Cellular immunotherapies (e.g. CAR-T cells) are primarily used as an autologous therapy to treat cancer. As such, these therapies are currently generated in small batches for each patient. To generate enough modified cells for a single treatment, cells are expanded in volumes from 1-10 L. In the final step of downstream processing (DSP) for immunotherapies, cells cultured in large volumes must be concentrated and reformulated into smaller volumes (e.g. 20-100 mL) suitable for delivery to patients.

In this post, we will outline the key steps for concentration and reformulation and highlight specialized equipment needed for this critical unit operation. CCRM’s team has leveraged a partnership with GE Healthcare (GEHC) to create an innovative process based on modular and closed unit operations that can be staggered to produce multiple doses of an autologous therapy simultaneously.

Steps for concentration and reformulation of immunotherapies:

1. Transfer from bioreactor: Traditionally the transfer of cells grown in suspension from one vessel to another would be accomplished by moving vessels such as bottles, tubes and flasks into and out of a biosafety cabinet (BSC), whereby fluid volumes are manipulated with pipettes. This process is open, manual, and not scalable. Instead, we have developed a fully process using wave-mixed bioreactors where cells are expanded in single-use bags (up to 10 L) that can be aseptically welded directly onto equipment for isolation, harvesting and final formulation of cells (e.g. Sefia Cell Processing System S-2000, kSep, LOVO, Rotea). 

2. Volume reduction, washing and final formulation: Currently, immunotherapy workflows rely on bulk centrifugation including labour-intensive, open manipulations in a BSC. In our process, these steps are carried out by one piece of equipment that combines several steps, reduces labour costs, and has built-in automation capabilities. This device uses single-use consumables, accommodates sterile welds for seamless unit-op to unit-op transfer, and can process 1 L of cell suspension down to 40 mL in less than an hour. This represents an important advancement over traditional methods using a centrifuge because system closure and automation can significantly reduce the risk of operator error and contamination while increasing process robustness. Final product formulation most commonly involves the addition of a cryoprotectant to cells under a BSC (often in cryovials) as cryopreservation is a critical unit operation and is almost always required for immunotherapies that will be shipped to hospitals from centralized manufacturing facilities. Equipment such as the Sefia CPS S-2000 can add cryoprotectant to bags, control rate of cryoprotectant addition, temperature, and mixing to minimize potential damage to the cells.

More information on the key steps above and methodologies compatible with industrial workflows for CAR-T manufacturing can be found here.

For a discussion about how we can provide the right solutions for your needs, please contact us.

Future process improvements for DSP of immunotherapies:

As immunotherapies evolve, so will the processes that therapy developers use to produce them. For instance, as we move towards the use of allogeneic therapies manufactured in large bioreactors, we will need to build DSP equipment that can accommodate much larger volumes.

While we have made significant strides in the development of closed and automated processes, there remain opportunities for further improvements. For example, a gap in the field is a method for rapid and high-throughput bag filling that would allow for the creation of master cells banks under completely closed conditions. Another key gap is the lack of a closed and automated system for removing air from cell cryopreservation bags prior to freezing. Air bubbles interfere with uniform and optimal cooling of cells, and currently are being removed manually in a biosafety cabinet by many developers, which opens the final product to contamination risk. An automated solution to overcome this challenge would save time and greatly reduce variability and risk.

Ultimately, the process developed at CCRM, in partnership with GEHC, was informed by the needs of industry. Our team will continue to work with clients to understand their priorities, build custom solutions, and improve our existing processes.