IN IN CELLULAR THERAPY STANDARDS IN FLOW CYTOMETRY OVERVIEW - PowerPoint PPT Presentation

PRODUCT CHARACTERIZATION IN IN CELLULAR THERAPY STANDARDS IN FLOW CYTOMETRY

OVERVIEW • Reproducibility & Variability in Pre-Clinical Research • Standards, Practices & Policies in Flow Cytometry (FCM) • Case Study: FCM for stem cell enumeration in Hematopoietic Stem Cell Therapy

FROM PRE-CLINIC ICAL RESEARCH TO IN INDUSTRY • Cellular Therapy recently entered biopharmaceutical sector • Cost-Benefit • Quality Assurance & Control • Product characterization for cell-based products in Cellular Therapy • Reliable • Accurate • Relevant characteristics

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CELL (P (PRODUCT) CHARACTERIZATION • Cell count • Cell size • Viability • Gene expression • Cell surface markers • Enzymatic activity • Intracellular markers • Oxidative burst • Cell cycle / DNA content • MDR

CELL CHARACTERIZATION BY FLOW CYTOMETRY • EX T ENSIVE • COMPLEX • EX P ENSIVE • VARIABLE

STANDARDS, , PRACT CTICES & POLIC ICIES IN IN FCM, , TODAY • Reagents (antibodies) • HLDA Workshops (1982) • Data file format • FCS files (1984) • Reporting (publications) • MIFlowCyt (2008) • Training & Proficiency • International Flow Cytometry Certification Examination (2011) • Calibration & Measurements • Instrument setup “(still) in progress” • Immunophenotyping

VARIA IABIL ILITY IN IN FLOW CYTOMETRY DUE TO: • Sample preparation • Reagents • Instrument • Instrument setup • Data analysis • Data interpretation

“What aspect of FCM contributes most to variability?” Main concerns for FCM in Industry/Biotech: - Analysis - Instrument - Calibration - Measurement

1995, IS ISHAGE: : STEM CELL ENUMERATION CMTE CASE • CD34 antigen expressed on the surface of almost all hematopoietic stem cells • Absolute number of CD34+ cells shown to correlate with engraftment potential in hematopoietic stem cell therapy STUDY • An accurate CD34+ cell count can help optimize the timing of apheresis collection • Flow cytometric enumeration of CD34+ cells – ANALYSIS, REAGENTS

EXIS ISTING STANDARD REFERENCE MATERIAL IN IN FCM • INSTRUMENT SETUP e.g. Rainbow Calibration Particles (RCP-30-5A) • PROCESS CONTROL e.g. Stem-Trol (KG1 cells) • DATA ANALYSIS e.g. Stem-Control (stabilized normal PBMCs)

• Provides a standard, single-platform flow cytometric method for enumerating CD34+ cells in PB, Leukapheresis, BM and CB • Step-by-step instructions with figures for demonstrating data analysis according to ISHAGE guidelines

CD34+ Cell Enumeration System Suitability Reference Standard

• RS is made from mobilized peripheral blood collected by apheresis of a G-CSF mobilized donor. • Contains human leukocytes, erythrocytes and CD34+ cells that have been fixed and lyophilized • Stable reference standard with CD34 and CD45 staining comparable to fresh G-CSF mobilized peripheral blood cells • Well-defined cell count and range assigned from a multi-laboratory collaborative study • Reconstitutes in water and is ready to use. No lysis steps required

USE OF USP CD34+ CELL ENUMERATION SYSTEM SUIT ITABILITY REFERENCE STANDARD • Used as a system suitability standard to assess the reagents used and ensure the correct gating during data (acquisition and) analysis for enumeration of CD34+ cells • Results should fall within the range provided in the USP CD34+ Cell Enumeration System Suitability RS certificate.

MAIN IN POIN INTS • Challenges for FCM in commercial cellular therapy • Calibration & Measurements • Some reference materials for calibration, process control and analysis in use in FCM • b ut no ‘standard’ • CD34+ cell enumeration by FCM, now standard for commercial cell therapy • USP Chapter <127> • USP CD34+ cell enumeration system suitability reference standard