Small molecule bioanalytical method development and transfer: is a “plug and play” approach possible?

Luca Ferrari Clinical Pharmacology and Bioanalytical R&D, pRED Pharmaceutical Sciences, Roche Innovation Center Basel EBF 10th Open Meeting, Barcelona, Nov. 15th, 2017

Outline

• Current trend in outsourcing bioanalytical services

• Common challenges during method transfer

• Factors affecting a successful method transfer

• Case studies (including mistakes!)

• Practical recommendations

PLUG & PLAY BIOANALYTICAL METHODS

Current trend in outsourcing bioanalytical services

Pharmaceutical companies are increasingly outsourcing bioanalytical work.

New methods are commonly developed internally, used to support a few in house studies and then transferred to CROs during the early phase of drug development.

Method transfer and subsequent validation at CRO should be performed efficiently to ensure that:

• Project timelines are met.

• An efficient workflow is implemented: no risk to loose assigned slots at CROs, no extra costs.

• Data quality and integrity is not compromised.

Our approach – known challenges

Due to resource constraints, methods are used to support a limited number of non-GLP preclinical and sometimes GLP studies under exceptional circumstances.

The assays are hence partially validated when they are transferred to the CRO. Some parameters (e.g. stability) are investigated to some extent as part of assay development. Issues may arise.

In-license compounds show added complexity (previous methods can be available but their reliability may be difficult to evaluate).

New therapeutic agents often require additional and unpredictable effort to establish the method.

How can we develop reliable, easily transferable methods?

Intended use of the method – target sensitivity

PhysChem properties (e.g. lipophilicity, pKa, solubility)

Stability of the drug and metabolites

Technology available in house and at CRO

Potential non-specific binding issues, carryover

Selectivity issues (e.g. vs. co-meds, metabolites)

Key factors to consider

Intended use of the method – target sensitivity

Methods used to support in house studies typically require high sensitivity to assess the PK/PD relationship.

These can be unnecessarily sensitive methods if applied to support toxicological studies.

Sample pre-treatment, chromatography and detection should be designed to achieve the sensitivity and selectivity required whilst keeping the analytical procedure as simple as possible.

Target sensitivity – real case 1

Method developed in preclinical matrices to support an ophthalmology project.

Topical administration, need to quantify up to 24hrs post dose. LLOQ = 10pg/mL in plasma.

A sensitive method based on Liquid Liquid Extraction, uHPLC chromatography, most advanced MS/MS system was developed and qualified in house.

Method ready to transfer to CRO in support of GLP Tox studies. 10 pg/mL LLOQ

Target sensitivity – real case 1

TK studies were re-designed, original method was too sensitive.

Method range had to be rapidly adjusted to avoid unnecessary dilutions into validated range. LLE volumes adjusted.

A much simpler bioanalytical method (e.g. based on protein precipitation) could have been implemented if specific requirements had been discussed by the time the method was first developed.

100 pg/mL LLOQ

Key learning: Define sensitivity requirement prior to method development! Avoid developing unnecessarily complicated methods.

Technology in use

The technology platform used in house should mimic that available at the external laboratory. The more similar the equipment and working processes are at both labs, the greater the chance of a smooth method transfer.

• Pump configuration, void volumes and the mixing capacity of LC systems should be carefully evaluated to ensure that their chromatographic performance is identical.

• MS parameters should be optimized. Sensitivity and linearity should be re-assessed if any differences between the brand or model used at the CRO and in house exist.

Technology in use – real case 2

Method established for a lipophilic compound.

Suitable for microsampling (5µL plasma).

Method fully qualified in house.

HPLC chromatography Column: core-shell, PFP, 110A, 2.6u, 2.1 x 50 mm

Technology in use – real case 2

Method transferred to CRO.

Chromatography had to be adjusted since backpressure >300 bar and not compatible with HPLC-MS/MS system used. Flow rate lowered to 0.65 mL/min - Gradient slope increased to improve peak shape. Lower k’ observed - Matrix factor potentially affected.

Method validated, but not plug and play!

Key learning: make sure that equivalent LC parameters can be set at both labs!

