The changing face of bioanalysis

Industry challenges and solutions for bioanalysis were top of the agenda for an exclusive bioanalysis seminar organised by Quotient Bioresearch The seminar  was attended by representatives from pharmaceutical and biotechnology organisations across Europe and featured debates and panel discussions from leading industry speakers on new techniques and hot topics including the latest industry.

Opening the seminar was Richard Houghton, principal scientist for Bioanalytical Sciences at Quotient Bioresearch, who sparked a discussion on the validation of Dried Blood Spot (DBS) methods for use in regulated bioanalysis. Houghton highlighted the advantages and disadvantages of DBS and talked about the relevant regulatory considerations addressing several specific issues including blood spot volume and hematocrit assessment.

He stressed the benefits of using DBS as reducing blood volume to allow multiple sampling in pre-clinical studies, lowering shipping costs, facilitating easier storage of samples and sample stability.

Consideration should be given to blood spot homogeneity, whole blood stability hematocrit assessment – as the hematocrit levels differ between male and female, infants and children, normal and disease etc, short term stability at 40°C and -20°C and how to dilute high concentration samples.

In order to harmonise the approach to DBS validation, Houghton outlined how the European Bioanalytical Forum (EBF) has set up a DBS topic team to discuss how to take the technique forward.

The first meeting was held at the recent EBF Large versus Small conference in Brussels and one of the issues raised was the nature of the FTA cards currently used as the medium on which to store the DBS. Concerns were raised as the FTA cards were originally designed for stabilising blood prior to DNA extraction and the denaturants used may not be wholly compatible with mass spectrometry.

Quotient Bioresearch’s Clinical Drug Product Optimisation Director John McDermott took to the podium next and discussed the approach to effective optimisation of oral dosage forms. He explained how traditionally it can take two to four years from First in Human to Proof of Concept and can involve multiple rounds of drug re-formulation.

McDermott used a case study of a PDE5 inhibitor to illustrate how clinical trial design could optimise a PK profile which minimised Cmax related adverse events whilst maintaining drug levels above the PD threshold over a 24 hour period post dosing. This was achieved in a 28 week project timeframe as opposed to the 12-18 months it would normally take.

The seminar continued with a presentation from David Little, Director of the MS systems evaluation laboratory at Waters who talked about the application of an integrated microfluidic device for high sensitivity analysis.

Little described how this would facilitate the analysis of low volume samples from individual small animals or paediatric study samples, DBS and unusual or rare matrices. He also presented a MIST (Metabolites in Safety Testing) case study of dichlofenac in plasma from humanised mice. Each humanised mouse costs $9,000 so by enabling multiple sampling from each mouse because of the reduced requirement for plasma it may provide a viable alternative to clinical trials for MIST.

Anders Fridström, EMEA Tactical Marketing Manager at Sigma-Aldrich took a different approach to the chromatography challenges and discussed the mobile phase considerations for peptide and protein analysis by LC-MS/MS. Data were presented showing that 0.1% formic acid at pH 3.5 (as opposed to pH 2.6) can improve peak shape and capacity and offer a real alternative to trifluoroacetic acid (TFA) for reverse phase chromatography of peptides.

Richard Houghton took part in a second presentation along with Andrew Roberts, Sector Manager for Bioanalytical Sciences at Quotient Bioresearch discussing recent advances in LC-MS/MS and ligand binding assays respectively, in the analysis of protein molecules.

Houghton used Factor Seven Activating Protein (FSAP), a relatively abundant protein in plasma, as an example of how LC-MS/MS can be used to discriminate between wild type protein and a single amino acid change (Marburg 1 mutation) present in 4-9% of the Caucasian population. Houghton also showed how LC-MS/MS has also been successfully used in a multiplex form to analyse up to 10 apolipoproteins in a single analytical run.

Roberts used case studies to demonstrate the use of the Gyrolab platform to develop a sensitive PK method for a monoclonal antibody biopharmaceutical, and how the correct choice of positive control was crucial in developing an MSD based assay for measuring an IgE anti-drug antibody response.

The seminar concluded that the industry is entering an interesting period; with pharmaceutical companies, CROs and regulators all working together towards global harmonisation of the bioanalytical guidance and promising to deliver huge benefits.

However the industry is still faced with a number of challenges including technology advances in the use of microfluidic devices which promise to deliver significant sensitivity gains for high-throughput bioanalysis and technical challenges for dried bloodspot analysis which need to be overcome before there is regulatory acceptance of the technique. The use of LC-MS/MS also offers selectivity gains over immunoassay for the analysis of large molecule proteins and peptides, but the challenge remains to improve the limits of quantitation using this technique.