Abstract... I. Zusammenfassung... II. 1 Aim of the work Introduction Short overview of Chinese hamster ovary cell lines...

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1 III Contents I II III Abstract... I Zusammenfassung... II Contents... III 1 Aim of the work Introduction Short overview of Chinese hamster ovary cell lines Unspecific mutations: A short survey of their use in cell line generation Selection methods for cell line generation of distinct phenotypes Identification of unknown regulatory cellular factors Proteomics Transcriptomics Pathway engineering towards high cell density cultures Cell cycle engineering Modulation of cell death factors Relevance of heat shock proteins, molecular chaperons and detoxifying enzymes Hypothermic factors Metabolic engineering towards high cell densities Increasing the maximal cell density by pathway engineering A summary Towards high cell density growing CHO cell lines: A theoretical approach Material and methods Cultivation Sampling III

2 3.2.1 Cell culture supernatant recovery for analytical purpose Cell pellet recovery for RNA and genomic DNA isolation Manual cell counting Cryopreservation and thawing Next generation sequencing Quantitative real-time PCR analysis Total RNA isolation cdna generation Quantitative real-time PCR analysis Preparative Methods Genomic DNA isolation Preparative PCR Custom gene synthesis Restriction digest DNA dephosphorylation DNA phosphorylation Isolation and Purification of DNA Fragments Ligation Transformation of competent E.coli DH5α cells E.coli cultivation Plasmid DNA preparation Linearisation of vectors and sterile DNA precipitation Vectors and cell line generation Overview on basal vectors and their derivatives IV

3 3.8.2 Nucleofection of CHO-S cell lines Selection of positively transfected cells and further cultivation Limited dilution and clonal cell line generation Analytics Fixation of cells and DAPI staining Agarose Gel Electrophoresis Plasmid DNA Sequencing Determination of glucose concentration higg ELISA SDS-PAGE Data compilation and computation Material Terms and acronyms Cell line terminology Results Generation of a clonal CHO cell line with capability growing at HCD Adaption of CHO-K1 cells to CM Chemical mutagenesis and mutated cell regeneration Selection of CHO-S-E400 towards growth at high osmolality and glutamine absence Single cell cloning of CHO-S-E400 HyOsm Selection of CHO-S-E400 HyOsm clones Single cell cloning of 25-CHO-S Transfection and productivity of CHO-S-E400/1.104 and 25-CHO-S/2.5C V

4 4.1.8 Accumulating cell culture CHO-S-E400/1.104 and 25-CHO-S/2.5C Optimised fedbatch cultures and sampling for factor identification Factor identification and verification Transcriptomics by HiSeq Factor verification using quantitative RT-PCR Factor validation in non-producing and producer cell lines Validation of cgrtprp in non-producing 25-CHO-S/2.5C8 cells Validation of LOC /TPR repeat-containing protein C10orf93-like Structural and homological relationship of C10orf93-like species Validation of cgrc10orf93-like in 25-CHO-S/2.5C8 and K20-3 cell line Validation of cgrgas5 variants and cgrsnord Identification, isolation and characterisation of cgrgas5 variants Validation of cgrgas5 variants in 25-CHO-S/2.5C Isolation and validation of cgrsnord78 in 25-CHO-S/2.5C8 as well as K20-3 cell line Single cell cloning of K20-3/cgrSnord Application of K20-3/cgrSnord78 variants in a production fedbatch process Validation of cgrttc Amplification of cgrttc Validation by overexpression in non-producing cell line 25-CHO-S/2.5C Further effect validation by overexpression in higg-producing cell line K Clonal variations in K20-3/cgrTtc Application of K20-3/cgrTtc36 variants in a production fedbatch process An excursus: The co-expression of cgrttc36 with cgrsnord78 and cgrtprp VI

5 5 Discussion Increase of genomic variation by mutagenesis and the selection process Mutagenesis and recovery of the 25-CHO-S cell line Selection towards growth at high cell densities Transcriptomic analysis and data verification The tetratricopeptide repeat-containing proteins The effect upon the overexpression of cgrtprp The putative function of cgrttc Prediction of secondary as well as tertiary structure, modifications and cellular localisation of cgrttc The interaction of cgrttc36 with Hsp70 and the impact thereof The mechanism behind cgrkmtttc36 fusion protein formation and action Putative mode of inhibition of cgrttc36-mediated effects The putative localisation as well as function of cgrc10orf93-like/cgrttc40-like The inhibition of cell aggregation in cgrttc36 and cgrc10orf93-like/cgrttc40-like overexpressing cell lines Arguments on cgrgas5 and its intronic sequence cgrsnord Insights into the function and structure of cgrgas Possible function and targets of cgrsnord Optimisation and industrial relevance of cgrsnord78 and cgrttc Conclusion and perspectives References Abbreviations VII

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Acknowledgements... 3 Contents... I Figures... VII Tables... IX Abbreviations... X

Acknowledgements... 3 Contents... I Figures... VII Tables... IX Abbreviations... X I Acknowledgements... 3... I Figures... VII Tables... IX Abbreviations... X Amino acids... XII Nucleobases... XII 1 Introduction... 1 1.1 Polyketides and non-ribosomal peptides... 1 1.2 Polyketide synthases...