Mammalian Cell Technology

Polymun in co-operation with the IAM gathered considerable know-how in mammalian cell culture that is kept at high standards due to the manifold activities in research and development.

The long term experience in classical mammalian cell culture is combined with up to date molecular biology for transfection and transformation. Together with the wide range of analytical tools we can create functional in vitro biological assays for various products and cell conditions. Each R&D project is thoroughly documented to allow clinical application later on, thereby profiting from Polymun′s own expertise with clinical trials. Polymun has established procedures and technologies to implement a broad range of products into Polymun′s established CHO cell based expression platform.

Transfection and Screening
An important topic is the expression of recombinant proteins via mammalian cells. Although working with various cell lines, CHO cells are used preferentially. The transfection and selection process was already tested under serum-free conditions. The vector construction is also employed for class-switch and humanization of monoclonal antibodies. Efforts are made to implement FACS-assisted procedures in the amplification and selection of high producing subclones in a time-saving combination with limited dilution.

Protein-Free Cell Culture
Polymun has established procedures to adapt various kinds of hybridomas (murine, xeno and human), NS0, SP2, CHO and other cell lines to defined, protein- and animal component-free propagation and production media. This development includes host cells for vaccine virus propagation. Such low cost formulations are also compounded by certified suppliers for our customers on the base of non-exclusive licenses. The optimization of protein-free media formulation is a permanent process also for new cell lines.

Protein-free Cell Preservation

The whole procedure of cell preservation in liquid nitrogen was optimized in order to eliminate all proteins and other animal derived materials. This method ensures enhanced value of master and working cell banks.

Fermentation
The development of mammalian cell bio-processes is an important focus as it represents a core competence of Polymun′s founder, Prof. Hermann Katinger. The cell line and product specific development includes processes employing batch, fed-batch, continuous and continuous perfused high cell density cultivation systems. The bioreactor types used are stirred tank, air-lift or fluidized bed. Various kinds of cell retention including ultrasonic devices can be incorporated in the fermentation process. Various types of microcarriers are available for adherent cell processes, partly developed in co-operation with GE Healthcare (former Amersham Biosciences).

Within our development pipeline we investigate new applications for reactor and process design. A new agitation system for advanced mixing, called Softmix® (Rütten Engineering), has been successfully applied for cultivating adherent cells susceptible to shear stress. We recently implemented a novel algorithm for the automated control of medium feed in perfusion and fed-batch systems. Mathematical tools as factorial design and mixing design are an integral part of our activities in the field of process medium optimization.

Patents:

Tumor and senescence marker
granted by: EP 1248839; US 7,202,352

Bioactive oligopeptides
granted by: US 7,101,664

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

Reisinger H, Sevcsik E, Vorauer-Uhl K, Lohner K, Katinger H, Kunert R (2007) Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles. Cytotechnology 54(3):157-68

Lattenmayer C, Löschel M, Schriebl K, Sterovsky T, Trummer E, Vorauer-Uhl K, Müller D, Katinger H, Kunert R (2007) Protein-free transfection of CHO host cells with an IgGFusion protein-selection and characterisation of stable high producers and comparison to conventionally transfected clones. Biotechnol Bioeng 96(6):1118-26

Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kunert R, Vorauer-Uhl K, Weik R, Borth N, Katinger H, Müller D (2006) Process parameter shifting: Part I. effect of DOT, pH and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol Bioeng 94(6):1033-44

Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kunert R, Vorauer-Uhl K, Weik R, Borth N, Katinger H, Müller D (2006) Process parameter shifting: Part II. biphasic cultivation - a tool for enhancing the volumetric productivity of batch processes using Epo-Fc expressing CHO cells. Biotechnol Bioeng 94(6):1045-52

Böhm E, Grillari J, Voglauer R, Gross S, Ernst W, Ferko B, Kunert R, Katinger H, Borth N (2005) Establishment of a strategy for the rapid generation of a monoclonal antibody against the human protein SNEV (hNMP200) by flow-cytometric cell sorting. J Immunol Methods 307(1-2):13-23

Borth N, Mattanovich D, Kunert R, Katinger H (2005) Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line. Biotechnol Prog 21(1):106-11

Zeng Y, Fest S, Kunert R, Katinger H, Pistoia V, Michon J, Lewis G, Ladenstein R, Lode HN (2005) Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice. Mol Immunol 42(11):1311-9

Kunert R, Wolbank S, Chang M, Voglauer R, Borth N, Katinger H (2004) Control of key parameters in developing mammalian production clones. BioProcess International 2(6):54-9

Naschberger S, Rütten K, Müller D, Katinger H (2003) Softmix, a new scalable mixing system for the cultivation of sensitive cells on microcarrier under protein-free conditions. Proceedings of the 18^th ESACT Meeting (Granada) - Animal Cell Technology: Animal Cell Technology Meets Genomics:735-8

Trummer E, Müller D, Steinfellner W, Kunert R, Steindl F, Hesse F, Katinger H (2003) Characterisation of CHO subclones showing profound changes in performance when propagated in different cultivation systems. Proceedings of the 18^th ESACT Meeting (Granada) - Animal Cell Technology: Animal Cell Technology Meets Genomics:529-32

