PRODUCT CHARACTERIZATION
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-CLINICAL RESEARCH TO 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
Kindly provided by
CELL (PRODUCT) CHARACTERIZATION
•
•
•
•
•
Cell count
Viability
Cell surface markers
Intracellular markers
Cell cycle / DNA content
•
•
•
•
•
Cell size
Gene expression
Enzymatic activity
Oxidative burst
MDR
CELL CHARACTERIZATION BY FLOW CYTOMETRY
• EXTENSIVE
• COMPLEX
• EXPENSIVE
• VARIABLE
STANDARDS, PRACTICES & POLICIES IN FCM, TODAY
• Reagents (antibodies)
• Data file format
• Reporting (publications)
• Training & Proficiency
• HLDA Workshops (1982)
• FCS files (1984)
• MIFlowCyt (2008)
• International Flow Cytometry
Certification Examination (2011)
• Calibration & Measurements
• Instrument setup
• Immunophenotyping
“(still) in progress”
VARIABILITY 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, ISHAGE: STEM CELL ENUMERATION CMTE
CASE
STUDY
• 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
• An accurate CD34+ cell count can help optimize the timing of
apheresis collection
• Flow cytometric enumeration of CD34+ cells – ANALYSIS, REAGENTS
EXISTING STANDARD REFERENCE MATERIAL 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
SUITABILITY 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 POINTS
• Challenges for FCM in commercial cellular therapy
• Calibration & Measurements
• Some reference materials for calibration, process control and analysis in use
in FCM
• but no ‘standard’
• CD34+ cell enumeration by FCM, now standard for commercial cell therapy
• USP Chapter <127>
• USP CD34+ cell enumeration system suitability reference standard