Over the past 20 years the demand for recombinant proteins has increased significantly. Mammalian cell lines have been extensively used to produce recombinant proteins. This expression system offers several advantages over microbial systems, mammalian cells have the cellular machinery to promote the secretion of the recombinant product and the posttranslational modifications, like glycosylation that is present in many of recombinant therapeutic proteins in the market. Human cell lines have emerged as a new and powerful alternative for production of such products. These cells are able to produce recombinant proteins with posttranslational modifications more similar to their natural counterparts, producing proteins with human-like glycosylation pattern avoiding immunogenic reactions against epitopes nonhumans. This review presents the available human cell lines that can be used in pharmaceutical industry, the advantages of this expression system and the main efforts made in this field.

Human cell lines have attracted great interest because they are capable of producing glycosylated proteins that are more similar to native human proteins, thereby reducing the potential for immune responses. However, these cells have not been extensively characterized and cultured under serum-free suspension conditions. In this work, we describe the adaptation, growth, and cryopreservation of the human cell lines SK-Hep-1, HepG2, and HKB-11 under serum-free suspension conditions. The results showed that both HKB-11 and SK-Hep-1 adapted to serum-free suspension cultures in FreeStyle and SFM II, respectively. Kinetic characterization showed that the HKB-11 and SK-Hep-1 cells reached cell densities as high as 8.6 × 10(6) and 1.9 × 10(6) cells/mL, respectively. The maximum specific growth rates (μ max) were similar for both cells (0.0159/h for HKB-11 and 0.0186/h for SK-Hep-1). The growth limitation of adapted cells does not appear to be associated with glucose or glutamine depletion, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production reached concentrations that are considered inhibitory to mammalian cells (2-5 mM). The adapted cells were also successfully cryopreserved under serum-free formulations. The SK-HEP-1 and HKB-11 cells that were adapted to serum-free suspension conditions might be suitable for use in the manufacturing of recombinant proteins, thereby eliminating the potential for the introduction of adventitious process contamination and greatly simplifying downstream protein purification.

Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100L scale for several biologics, including recombinant viruses. Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.