Calarese DA, Scanlan CN, Zwick MB, Deechongkit S, Mimura Y, Kunert R, Zhu P, Wormald MR, Stanfield RL, Roux KH, Kelly JW, Rudd PM, Dwek RA, Katinger H, Burton DR, Wilson IA (2003) Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300(5628):2065-71

Dürrschmid M, Landauer K, Simic G, Klug H, Keijzer T, Trampler F, Oudshoorn A, Grüschl M, Müller D, Doblhoff-Dier O (2003) Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture. Biotechnol Prog 19(3):1045-8

Jursik C, Voglauer R, Grillari J, Hemetsberger T, Jungfer H, Katinger H (2002) Killer T cells: a promising new tool for adoptive immunotherapy. Transplant Proc 34(7):2879-80

Kunert R, Katinger H (2002) Production of immunoglobulins and monoclonal antibodies targeting infectious diseases. In: Infectious Diseases; Ed.: Georgiev V, Humana Press, pp. 63-99

Grillari J, Fortschegger K, Grabherr RM, Hohenwarter O, Kunert R, Katinger H (2001) Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells. J Biotechnol 87(1):59-65

Borth N, Zeyda M, Kunert R, Katinger H (2000) Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting. Biotechnol Bioeng 71(4):266-73

Franek F, Hohenwarter O, Katinger H (2000) Plant protein hydrolysates: Preparation of defined peptide fractions promoting growth and production in animal cells cultures. Biotechn Prog 16(5):688-92

Kunert R, Steinfellner W, Purtscher M, Assadian A, Katinger H (2000) Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells. Biotechnol Bioeng 67(1):97-103

Müller D, Unterluggauer F, Kreismayr G, Wiederkum S, Hohenwarter O, Schmatz C, Vorauer-Uhl K, Assadian A, Doblhoff-Dier O, Katinger H (2000) Online controlled perfused fluidized bed and fed-batch production. Proceedings of the Cell Culture Engineering VII, Santa Fe, USA

Borth N, Strutzenberger K, Kunert R, Steinfellner W, Katinger H (1999) Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by northern blot and flow cytometry. J Biotechnol 67(1):57-66

Müller D, Doblhoff-Dier O, Katinger H (1999) Non-invasive adaptive control of the feed rate for hich cell density CHO cultures according to the pO2 profile. Proceedings of the 16th ESACT Meeting (Lugano) - Animal Cell Technology: Products from Cells, Cells as Products:171-3

Strutzenberger K, Borth N, Kunert R, Steinfellner W, Katinger H (1999) Changes during subclone development and ageing of human antibody-producing recombinant CHO cells. J Biotechnol 69(2-3):215-26

Zeyda M, Borth N, Kunert R, Katinger H (1999) Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines. Biotechnol Prog 15(5):953-7

Müller D, Schmatz C, Weik R, Wiederkum S, Katinger D, Unterluggauer F, Kreismayr G, Koller G, Doblhoff-Dier O, Katinger H (1998) Perfused fluidised bed production, expanded bed affinity purification and validation (Contract or licensing) Manufacture of clinical grade (CHO-recombinant human) mAbs or other recombinant proteins. Proccedings of the 10th Annual Meeting of Japanese Association for Animal Cell Technology JAACT

Kunert R, Rüker F, Katinger H (1998) Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5. AIDS Res Hum Retroviruses 14(13):1115-28

Klima G, Kreismayr G, Müller D, Schmatz C, Wiederkum S, Steinfellner W, Assadian A, Lhota G, Blüml G, Doblhoff-Dier O, Katinger H (1997) Process development for the large scale production of clinical grade human monoclonal antibody using a Cytopilot fluidised bed reactor. Proceedings of the 14th ESACT Meeting (Vilamoura): Animal Cell Technology: From Vaccines to Genetic Medicine:447-50

Müller D, Kreismayr G, Klima G, Assadian A, Schmatz C, Wiederkum S, Steinfellner W, Blüml G, Doblhoff-Dier O, Katinger H (1997) Scale-up optimisation based on the development of a perfusion rate control for CHO cells using a Cytopilot Mini fluidised bed reactor. Proceedings of the 14th ESACT Meeting (Vilamoura): Animal Cell Technology: From Vaccines to Genetic Medicine:451-4

Katinger H, Assadian A, Blüml G, Borth N, Buchacher A, Doblhoff-Dier O, Gaida T, Reiter M, Schmatz C, Strutzenberger K, Steinfellner W, Unterluggauer F, Zach N (1996) Long term stability of continuously perfused animal cells immobilized on novel macroporous microcarriers. Advances in Molecular and Cell Biology 15A:193-207

Buchacher A, Predl R, Strutzenberger K, Steinfellner W, Trkola A, Purtscher M, Gruber G, Tauer C, Steindl F, Jungbauer A (1994) Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res Hum Retroviruses 10(4):359-69

Unterluggauer F, Doblhoff-Dier O, Tauer C, Jungbauer A, Gaida T, Reiter M, Schmatz C, Zach N, Katinger H (1994) Stable continuous large scale production of human monoclonal HIV-1 antibody using a computer controlled pilot plant. Biotechniques 16(1):140-7