The impact of ligand binding based assays critical reagent characterization and storage.

Biological critical reagents are the foundation of many bioanalytical methods and often chemically modified or conjugated with various chemical tags. As such, the quality and performance of these methods are inherently tied to the quality and stability of critical reagents. This article will outline recommendations for conjugated critical reagent development and characterization. Examples of the impact of regent quality will be discussed for the two common bioanalytical assays in support of drug development for biotherapeutics. Finally, a brief discussion of conjugated reagent stability and recommendations for storage and testing will be presented.

Effect of 'Candidatus Phytoplasma pruni' Infection on Sweet Cherry Fruit.

In sweet cherry (Prunus avium), infection by 'Candidatus Phytoplasma pruni' results in small fruit with poor color and taste, rendering the fruit unmarketable. Yet the disease pathology is poorly understood, particularly at the cultivar level. Therefore, in this study we examined the physiological effects of Ca. P. pruni infection across a range of cultivars and locations in eastern Washington. We found that infection could be separated into early and established stages based on pathogen titer, which correlated with disease severity, including fruit size, color, and sugar and metabolite content. Furthermore, we observed that the effects of early-stage infections were largely indistinguishable from healthy, uninfected plants. Cultivar- and location-specific disease outcomes were observed with regard to size, color, sugar content, and citric acid content. This study presents the first in-depth assessment of X-disease symptoms and biochemical content of fruit from commercially grown sweet cherry cultivars known to be infected with Ca. P. pruni.

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

New insights on critical reagent optimization for antidrug antibody assays.

Aim: Conjugated critical reagents are a pillar of ligand binding analysis. Although good practices for characterization have already been discussed, little is known about how assays are affected by specific conjugation parameters. Results: Here we developed, characterized and screened a toolset of bioconjugates that provided new insights about the optimization of conjugated critical reagent attributes. Biotinylated and sulfo-tagged trastuzumab were utilized as capture and detection antibodies, respectively, in an antidrug antibody (ADA) assay. The optimal conjugation window was identified by functional assessment. Excess of unlabeled biotin, but not sulfo-tag, affected the assay performance. An increase in the assay baseline was observed when sulfo-tagged trastuzumab underwent increasing freeze-thaw cycles. Conclusion: Upfront systematic screening and characterization of conjugated critical reagent attributes benefit assay robustness.

Interactions of cytochrome C with N-acylated phosphatidylethanolamine lipids.

N-acylphosphatidylethanolamines (NAPEs) are naturally occurring derivatives of phosphatidylethanolmine (PE) in which the PE amino group is attached to an acyl chain. Given their occurrence in natural systems, there is interest in knowing the effect of NAPEs on membrane dynamic structure and function. This study examines the ability of NAPEs to affect the association of the cytochrome c and Zn-heme cytochrome c with the surface of bilayer membranes. Fluorescence titration experiments show that cationic cytochrome c has the same high affinity for the surfaces of anionic vesicles that are rich in NAPEs or diplalmitoyphosphatidylglycerol (DPPG) but the protein/membrane interaction in each case is quite different. Cytochrome c adsorption to DPPG membranes is relatively irreversible due to the DPPG molecules adopting an extended conformation that promotes strong hydrophobic contact with the adsorbed protein. In contrast, cytochrome c association with N-acyl DPPE membranes is due primarily to reversible electrostatic interactions with the anionic headgroup, and not hydrophobic contact with the N-acyl chain. The presence of a small mole fraction of an N-propionyl derivative of DPPE (N-C3:0-DPPE) diminishes cytochrome c affinity for vesicles containing a large amount of DPPG apparently by relieving the membrane packing strain that drives the extended DPPG conformation.

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.

Impact of extended maceration and regulated deficit irrigation (RDI) in Cabernet Sauvignon wines: characterization of proanthocyanidin distribution, anthocyanin extraction, and chromatic properties.

