Conference Report: Bioanalytical sessions at the 2011 Eastern Analytical Symposium

The Eastern Analytical Symposium (EAS) and Exposition is a regional meeting dedicated to all fields and applications of Analytical Chemistry. The 2011 edition marked the 50th anniversary of EAS and was held at the Garden State Exhibit Center (NJ, USA). The technical program was rich in oral sessions relevant to bioanalytical scientists and was complemented by short courses and poster presentations. All areas of bioanalysis, from small-molecule drug and metabolite analysis to biomarkers and protein therapeutics were featured. The following are highlights from several invited and contributed sessions.

The EAS Award for Outstanding Achievements in MS was given to Ian Blair from the University of Pennsylvania (PA, USA), whose work in the area of biomarkers is well renowned. The session highlighted different aspects of biomarker analysis by MS and more broadly of translational medicine. Many of the speakers were Blair’s former students. Ajai Chaudhary from Merck gave an overview of Blair’s accomplishments in the area of biomarker research. Blair, in his address, spoke of the evolution of MS approaches to achieve the sensitivity required for many biomarker assays. One approach that has been very successful is the formation of pre-ionized derivatives for electrospray ionization MS, which allowed, for example, the determination of steroids in plasma in the amol/ml range.

Also related to translational medicine, the EAS Award for Outstanding Achievements in Chemometrics was honoring Beata Walczak, from University of Silesia (Katowice, Poland), and focused on proteomics and metabolomics. There was also a contributed session related to biomarkers, demonstrating the strength of the research in both industry and academia. Applications went from neurotransmitters to peptide hormones, and included assays by LC–MS, sensors and immunoaffinity capillary electrophoresis.

There was an entire session titled ‘Sensitivity for Bioanalysis’. Xiaohui Xu from Bristol-Myers Squibb (BMS) presented her evaluation of accelerated MS (AMS) to support an absolute bioavailability study. One approach for clinical absolute bioavailability studies is to perform intravenous microdosing; however, this requires a very sensitive bioanalytical assay. AMS was evaluated as an ultra-sensitive technique when LC–MS/MS was not sensitive enough to provide results for these study samples. AMS is more beneficial for sensitivity and has comparable results with LC–MS/MS sample analysis, while meeting incurred sample repeat analysis requirements. AMS has a wider linear dynamic range without the use of an internal standard than LC–MS/MS assays; however, AMS has low-throughput, higher costs and limited instrument vendors. The second speaker, Rand Jenkins of PPD Inc., presented the considerations and pitfalls in developing an ultra-sensitive LC–MS/MS bioanalytical method. One of the major challenges presented involved the use of inhaled drugs that were collected for bioanalytical analysis. The specimen tubes that were shipped to the bioanalytical laboratory testing site were found to be contaminated with the drug on the cups and outside of the tubes, causing high concentration level results as the powder was being transferred to the sample extraction plate during the extraction. His recommendation was that for assays that require ultra-sensitive methods, the sample collection tubes and any other high concentration solutions must be handled in a separate area from the sample analysis and several glove changes are required when handling these solutions.

In the ‘LC–MS Analysis for Pharmaceuticals, Biomarkers and Petroleum Products’ session, Jessalynn Wheaton of Waters presented a bioanalytical dried blood spot (DBS) analysis method to remove phospholipids. Wheaton compared the results for analyte recovery and phospholipid removal by extracting DBS samples using the traditional in-well approach compared to using a 96-well phospholipids removal (PLR) plate, Ostro, provided by Waters. The PLR plate is a one-step method where the DBS sample is punched directly into the plate and the extraction solution added subsequently. The samples are collected in a collection plate (using vacuum) and no further processing is required. Following the PLR plate extraction, 99.9% of the phospholipids relative to the traditional DBS sample extraction method were removed. The PLR extraction method was shown to meet the required 15% acceptance criteria for small-molecule analysis. The established LOQ was 0.05–0.1 ng/ml for resperidone and its metabolites. In the same session, Michelle Dawes from BMS presented a design-of-experiment approach for optimizing the extraction procedure of a drug and its metabolite. Hao Jiang, also from BMS, presented an ultra-sensitive LC–MS/MS assay for the determination of a stable-labeled drug in a microdosing PK study.

