Flow cytometry and pharmacokinetics

Bioanalysis ◽  
2021 ◽  
Author(s):  
Kevin Lang ◽  
Katie Matys ◽  
Patrick Bennett ◽  
Vellalore N Kakkanaiah
Keyword(s):  
Flow Cytometry ◽  
Method Development ◽  
Positive Control ◽  
Analysis Tool ◽  
Cell Therapies ◽  
Multiparametric Flow Cytometry ◽  
Cellular Kinetics ◽  
Cellular Analysis ◽  
Sample Stability ◽  
Car T

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.

Considerations in the development and validation of real-time quantitative polymerase chain reaction and its application in regulated bioanalysis to characterize the cellular kinetics of CAR-T products in clinical studies

Bioanalysis ◽  
2020 ◽  
Author(s):  
Tong-yuan Yang ◽  
Rajitha Doddareddy
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Method Development ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Cellular Kinetics ◽  
Car T ◽  
Polymerase Chain ◽  
Development And Validation ◽  
Kinetics Of

Real-time quantitative polymerase chain reaction (qPCR) has become the standard method for monitoring cellular kinetics of CAR-T therapies with measurement of the CAR transgene copy numbers in peripheral blood mononuclear cells isolated from patients receiving the treatment. Unlike other biophysical and immunological methodologies for bioanalytical characterization of conventional small molecule drugs or protein biologics, there is no relevant regulatory guidance to date on the method development and validation for quantitative qPCR assays employed during clinical development of CAR-T products. This paper will provide an overview and considerations in the development and validation of a qPCR assay from sample extraction to assay parameters and its implementation in regulated bioanalysis for CAR-T or other types of cell therapies.

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

Bioanalysis ◽  
2021 ◽  
Author(s):  
Vellalore N Kakkanaiah ◽  
Katie Matys ◽  
Patrick Bennett
Keyword(s):  
Flow Cytometry ◽  
Drug Discovery ◽  
Drug Development ◽  
Ligand Binding ◽  
Specific Interaction ◽  
Flow Cytometer ◽  
Analysis Tool ◽  
Binding Assays ◽  
Cellular Analysis ◽  
Main Components

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.

Monitoring CAR‐T cell kinetics in clinical trials by multiparametric flow cytometry: Benefits and challenges

2020 ◽  
Author(s):  
Ghanashyam Sarikonda ◽  
Anil Pahuja ◽  
Creton Kalfoglou ◽  
Kerri Burns ◽  
Kevin Nguyen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Clinical Trials ◽  
T Cell ◽  
Cell Kinetics ◽  
Multiparametric Flow Cytometry ◽  
Car T Cell ◽  
Car T

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

Bioanalysis ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 1881-1884 ◽  
Author(s):  
Vellalore N Kakkanaiah ◽  
Fan Pan ◽  
Patrick Bennett
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Room Temperature ◽  
Basophil Activation Test ◽  
Blood Collection ◽  
Blood Samples ◽  
Human Whole Blood ◽  
Cellular Kinetics ◽  
Sample Stability ◽  
Blood Collection Tubes

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.

scholarly journals Flow cytometry and pharmacokinetic studies

2019 ◽  
Vol 4 (3) ◽  
pp. IPK06
Author(s):  
Vellalore N Kakkanaiah
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cell Therapy ◽  
Adoptive Cell Therapy ◽  
Pharmacokinetic Studies ◽  
Cellular Kinetics ◽  
Car T Cells ◽  
Developing Flow ◽  
Car T ◽  
Kinetics Of

Biography Vellalore Kakkanaiah, PhD serves as the director of the flow cytometry group, which is part of the Biomarker Laboratories at PPD Richmond, VA. His team is focused on the development of flow cytometry methods to support drug development. Recently, his team developed a simple whole blood assay to study the cellular kinetics of CAR-T cells after adoptive cell therapy in multiple myeloma patients. Prior to joining PPD, Dr Kakkanaiah was a senior scientist at SurroMed, Inc. where he developed micro volume laser scanning cytometry assays. He earned a doctorate in immunology from Madurai Kamaraj University in India and then served as a postdoctoral fellow at Virginia Tech. His postdoctoral training involved the first identification of mature CD4-CD8 T cells by flow cytometry from the thymus of an autoimmune mouse model. He was also a fellow of the Arthritis Foundation at the UNC-Chapel Hill. Vellalore N Kakkanaiah speaks to the International Journal of Pharmacokinetics about his experience in developing flow cytometry assays for measuring cellular kinetics in adoptive cell therapies. Flow cytometry is new to bioanalysis and is being used for measuring the cellular kinetics of infused cells from adoptive cell therapy. Here, he discusses the challenges in developing flow cytometry methods for monitoring the infused CAR-T cells.