The impact of extended maceration (EM) was studied in Cabernet Sauvignon grapes sourced from a vineyard subjected to four regulated deficit irrigation (RDI) treatments: (I) 100% replenishment of crop evapotranspiration (100% ETc), (II) 70% ETc, (III) 25% ETc until véraison, followed by 100% ETc until harvest, and IV) 25% ETc. Each vineyard replicate was made into wine with two replicates designated as controls (10-day skin contact) and two as extended maceration (EM, 30-day skin contact). The mean degree of polymerization (mDP), size distribution, concentration, and composition of wine proanthocyanidins (PAs) and monomeric flavan-3-ols of 90 fractions were characterized by preparative and analytical HPLC techniques. The maceration length imparted a larger effect on most chemical parameters. The RDI treatment had no effect on the extraction patterns of anthocyanins, PAs, and/or on the origin of the PAs extracted into the wines. Conversely, EM led to anthocyanin losses and increased PA extraction during maceration, with ~73% of seed-derived PAs. Accordingly, the concentration of monomeric flavan-3-ols, oligomeric (2 ≤ mDP < 5) and polymeric PAs (mDP ≥ 5) was higher in EM wines. The size distribution of the wines' PAs revealed two major peaks as a function of concentration at mDP 2 (22-27% of total PAs mass) and at mDP 6-7 (12-17% of total PAs mass) and was found to follow a non-normal Rayleigh-type distribution.

Lactate uptake contributes to the NAD(P)H biphasic response and tissue oxygen response during synaptic stimulation in area CA1 of rat hippocampal slices.

Synaptic train stimulation (10 Hz x 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO(2) decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO(2) response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150-250 micromol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150-250 micromol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO(2) response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.

Effects of relative hypoglycemia on LTP and NADH imaging in rat hippocampal slices.

Cognitive and neuronal impairment in diabetes may be associated with iatrogenic hypoglycemia, particularly at low serum glucose levels (<3 mM). To evaluate cellular impairment, we assessed acute hippocampal slice functioning during decreased ambient glucose, by monitoring evoked field excitatory post-synaptic potentials (fEPSP), and slice nicotinamide adenine dinucleotide (NADH) fluorescence. The effects of lowered glucose levels (60 min) were analyzed by examining the induction and maintenance of long-term potentiation (LTP), and NADH metabolic imaging in the CA1 region. The basal fEPSP response was reduced by lowered ambient glucose, an effect that was reversible in 2.5 mM glucose, partially reversible in 1.25 mM glucose and irreversible in 0 mM glucose, after 25 min recovery. LTP induction and maintenance declined during glucose restriction, demonstrating reversibly failed maintenance in 5 mM and 2.5 mM ambient glucose, and absent induction in 1.25 mM glucose. Peak NADH levels observed during train-induced biphasic transients were significantly reduced during 1.25 mM and 2.5 mM glucose. Significant functional compromise in our slice model occurred at 2.5 mM ambient glucose, equivalent to <1 mM tissue glucose in the slice center, due to diffusion limitations and active glucose utilization. This tissue glucose level correlates with human observations of a serum threshold of <3 mM for cognitive impairment, since brain tissue glucose is approximately one third of serum levels. The physiological effects of hypoglycemia in our slice model, assessed through fEPSP, LTP, and NADH responses, replicate closely the in vivo situation, confirming the usefulness of this model in assessing consequences of relative hypoglycemia.

Requirement for the RIIbeta isoform of PKA, but not calcium-stimulated adenylyl cyclase, in visual cortical plasticity.

The cAMP-dependent protein kinase (PKA) signaling pathway plays a key role in visual cortical plasticity. Inhibitors that block activation of all PKA regulatory subunits (RIalpha,RIbeta, RIIalpha, RIIbeta) abolish long-term potentiation (LTP) and long-term depression (LTD) in vitro and ocular dominance plasticity (ODP) in vivo. The details of this signaling cascade, however, including the source of PKA signals and which PKA subunits are involved, are unknown. To investigate these issues we have examined LTP, LTD, and ODP in knock-out mice lacking either the two cortically expressed Ca2+-stimulated adenylyl cyclases (AC1 and AC8) or the predominant neocortical subunit of PKA (RIIbeta). Here we show that plasticity remains intact in AC1/AC8-/- mice, whereas ODP and LTD, but not LTP, are absent in RIIbeta-/- mice. We conclude that (1) plasticity in the visual cortex does not require the activity of known Ca2+-stimulated adenylyl cyclases, (2) the PKA dependence of ODP and LTD, but not LTP, is mediated by RIIbeta-PKA, and (3) multiple isoforms of PKA contribute to LTD.