A: Many common perfusion platforms utilize hollow fibers as a cell retention mechanism in order to be able to flow media at an acceptable rate. ATF tank attachments, for example, are a common perfusion system that use HFs like a filter, retaining and circulating the cells back into the tank while passing the cell product through the membrane into a holding tank. However, there are several distinct advantages to C3’s HF perfusion bioreactor design when comparing to ATF tank systems:

• Homogenous Environment: Cells are grown in the extra-capillary space of the hollow fibers, while bulk media flow brings nutrients through the intra-capillary space. This allows for cells to be grown to a high density (~10E9 cells/mL) without exposure to shear force. In ATF systems, the cells are freely moving through the tank and ATF space with the flow of media and thus exposed to shear. This is important, since cells experiencing too much shear could produce low-quality protein products.
• Save on Media Use: C3’s bioreactors use less media than ATF systems. C3’s bioreactors are designed as high surface area cartridges where cells can grow to high densities in a relatively small space. While media is constantly flowing throughout the bioreactor run, total media usage remains low compared to ATF systems. An ATF-equipped tank is designed to grow cells in a comparatively large tank with very low surface area, where several vessel volumes per day (VVD) of media may be needed. Additionally, to keep the cells alive and producing product, ATF tanks need to be “bled” every day to limit the cell count, which can be up to 30% VVD. C3’s bioreactors do not need to be bled, because the cell mass is embedded in the fiber bundle and takes on the density and consistency of live tissue. Our bioreactors are therefore designed to maintain the cells in a fixed static space with the capillary fibers providing the dynamics to deliver nutrients and remove waste.
• Long-term, stable production: C3’s bioreactors can maintain cell health and protein production for up to 100 days (or longer). We have data to show that C3’s bioreactors maintain a consistent environment for the cells as waste is constantly removed and parameters such as pH and lactate are automatically monitored and controlled. This leads to longer periods of protein production, which is especially important for difficult-to-express proteins or low-producing cell lines. ATF systems can support protein production for 30-60 days before cell viability (and thus protein production) is dramatically reduced due to membrane fouling and accumulation of dead cells.
• Easy Protein Retention: In C3’s bioreactors, protein product is retained and collected from the extra-capillary (or cell) space. It does not have to cross the hollow fiber membrane to be collected. In contrast, protein produced in ATF systems must cross the hollow fiber membrane, and collection is therefore subject to clogging and fouling.
• Consistency: The homogenous environment in C3’s bioreactors leads to consistent protein product. In an ATF tank system, cells are freely floating in the tank, and cells near where the ATF attachment draws media out may be subjected to more stress than cells in other parts of the tank. This leads to non-homogenous cell growth and protein production, which could affect the quality of product as a run progresses.

A: C3 is capable of producing proteins in both a GMP and RUO manner in quantities ranging from a few mg to up to 500g of protein product. C3’s hollow fiber bioreactors are most cost-effective in the 100s of mg to 500g range. For smaller quantities of protein, C3 can offer use of more traditional cell culture platforms such as flasks or roller bottles under either GMP or GLP guidelines.

A: C3 has performed a leachable/extractable study on the the Autovaxid bioreactor system. The results showed that all levels of extractables and leachables were acceptable for the process supporting a Phase III human therapeutic drug. As with other single use bioreactor systems, each process requires testing as different media, cells and culture conditions (temperature, pH, lactic acid level, time) will have an impact on the L&E levels.

A: C3’s HFBR technology is ideal for challenging and small- to mid-scale protein production. Tank-based bioreactor systems are very effective for high-yielding CHO cell lines where IgG production of multiple kg/year is required. However, tanks are often not cost-effective for the production of certain types of challenging molecules and small- to mid-scale projects. Pre-clinical, toxicology, ex vivo, and clinical trial protein production is challenging to cost-effectively accomplish in a GMP environment. C3’s bioreactors are cost-effective and ideal for the mg to kg range of protein production. The small bioreactor footprint, high level of automation, and design of the bioreactors save money on facility usage, labor, and raw materials.

We have previously shown that C3’s HFBR system can effectively produce difficult-to-express proteins, like many recombinant, chimeric, or fusion proteins. Additionally, low-yielding cell lines, cell lines sensitive to shear stress, or cell lines exhibiting feedback inhibition can produce higher yields in our perfusion bioreactors than they can in tank culture.

The small bioreactor footprint and fast turnaround time allowed by the single-use cartridges are also ideal for autologous applications.

A: C3 has a number of case studies demonstrating that production of challenging proteins and cell lines can be dramatically improved by use of C3’s perfusion technology:

• One customer (HC3 recombinant protein data) had a low-expressing cell line that was determined to be due to feedback inhibition of the expressed protein. In this case, the continuous harvest of protein product accomplished by C3’s technology solved the problem. Others have observed benefits to having constant waste removal.
• High cell density, low shear stress, and consistent metabolic parameters lead to high cell viability and consistent protein production. For cell lines that are proving challenging in tank systems, this can significantly improve yield.

A: We frequently work with customers who have specific analytical needs. If you have an assay or require a test that is not listed on this site, we’d be more than happy to discuss your options here at C3!