The N-glycans of human serum transferrin produced in Trichopulsia ni cells were analyzed to examine N-linked oligosaccharide processing in insect cells. Metabolic radiolabeling of the intra- and extracellular protein fractions revealed the presence of multiple transferrin glycoforms with molecular weights lower than that observed for native human transferrin. Consequently, the N-glycan structures of transferrin in the culture medium were determined using three-dimensional high performance liquid chromatography. The attached oligosaccharides included high mannose, paucimannosidic, and hybrid structures with over 50% of these structures containing one fucose, alpha(1,6)-, or two fucoses, alpha(1,6)- and alpha(1,3)-, linked to the Asn-linked N-acetylglucosamine. Neither sialic acid nor galactose was detected on any of the N-glycans. However, when transferrin was coexpressed with beta(1,4)-galactosyltransferase three additional galactose-containing hybrid oligosaccharides were obtained. The galactose attachments were exclusive to the alpha(1, 3)-mannose branch and the structures varied by the presence of zero, one, or two attached fucose residues. Furthermore, the presence of the galactosyltransferase appeared to reduce the number of paucimannosidic structures, which suggests that galactose attachment inhibits the ability of hexosaminidase activity to remove the terminal N-acetylglucosamine. The ability to promote galactosylation and reduce paucimannosidic N-glycans suggests that the oligosaccharide processing pathway in insect cells may be manipulated to mimic more closely that of mammalian cells.

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Transferin manusia (hTf) memainkan peranan yang penting dalam fungsi bakteriostatik dan pengangkutan ferum dari bahagian penyimpanan ke sel–sel yang membiak melalui proses endositosis janaan reseptor. Sistem ekspresi bakulovirus sel serangga telah dipakai secara meluas sebagai sistem alternatif dalam penghasilan Transferin manusia rekombinan (rhTf). Kajian ini ditumpukan ke atas pengoptimuman glutamina, glukosa dan campuran lipid 1000x yang dapat meningkatkan penghasilan rhTf. Reka bentuk eksperimen yang melibatkan 17 eksperimen reka bentuk komposit berpusat (CCD) telah digunakan dan hasil kajian dianalisis oleh Statistika (Statsoft v. 5.0). Metodologi permukaan tindak balas (RSM) telah mengenalpasti nilai optimum parameterparameter yang dikaji iaitu glutamina=2211.20 mg/L, glukosa=1291.95 mg/L, dan campuran lipid 1000x=0.64 %v/v. Hasil optimasi menunjukkan peningkatan hasil rhTf sebanyak tiga kali ganda, iaitu daripada 19.89 μg/ml kepada 65.12 μg/ml. Kata kunci: Transferin manusia; bakulovirus sel serangga; reka bentuk eksperimen; reka bentuk komposit berpusat; metodologi permukaan tindak balas Human Transferrin (hTf) plays a big role in providing bacteriostatic functions as well as to transport iron from the storage part to all proliferating cells by receptor mediated endocytosis. Insect cells baculovirus expression system has been widely used as an alternative expression system for the production of recombinant human Transferrin (rhTf). This work focused mainly on the optimization of glutamine, glucose and lipid mixtures 1000x to increase rhTf yield. An experimental design involving 17 central composite design (CCD) experiments was employed and results were analyzed by Statistica (Statsoft v. 5.0). The response surface methodology (RSM) had identified the optimum values where glutamine=2211.20 mg/L, glucose=1291.95 mg/L, and lipid mixtures 1000x=0.64 %v/v. Using the optimized parameters, the studies demonstrated an increase in the rhTf yield by three–fold from 19.89 μg/ml to 65.12 μg/ml. Key words: Human transferrin; insect cells baculovirus; experimental design; central composite design; response surface methodology

In conventional fed batch process development approaches, batch operating parameters (such as pH, temperature, seeding density, dissolved oxygen concentration) are kept constant and only feeding parameters such as feeding time, post-feed concentration are manipulated. The batch and fed batch operating parameters are assumed to be independent of each other. This approach to process development ignores any interactions that might exist between the batch and fed-batch operating parameters are therefore not evaluated. However in a complex bioprocess, none of the factors affecting the process can be assumed to be independent of each other and mutually exclusive. Therefore in this study an attempt was made to study the interaction between fed-batch operating parameter-post feed glucose concentration (A) and batch operating parameters-seeding density (B), temperature (C), and dissolved oxygen concentration (D) by their simultaneous manipulation, as well as the effect of these interactions on cell growth and monoclonal antibody (mAb) production. NS0 cell line producing the mAb's against carcino-embryogenic antigen (Anti-CEA) was used. The final mAb concentration, viable cell density and integral of viable cell concentration (IVCC) were the responses evaluated. Statistical analysis experimental data showed that parameter (A) and its interaction with parameter (B) were the main factors affecting both the response variables. In comparison to the batch run which yielded 5.21 mg/L mAb, the developed fed batch process increased the mAb titer by 10 fold (59.40 mg/mL), and the IVCC was increased by 7 fold. The maximum VCD value (3.46×10 6 cells/mL) of the developed fed batch process was 1.25 over fold the value for batch.

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