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.

Modeling of cell culture damage and recovery leads to increased antibody and biomass productivity in CHO cell cultures.

The development of an efficient and productive cell-culture process requires a deep understanding of intracellular mechanisms and extracellular conditions for optimal product synthesis. Mathematical modeling provides an effective strategy to predict, control, and optimize cell performance under a range of culture conditions. In this study, a mathematical model is proposed for the investigation of cell damage of a Chinese hamster ovary cell culture secreting recombinant anti-RhD monoclonal antibody (mAb).

Modeling the coupled extracellular and intracellular environments in mammalian cell culture.

The regulation of metabolism in mammalian cell culture is closely linked to the process of apoptosis-programmed cell death. Apoptosis negatively impacts culture viability, product yield, and quality. An improved understanding of the interaction between apoptosis and metabolism will give rise to better control over the culture process, and thus improvements in product yield.

Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification.

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression.

Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC).

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system.

Population-based modeling of the progression of apoptosis in mammalian cell culture.

The production of biopharmaceuticals from mammalian cell culture is hindered by apoptosis, which is the primary cause of cell death in these cultures. As a tool for optimization of culture yield, this study presents a population-based model describing the progression of apoptosis in a monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell culture. Because mAb production does not cease when apoptosis begins, the model was designed to incorporate subpopulations at various stages in the progression of apoptosis.

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.

Development of a mathematical model for evaluating the dynamics of normal and apoptotic Chinese hamster ovary cells.

A metabolic flux based methodology was developed for modeling the metabolism of a Chinese hamster ovary cell line. The elimination of insignificant fluxes resulted in a simplified metabolic network which was the basis for modeling the significant metabolites. Employing kinetic rate expressions for growing and non-growing subpopulations, a logistic model was developed for cell growth and dynamic models were formulated to describe culture composition and monoclonal antibody (MAb) secretion.

Potential application of hydrogel-based strong anion-exchange membrane for plasmid DNA purification.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid recovery yield of ∼90%.

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line.

A Chinese Hamster Ovary cell line, CHO1-15(500), producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.

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.

Statistical methods in media optimization for batch and fed-batch animal cell culture.

Hybridoma 130-8F producing anti-F monoclonal antibodies (MAb) were grown in batch and fed-batch mode with glutamine as the limiting substrate. The initial concentration of glucose varied between 10 and 25 mM but was not growth limiting. Monoclonal antibody production was identified as being partially growth associated.

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.