Automating Blood Cell Population Separation

Industry collaboration delivers good manufacturing practices approach to peripheral blood mononuclear cells separation

By Arnaud Foussat, VP research and new products, TxCell; and Sébastien Olivier, sales manager, Biosafe

Cellular immunotherapy is a rapidly developing field that holds great promise in oncology, autoimmunity and chronic inflammation as well as transplantation. As one example, cancer immunotherapy research was awarded the scientific breakthrough of the year by the journal Science. In addition, the recent deals between biotechs and academic centers developing cellular immunotherapies and pharmaceutical companies highlight the interest of pharmaceutical industry stakeholders for these new therapeutic approaches.

Cellular immunotherapy uses the properties of cells from the immune system either to fight tumors through cytotoxicity mechanisms involving effect or T cells or to decrease the immune mediated pro-inflammatory processes in patients with autoimmune and chronic inflammatory diseases using regulatory T cells. A recent boost in the field has been observed with significant results of remission in late stage lymphoma patients treated with engineered T cells. This is the most recent demonstration of the huge promise of immunotherapies where classical treatments have failed.

ONE KEY CHALLENGE
One key challenge faced by cellular immunotherapies is the standardization of the cell processing procedures. These procedures are an integral part of the manufacturing process. So far there are few tools that allow cell therapy production methods to achieve a high scale production capacity that includes process robustness and pharmaceutical grade.

Peripheral blood mononuclear cells (PBMC) represent the most easily accessible source of immune cells. Consequently, the common first manufacturing step for many cellular immunotherapies is the isolation of PBMCs from whole blood collections. The classical method for PBMC separation is performed using centrifugation in gradient density media such as Ficoll, Percoll or Optiprep. This allows the separation of blood cell populations according to their respective density.

In the Ficoll procedure, the platelets, red blood cells, granulocytes and blood dendritic cells are pelleted in the bottom of the centrifuge tube. A ring of mononuclear cells comprising T cells, B cells, NK cells and monocytes are kept in suspension in the diluted centrifuged Ficoll medium allowing their harvest for further manufacturing steps. This procedure is routinely used manually worldwide as no specific device has yet been set up to standardize this separation procedure.

As a result, however, operator-dependent variability can be observed on top of the starting material variability, in terms of cellular impurities (i.e., residual red blood cells and granulocytes in the mononuclear cell ring) and separation performance (mononuclear cell yield). Such variation can dramatically impair the manufacturing robustness of cellular immunotherapy products. Indeed, in these types of products, where significant donor-derived variability can be already expected due to starting material, any further variability caused by the manufacturing procedures should be avoided.

TEAMED TO TACKLE THE CHALLENGE

TxCell and Biosafe, two companies well-established in the field of cell therapy decided to jointly tackle this challenge with the aim of offering a standardized and automated solution for this common first step of most cellular immunotherapies. Indeed, besides the need to increase the robustness and productivity of cell therapy production processes through automation, the regulation of medicinal cell therapy products as part of the Advanced Therapy Medicinal Product (ATMP) directives and guidelines requires production of these innovative treatments under Good Manufacturing Practices (GMP). Full environmental monitoring and control for the absence of microbiological contamination are critical elements for GMP production of cell based ATMP. This is because these products are made from living materials and cannot be sterilized before release. As a consequence, standardization of cell processing procedures should include, in addition to automation, systems to control these parameters, for example, by implementing closed systems.

French biotech company TxCell is developing economically viable, autologous cellular immunotherapies for chronic inflammation and autoimmune diseases. TxCell’s products are based on the anti-inflammatory and immuno-modulatory properties of a subtype of blood leucocytes called antigen-specific regulatory T cells (Ag-Treg). Through ASTrIA, its proprietary discovery and manufacturing platform of Ag-Tregs, TxCell has already advanced its first Ag-Treg product candidate, Ova save, into the clinic stage. TxCell has also demonstrated the tolerability and potential benefit of this product for Crohn’s disease patients refractory to existing treatments. TxCell is also already authorized by the French regulatory agency (ANSM) to produce cellular immunotherapy products for clinical use in its GMP-certified manufacturing site in Besançon, France.

With 17 years of experience, Swiss company Biosafe’s cell-processing technology supports the automation and standardization of cellular immunization therapy production. SEPAX 2, Biosafe’s automated technology is commonly used for stem cell processing and is a recognized and efficient tool for stem cell isolation. More recently, pharmaceutical and biotech companies working in the cell therapy area are increasingly adopting SEPAX 2 technology.

KNOWN AS POSITIVE
Known as “POSITIVE,” the TxCell and Biosafe project includes the participation of the cell and gene therapy unit (CGTU) of the CHU (University Hospital) of Nice, France. It aims at standardizing the PBMC separation procedure using Biosafe’s device to produce cellular immunotherapies under GMP. The project has been awarded a grant by the French Provence-Alpes-Côte d’Azurregion.

Biosafe is contributing its expertise in automated cell processing and cell separation techniques. This will allow fine-tuning of the device software and processing kits for GMP manufacturing of therapeutic immune cells. TxCell will add its expertise in clinical-grade GMP manufacturing of regulatory T cells from peripheral blood. Lastly, the Nice CGTU will offer its expertise on clinical grade production of effector T cells.

The primary objective of the project is to bring performance consistency and promote separated PBMC purity levels. This will ensure suitable properties of the PBMC preparations for further cell processing steps that can involve cell-specific stimulation steps. It will also enable GMP compliance by ensuring aseptic handling of cells in a closed system.

One of the first R&D steps will evaluate the parameters to be fine-tuned as well as the suitability of the procedure for immune cell therapy production. A second step involves concentrating on validating the new, improved PBMC separation method in one of TxCell’s upcoming clinical trials.

Ultimately, the standardization of this procedure will benefit all developers and manufacturers of cellular immunotherapies, as standardization will allow scale-up with robust and automated cell processing methods.

By bringing together an industrial developer of innovative treatments, an academic cell therapy unit and a developer of devices, the project provides a demonstration of how different stakeholders can overcome a scientific and industrial challenge by sharing their strengths for a common goal. This collaboration demonstrates how teamwork can push forward a field like cellular immunotherapy to fulfill the requirements of pharmaceutical industrial commercialization and distribution. Most importantly, the project leaders hope the project will prove an ideal route to ultimately bring new, innovative treatment solutions for patients.

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