Montoya N, González N, Sánchez I, Pimentel R, Basabe L, Basulto RM, Galdós L, Mora N, Salazar E, Salinas D, Pérez C, Moreira A, Segura R

Language: English
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ABSTRACT

The recombinant MY32/Ls protein is used as a vaccine antigen against sea lice Lepeophtherius salmonis. It is produced from the fermentation of the E. coli BL21 (DE3)-pET28a-my32/Ls expression system, which is stored frozen at -70 ºC in a pure and homogeneous Primary Cell Bank (PCB). This work was aimed to analyze the genetic stability of cells from this PCB grown for a high number of generations, and how it influences cell growth rate, doubling time and the expression of the protein of interest, either in shake flasks or cultured in a 200-L bioreactor. It was found that in this large scale process, the strain replicates for 46 generations. It was demonstrated that the number of generations did not affect the growth rate (μ) and the doubling time. Similarly, plasmid stability and protein expression remained unaffected and a 100 % identity match was obtained for the my32/Ls gene sequences in the expression plasmid isolated from PCB samples and after nearly 100 generations. It was also shown that the expression system of the MY32/Ls protein is genetically stable throughout the production process, supporting the development of a working cell bank from the characterized PCB that guarantees the stability, integrity and safety of large scale productions of this recombinant protein.