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.Therapeutic proteins account for more than $99 billion of drug revenues annually worldwide (14) and account for a quarter of all new drugs for the treatment of diseases ranging from autoimmunity to cancer (1, 15). The pharmaceutical industry uses both microbial and cultured mammalian cells to produce these biologic drugs in large quantities. Fermentation titer remains a dominant determinant of the cost of recombinant proteins (3), and thus, identifying clonal master cell lines that maximize protein expression and secretion is critical for bioprocess development (1, 11).Advances in robotics, automated liquid handling, and highthroughput imaging of microtiter plates have enabled new technologies for microbial colony picking. For example, the Genetix QPix and Aviso CellCelector can pick thousands of microbial colonies per day. Both, however, require highly sophisticated, costly equipment, and neither system allows the screening of colonies directly for secreted proteins. Furthermore, cells must be incubated in semisolid medium and cannot be screened immediately after cultivation by fermentation. Flow cytometry (FC) has also been used to select both mammalian and microbial strains based on intracellular, surfacebound, or locally encapsulated reporting molecules that link protein production with the expression of fluorescent markers (8, 12). These approaches are rapid and relatively inexpensive, but the analysis required to identify and translate beneficial genetic factors into production strains can be costly and require significant effort.Here we report a method that uses microengraving for clonal selection to screen production-ready hosts rapidly for the secretion of a desired recombinant protein. Microengraving is a bioanalytical process that isolates and quantitatively measures the rates of protein secretion for thousands of single cells simultaneously (6). Cells are deposited into an array of subnanoliter wells at a density of ϳ1 cell per well, and the array is then sealed to a glass slide uniformly coated with a reagent to capture specific secreted products of interest (e.g., antibodies). Following a short incubation of the sealed array (1 to 2 h), the glass is removed to yield a protein microarray comprising the secreted proteins captured from each individual well. Using this method, cells with desired phenotypes are identified and recovered in less than 24 h using off-the-shelf components typically available in university core facilities for microscopy or microarray processing. The miniaturized format of each microengraving assay also helps conserve reagents and thus limits costs-a typical screen costs less than a single enzyme-linked immunosorbent assay (...

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…A compromise between the uniformity and speed of analysis by FACS and traditional cultivation (with its loss of information at the single cell level) is provided by the technique of micro-engraving, an emerging method for highthroughput analysis of secreted products from single cells (Love et al, 2006). Microengraving is a soft lithographic method based on intaglio printing to generate microarrays comprising secreted products from single cells, and has been used for screening a variety of secreted recombinant proteins (Panagiotou et al, 2011) as well as to characterise the secretome of B cells (Story et al, 2008). Interestingly, this method provides useful insights into secretion dynamics, showing, for example, how such a process is not linked to the cell cycle, but varies stochastically within a single cell during cell duplication.…”

Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms

“…The methylotrophic yeast secrete protein more effectively than S. cerevisiae because they have a stacked Golgi apparatus adjacent to the ER, better organized to facilitate secretion (Preuss, Mulholland, Franzusoff, Segev, & Botsteint, ). Biological studies and strain engineering, however, have shown that there is potential to improve other bottlenecks in secretion by P. pastoris (Idiris, Tohda, Kumagai, & Takegawa, ; Panagiotou et al, ). In contrast, filamentous fungi are very effective secretors.…”

Stochastic Particle Barcoding for Single-Cell Tracking and Multiparametric Analysis

“…As the final step in optimizing the SPB workflow, we iterated process variables that could affect cell viability during processing and recovery from hydrogel blocks. Viable cell isolation is crucial for the recovery of usable biological materials for downstream assays and single‐cell or clonal growth, which are of broad interest for many biological applications, such as selecting yeast and bacteria for bioproduction, and the analysis of various clonal populations in biology, such as B and T cells in immunology or circulating tumor cells in oncology . Notably, MWAs have been used to screen and clone cells, but the upper limit has remained approximately 100 cells per array by a manual recovery method .…”