Tumor-based Bioreactor

The primary means of large-scale production of therapeutic proteins in the pharmaceutical industry is through the use of bioreactors containing cells that have been modified to secrete the protein of interest. Typically, a cell, like e. coli or yeast is transfected with a designed plasmid. The cells own machinery is utilized to synthesize the protein from the DNA. The primary problem with this approach is the inability of these cells to perform post-translational modifications such as attaching sugar molecules to the protein surface. As a result, some proteins cannot be synthesized. One method to overcome this problem is to use a tumor cell line or spleen cell/carcinoma combination called a hybridoma. These cells, since they are derived from mammals, can perform additional modifications. They usually have desirable properties, like fast growth and immortalization, that bacteria and yeast have.

Different types of bioreactor have been developed to grow these cells. The most basic is the batch reactor. In this type, cells are placed in the bioreactor with liquid media for food and allowed to expand over a certain period of time. At the end, the cells are entire contents are removed and the protein recovered. For constant operations, the main alternative is the continuous stirred tank reactor (CSTR). Cells and fresh media are fed slowly fed into the reactor at the same rate that product is removed. An extension of this type is the perfusion reactor where the cells are actively retained in the bioreactor while cell free product is removed. A separate stream is typically drawn off to help remove any dead cells. Methods for cell retention include membrane separation as well as settling by gravity. From batch to CSTR to perfusion, the bioreactor increases in complexity. Also, perfusion bioreactors (5x10E7 cells/ml) can obtain a much higher cell density than the CSTR(10E7) or batch (10E6). The higher cell density allows for more efficient use of media and smaller bioreactor volume. As the cell density increase, oxygen and nutrient deprivation becomes a problem. Overcoming this problem typically involves increased either mixing or oxygen flow rate. Both have their limitations and may even lead to problems such as shear induced cell death.

A unique cell line has been isolated from a subcutaneous melanoma by researchers at the University of Iowa. These cells have been shown to develop their own tubular network that mimics the blood vessels found in tissue (also known as vasculogenic mimicry). Since carcinomas can also be transfected to secrete a therapeutic protein, these cells can be used as part of a novel bioreactor to create a tissue dense (10E8-10E9 cells/ml) culture. Initial studies will involve studies to determine cell growth rates, oxygen and nutrient uptake rates, as well as fluid permeation rates through the tissue. The basic design of the bioreactor will be a hollow fiber membrane system (Figure 1). Cells will be fed into the shell side of the bioreactor. Fresh media will be fed into half of the fibers that will be blocked on one end. The fluid will permeate through the cells and enter the other half of the fibers (also blocked on one end) before leaving the bioreactor. These cells will also be transfected to produce a therapeutic protein. Studies will be performed to examine seeding density, cell growth protocol, flow rates, cell death rates, uptake rates, and production rate.

Specific Projects:

• Optimization of media ingredients to reduce the need of serum

• Determine factors to encourage tissue formation without the need of a collagen supoort matrix

• Evaluation of cell growth, and nutrient consumption within the bioreactor

• Transfection of a gene into the cells

• Scale up of bioreactor with model simulation

• Economic design of bioreactor and purification system applied to orphan drug synthesis

Figure 1: Schematic of tumor based bioreactor. The cells are placed into the central volume of the chamber separated by a porous membrane at the top and bottom  Media is pumped through the bottom of the bioreactor through the tumor tissue.