Manufacturing Processes for Engineered T-Cell Therapy – CAR-T

Why T cells are important

They recognize host vs. non-host cells to initiate an immune response
They’re the primary immune system agents that attack diseased cells or infection
They’re used in several cell-based therapies:
- Transplantation of donor T-cells - a bone marrow transplant, in which a matched donor’s cells are used to replace the patient’s cells, is the most widely used approach—typically for diseases that originate in the immune system (e.g., leukemia).
- Isolation and enrichment of a patient’s T-cells – an approach considered the safest because it enhances a patient’s own immune response to disease.
- CAR-T therapy – a more recent approach in which T-cells are removed, genetically engineered to express a synthetic receptor against specific cancer cells, then infused back into the patient’s bloodstream; this therapy is effective because many cancer cells “hide” from the immune system by appearing to be normal cells, thereby triggering no immune response.

Examples of parameters collected during cell therapy production

Data Collected	Why and When
Viable Cell Count	Used throughout process to monitor cell viability and impact of any cell manipulation events.
Cell Size and Volume	Used as label-free method to monitor cell population during expansion, differentiation and/or transduction stages. Cell volume is a critical indicator of the activation state of the T-cells.
Cell Count	General population monitor, particularly during expansion stages and when sample volume is manipulated (e.g., during concentration and packaging)
Aggregate Detection	Primarily a concern during packaging and prior to administration
Gene Expression	Looking for specific surface marker expression or reporter gene expression
Metabolic Indicators	General monitoring during expansion stages when cells are under continuous culture
Immunophenotyping	Critical to ensure correct cell types are isolated and enriched in therapy population; often used to define purity

CAR-T Production Workflow

*The DART data manager captures data from devices integrated into a Biomek robotic workstation. The Vi-CELL BLU and CytoFLEX can both be optionally integrated to a Biomek to enable this data capture and association with a sample ID.

T-Cell Isolation

Output material generated in CAR-T cell manufacturing can be significantly affected by the starting raw materials. Leukapheresis is used to prepare/concentrate whole blood components (e.g., T-cells separated from red blood cells).

To maximize output of the CAR-T cell manufacturing process, samples are assessed for critical criteria, including:

Cellular viability
Change in cell volume
Cell counts
Rapid microbial testing
Mycoplasma testing
Immunophenotype

MULTISIZER 4e AND VI-CELL BLU ANALYZERS²

Defining conditions for cell viability and cellular proliferation are critical indicators of isolation conditions.

Evaluate incoming samples
Accurately analyze change in cell volume/size
Rapidly determine cell count
Supports 21 CFR Part 11 compliance

AVANTI J-26S XP HIGH-PERFORMANCE CENTRIFUGE FOR ELUTRIATION

The counterflow centrifugation elutriation system is a gentle, yet powerful technique for harvesting large populations of living cells, resulting in high viability rates.

Collects/separates cells using the JE-5.0 elutriation rotor
Lets users select chamber flow rate and centrifugal speed based on cell size
Uses counterflow to separate based on density/size
Pushes cells into flask for easy, high-yield collection
Uses centrifugal force versus flow pump

BIOMEK i5 LIQUID HANDLER

Dispensing raw material inputs into unitized formats reduces manual manipulation steps, thereby reducing variability. Biomek iSeries workstations further reduce operator-induced variability by shuttling samples to integrated end-point analysis platforms (e.g., cell counters, flow cytometers).

Start-to-end automation
Innovative integration options
Supports 21 CFR Part 11 compliance

CYTOFLEX FLOW CYTOMETER¹

Integrated CytoFLEX flow cytometers can phenotype and quality check raw materials for the manufacturing process to ensure standardization of protocols.

Immunophenotyping of cells to characterize target T-cell populations
Supports 21 CFR Part 11 compliance

DURA INNOVATIONS¹

Standardized dry antibody panels for comprehensive immunophenotypic characterization of source materials.

Reduction of error-prone antibody pipetting
Room temperature-stable unitized format, minimizing reagent inventory management
Automation-friendly streamlined staining protocols
GMP-compliant manufacturing

T-Cell Activation

T-cell activation and population expansion are critical for viral vector delivery and increased autologous dosage availability. Cell volume is a critical indicator of the activation state of the T-cell.

MULTISIZER 4 e COULTER SIZE ANALYZER²

Monitor:

Cell volume change after activation to determine cell state
Cell proliferation during expansion

VI-CELL BLU AND VI-CELL METAFLEX ANALYZERS²

Viable cell count using trypan blue if required/desired
Rapid metabolite analysis to monitor bioreactor health

GENE TRANSFER: VIRAL VECTOR DELIVERY

Transformation of endogenous T-cell populations that carry a diverse array of antigen receptors into a homogeneous population with a target-specific receptor is the hallmark of CAR-T cell technology.

The process for efficient viral vector delivery presents significant challenges when performed manually. Viral vector delivery for CAR is achieved in small doses and by incremental up-scaling to improve transduction efficiency. Furthermore, culture conditions must be continuously monitored for in-process control.

For example the culture media is routinely assessed for bioanalytes such as pH, pO₂, pCO₂, glucose, lactate, electrolytes and more.

BIOMEK i5 LIQUID HANDLER

Start-to-end automation
Innovative integration options
Supports 21 CFR Part 11 compliance

MULTISIZER 4 e COULTER ANALYZER²

Monitor:

Cell volume change after transduction with CAR vector
Cell proliferation during expansion

VI-CELL METAFLEX²

Rapid in-process control
Rapid metabolite analysis to monitor bioreactor health