP = 2 6

Trastuzumab(Herceptin)

CisplatinMW 835.938, LogD 3.3, pKa=12.0&12.5, PB=90-97%

logP 2.6pKa = 2.9 MW = 284

(Herceptin)

DiclofenacpKa=4 15 logP=4 98 Mol

Donepezil EPA(endogenous fatty acid)

one from:bevacizumab

OxaliplatinpKa 4.15, logP 4.98, Mol wt=296.148, PB >99%

(endogenous fatty acid) Log D 4, MW 302

bevacizumabcetuximabadalimumabtocilizumab

Fasiglifam Iohexol Methyl BlueFasiglifamMW=524.638, LogD=2.52

IohexolpKa n.a., log P -3.1, MW 821, PPB <5%, B/P ratio 0.63

Methyl Blue Log D -0.6, Quaternary amine MW 284

Midazolam Norbuprenorphine OmeprazoleMidazolam, MW 325.0782, logP 4.13, pKb1=5.61 pKb2=4.69

NorbuprenorphineMW=413.55, pKa=9.14/9.77, LogD=-1.9

OmeprazoleMW345.1147, logP 1.45, pKa1=13.72 pKb1=6.68 pKb2=4.04

Paracetamol Rosuvastatin TolterodineParacetamolpKa=9.5, logP=0.49, Mol wt=151.163, PB=10-25%

RosuvastatinMW=481.54, pKa=4.25, LogD=0.89/-2.86protein binding 88%

TolterodinepKa 9.7, Log P 5.6,MW 325

Trastuzumab Verapamil Warfarin


TrastuzumabpI 8.5, hydrophobicity -0.4, MW 145531, PPB n.a. B/P ratio 0.40

VerapamilpKa 8.92, Log P 3.79, MW 454

WarfarinpKa= 4.5, MW=308.33, PPB~99%, logP=3.42

Emerging Data L b 1 Vi C ill iLab 1 - Vitrex Capillaries

l ll Mid l Vit C ill i20.0%

Omeprazole ‐ Vitrex Capillaries20.0%

Midazolam ‐ Vitrex Capillaries

15.0%

tage

15.0%

tage

5.0%

10.0%

Percen

5.0%

10.0%

Percen

t

0.0% 0.0%

% diff from Control CV (%) % diff from Control CV (%)


Emerging Data L b 1 D d C ill iLab 1 - Drummond CapillariesOmeprazole Drummond Capillaries Mid l D d C ill i

15.0%

20.0%

Omeprazole ‐ Drummond Capillaries

15.0%

20.0%

Midazolam ‐ Drummond Capillaries

5.0%

10.0%

tage 5.0%

10.0%

age

‐10.0%

‐5.0%

0.0%

Percen

t

10 0%

‐5.0%

0.0%

Percen

ta

‐20.0%

‐15.0%

‐20.0%

‐15.0%

‐10.0%

% diff from Control CV (%)% diff from Control CV (%)


Next stepsNext steps

Complete experimental work across each member Complete experimental work across each member laboratory

Consolidate and interrogate data

Share findings within the EBF community Share findings within the EBF community

Share findings with wider community via an EBF C ti bli tiConsortium publication


Closing RemarksClosing Remarks

Await further experimental data before drawing Await further experimental data before drawing conclusions on small volume manipulation & homogeneityhomogeneity

Appropriate experimental evidence during assay validation and production use will validate the sampling technique used

QCs prepared in same volume and handled in the same way as samples will highlight any issues (orsame way as samples will highlight any issues (or lack of)


Acknowledgementsg

Iain Love Charles River Karina Joyce - LGCIain Love – Charles River

Tim Sangster – Charles River

N il S Gl S ithKli

Karina Joyce - LGC

Valerie Boutet - Sanofi

Katrin Schroeter SanofiNeil Spooner – GlaxoSmithKline

Nancy Papastefanou - GlaxoSmithKline

Li Dill J R&D

Katrin Schroeter – Sanofi

Nico van de Merbel – PRA

K W d A t ZLieve Dillen – Janssen R&D

Philip Timmerman – Janssen R&D

Karen Woods - Astra Zeneca

Glen Hawthorne – Astra Zeneca

Morten Anders Kall – Lundbeck

Morten Rohde - Lundbeck

Marion Kraneborg - QPS

Elwin. Verheij – TNO

Nick Gray – Covance

Stuart McDougall – Covance

Graeme Smith – Envigo

Pratap Davuluri- Envigo

Zoe Cobb – LGC


update from the ebf liquidupdate from the ebf liquid ...li idliqu id m iliicrosamp ling. bi l...

Documents