Effects of nutrient levels and average culture pH on the glycosylation pattern of camelid-humanized monoclonal antibody.

The impact of operating conditions on the glycosylation pattern of humanized camelid monoclonal antibody, EG2-hFc produced by Chinese hamster ovary (CHO) cells has been evaluated by a combination of experiments and modeling. Cells were cultivated under different levels of glucose and glutamine concentrations with the goal of investigating the effect of nutrient depletion levels and ammonia build up on the cell growth and the glycoprofiles of the monoclonal antibody (Mab). The effect of average pH reduction on glycosylation level during the entire culture time or during a specific time span was also investigated.

Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation.

The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells.

Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation.

Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression.

Metabolic flux-based modeling of mAb production during batch and fed-batch operations.

This paper proposes mathematical models that predict the physiology, growth behavior and productivity of hybridoma cells in both batch and fed-batch systems. Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was employed as a model system. A systematic approach based on metabolic flux analysis (MFA) was utilized to yield a dynamic model for predicting the concentration of significant metabolites over time.

Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66.

A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin 3 (rHuIL-3) by Streptomyces lividans. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of 20 to 8. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation.

Dynamic metabolic modeling for a MAB bioprocess.

Production of monoclonal antibodies (MAb) for diagnostic or therapeutic applications has become an important task in the pharmaceutical industry. The efficiency of high-density reactor systems can be potentially increased by model-based design and control strategies. Therefore, a reliable kinetic model for cell metabolism is required.

Biodegradation kinetics of 2,4,6-trichlorophenol by an acclimated mixed microbial culture under aerobic conditions.

The objective of this study was to achieve a better quantitative understanding of the kinetics of 2,4,6-trichlorophenol (TCP) biodegradation by an acclimated mixed microbial culture. An aerobic mixed microbial culture, obtained from the aeration basin of the wastewater treatment plant, was acclimated in shake flasks utilizing various combinations of 2,4,6-TCP (25-100 mg l(-1)), phenol (300 mg l(-1)) and glycerol (2.5 mg l(-1)) as substrates.

Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli.

The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the T7 promoter in Escherichia coli. PAC, with its complex formation mechanism, serves as a unique target protein for demonstration of several key strategies for enhancing recombinant protein production. The current T7 system for pac overexpression was fraught with various technical hurdles.

Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli.

Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli.

Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli.

Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli.

Fungal glucoamylases.

Fungi are employed to produce industrially important glucoamylases. Most glucoamylases are glycosylated. Glycosylation enhances the enzyme stability.