Technology in use – real case 3

Clinical method developed for a basic, lipophilic compound – topical administration.

LLOQ = 10pg/mL in plasma.

Sensitive method based on Liquid Liquid Extraction, uHPLC chromatography, front-end MS/MS system developed in house.

Method transferred to CRO to support FTiH study.

10 pg/mL LLOQ

Technology in use – real case 3

Key learning: Same equipment does not necessarily mean identical methods in place – consider all factors!

Method performance ok but carryover was found.

Original method did not exhibit any.

Same uHPLC-MS/MS system, same chromatography implemented: need to troubleshoot!

Sensitivity was not ok for a proper assessment of carryover, so sample volume had to be increased to get a similar S/N ratio.

Further investigations revealed that the equipment was identical but there was a difference in the firmware installed therefore the same autosampler wash could not be implemented.

Technology in use – carryover

For particularly challenging compounds, we may spend more time to eliminate carryover than to develop the entire method!!! Our strategy at Roche is to fully evaluate carryover and to determine which portion originates in: • Autosampler • Analytical column

Autosampler and column carryover are addressed separately: in case of complex problems the contribution from both parts can make root cause analysis impossible.

Column carryover is addressed first and no residual column carryover should be present – the method can then be sent to the CRO, where the protocol for autosampler wash can then be optimized if the equipment is different.

Time factor

Timely, effective communication is key to success.

Both internal lab and CRO lab operate at full capacity, any delays can have a huge impact on the workflow.

Risk in missing time slots for method transfer and validation.

• Method details should be discussed upfront in a timely manner, possibly through lab staff interactions at both sites.

• Enough time should be allowed to evaluate method details and to ensure availability of all necessary tools ahead of method transfer.

Time factor – real case 4

Method developed for parent compound and 3 metabolites.

Chromatography was optimized to have baseline separation of all analytes.

HPLC chromatography, API5000 MS.

Method qualified in house, no issues. Transferred to CRO.

Tight timelines!

Time factor – real case 4

Analytical column proved difficult to procure, switched to another C18 column due to local unavailability of original one. Gradient adjusted.

Good separation achieved, however deviation from linearity was observed.

Same MS system, different performance? Different MS linearity due to change in chromatography? No time to investigate.

Method validated over a narrower range.

Key learnings: Timely communication! Allow enough time to procure all consumables. Parameters set in one lab do not necessarily mean optimized conditions in another one.

Method B

Method A

Knowledge of the analyte – case study 5

Method developed for a backup compound.

Method for lead compound already successfully validated – a “copy and paste” approach was adopted for method establishment.

Method based on protein precipitation with perchloric acid, direct injection of supernatant onto HPLC-MS.

Method qualified in house, used in support of >10 preclinical studies, no issues.

Method transferred to CRO.

Knowledge of the analyte – case study 5

LLOQ was raised, pre-validation failed, all low QCs negatively biased, evidence for non-specific binding during sample processing.

Method adjusted by adding organic solvent to extracts.

Why did that happen???

• Back-up molecule slightly more hydrophobic than lead compound (higher logP value). Different polypropylene tubes may have caused NSB at CRO.

• Extracts had to be stored at room temperature but the info was omitted by mistake in the bioanalytical method description.

Key learnings: Do not copy/paste, always evaluate physico- chemical properties before developing a new method.

Missing details are key to failure!

To transfer or not to transfer?

We still want to maintain our knowledge of the analytes and their potential liabilities in house as much as possible, in particular for novel therapeutic agents.

We believe that developing easily transferable methods is possible if all the key factors previously described are considered.

Effective communication and knowledge of technology and procedures at both labs is key to establish an efficient workflow.

Direct scientist-to-scientist communication (possibly including laboratories visits) is the most efficient way to prevent issues and solve them if they arise.

Should we establish methods in house or let external partners do this?

Acknowledgments

Katja Heinig

Enric Bertran

Andreas Gloge

Julian Potter

Denis Herzog

Thomas Wirz

Franz Bucheli

Daniela Fraier

Shaolian Zhou

Lisa Benincosa

Homogenize

Thank you for your attention!

Doing now what patients need next