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

• Calibration & Measurements • Instrument setup

• Immunophenotyping

• HLDA Workshops (1982)

• FCS files (1984)

• MIFlowCyt (2008)

• International Flow Cytometry Certification Examination (2011)

“(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

CASE STUDY

1995, ISHAGE: STEM CELL ENUMERATION CMTE

• 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