Expression of Recombinant Antibodies

Frenzel, André; Hust, Michael; Schirrmann, Thomas

Eukaryotic cells have developed an advanced folding, posttranslational, and secretion apparatus which enhances the secretory production of antibodies, including full immunoglobulins compared to bacteria. Yeasts combine the properties of eukaryotic cells short generation time and ease of genetic manipulation with the robustness and simple medium requirements of unicellular microbial hosts. Moreover, yeasts have been used for fermentation in food production for several millennia in human history; they do not produce bacterial endotoxins and have gained the GRAS status paving the way toward production of therapeutic proteins (90, 91). Pichia pastoris represents the major yeast strain used for recombinant antibody production (92). Other yeasts like Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe (93, 94), Schwanniomyces occidentalis, Kluyveromyces lactis, and Yarrowia lipolytica (95) have also been described for protein production but have played only a minor role. P. pastoris shows overall optimal capacity for the production and secretion of heterologous proteins than S. cerevisiae and does not secrete large amounts of its own protein which simplifies the downstream processing. Moreover, P. pastoris prefers respiratory growth resulting in high-cell densities of more than 100 g/L dry weight (96). Probably the most prominent feature of P. pastoris is the metabolization of methanol as sole carbon source. The alcohol oxidase 1 (AOX1) promoter is strictly controllable by methanol and commonly used for recombinant protein expression. The secretory production of heterologous proteins including antibodies requires an aminoterminal signal sequence targeting the yeast's secretory pathway. S. cerevisiae mating factor alpha (alpha-factor) pre-pro peptide is the most commonly used secretory signal sequence and is followed by appropriate proteolytic cleavage sites sensitive for the Golgi resident endoprotease KEX2 for efficient release of antibodies during secretion, which is often used in combination with ST13 exoprotease sites (97).