6.2 High Cell Density Cultivation Process

What is it about?

Since human tissue plasminogen activator (tPA) became in 1987 the first approved biotherapeutic obtained from mammalian cells using recombinant DNA technology1, the biopharmaceutical market rapidly expanded based on a development pipeline of several mammalian cell culture-derived drugs. Even more, mammalian cells are still the dominant recombinant protein production systems for clinical applications2. In this scenario, high-throughput production processes of therapeutics, expressed in mammalian cells, are necessary to fulfill the market demands. The success of a process depends both on cell productivity (defined as the quantity of the interest protein produced by cell) and the viable cell density in the culture. Considering these parameters, one of the applied strategies to obtain a high recombinant protein yield is the development of culture systems that reach high cell densities and sustain cell viability, leading to an increment of the volumetric productivity. Animal cells can be grown using different modes of operation, and the maximum cell density that can be achieved will depend on that selection. The method of cultivation defines the environmental and nutritional conditions under which cells will proliferate and synthesize the protein of interest, determining the success of attaining high viable cell density and, consequently, high concentration of the desired product. A general classification distinguishes discontinuous modes (batch, repeated batch and fed-batch) and continuous ones (with cell retention, perfusion or without cell retention, chemostat). Despite the fact that suspension cultures are by far the most common cell culture format for recombinant protein expression in mammalian cells3, anchorage-dependent cell lines are also used in the industry for the mentioned purpose. Consequently, the scaling up of a process where cells grow in adherence can be carried out by cultivating them as monolayer in static or gently agitated surfaces or by growing cells attached to polymers spheres that are introduced in bioreactors to maintain them in suspension at high density cultures.

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Why is it important?

The present chapter comprehensively describes the approaches for high cell density cultivation of animal cells taking into account strategies to grow them in suspension or immobilized into solid supports.