European Bioanalysis Forum recommendation on singlicate analysis for ligand binding assays: time for a new mindset

Bioanalysis ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 273-284 ◽  
Author(s):  
Matthew Barfield ◽  
Joanne Goodman ◽  
John Hood ◽  
Philip Timmerman
Keyword(s):  
Ligand Binding ◽  
Pharmacokinetic Analysis ◽  
Pharmacokinetic Parameters ◽  
Binding Assays ◽  
Bioanalytical Method Validation ◽  
Regulatory Guidelines ◽  
The Status ◽  
Analysis Strategy ◽  
Assay Methods ◽  
Pharmacokinetic Assay

It is well accepted that chromatographic assay methods employ singlicate analysis for toxicokinetic and pharmacokinetic analysis. While conversely, it has been the norm for ligand-binding assays to be run in at least duplicate analyses, stemming mainly from concerns over inherent assay variability and reagent quality. Regulatory guidelines and guidance on bioanalytical method validation has, in the most part, recommended multiple replicates for immunoassays and this has led to the industry being comfortable and familiar with duplicate analysis. Over the last few years, the discussion on whether singlicate analysis is acceptable for ligand-binding assays has grown and the status quo is being challenged for regulated bioanalysis performed using immunoassays. Through interrogation of preclinical and clinical pharmacokinetic assay data from the European Bioanalysis Forum community, the application of a singlicate analysis strategy has shown to have no impact on toxicokinetic and pharmacokinetic parameters when compared with duplicate analysis from the same studies. Therefore, now is the time to adopt a new mindset when it comes to sample analysis for toxicokinetic and pharmacokinetic ligand-binding assays and embrace singlicate analysis in the regulated environment.

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

Bioanalysis ◽  
2019 ◽  
Vol 11 (22) ◽  
pp. 2075-2086 ◽  
Author(s):  
Douglas Donaldson ◽  
Shobha Purushothama ◽  
Eric David ◽  
Kristopher King ◽  
Shuguang Huang ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Test Tube ◽  
Comparable Data ◽  
Binding Assays ◽  
Robust Performance ◽  
Liquid Handling ◽  
Accuracy And Precision ◽  
Regulatory Guidelines ◽  
Sample Testing ◽  
Pharmacokinetic Assay

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.

scholarly journals EBF recommendation on practical management of critical reagents for antidrug antibody ligand-binding assays

Bioanalysis ◽  
2019 ◽  
Vol 11 (19) ◽  
pp. 1787-1798 ◽  
Author(s):  
Susanne Pihl ◽  
Barry WA van der Strate ◽  
Michaela Golob ◽  
Janka Ryding ◽  
Laurent Vermet ◽  
...  
Keyword(s):  
Life Cycle ◽  
Ligand Binding ◽  
Crucial Role ◽  
Binding Assays ◽  
Antidrug Antibody ◽  
Regulatory Guidelines ◽  
Minimum Requirements ◽  
Antidrug Antibodies ◽  
Immunogenicity Assays

Immunogenicity (ISI) assays are required to measure antidrug antibodies that are generated against biotherapeutic modalities. As for any ligand-binding assays, critical reagents (CR) play a crucial role in immunogenicity assays, as the robustness and reliability of an assay are defined by the quality and long-term availability of these reagents. The current regulatory guidelines do not provide clear directions on how to implement and verify lot-to-lot changes of CR during an assay life cycle, or the acceptance criteria that should be used when implementing new lots of CR. These aspects were extensively discussed within the European Bioanalysis Forum community. In this paper, CR for immunogenicity assays are identified and the minimum requirements for introducing new lots of CR in immunogenicity assays are described.

Recommendations for the Bioanalytical Method Validation of Ligand-Binding Assays to Support Pharmacokinetic Assessments of Macromolecules

2003 ◽  
Vol 20 (11) ◽  
pp. 1885-1900 ◽  
Author(s):  
Binodh DeSilva ◽  
Wendell Smith ◽  
Russell Weiner ◽  
Marian Kelley ◽  
JoMarie Smolec ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Method Validation ◽  
Binding Assays ◽  
Bioanalytical Method Validation ◽  
Bioanalytical Method

Japanese bioanalytical method validation guideline: the world's first regulatory guideline dedicated to ligand-binding assays

Bioanalysis ◽  
2015 ◽  
Vol 7 (9) ◽  
pp. 1151-1156 ◽  
Author(s):  
Mami Imazato-Hirano ◽  
Yoshitaka Taniguchi ◽  
Masaaki Kakehi ◽  
Yoji Kuze ◽  
Takahiro Nakamura ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Method Validation ◽  
Binding Assays ◽  
Bioanalytical Method Validation ◽  
Bioanalytical Method ◽  
Regulatory Guideline

scholarly journals High-throughput screening of natural compounds and inhibition of a major therapeutic target HsGSK-3β for Alzheimer’s disease using computational approaches

