Browsing by Author "Demirhan, Deniz"

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    Comparative systeomics to elucidate physiological differences between CHO and SP2/0 cell lines
    (NATURE PORTFOLIO, 2022-01-01) Demirhan, Deniz; Kumar, Amit; Zhu, Jie; Poulsen, Pi Camilla; Majewska I, Natalia; Sebastian, Yinong; Chaerkady, Raghothama; Yu, Wen; Zhu, Wei; Zhuang, Li; Shah, Punit; Lekstrom, Kristen; Cole, Robert N.; Zhang, Hui; Betenbaugh, Michael J.; Bowen, Michael A.
    Omics-based tools were coupled with bioinformatics for a systeomics analysis of two biopharma cell types: Chinese hamster ovary (M-CHO and CHO-K1) and SP2/0. Exponential and stationary phase samples revealed more than 10,000 transcripts and 6000 proteins across these two manufacturing cell lines. A statistical comparison of transcriptomics and proteomics data identified downregulated genes involved in protein folding, protein synthesis and protein metabolism, including PPIA-cyclophilin A, HSPD1, and EIF3K, in M-CHO compared to SP2/0 while cell cycle and actin cytoskeleton genes were reduced in SP2/0. KEGG pathway comparisons revealed glycerolipids, glycosphingolipids, ABC transporters, calcium signaling, cell adhesion, and secretion pathways depleted in M-CHO while retinol metabolism was upregulated. KEGG and IPA also indicated apoptosis, RNA degradation, and proteosomes enriched in CHO stationary phase. Alternatively, gene ontology analysis revealed an underrepresentation in ion and potassium channel activities, membrane proteins, and secretory granules including Stxbpt2, Syt1, Syt9, and Cma1 proteins in M-CHO. Additional enrichment strategies involving ultracentrifugation, biotinylation, and hydrazide chemistry identified over 4000 potential CHO membrane and secretory proteins, yet many secretory and membrane proteins were still depleted. This systeomics pipeline has revealed bottlenecks and potential opportunities for cell line engineering in CHO and SP2/0 to improve their production capabilities.
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    Evaluation of Feed Strategy for High Quality Biosimilar IgG Production in CHO Cell Fed-batch Process
    (Acıbadem Mehmet Ali Aydınlar Üniversitesi, 2022-01-01) Üstüner, Berna; Dayankaç Ünver, Seçil; Turgut, Tunç; Demirhan, Deniz; Ayyıldız Tamış, Duygu
    ABSTRACT Purpose: Chinese Hamster Ovary (CHO) cells are currently the leading hosts for biosimilar Immunoglobulin G (IgG) production in the biopharmaceutical industry. Most eukaryotic proteins are glycosylated, and charge variants affect both the in vivo and in vitro properties of monoclonal antibodies (mAb). Adjusting the N-glycosylation patterns and charge variants while achieving high antibody titer is a production challenge. In this study, the effects of feed type and strategy on cell growth, product titer, glycosylation and charge variation were investigated using different CHO clones producing different IgG mAbs. Methods: Cultivated CHO cells were supplemented with different feeding schemes, under fed-batch productions of 14 days. Screenings were conducted in spin-tubes and further investigated in 3L bioreactor systems. Results: Change in feed strategy decreased productivities by 10.4% (P < 0.05), while it increased non-fucosylated glycoforms by 33.3% and enhanced galactosylation up to 3-folds. Basic variants were observed to increase 2.5 folds. Conclusion: These remarkable alterations are of great importance in terms of mAb quality, in a manufacturing point of view, as they provide modulation of efficacy and safety. This reveals that feed strategy is a major driving force that significantly impacts culture longevity, galactosylated glycoforms, high-mannose glycan contents and charge variants.