Flow cytometry and pharmacokinetics.

Multiparametric flow cytometry is a powerful cellular analysis tool used in various stages of drug development. In adoptive cell therapies, the flow cytometry methods are used for the evaluation of advanced cellular products during manufacturing and to monitor cellular kinetics after infusion. In this report, we discussed the bioanalytical method development challenges to monitor cellular kinetics in CAR-T cell therapies. These method development challenges include procuring positive control samples for the development of the method, flow cytometry panel design, LLOQ, prestain sample stability, staining reagents and data analysis.

Critical reagents in flow cytometry, instrumentation and application in drug discovery development.

Flow cytometer is a powerful cellular analysis tool consists of three main components; fluidics, optics and electronics. Flow cytometry methods have been used in all stages of drug development as like ligand binding assays (LBA). Both LBA and flow cytometry methods require specific interaction between the critical reagents and the analytes. Antibodies and their conjugates, viable dyes and permeabilizing buffer are the main critical reagents in flow cytometry methods. Similarly, antibodies, engineered proteins and their conjugates are the main critical reagents in LBA. The main difference between the two methods is the lack of true reference standards for flow cytometry cellular analysis.

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.

Evaluation of sample stability for cellular kinetics and pharmacodynamic flow cytometry methods.

We evaluated the sample stability for a cellular kinetics and a pharmacodynamic flow cytometry methods. First, the blood collection tubes were compared for the enumeration of chimeric antigen receptor-T cells in human whole blood. Blood samples with chimeric antigen receptor-T cells were stable up to 3 days at room temperature in both conventional EDTA and Cyto-Chex® blood collection tubes (Streck Laboratories, NE, USA), but with better consistency in Cyto-Chex-BCT than conventional EDTA tubes. Second, sample storage temperatures were compared for the basophil activation test in human whole blood samples. The samples were stable up to 3 days for basophil activation test when stored at refrigerator temperature, but not stable when stored at room temperature. It is crucial during the development of method to evaluate all the variables which might impact sample integrity.

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity. Conclusions and consensus from discussions of these topics are included in this article.

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods. Contract research organizations regularly use commercially available immunoassay kits to validate biomarker methods. However, adaptation of such kits in a regulated environment presents significant challenges and was one of the key topics discussed during the 12th Global Contract Research Organization Council for Bioanalysis (GCC) meeting. This White Paper reports the GCC members' opinion on the challenges facing the industry and the GCC recommendations on the classification of commercial kits that can be a win-win for commercial kit vendors and end users.

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the 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 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

Implementation of highly sophisticated flow cytometry assays in multicenter clinical studies: considerations and guidance.

Flow cytometry is increasingly becoming an important technology for biomarkers used in drug discovery and development. Within clinical development flow cytometry is used for the determination of PD biomarkers, disease or efficacy biomarkers or patient stratification biomarkers. Significant differences exist between flow cytometry methodology and other widely used technologies measuring soluble biomarkers including ligand binding and mass spectrometry. These differences include the very heavy reliance on aspects of sample processing techniques as well as sample stabilization to ensure viable samples. These differences also require exploration of new approaches and wider discussion regarding method validation requirements. This paper provides a review of the current challenges, solutions, regulatory environment and recommendations for the application of flow cytometry to measure biomarkers in clinical development.

Hypoestoxide, a natural nonmutagenic diterpenoid with antiangiogenic and antitumor activity: possible mechanisms of action.

We have shown previously that hypoestoxide (HE), a natural diterpenoid [a bicyclo (9, 3, 1) pentadecane], is a potent nonsteroidal anti-inflammatory drug. In this report, we demonstrate that HE also inhibits the growth of a variety of human and murine tumor cell lines in vitro at concentrations ranging from 0.3 to 10 microM and was inactive as a mutagen in the Ames test. HE exhibited highly potent (0.3-10 mg/kg dose ranges) activities against B16 melanoma growth in C57BL/6 mice and P388D1 leukemia in C57BL/6 x DBA/2 F(1) mice, respectively. At a low maximal effective dose of 5 mg/kg, HE induced significant in vivo antitumor activities that were better than or comparable with most of the standard chemotherapeutic antiangiogenic agents tested: cortisone acetate, vincristine, bleomycin, Adriamycin, 5-fluorouracil, cyclophosphamide, and etoposide. All of the agents, except vincristine, had much higher maximal effective doses than HE. HE arrested the growth of human Burkitt lymphoma CA46 cells and HeLa (cervical epitheloid carcinoma) cells in the G2-M phase of the cell cycle, which was caused by interference, either direct or indirect, with actin assembly. Thus, the cell cycle arrest occurred at cytokinesis, as demonstrated by an increase in the number of binucleate cells. Moreover, HE inhibited vascular endothelial growth factor-induced cell proliferation in vitro, with an IC(50) of 28.6 microM, and it significantly inhibited basic fibroblast growth factor-induced angiogenesis on the chick chorioallantoic membrane, with an IC50 of 10 microM. Furthermore, HE inhibited endothelial cell migration on vitronectin, collagen, and fibronectin. Besides its activity as a nonsteroidal anti-inflammatory drug, HE also has promise for the chemotherapy of cancer.