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Rohit Shukla ◽  
Tiratha Raj Singh
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
High Throughput Screening ◽  
Complex Disease ◽  
Glycogen Synthase ◽  
Binding Free Energy ◽  
Drug Candidate ◽  
Pharmacokinetic Analysis ◽  
Pharmacokinetic Parameters ◽  
Computational Approaches

Abstract Background Alzheimer’s disease is a leading neurodegenerative disease worldwide and is the 6th leading cause of death in the USA. AD is a very complex disease and the drugs available in the market cannot fully cure it. The glycogen synthase kinase 3 beta plays a major role in the hyperphosphorylation of tau protein which forms the neurofibrillary tangles which is a major hallmark of AD. In this study, we have used a series of computational approaches to find novel inhibitors against GSK-3β to reduce the TAU hyperphosphorylation. Results We have retrieved a set of compounds (n=167,741) and screened against GSK-3β in four sequential steps. The resulting analysis of virtual screening suggested that 404 compounds show good binding affinity and can be employed for pharmacokinetic analysis. From here, we have selected 20 compounds those were good in terms of pharmacokinetic parameters. All these compounds were re-docked by using Autodock Vina followed by Autodock. Four best compounds were employed for MDS and here predicted RMSD, RMSF, Rg, hydrogen bonds, SASA, PCA, and binding-free energy. From all these analyses, we have concluded that out of 167,741 compounds, the ZINC15968620, ZINC15968622, and ZINC70707119 can act as lead compounds against HsGSK-3β to reduce the hyperphosphorylation. Conclusion The study suggested three compounds (ZINC15968620, ZINC15968622, and ZINC70707119) have great potential to be a drug candidate and can be tested using in vitro and in vivo experiments for further characterization and applications.

scholarly journals The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kenichi Kamata ◽  
Kenji Mizutani ◽  
Katsuya Takahashi ◽  
Roberta Marchetti ◽  
Alba Silipo ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Ligand Binding ◽  
Steric Hindrance ◽  
Sequence Similarity ◽  
Binding Assays ◽  
Subunit Interface ◽  
Nmr Techniques ◽  
And Control ◽  
Asialo Gm1

AbstractSeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac$$\alpha$$ α (2-3)Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc) and its precursor, asialo-GM1 (Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the $$\beta$$ β -trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

scholarly journals Pharmacokinetics and toxicity of carboplatin used for hyperthermic intraperitoneal chemotherapy (HIPEC) in treatment of epithelial ovarian cancer

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Mette Schou Mikkelsen ◽  
Jan Blaakaer ◽  
Lone Kjeld Petersen ◽  
Luise Gram Schleiss ◽  
Lene Hjerrild Iversen
Keyword(s):  
Ovarian Cancer ◽  
Epithelial Ovarian Cancer ◽  
Intraperitoneal Chemotherapy ◽  
Hyperthermic Intraperitoneal Chemotherapy ◽  
Systemic Exposure ◽  
Hematological Toxicity ◽  
Pharmacokinetic Analysis ◽  
Pharmacokinetic Parameters ◽  
Severe Toxicity ◽  
Perfusion Time

AbstractObjectivesCarboplatin is frequently used in various doses for hyperthermic intraperitoneal chemotherapy (HIPEC) in the treatment of epithelial ovarian cancer (EOC) although its pharmacokinetics, including focus on the perfusion time, has not been evaluated when used in modern era cytoreductive surgery (CRS). The aim was to evaluate the pharmacokinetics and hematological toxicity of carboplatin used for HIPEC with a perfusion time of 90 min.MethodsFifteen patients with stage III–IV primary EOC received CRS and 90 min of HIPEC with carboplatin at dose 800 mg/m2. For the pharmacokinetic analysis, perfusate and blood samples were obtained during HIPEC and up to 48 h after HIPEC (blood only). Hematological toxicity within 30 days was graded according to Common Terminology Criteria for Adverse Events. Severe toxicity (grades 3–5) is reported.ResultsMean maximum concentration of carboplatin was 12 times higher in perfusate than plasma (mean CmaxPF=348 µg/mL (range: 279–595 µg/mL) versus mean CmaxPL=29 µg/mL (range: 21–39 µg/mL)). Mean terminal half-life of carboplatin in perfusate was 104 min (range: 63–190 min) and mean intraperitoneal-to-plasma area under the concentration-time curve (AUC) ratio was 12.3 (range: 7.4–17.2). Two patients (13%) had grade 3 neutropenia within 30 days. No grade 4–5 hematological toxicities were identified.ConclusionsCarboplatin has a favorable pharmacokinetic profile for 90 min HIPEC administration, and the hematological toxicity was acceptable at dose 800 mg/m2. Large interindividual differences were found in the pharmacokinetic parameters, making risk of systemic exposure difficult to predict.

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