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Background: A thorough understanding of the biological role of oxyntomodulin (OXM) has been limited by the availability of sensitive and specific analytical tools for reliable in vivo characterization. Here, we utilized immunoaffinity capture coupled with high-resolution accurate mass LC-MS detection to quantify OXM and its primary catabolites., Results: Quantification of intact OXM 1-37 in human and rat plasma occurred in pre- and post-prandial samples. Profiles for the major catabolites were observed allowing kinetic differences to be assessed between species., Conclusion: A validated assay in human and rat plasma was obtained for OXM 1-37 and its catabolites, 3-37 and 4-37. The value of full scan high-resolution accurate mass detection without selected reaction monitoring for low-abundance peptide quantification was also demonstrated.

Crystal City VI Workshop on Bioanalytical Method Validation of Biomarkers, Renaissance Baltimore Harborplace Hotel, Baltimore, MD, USA, 28-29 September 2015 The Crystal City VI workshop was organized by the American Association of Pharmaceutical Scientists in association with the US FDA to continue discussion on the bioanalysis of biomarkers. An outcome of the Crystal City V workshop, convened following release of the draft FDA Guidance for Industry on Bioanalytical Methods Validation in 2013 was the need to have further discussion on biomarker methods. Biomarkers ultimately became the sole focal point for Crystal City VI, a meeting attended by approximately 200 people and composed of industry scientists and regulators from around the world. The meeting format included several panel discussions to maximize the opportunity for dialogue among participants. Following an initial session on the general topic of biomarker assays and intended use, more focused sessions were held on chromatographic (LC-MS) and ligand-binding assays. In addition to participation by the drug development community, significant representation was present from clinical testing laboratories. The experience of this latter group, collectively identified as practitioners of CLIA (Clinical Laboratory Improvement Amendments), helped shape the discussion and takeaways from the meeting. While the need to operate within the framework of the current BMV guidance was clearly acknowledged, a general understanding that biomarker methods validation cannot be adequately depicted by current PK-centric guidelines emerged as a consensus from the meeting. This report is not intended to constitute the official proceedings from Crystal City VI, which is expected to be published in early 2016.

Background: Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention., Results: Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation., Conclusion: A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.

Background: Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation., Results: In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach., Conclusion: For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

Background: Accurate measurement of a total protein target (free plus bound) is essential to optimize dose selection for monoclonal antibody drugs. Herein, we describe a novel sandwich immunoassay format in which the biotherapeutic antibody itself serves as the primary detection antibody. A signal is then generated through the addition of a labeled secondary antibody that recognizes the biotherapeutic antibody. The secondary antibody is conjugated with ruthenium to facilitate electrochemiluminescent analysis., Results: Data are presented from the analysis of two protein biomarkers having disparate size and structure; a 4.5 kDa peptide and a 60 kDa protein. In both cases, validated, highly specific assays were developed and shown to be tolerant to elevated levels of the therapeutic monoclonal antibody in question., Conclusion: Our novel format allows drug-tolerant measurement of soluble protein biomarkers targeted by monoclonal antibodies when only two independent epitopes for antibody binding are available and one is recognized by the therapeutic antibody.

Background: Immunogenicity testing is required for safety assessment of biotherapeutic drugs. Because levels observed during biotherapeutic administration can approach the mg/ml range, establishing drug tolerance is significantly important for assay development., Results: Three assay formats for immunogenicity assessment were tested with respect to drug tolerance: Meso Scale Discovery(®) bridging (MSDB), solid-phase extraction with acid dissociation (SPEAD) and affinity capture elution (ACE). Six biotherapeutic drugs were analyzed by the three methods; four monoclonal antibodies, one Fc fusion protein and one Pegylated protein. Overall, ACE performed best for assays involving therapeutic monoclonal antibodies and also functioned well for therapeutic proteins. Despite several advantages, the MSDB assays displayed a potentially significant hook effect. SPEAD was comparable in performance to ACE for the biotherapeutic drugs tested, but suffers the disadvantage of being reagent-intensive., Conclusions: Novel assay formats offer significant advantages for immunogenicity testing, particularly in the design of assays that are tolerant to circulating levels of the biotherapeutic drug.