Mammalian Cell Culture Clarification
Mammalian cells are cultured by a variety of methods at a range of volumes for the production of therapeutic and diagnostic proteins. The cells are grown in a bioreactor or fermentation vessel. The first step in the purification process is the removal of whole cells, cell debris, and particulates prior to further downstream unit operations.
Understanding Cell Lines
Cell lines typically employed for mammalian cell culture include CHO, NS0 hybridoma cells, baby hamster kidney (BHK) cells, and PER.C6™ human cells. The most commonly employed mammalian cell line used for mammalian cell culture at production scale volumes are the CHO and NS0 cell lines. These cell types are relatively easy to genetically engineer, have been extensively characterized, are relatively easy to grow at large scale, and can excrete high titers of recombinant proteins in solution. Both CHO and NS0 cell lines can produce high protein expression levels, however, cell viability tends to decline with high protein expression levels. Low cell viabilities burden downstream purification with cell debris, DNA, host cell protein and other impurities. NS0 cell lines require additional media components to support cell growth (i.e. cholesterols, lipids, etc.) that can affect downstream processing. Hybridoma cells have relatively low protein expression levels (<100 mg/L) and are typically grown to low cell viabilities (<30%) which complicates the harvest operation.
Cell Culture Media Types
Cell culture medium typically contains amino acids, vitamins, electrolytes and may contain hormones, growth factors, plant hydrolysates, animal serum, antibiotics, antioxidants, antifoams, cell stabilizers and other components. Types of media include serum based, serum free, animal free, and protein free or chemically defined. Serum based and serum free media are widely used since they can be used to grow a broad range of cell types and cell lines such as CHO or NS0 cells.
Comparison of Cell Culture Operating Modes
| Operating Mode | Batch | Fed Batch | Perfusion |
| Media Addition | Initial | Periodic | Continuous |
| Fluid Harvest | Final | Final | Continuous |
| Benefits | Less cell debris, lower risk of contamination | High titer | High productivity Low capital cost |
| Disadvantages | Fair productivity Frequent equipment turnover | Maintaining sterility in bioreactor Medium cell debris | Maintaining sterility in bioreactor and in the primary clarifier High cell debris |
Cell Culture Characterization
Evaluating the cell culture characteristics is a critical step in developing an effective process. Below are some generally accepted culture properties that can be used to help guide the technology selection.
| Culture Type | Batch 3-7 Days | Fed Batch 7-15 Days | Perfusion >20 Days |
| PSV (Solids) | Low <1% | Medium 2–3% | High >3% |
| Cell Density | Low <3 x 106 cells/mL | Medium to High 5–15 x 106 cells/mL | High >10–15 x 106 cells/mL |
| Cell Viability | High >90% | Medium to High 20–90% | Medium to Low <50% |
| Colloids | Low | Medium to High | High |
| Turbidity | Low <200 NTU | High 500 to >1,000 NTU | Very High >>1,000 NTU |
| Ease of Clarification | Easy | Medium | Difficult |
