Serum-Free Suspension Adaptation of HEK-293T Cells: Basis for Large-Scale Biopharmaceutical Production

Abstract Human Embryonic Kidney 293T cells (HEK-293T) are the most common host for viral vector production and are also widely employed for recombinant protein production. These cells are typically cultured in monolayer (adherent culture) using culture medium containing fetal bovine serum (FBS), which impairs batch-to-batch reproducibility and scale-up. The adaptation of adherent cell culture to suspension culture in chemically defined serum-free culture medium is an attractive approach for large-scale bioprocess implementation while aiming for a Good Manufacturing Practice (GMP) compliant production process. Therefore, in the present study, our goal was to adapt HEK-293T cells to serum-free suspension culture conditions and evaluate the feasibility of adapted cells to be transfected using different plasmid vectors for recombinant protein production. Firstly, the cells were efficiently adapted to serum-free conditions by sequential adaptation (FBS-containing medium weaning). During the whole process, parameters such as cell growth, viability and doubling time were evaluated and compared to the control (adherent serum-supplemented HEK-293T cell culture). Afterwards, these cells were adapted to suspension culture by using Erlenmeyer flasks in an orbital shaker platform, being able to achieve meaningful cell density with high viability. Adapted cells presented a transfection efficiency of approximately 50% for all vector constructs used (1054-GFP, Factor-VIII and Factor-IX). Overall, it was possible to successfully adapt HEK-293T cells to suspension and serum-free conditions, which represents an important step towards the development of a scalable and GMP-compliant production process. In addition, adapted cells efficiently expressed the different transgene tested, opening up possibilities for its use in recombinant protein production.

Strategies to Suspension Serum-Free Adaptation of Mammalian Cell Lines for Recombinant Glycoprotein Production.

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.

Purification and biochemical characterization of an extracellular serine peptidase from Aspergillus terreus.

Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S2 did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S1 showed higher catalytic efficiency than the S2 and S3 subsites.

Serum-free suspension culturing of human cells: adaptation, growth, and cryopreservation.

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.

Production of recombinant therapeutic proteins in human cells: current achievements and future perspectives.

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.