Effect of two preservation methods on the viability and enzyme production of a recombinant Komagataella phaffii (Pichia pastoris) strain

• Published in: Cryobiology Vol. 105; pp. 32 - 40
• Main Authors: Alvarado-Fernández, Angela María, Rodríguez-López, Edwin Alexander, Espejo-Mojica, Angela Johana, Mosquera-Arévalo, Angela Rocío, Alméciga-Díaz, Carlos Javier, Trespalacios-Rangel, Alba Alicia
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 01-04-2022
• Subjects: Cell survival; Cryopreservation; Cryopreservation - methods; Disaccharides; Freeze drying; Glycerol - pharmacology; Hexosaminidase A; Humans; Komagataella phaffii; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Saccharomycetales; Sorbitol - pharmacology; Yeast; Freeze drying; Komagataella phaffii; Hexosaminidase A; Cell survival; Yeast; Cryopreservation
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/j.cryobiol.2021.12.004

The methylotrophic yeast Komagataella phaffii, previously known as Pichia pastoris, has been reported as a host for producing human recombinant lysosomal enzymes intended for enzyme replacement therapy. K. phaffii has advantages such as easy genetic handling, rapid growth, cost-effective mediums, and the ability to develop mammalian-like post-translational modifications. To maintain cell viability and enzyme activity over time, it is important to consider the bioprocess optimization and the proper selection and preservation of clones. In this study, we evaluated the effect of glycerol and skim milk in cryopreservation at −80 °C, as well as the use of skim milk or its combination with NaCl, disaccharides or sorbitol in freeze-drying on the cell viability and activity of a recombinant lysosomal enzyme (i.e., human β-hexosaminidase-A) produced in K. phaffii GS115 strain. The results showed that cryopreservation with glycerol and skim milk, as well as freeze-drying using disaccharides and sorbitol with skim milk, maintained the viability above 80%. Although variations in enzyme activity among treatments were found, the use of disaccharides had a positive effect on the enzymatic activity levels. This is the first report of the evaluation of two suitable methods to preserve a recombinant K. phaffii strain, preventing the loss of viability and maintaining the activity of the recombinant protein.
•Cryopreservation and freeze-drying were evaluated in a recombinant yeast K. phaffii.•Ten different treatments using cryoprotectants alone or mixed were done.•Both methods maintained the viability and the biological activity over a year.•Inserted genes remained stable over time.•First report of preservation methods for a recombinant K. phaffii strain.