Impact of Sample Handling and Processing on Bioanalycial Outcome

IMPACT OF SAMPLE HANDLING AND

PROCESSING ON BIOANALYTICAL

OUTCOME

Haiko Pillu

Head Technical Operations

CPU Antwerpen

SAFETY & EFFICACY CLINICAL TRIAL SOLUTIONS SGS Life Science Services Biopharm Day Seminar – Antwerp, October 29, 2015

2 SGS LIFE SCIENCE SERVICES BIOPHARMA DAY – OCTOBER, 29 2015

INTRODUCTION

Data from clinical assays (biomarkers, PK, PD, and

immunogenicity) are often key outcomes from clinical trials

Implementing these endpoints in clinical trials is very :

Costly

time - and resource-consuming

Ensuring appropriate measures are taken from the sample

collection until the completion of laboratory testing is

paramount.


SAMPLES WITHIN CLINICAL TRIALS

Past Clinical studies sample

treatments

PK samples

Low amount of PD samples

Sample treatment

1 step handling

Amount of time per sample

= limited

Techniques

Centrifugation

Aliquoting

Freezing

Occasional sample preparations

with larger sample

treatments/procedures

Present Clinical studies larger/ expanded sample

treatments

PK assesments

PD assesments : more specific

treatments on sample preparation

Sample treatments

Multiple steps during treatments

sample treatments up to 5h for each

sample /batch of samples

Large amount of samples/aliquots

up to 10 different assays on 1 time

point

Specific conditions

Time consuming treatment schedules

New processes new techniques

Implementation new techniques

Need for training, specific qualifications

Running pilot studies

evolution



Past Clinical studies sample

treatments

PK samples

Low amount of PD samples

Sample treatment

1 step handling

Amount of time per sample

= limited

Techniques

Centrifugation

Aliquoting

Freezing

Occasional sample preparations

with larger sample

treatments/procedures

Present Clinical studies larger/ expanded sample

treatments

PK assesments

PD assesments : more specific

treatments on sample preparation

Sample treatments

Multiple steps during treatments

sample treatments up to 5h for each

sample /batch of samples

Large amount of samples/aliquots

up to 10 different assays on 1 time

point

Specific conditions Time consuming treatment schedules

New processes new techniques

Implementation new techniques

Need for training, specific qualifications

Running pilot studies

evolution



Lab manuals getting more specific/demanding

Specific conditions/requests

Timelines to centrifugation

Timelines after centrifugation

Timelines to storage

Storage conditions

Matrix used

Additional handling steps

….

All this makes it more challeging to maintain good

sample quality and to plan resources


WHAT’S ON THE OTHER END?

Why are all these parameters set?

How do we get to these (more complex) sample treatments?

What are the thoughts/reasoning behind it?

How is it proven to be effective?

How will information passed by from lab to site?


SETTING THE RIGHT

PARAMETERS

Source :” The effects of anticoagulant choice and sample

processing time on hematologic values of juvenile

whooping cranes” (Joan Maurer,Betsy Reichenberg, Cristin Kelly,Barry K. Hartup)

Case study describes collection blood and the outcome

with following factors

2 anti- Coagulants used (K3 EDTA and LiHE)

Slides made immediatly versus 4-6h delay

Questions

Does the anti coagulatant has an impact on results?

Does the processing time have an impact on results?

Is there any correlation of factors between anti-Coalgulant

and/or sample processing time?

1


SETTING THE RIGHT PARAMETERS

The total granulocyte concentration(heterophils and

eosinophils; H/E concentration) of each of the divided

samples



The relative (%) leukocyte counts of each of the divided

samples



In this specific case study no effect has been seen on

results depending on your anti-coagulant if an immediate

sample processing was feasible.

However when having a time delay (sample processing) this

have an impact

Validation of techniques on specific parameters is key to

check if :

• goals are reached

• final results provided are fully “appropriate and correct” to

analysis demands and results


VALIDATION –

TIME & RESOURCE

Part 1 : Method Implementation and qualification

Set up and testing of PBMC protocol for preparation and

testing

Testing of PBMC stimulation procedure

Testing labelling procedures

Lysis, fixation, permeabilization buffer selection

Acquisition and analysis templates/gating strategy

Preliminary testing of method reproducibility

Bio

analy

tical Lab

1 m

onth

2


Part 2 : In-vitro feasibility testing of the effect of

product on CD4 Th1/Th2 and Treg response

Set-up of culture conditions and stimulation (reagents,

stimulus, duration of culture, of stimulation)