scholarly journals Insights on Droplet Digital PCR–Based Cellular Kinetics and Biodistribution Assay Support for CAR-T Cell Therapy

2021 ◽  
Vol 23 (2) ◽  
Author(s):  
Hiroshi Sugimoto ◽  
Susan Chen ◽  
Jean-Pierre Minembe ◽  
Johara Chouitar ◽  
Xingyue He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Regulatory Requirement ◽  
T Cell Therapy ◽  
Immunodeficient Mice ◽  
Cellular Kinetics ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractCharacterizing in vivo cellular kinetics and biodistribution of chimeric antigen receptor T (CAR-T) cells is critical for toxicity assessment, nonclinical and clinical efficacy studies. To date, the standardized assay to characterize CAR-T cell distribution, expansion, contraction, and persistence profiles is not readily available. To overcome this limitation and increase comparability among studies, we have established a universal protocol for analysis. We established a duplexing ddPCR protocol for the CAR-T transgene and reference gene to normalize the genomic DNA input prepared from mouse blood and tissues. The high-throughput gDNA extraction method enabled highly reproducible gDNA extraction while eliminating labor-intensive steps. The investigational CAR-T cells were intravenously injected into immunodeficient mice bearing human colorectal cancer xenografts. The blood and tissue samples were collected to measure the cellular kinetics by ddPCR and flow cytometry. The standard curves were linear throughout the calibration range with acceptable intra- and inter-day precision and accuracy. The gDNA recovery study performed by spiking in the exo-gene plasmid DNA or CAR-T cells revealed that the recovery ranged from 60 to 100% in blood and tissue homogenates. The use of both units of copy/μg gDNA and copy/μL blood met the current regulatory requirement and allowed for a systematic understanding of CAR-T cell expansion and a direct comparison with the flow cytometry data. A standardized ddPCR assay, including automated gDNA extraction procedures, has been established for evaluating cellular kinetics and biodistribution in CAR-T cell therapies.

OP206 Expert Elicitation Of Probabilistic Distributions to Inform Survival Modelling of CD19 Chimeric Antigen Receptor T-Cell Therapies

2020 ◽  
Vol 36 (S1) ◽  
pp. 3-3
Author(s):  
Niamh Carey ◽  
Conor Hickey ◽  
Laura Mc Cullagh ◽  
Michael Barry
Keyword(s):  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Expert Elicitation ◽  
Cell Transplant ◽  
Major Advance ◽  
Cell Therapies ◽  
Risk Of Death ◽  
Car T Cell ◽  
Car T

IntroductionIn 2018, the National Centre for Pharmacoeconomics (NCPE) was commissioned to conduct a health technology assessment (HTA) of one of the first commercially available chimeric antigen receptor (CAR) T-cell therapies, tisagenlecleucel. CAR T-cells are a major advance in personalized cancer treatment, demonstrating promising outcomes in relapsed/refractory pediatric acute lymphoblastic leukemia (pALL). However, the results are based on short-term follow up, limiting their value in predicting long-term survival and leading to uncertainty about the most appropriate survival modeling method to employ. This study aimed to address these limitations by means of expert elicitation.MethodsAn expert elicitation method, the histogram technique, was employed. A predefined discrete numerical scale was presented in Microsoft Excel® and the expert was asked to place twenty crosses on a frequency chart. These crosses represented the expert's beliefs about the distribution of particular quantities. Each cross represented five percent of the probabilistic distribution. Individual distributions were then aggregated across experts using linear pooling.ResultsA total of seventeen experts were invited to take part; eight agreed to participate and five completed the exercise. Three experts did not consider tisagenlecleucel to be a “curative” therapy because patients had a higher risk of death, compared with the age- and sex-matched general population. The aggregated distributions indicated the five-year overall survival rate to be thirty-three percent (95% CI 8.65–56.88) in patients who do not receive a subsequent stem cell transplant and twenty percent (95% CI 2.38 -52.04) in those who do.ConclusionsThe results of this study will be used to calibrate CD19 CAR T-cell therapy survival estimates presented in HTA submissions to the NCPE to ensure more robust assessments. They will also be used to inform the construction of a de novo cost-utility model for examining the cost effectiveness of CD19 CAR T-cell therapies for relapsed/refractory pALL in the Irish healthcare setting.

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