The analysis of therapeutic biologics was presented in a session titled ‘Immunogenicity Assessment in Biopharmaceutical Development’. Sheng Dai from Centocor talked about the risk assessment for biological drugs. The potential to induce antidrug antibodies (ADAs) after treatment with biologics has become a safety concern and must be assessed carefully during drug development and for regulatory filings. Dai indicated the regulatory guidelines are not as well developed as for pharmacokinetics evaluation, and regulatory agencies are recommending that immunogenicity be studied by sponsors using their own risk-management plans. The risk can be assessed and classified as low, medium and high by two elements:

• ▪ The ADA incidence rate;

• ▪ The clinical safety consequences caused by those ADAs.

As part of the development of new biopharmaceutical products (BPs), the ability to assess, understand or even predict the immunogenicity potential of each new product has become essential. Dan Sikkema from GSK proposed that by studying the immunogenicity of the current BPs, it may be possible to get a basic understanding of the mechanism of immunogenicity that can be broadened to new BPs, and may help reduce the failure of BPs in clinical development. ADA can be categorized into neutralizing antibody (NAB) or non-neutralizing antibody. NABs could inhibit or reduce drug efficacy. Shawn Li from Alliance Pharma introduced a cell-based NAB assay with discussions of its design, development, validation and application. Finally, for biological therapeutics against soluble receptors, the soluble target may result in an increase in assay background; it may also bind to the detection and capture reagents in immunogenicity assay and induce false-positive signals. A method involving pretreatment of samples to disrupt the interferences, was introduced by Dong Geng and several case studies were discussed.

A session on ‘LC–MS Techniques for Studying Xenobiotic and Endogenous Metabolites’ was particularly relevant for scientists in industry as speakers described their experience with high-resolution MS (HRMS) for qualitative/quantitative measurements of metabolites and approaches for compliance with the Metabolite in Safety Testing guidance. The most common strategy in assessing human metabolite profile is to start typically in Phase II of clinical development, using a single-dose ADME study with a radiolabeled drug. However, this strategy does not meet the steady-state and early evaluation requirements of the US FDA guideline. Jose Castro-Perez from Merck reviewed a newer approach in the detection, identification and quantification of drug metabolites from first-in-human studies, which will give information of human unique metabolite and metabolite accumulation. Qian Ruan from BMS talked about the use of HRMS for analysis of reactive metabolites in in vitro assays and in vivo studies, demonstrating significant advantages in sensitivity and accuracy. Multiple data-mining tools based on HRMS such as isotope and mass-defect filter have been developed.

Another session that touched on topics of interest to LC–MS bioanalysts, was ‘Adjusting to the Requirements of Fast Chromatography’. Jason Anspach from Phenomenex addressed frictional heating in sub-2-µm particle columns. Frictional heating is known to cause a loss in resolution and degradation of column performance equivalent to approximately 20% in ethylene bridged hybrid (BEH) sub-2-µm particle size columns. According to Anspach, core-shell particles, in comparison to sub-2-µm particles, provide higher plate counts; therefore, shorter columns can be used to speed up the separation of analytes without losing performance. Core-shell particle phases are similar in properties to sub-2-µm phases; however, these columns are less prone to frictional heating and have the added benefit of efficiency and speed with lower backpressure.

Among the short-course offerings, ‘The Role of Chromatography in the Analysis and Characterization of Protein Therapeutic Drugs’, lectured by C David Carr of Bioanalytical Technologies, reviewed protein properties relevant to the therapeutic activity of protein drugs that must be characterized and controlled, such as deamidation, oxidation, glycosylation, charge state variants, aggregation and denaturation, as well as PEGylation and other forms of conjugation. The basic concept and process of peptide mapping was introduced. A number of chromatographic separation techniques, including reversed-phase HPLC, ion exchange and size exclusion, which play key roles in the analysis and characterization of protein therapeutic drugs, were also discussed.

With the current heightened interest in protein therapeutics, translational medicine and omics, bioanalytical fields are expected to be a growing area of focus for EAS in coming years.