The development and optimization of processes for the expression of sialylated recombinant human therapeutic glycoprotein in insect cell-baculovirus system.

• Main Author: Mohd. Hani, Mohamed Khalil
• Format: Monograph
• Language: English
• Published: Universiti Teknologi Malaysia 2001
• Subjects: TK Electrical engineering. Electronics Nuclear engineering
• Online Access: http://eprints.utm.my/id/eprint/2932/1/74092.pdf; http://eprints.utm.my/id/eprint/2932/

proteinbased therapies (cancer, HIV, autoimmune disease, neuromuscular, organ rejection and allergic diseases). Most human proteins, glycoproteins with ‘complex type’ oligosaccharide moieties attached to an asparagine residue, usually terminated with sialic acid, an important determinant for biological activity, solubility and blood circulation time. Insect cells-baculovirus system, an excellent artificial systems for the expression of recombinant glycoproteins, offers a very promising alternative to the mammalian cell lines which suffer from high cost of operation, as well as slow growth and low yield. Unfortunately the Nglycan structure of the recombinant glycoprotein produced in insect cell culture is slightly different from the ones naturally produced in human, usually unsialylated and sometimes nongalactosylated, suggesting the nonexistence of several genes in insect cells. Metabolic engineering is one of the approaches to mimic the biosynthesis pathway that is naturally occurring in human. The used of stably transformed cells with human enzymes or co-infection with baculoviruses carrying mammalian β- (1-4)galactosyltransferase (1) and human CMP-Neu5Ac sugar nucleotide (2) were able to push the pathway towards humanlike biosynthesis pathway. The addition of nucleotide sugar precursor into culture media also influences the final oligosaccharide structure (3). Apart from that, the existence of β-N-acetylglucosaminidase in native cell lines is believed to be another reason for the deviation from human glycoprotein synthesis pathways (4). Recently, we have cloned, expressed and characterized three D. melanogaster genes for β-N-acetylglucosaminidase and the inhibition study of these enzymes is underway (5). Since the complete pathway involves several enzyme and sugar nucleotides biosynthesis, process optimization for the expression of sialylated (human-like) recombinant glycoprotein is to be carried out in this project, specifically integrating molecular biology and chemical engineering approach. Missing enzymes will be introduced by means of metabolic engineering, in combination with viral co-expression, culturing condition and bioreactor design. Statistical approaches with the utilization of Response Surface Methodology and Taguchi Method will be applied to analyze several important parameters for this process.