Ex Vivo Sample Preparation and Analysis Techniques

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As drug discovery programs evolve, the analysis of efficacy, pharmacokinetics, and pharmacodynamics becomes critical for lead optimization. Here Director of ADME / DMPK Laie Abello and Senior Bioscience Research Scientist, Rachel Doidge share their thoughts on streamlining this process.

They outline the preparation of animal study samples, xenograft models, and patient samples for analysis within our integrated departments. The key benefit of this setup is that it allows monitoring of signaling pathway activity and protein biomarker levels in Bioscience while evaluating compound concentrations in DMPK. The result is a powerful diagnostic tool to assess therapeutic effects.

Ex Vivo Sample Preparation

Laie explains how she goes about sample preparation.

I begin with the Precellys® Evolution Touch tissue homogenizer, which we use because of its efficiency and versatility for sample prep. This homogenizer uses glass or metal beads to grind, lyse, or homogenize a wide range of tissues, accommodating various tube sizes and even the most challenging samples. When coupled with the Cryolyos® Evolution, which cools samples down to 0˚C, it becomes ideal for biomarker analysis and the preparation of sensitive samples.

Within ADME / DMPK samples are homogenized in a buffer such as PBS and analyzed against a matrix-matched standard curve to determine compound concentration.  A Thermo Scientific™ Exploris 120 Quadrupole Orbitrap mass spectrometer (MS) with a Thermo Scientific Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) system is used for this. 

Ex vivo Sample Preparation

In conjunction with biology the same tissue can be prepared in lysis buffer ready for biomarker analysis. Here we show how tissue samples analyzed with Simple Western™ Jess immunoblotting to evaluate target protein levels. 

Samples are first homogenized, then a fixed quantity of protein, determined by BCA, is loaded and analyzed with specific antibodies. We have extensive experience preparing various tissue types, including liver, lungs, brain, muscle, kidney, and tumors, which are subsequently analyzed in immunoblotting assays.

I find that this integrated approach ensures precise and comprehensive analysis of both protein targets and compound concentrations.

Analysis of Phospho-Proteins in Glucose-Challenged Rats

Rachel continues by illustrating how biomarker analysis plays an important role. 

Determining whether a new drug reaches the relevant tissue at a therapeutic dose and achieves the desired biological effect is crucial for advancing a drug discovery project toward clinical trials. By harvesting the liver and brain from rats subjected to a glucose challenge, biomarker analysis was used to assess protein phosphorylation levels within the signaling cascade under investigation.

After establishing an optimized homogenization and lysis protocol, the samples were processed using Simple Western Jess and probed for target phospho-protein levels. The results demonstrated that the drug was highly effective in the liver (Figure 1) but did not alter biomarker levels in the brain (Figure 2). This discrepancy highlights the importance of tissue-specific analysis in evaluating drug efficacy and guiding further development.

Ex vivo_Case Study_results_rat liver
Figure 1. Quantification of the phosphorylated target normalised to the total target derived from. Pseudoblot from the Simple Western™ showing replicates of sample groups.
Ex vivo_Case Study_results_rat brain
Figure 2. Quantification of the phosphorylated target normalised to the total target derived from. Pseudoblot from the Simple Western™ showing replicates of sample groups.

Integrated Approach at Charnwood Discovery

At Charnwood Discovery, we take an integrated and meticulous approach to sample preparation and analysis. This ensures that we can effectively navigate the complexities of drug discovery, driving projects forward with precision and reliability.

About the Authors

Laie Abello

Director of ADME / DMPK

After graduating with a degree in Biology from Pompeu Fabra University in Barcelona Laie went on to gain a Master’s degree in Pharmaceutical and Biotechnology Industry. Laie started her career at Almirall in Barcelona in 2010 as a Junior Scientist in DMPK. She moved to Almac Discovery in Belfast in 2012 where she was involved in setting up their new DMPK department, before moving to Xenogesis at BioCity, Nottingham in 2015, which was then acquired by Sygnature Discovery in September of 2020. She then joined Charnwood Discovery in 2022 where she set up the new ADME / DMPK department.

Rachel Doidge

Senior Research Scientist

Rachel graduated with a degree in Biological Sciences from the University of Warwick before going on to complete a PhD in gene regulation at the University of Nottingham, School of Pharmacy. During her career she has worked as a research biologist for Public Health England studying bovine spongiform encephalopathy and worked for a small start-up biotech researching STi testing. Rachel joined Aurelia Bioscience at BioCity, Nottingham in 2014 as a senior research biologist working on a range of different drug discovery projects. Aurelia bioscience was acquired by Charnwood Discovery in 2022.

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