Mammalian cell lines

The Chinese Hamster Ovary (CHO), myeloma, and hybridoma cells are the most used mammalian cell lines for biopharmaceutical production. Significant progress in optimizing these systems, particularly the NS0 myeloma and CHO cell lines, makes them more attractive for use. Both expression systems are currently predominant in biopharmaceutical protein production.

Other factors too add to the advantages, resulting in high-specific productivities ranging 20–60 pg × cell–1× day–1. They are:

• Fine-tuning of vector construction;
• Identification and use of selectable markers;
• Optimization of transfection in production media;
• Gene targeting;
• High-throughput screening;
• Shortened lead-time (3-5 months) for clone generation for certain clones.

Other mammalian cell lines used for biopharmaceutical and vaccine production are: CHO, DHFR-, DG44, DUK-B11, NS0, HEK 293, BHK, COS-1, COS-7, Sp2/0, PER.C6

Over the past few years, improvements in mammalian expression systems have focused on genetic modifications of host cells. This helps improve their ability to produce more recombinant proteins with the proper Post-Translational Modifications or PTMs (for product quality). Many studies have also focused on metabolic control of cell growth: regulating life cycles and limiting apoptotic death.

Processes

A standard media formulation supports the growth of different types of cells and cell lines; but cell growth is no longer a sufficient criteria for cell culture researchers. They look for more specialized media that support the growth of healthy cells with normal morphology and activity, such as protein expression; i.e., media tailored to a specific cell line and application.

Current regulatory requirements strongly discourage or ban the use of products derived from animals, specially bovine sources, in the culture media. Therefore, the use of bovine serum, common earlier in mammalian cell culture, has been discontinued. Significant efforts have been made to develop cell culture media that is free from animal-derived products. Trends suggest a growing use of chemically defined media to minimize the risk associated with the addition of animal-derived components. CHO and other host cell lines used for antibody production are now selected for their ability to grow and produce product at high-levels in chemically defined media.

Initially, perfusion technology – in which the media is continuously removed from the bioreactor and replaced afresh – was used to increase the yield of antibody products from a single bioreactor. Its underlying rational is that cells in culture could continue to produce proteins over several weeks if the conditioned media, containing the antibody product along with potentially growth limiting metabolites, were replaced regularly with fresh media and growth factors.

Years of comparative work have shown that perfusion cultures can achieve higher volumetric productivities than fed-batch cultures at the expense of lower product titres per liter of medium consumed. Moreover, the continuously changing media conditions and long culture times required for perfusion production frequently lead to inconsistent processes, variable glycosylation, and other PTMs in the product, over time, in culture. Contamination risk also increases.

However, perfusion operations tend to be complex, difficult to scale-up, and generally less robust than fed-batch processes. Therefore, fed-batch culture is now popular for robust, reproducible, and reliable manufacturing processes. While the capital investment in a manufacturing facility using fed-batch culture is higher than in a perfusion-based facility, the overall cost of goods for both processes are similar.

The challenges

Biologics produced from mammalian cells present two challenges: purification and consistent delivery of quality. At Inbiopro, we have reliable solutions for both.

• Our resin library is continuously updated with valuable resins. We proactively use the latest resins to benefit from better selectivity and resolution. This helps reduce charge variants and glycan-related heterogeneity. We have optimized processes for the leading MAb with reduced heterogeneities, which are important for biosimilarity and influence the biological functions.
• We follow a Quality by Design (QBD) based platform for designing process parameters and combining effective process and facility validations. We are committed to delivering batch-to-batch reproducible material quality and evaluate different formulations of the product to conform to international standards.