Generation and screening of Pichia pastoris strains with enhanced protein production by use of microengraving

Abstract

The selection of highly productive cell lines remains a key step for manufacturing therapeutic proteins. Microengraving was used to screen chemically mutagenized populations of Pichia pastoris for increased production of an Fc fragment. Clones retrieved following three rounds of mutagenesis yielded titers 2.65-fold greater than those of the parental strain.

Fig. 1.

(A) Iterative process of mutagenesis and microengraving enables generation and identification of single cells with improved rates of heterologous protein secretion. Wild-type P. pastoris cells constitutively secreting a human Fc fragment were subjected to chemical mutagenesis and then analyzed by microengraving to characterize the rates of protein secretion from single cells quantitatively. Cells with the highest rates of secretion were recovered by micromanipulation, grown, and frozen. These clonal stocks were regrown and pooled before each subsequent round of mutagenesis and screening. PDMS, polydimethylsiloxane. (B) Distribution of single-cell rates of secretion obtained by microengraving using polyclonal populations comprising 10 clones recovered in each round of chemical mutagenesis. Frozen stocks of recovered clones were used to inoculate separate 10-ml cultures of YPD and grown to mid-log phase before pooling and analysis by microengraving. The black arrow indicates a rate five times the median rate of secretion of the parental strain.

Fig. 2.

Comparison of relative productivities of clonal strains recovered after three rounds of chemical mutagenesis. Fc titers in culture supernatants were assessed by ELISA following the growth of each clone and compared to that of the parental strain. Each bar represents the mean ratio of a given clone's titer relative to that of the parental strain from seven independent outgrowth experiments. Error bars indicate standard errors. The dashed line shows the ratio above which a clone's secretion output was considered improved. The significance of the increased productivity was determined by Student's t test (*, P < 0.1; **, P < 0.05; ***, P < 0.01).