Dose- and time effect of product (n= up to 6 subjects)

Test in-vitro effect on both CD4 Th1/ Th2 and Treg responses

Culture conditions in duplicate

FACS testing in duplicate

Data processing and statistical analysis

Bio

analy

tical Lab

1 m

onth

VALIDATION –

TIME & RESOURCE


Part 3 : Validation of a FACS method for analysis of

CD4 Th1/ Th2 subset (CD3, CD4, IFN-Gamma, IL-4) and

T Regulatory (CD3, CD4, CD25, FoxP3, CD127)

Sensitivity

Reproducibility:

• Between replicates;

• Between runs ;

• Between analysts;

• Between donors;

• Between two FACS systems.

Stability testing (e.g. storage of PBMC and effect of

cryopreservation, stability of reagents)

Robustness

Bio

analy

tical Lab

1 m

onth

VALIDATION –

TIME & RESOURCE


Part 4 : transition of the method to the clinical site

Providing procedure

Providing training

• Handling steps

• Conditions

• Go’s don’t go’s

Running pilot study qualification staff

• Between replicates Analyst evaluation

• Between analysts method/training validation

Reviewing results

Running clinical trial

Clin

ical S

ite

1 m

onth

VALIDATION –

TIME & RESOURCE


VALIDATION –

TIME & RESOURCE

Part 5 : Testing of CD4 Th1/ Th2 subset (CD3, CD4,

IFN-g, IL-4) and T Regulatory (CD3, CD4, CD25, FoxP3,

CD127) in clinical study samples

PBMC stimulation / 230 samples

CD4 Th1/ Th2 (CD3, CD4, IFN-Gamma, IL-4) FACS analysis/

230 samples

T Regulatory (CD3, CD4, CD25, FoxP3, CD127) FACS

analysis/ 230 samples

Bio

analy

tical Lab

X m

onth

s


OTHER EXAMPLES

Urine collections : adding of additional products as Tween

or BSA to avoid interference on tubes

Determination of Cytokines : use of a non standard blood

collection tube such as Tru Culture tubes

Sputum induction: effect of using Sputolysin during

handling on end parameters

Use of matrix : effect of presence of binding factors on

specific compounds/ parameters

Storage conditions : use of snapfreezing samples, storage

at -20°C or -70°C

…


CONCLUSION

Choose wise on parameters such as

Sampling tubes (anti coagulant)

Conditions

Sample processing times

Methods

…..

Validation of techniques is crucial

Implementation time consuming

Communication/training between bioanalytical lab and site

is key to :

get good final results

understand potential pitt falls for bioanalytical outcome


HOW DO WE ASSIST YOU IN THIS PROCESS?

Validation and choosing the right method is key for good

results

How do we achieve a good technique to deliver very good

Data when running a clinical trial?

Specialists on the bioanalytical part, Set up/validation

techniques

Specialists on the site part excecution

Running pilot studies between site and the BAN lab

• Handling of samples?

• Conditions?

• Experience

• Communication flow


PHARMACOLOGY SYNERGIES

- FROM LAB TO CLINICAL (AND VICE VERSA) -

A Clinical Pharmacology Unit with GMP pharmacy

Class II GCP laboratory

Fahmp & mec agreement

Mass Spectrometry & Immunoassays Experts

4 GLP/GMP Bioanalytical Laboratories 25 years experience

700 methods validated

31 LC-MS/MS

Services for small and large molecule testing in TK, PK and PD Online clinical samples dosing with CPUs

Discovery biomarkers translated in clinical research

Immune function testing

• Immunogenicity, flow cytometry, cytokine multiplexed ELISA

Biopharmaceuticals - Cell characterization

Metabolite profiling and mass balance studies (14C-labelled drug)


Life Science Services Pillu Haiko

Head Technical Operations

SGS Belgium NV Phone: + 32 3 217 25 77

Clinical Pharmacology Unit Fax: +32 (0) 3 217 25 81

Lange Beeldekensstraat 267 E-mail : [email protected]

2060 Antwerpen

Belgium Web : www.sgs.com/lifescience

THANK YOU FOR YOUR ATTENTION

+ 41 22 739 9548

+ 1 866 SGS 5003

+ 65 637 90 111

+ 33 1 53 78 18 79

+ 1 877 677 2667

+ 33 1 41 24 87 87


QUESTIONS ?


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Impact of Sample Handling and Processing on Bioanalycial Outcome