How Flow Cytometry Is Used

Flow assays are not limited to a single target or process. Multiple parameters can be measured in a single sample and samples can be cultured or treated and monitored for changes over time.

For example, flow cytometry endpoints are utilized for the following common culture assays:

• Immunogenicity Assessments
  • Pair flow readouts such as proliferation and viability with MLR culture assay
• Activity and Dose Response Profiles
  • Characterize the immunomodulatory properties of therapeutics by pulse treatment followed by flow phenotypic assessments, receptor binding, viability, and kinase activity
• T-cell activation and checkpoint inhibitors
  • Pre-stimulate T-cells, followed by culture with checkpoint inhibitors, then assess viability, activation markers, and proliferation by flow cytometry.

These assay types are excellent tools for assessing proof of concept and/or safety of a therapeutic and can be utilized in support of preclinical studies, or clinical trials for cellular therapies.

How does Flow Cytometry fit into industry guidance?

Flow cytometry is incredibly versatile. In addition to typical flow assays (e.g. phenotyping, receptor occupancy, etc.) flow cytometry can serve as an endpoint for many cell-based culture assays like the MLR, ADCC, phagocytosis assays, and more. As such, it is well positioned to be an invaluable tool for nearly any stage of the product development pipeline. 

Below we discuss how flow cytometry can fit into different stages of product development based on recommended regulatory guidance. It should be noted that regulatory guidance for industry rarely recommends one specific assay or technique that should be used to evaluate the properties of a therapeutic; however, the information below is our best interpretation of where flow cytometry can be best utilized to fulfill recommended guidance criteria independently or coupled with other cell-based assays associated with the ex vivo evaluation of immunological drug safety or efficacy.

Discovery:

• Investigate Bioactivity, Dose Response
• Investigate Cellular Identity (Phenotype)
• Investigate Binding Kinetics / Receptor Occupancy

Preclinical:

• Proof of Concept
  • Characterization ^{2, 5, 9}
  • Efficacy, Response Profile, & Activity ^{2, 3, 7, 8}
  • Mechanism of Action ^{2, 5}
  • Stability and Viability (Cellular Therapies) ⁵
• Safety
  • Biological Activity/Immunogenicity (affect to host phenotype, T-cell proliferation, B-cell activation, potency) ^{1, 3, 4, 6, 7, 8, 9}
  • Identity (phenotype of Cellular Therapies) ^{2, 5, 7, 8}
  • Impurities (IIRMIs) ⁴
  • Pharmacodynamics ¹
  • Receptor Occupancy ¹
  • Receptor Binding, Affinity, Cross Reactivity ^{1, 9}

Clinical:

• Persistence (phenotype) and Proliferation of Cellular Therapy ⁷
• Immunogenicity of Cellular Therapies ⁷

CMC Manufacturing:

• Product Comparability / Bridging Studies (activity and potency) ^{2, 9}
• In-process testing for Cellular Therapies (viability, cell number, phenotype, receptor expression) ⁷
• Stability and Viability of Cellular Therapies ^{7, 8}
• Lot Release (Alloreactivity, Immunogenicity) ⁷

Referenced Guidance Documents:

1. S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals
2. Preclinical Assessment of Investigational Cellular and Gene Therapy Products
3. Human Gene Therapy Products Incorporating Human Genome Editing
4. Immunogenicity Assessment for Therapeutic Protein Products
5. Potency Tests for Cellular and Gene Therapy Products
6. Content and Format of Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-derived Products
7. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products
8. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)
9. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use