Microbial Cell Technology

Polymun in co-operation with the IAM is experienced in cloning and expression of proteins in E. coli and yeasts. Various cloning systems have been applied successfully. An integrated approach within all developments considers the expression level as well as proper processing, specific activity and the ease of purification.

The combination of skills enables us to achieve recombinant protein titers of 3 kg/m³ and more depending on the kind of protein. This is backed by careful product characterization using the broad range of analytical tools and comprehensive documentation.

In 2008, Polymun was performing a part of the manufacturing process (fermentation of recombinant bacteria) of GMP-material for a multi-valent recombinant outer surface protein A (OspA) Lyme Borreliosis Vaccine for Baxter Innovation GmbH.

Cloning and Screening
Cloning genes of interest into E. coli and yeasts is performed using long term experience in molecular biology. The screening for optimized strains can be supported using FACS technology. Screening of our strain collections with a wide variety of specific properties is the other way of deriving production clones.

Protein-Free Cultivation Media
The optimization of media and induction procedures is a permanent project aiming at low cost and protein-free media and the balancing of the metabolic flux. Media are designed individually for specific production clones.

Cell Preservation
Procedures for GMP-compliant and well documented master and working cell banks are established that warrant their later industrial application.

Fermentation
Processes are designed for E. coli and yeast with fed-batch and continuous mode with up to 30 liter capacity in the research laboratories. GMP-material can be fermented with up to 50 liter net fermentation volume. Non-invasive strategies of process monitoring are applied for process control. Polymun can rely on its experience in downstream processing for the further processing of the harvest.

Patents:

Method for the manufacture of recombinant trypsin
granted by: EP 1 250 442 (AT BE CH/LI DE FR GB IE IT NL SE); SG 90540; US 7,351,549
Expression system
pending in: WO 2008/128701

Publications (selection):

Kunert R, Gach J, Katinger H (2008) Expression of a Fab Fragment in CHO and Pichia pastoris. BioProcess International 6(6):34-40

Gasser B, Sauer M, Maurer M, Stadlmayr G, Mattanovich D (2007) Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. Appl Environ Microbiol 73(20):6499-507

Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D (2006) Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng 94(2):353-61

Maurer M, Kuhleitner M, Gasser B, Mattanovich D (2006) Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris. Microb Cell Fact 5:37

Porro D, Sauer M, Branduardi P, Mattanovich D (2005) Recombinant protein production in yeasts. Mol Biotechnol 31(3):245-59

Valli M, Sauer M, Branduardi P, Borth N, Porro D, Mattanovich D (2005) Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry. Appl Environ Microbiol 71(3):1515-21

Sauer M, Branduardi P, Gasser B, Valli M, Maurer M, Porro D, Mattanovich D (2004) Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation. Microb Cell Fact 3(1):17

Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113(1-3):121-35

Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2004) Effects of gene dosage, promoters and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85:367-75

Hohenblum H, Vorauer-Uhl K, Katinger H, Mattanovich D (2004) Bacterial expression and refolding of human trypsinogen. J Biotechnol 109:3-11

Porro D, Mattanovich D (2004) Recombinant protein production in yeast. In: Recombinant Gene Expression Protocols, 2^nd edition; Ed.: Balbas P; Humana Press, Totowa, NJ, USA

Hohenblum H, Borth N, Mattanovich D (2003) Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J Biotechnol 102:281-90

Soriano E, Borth N, Katinger H, Mattanovich D (2002) Optimization of recombinant protein expression level in Escherichia coli by flow cytometry and cell sorting. Biotechnol Bioeng 80:93-9

Hohenblum H, Naschberger S, Weik R, Katinger H, Mattanovich D (2001) Production of recombinant human trypsinogen in Escherichia coli and Pichia pastoris. A comparison of expression systems. In: Production of recombinant proteins with prokaryotic and eukaryotic cells. Eds.: Merten OW, Mattanovich D, Cole J, Lang C, Larsson G, Neubauer P, Porro D, Postma P, Teixeira de Mattos J, Kluwer Academic Publ, pp. 339-46

Soriano E, Borth N, Katinger H, Mattanovich D (1999) Flow cytometric analysis of metabolic stress effects due tore combinant plasmids and proteins in Escherichia coli production strains. Metabolic Eng 1:270-4

Weik R, Striedner G, Francky A, Raspor P, Bayer K, Mattanovich D (1999) Induction of oxidofermentative ethanol formation in recombinant cells of Saccharomyces cerevisiae yeasts. Food Technol Biotechnol 37:191-4

Weik R, Francky A, Striedner G, Raspor P, Bayer K, Mattanovich D (1998) Recombinant expression of alliinlyase from garlic (Allium sativum L.) in bacteria and yeasts. Planta Medica 64:387-8

Borth N, Mitterbauer R, Mattanovich D, Kramer W, Bayer K, Katinger H (1998) Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells. Cytometry 31:125-9

Mattanovich D, Kramer W, Lüttich Ch, Weik R, Bayer K, Katinger H (1998) Rational design of an improved induction scheme for recombinant Escherichia coli. Biotechnol Bioeng 58:296-8

Kramer W, Elmecker G, Weik R, Mattanovich D, Bayer K (1996) Kinetic studies for the optimization of recombinant protein formation. Ann NY Acad Sci 782:323-33

Mattanovich D, Weik R, Thim S, Kramer W, Bayer K, Katinger H (1996) Optimization of recombinant gene expression in Escherichia coli. Ann NY Acad Sci 782:182-90

Mattanovich D, Kramer W, Lüttich Ch, Bayer K, Katinger H (1993) Galactose can strongly induce the lac promoter in galmutants of Escherichia coli. Life Science Adv - Mol Biol 12:121-5