Clonal Selection and Characterization of Epigenetic Variation in Pichia Pastoris by

Recombinant proteins produced by different host organisms have been broadly used as therapeutics. Considering the demand for large quantities of protein drugs, methods are needed to increase reactor titers in a timely and cost-effective manner. We used random chemical mutagenesis to modify a wild-type strain of the heterologous protein production host Pichia pastoris, which resulted in overall improvement of the secretion rate of the mutated population. More than 4000 single-cells were simultaneously screened for high secretion of a human Fc fragment using microengraving and the top-producing clones were retrieved. Future characterization of these improved clones by transcript profiling should yield information about networks of genes central in heterologous protein secretion in the yeast P. pastoris. 1 C H A P T E R 1 ........................................................................................................................................... 6 1 .1 In tro d u ctio n ............................................................................................................................................................ 7 1.2 The use of recom binant proteins as therapeutics................................................................ 8 1.2.1 M onoclonal Antibodies used as therapeutics............................................................. 10 1.3 Different host organism s used as production system s ...................................................... 13 1.3.1. Escherichia Coli as a host organism ............................................................................ 13 1.3.2. CHO cells as a host organism ........................................................................................ 14 1.3.3. Saccharomyces cerevisiae as a host organism ............................................................. 15 1.3.4. Pichia pastoris as a host organism ............................................................................... 16 1.3.4.1 The Pichia pastoris expression system ........................................................................ 17 1.3.4.2 Recom binant protein expression in Pichia pastoris .................................................... 19 1.4 Im provem ent of host properties........................................................................................... 20 1.5 Heterogeneity am ong cell populations .............................................................................. 24 1.6 Clonal selection of best producers ..................................................................................... 25 1.6.1 Flow cytom etry ......................................................................................................................... 26 1.6.2 Gel m icrodrop technology (GM D) ....................................................................................... 28 1.6.3 Matrix-based secretion assays ........................................... 30 1.6.4 Microfluidic Devices .................................................................................................................. 31 1.6.5 Com m ercial High-speed m achines for single-cell m easurem ents .................................... 33 1.7 Dissertation objectives ....................................................................................................... 35 2 C h a p te r 2 .............................................................................................................................................. 3 6 2 .1 In tro d u ctio n .............................................................................................................................. 3 7 2.2 W hat is m icroengraving? ...................................................... .. . . .. . . . .. . .. . . .. . . . .. . . .. . . . .. . . .. . . . .. . .. . . . 38 2.3 Pichia pastoris as a host organism for heterologous protein secretion..............42 2.3.1 Dynam ics in P. pastoris protein secretion over tim e ........................................................ 42 2.4 "Fishing" for high producers through clonal selection .................................................... 44 2.5 Engineering a high producer ............................................................................................ 48 2.6 Discussion and Conclusions ................................................................................................. 56 2.7 M aterials and m ethods ........................................................................................................ 57 3 C h a p te r 3 :........................................................................................................................................................................ 6 1 3.1 Thesis Sum m ary ....................................................................................................................... 62 3.2 Discussion Conclusions ..................................................................................................... 63 3 .3 F u tu re p la n s ............................................................................................................................... 6 5 4 R e fe re n ce s ........................................................................................................................................ 6 6