UNIT OPERATION / PRODUCTION PROCESS


Adherent Bioprocessing

Cell therapy bioprocessing activity mainly focuses to accelerate the safe, cost- effective translations and clinical efficacious of cell therapies into commercial products. This activity covers the entire range of cell therapy activities as well as tissue engineering. For biopharmaceuticals that are derived from microorganisms, adherent cells or suspended cells, a specific bioreactor must be used for each mentioned type of cells. Adherent cells are usually cultured in packed-bed bioreactor system, planar cell culture systems, and suspension platforms such as microcarriers and aggregate cultures.

Packed-bed bioreactor system

Cultures based on packed-bed bioreactors (PBRs) are systems in which the adherent cells are immobilized to a substrate enclosed in a packed-bed within a bioreactor system. A wide array of substrate materials and configurations can be used in a packed bed, it can be beads, porous structures, fibers, and hollow fibers. They are advantageous for scaling-up adherent cell production due to the following reasons: ability to reach high densities of cells per milliliter of packed-bed volume, real-time ability to control cell culture parameters (pH, dO­2, temp, perfusion, agitation), low shear-stress forces on cells during culture, beneficial biological attributes of cell cultures in 3D scaffolds.

Culturing adherent cells in a PBR includes three main stages: (1) cell seeding, (2) cell culture, and (3) cell harvest. Each step must be optimized based on the cell type, carrier properties, and packed-bed volume to achieve an effective process and optimal cell number and quality.

Factors that affect cell seeding efficiency:

  • Adherence properties of the cells
  • Physical properties of the carrier
  • Chemistry and electrical charge of the carrier
  • Rate and pattern of media flow during seeding
  • Dimensions of the packed bed

During the culture stage, online control of culture conditions must be optimized so as to ensure efficient cell proliferation and maintenance of cell health at high cell densities.

The most challenging stage in adherent-cell PBR culture is the cell harvest. This phase must be optimized to attain high yield while maintaining cell health and properties.

All of the three phases must be analyzed as well as optimized when increasing packed-bed size. The PBR system has a capacity for high cell density and control over culture parameters. One possible limitation is that the nutrient gradients in culture limit the height of the bed. Overall, this system is a promising platform technology for highly controlled and efficient commercial scale culture of adherent therapeutic cells.

Planar cell culture systems

Two-dimensional (2D) cell cultures or planar cell culture systems have been the mainstay expansion platform for adherent cells when handling low to moderate cell quantities. These culture methods are well established and easy to handle. The procedure is labor intensive and shows limited scale-up potential due to restricted available growth surface area. Maximizing the total area and the density of the cells are the main parameters to increase lot size of planar cell culture systems. For the scale up of these 2D systems, the following concepts must be applied: The larger the total surface area of the unit that has to be handled during the manufacturing process (number of layers and the size of each layer), the larger the possible harvest. For scaling up, use multiple of these units, but it has to be considered that the overall handling time is as short as possible in order to achieve constant and comparable results.

Suspension microcarrier and aggregate cultures

A potential benefit of using microcarriers for large-scale production is that the surface-area-to-volume ratio is greatly increased over traditional 2D cell culture methods. Many different types of microcarriers are commercially available. It can be made from polystyrene or from cross-linked dextran.

Different properties of microcarriers may significantly affect expansion rates and cell multi- or pluripotency. Some surface chemistry modifications can improve cell adhesion. These methods include applying positive or negative charges and coating with extracellular matrix proteins.

One advantage of using microcarriers is the increased control of a culture’s environment in a bioreactor based system. Technology borrowed from the development of single-use bioreactors for biopharmaceutical processes can be applied to growing therapeutic adherent cells. Bioreactor technology offers the ability to precisely control process parameters such as gas exchange, nutrient feeding, and pH. Following expansion, cells must be efficiently removed and separated from the growth surface, a process that is increasingly complex when moving from planar to microcarrier-based bioreactor cultures with large working volumes. The system chosen, microcarrier, and cell phenotype and differentiation potential is critical.

Cell harvesting and yield of microcarrier-based methods depends on efficiency of cell dissociation and separation from beads. Enzymatic treatment using commercially available recombinant animal-origin-free protease is commonly used to remove cells from microcarriers.

An alternative approach for harvesting is culturing these cells as aggregates. The advantage is that it does not need a carrier or extracellular matrix. The key to ensuring the success of this method is using single-cell seeding and maintaining high viability.

Regardless of the method used for harvest and cell concentration, robust quality control (QC) assays will be necessary to demonstrate product consistency and efficacy. So suspension systems can achieve homogenous cultures of high densities, but that will require fine control to maintain stem cell function as the volumes increase.

 

References:

  1. Gunther, C. et al., 2016. Advances in Pharmaceutical Cell Therapy: Principles of Cell-Based Biopharmaceuticals. Singapore: World Scientific Publishing Co. Pte. Ltd., pp. 128-133
  2. Rowley, J. et al., 2012. Cell Therapies Manufacturing: Meeting Lot-Size Challenges of Manufacturing Adherent Cells for Therapy. BioProcess International, pp. 16-22
  3. Schnitzler, A. et al., 2017. Harvest of Human Mesenchymal Stem Cells from Microcarriers. BioPharm International