Well-developed ligand-binding assays demonstrate robust performance using singlet analysis.

Aim: Replicate sample testing has long been regarded as a necessity for bioanalytical laboratory testing, especially in the realm of ligand-binding assays (LBAs). In an era in which results were derived from crude test tube-based assays, the replication of results was warranted. Those assays were often imprecise and required multiple replicates to arrive at results that approached accuracy. However, given technological advancements and excellent accuracy and precision of many modern LBAs, the practice of replicate testing should be re-evaluated. Although most regulatory guidelines allow for singlet testing when sufficient robustness and precision are demonstrated during validation, duplicate testing is still common practice. Recently however, several articles have been published that support singlet analysis for LBAs performed on a platform with automated liquid handling. Results: Data from five pharmacokinetic assay validations and five clinical and preclinical studies originally run in duplicate were re-evaluated in singlet and found to be nearly identical to the original duplicate results. Conclusion: We confirm that well-developed LBAs produce comparable data whether evaluated in singlet or duplicate. Additionally, automation is not requisite for singlet testing.

Perspectives on gender parity in bioanalysis: an interview with Lauren Stevenson.

Biography Lauren is Senior Director, Development Biomarkers & Bioanalytical Sciences at Biogen, Cambridge, MA, USA where she leads a team of talented scientists, setting bioanalytical and biomarker strategies and developing pharmacokinetics (PK), immunogenicity and biomarker assays in support of programs at all stages of development, from discovery to post-marketing. Lauren's team currently supports over 40 therapeutic programs, delivering PK and immunogenicity strategy and execution for Biogen's large molecule, antisense oligonucleotide (ASO0 and gene therapy portfolio as well as developing biomarkers in support of multiple sclerosis, immunology, fibrosis, rare and neurodegenerative disease indications. Externally, Lauren engages the broader industry and regulatory agencies as an invited speaker and course instructor at multiple conferences and workshops each year. In concert with her scientific role, Lauren is passionate about people development, has received certification as a Strengths coach and is active in mentorship and career development programs.

Parallelism: considerations for the development, validation and implementation of PK and biomarker ligand-binding assays.

The goal of this article is to discuss the fundamental key questions around parallelism assessments: why to do it, when to do it and how to do it, with consideration for different molecule types and the scientific rationale that drives different approaches. Current practices for both PK and biomarker assays regarding which samples to pick, whether to pool or not pool samples, as well as generally accepted acceptance criteria are discussed, while also highlighting the many outstanding questions that remain. In order to reach the long-term goal of understanding and developing best practices for implementation of parallelism testing for both PK and biomarker assays, industry and regulators will need to keep the conversations going, and commit to generating and reviewing data for the purposes of our own education. Means to easily share data in open forums to facilitate and build common understandings should continue and will be necessary to expedite resolution of many of these questions.

Large molecule run acceptance: Recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

The L1 Global Harmonization Team provides recommendations specifically for run acceptance of ligand binding methods used in bioanalysis of macromolecules in support of pharmacokinetics. The team focused on standard curve calibrators and quality controls for use in both pre-study validation and in-study sample analysis, including their preparation and acceptance criteria. The team also considered standard curve editing and the concept of total error.

The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.

Mdm2 is an E3 ubiquitin ligase that promotes its own ubiquitination and also ubiquitination of the p53 tumour suppressor. In a bacterial two-hybrid screen, using Mdm2 as bait, we identified an Mdm2-interacting peptide that bears sequence similarity to the deubiquitinating enzyme USP2a. We have established that full-length USP2a associates with Mdm2 in cells where it can deubiquitinate Mdm2 while demonstrating no deubiquitinating activity towards p53. Ectopic expression of USP2a causes accumulation of Mdm2 in a dose-dependent manner and consequently promotes Mdm2-mediated p53 degradation. This differs from the behaviour of HAUSP, which deubiquitinates p53 in addition to Mdm2 and thus protects p53 from Mdm2-mediated degradation. We further demonstrate that suppression of endogenous USP2a destabilises Mdm2 and causes accumulation of p53 protein and activation of p53. Our data identify the deubiquitinating enzyme USP2a as a novel regulator of the p53 pathway that acts through its ability to selectively target Mdm2.

Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo.

p53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNA-mediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity.