Janice A. Phillips

In Situ Monitoring of an Escherichia coli Fermentation Using a Diamond Composition ATR Probe and Mid-infrared Spectroscopy

A diamond composition ATR probe was used in situ to obtain IR spectra on replicate Escherichia coli fermentations involving a complex medium. The probe showed excellent stability over a 6‐month operating period and was unaffected by either agitation or aeration. The formation of an unknown was observed from IR spectra obtained during the sterilization; subsequent experiments proved this to be a reaction product between yeast extract and the phosphates used as buffer salts. Partial‐least‐squares‐based calibration/prediction models were developed for both glucose and acetate using in‐process samples. The resulting models had prediction errors of ±0.26 and ±0.75 g/L for glucose and acetic acid, respectively, errors which were statistically equivalent to the estimated experimental errors in the reference measurements. Relative concentration profiles for the unknown formed during sterilization could be generated either by tracking peak height at an independent wavelength or by self‐modeling curve resolution of the spectral region overlapping that of glucose. These profiles indicated that this compound was metabolized simultaneously with glucose; upon depletion of the glucose, when the microorganism switched to consumption of acetic acid, utilization continued but at a lower rate. The data presented provide an extensive characterization of the performance characteristics of this in situ analysis and clearly demonstrate its utility not just in the quantitative measurement of multiple known species but in the qualitative evaluation of unknown species.

Production of Small, Monodispersed Alginate Beads for Cell Immobilization

Gel entrapment retains suspension cells in a bioreactor for high‐density operations without the difficulties of cell separation. To achieve and sustain high cell densities in animal cell culture, beads under 1 mm in diameter are desirable to minimize diffusional limitations. The challenge is to develop a controllable, scaleable process to produce small, monodispersed beads for cell immobilization. In this work, alginate bead populations were produced in the 0.5–1 mm size range with less than 10% standard deviation by destabilizing a viscous jet with a controlled disturbance. Systematic study of the drop formation with this “vibration” method demonstrated that Webers theory for the instability of viscous jets qualitatively applies to the alginate system and provides a predictive tool for selecting the wavelength of disturbance for controlled bead formation. Nonlinear theory also provided guidance in establishing proper operating conditions to avoid satellite drop formation which decreases bead uniformity. A viscosity range 150–500 cp was found to be optimum for bead formation and served to rapidly establish the appropriate alginate concentration for the wide variety of commercial alginates. Through an understanding of the interaction of viscosity, linear velocity, nozzle diameter, and frequency of disturbance used to destabilize the jet, the predictable formation of small, uniform alginate beads was demonstrated with this method. As an example of scaleup, a 10‐nozzle manifold can process 3–50 L of alginate/h, depending on the desired bead size.

Porous Alginate−Poly(ethylene glycol) Entrapment System for the Cultivation of Mammalian Cells

A novel gel entrapment method has been developed where macropores are created within alginate beads to provide an environment for high‐density growth of mammalian cells. The method takes advantage of an interaction between poly(ethylene glycol) (PEG) and alginate to provide a network of pores within the bead for growth while the surrounding alginate matrix retains the integrity of the bead and minimizes cell leakage. Hybridomas were grown to a density approaching 108 cells/mL of beads in this system, while conventional alginate restricted growth to a maximum of 2 × 107 cells/mL of beads. In addition, cell leakage was minimal even at high cell densities, which was not the case with the conventional alginate system. Study of the conventional system determined that cell growth was limited by the alginate matrix; increasing the alginate concentrations resulted in lower final cell densities. In contrast, the PEG−alginate system permits growth in pores so the alginate matrix serves only as a structural matrix for cells. The pore size can be varied as a function of PEG concentration (10–20 wt % PEG) to provide radially defined areas for cell growth and radial diffusion pathways for nutrients/products in the adjacent alginate matrix. Because the PEG−alginate entrapment process does not require additional chemical reactions or temperature changes, the system offers a simple alternative to attain high cell densities in an immobilized bead system. As an illustration of the concept, cells entrapped in this system were grown to high density in both batch and perfusion modes for the production of monoclonal antibodies. Using the suspension batch culture as the base case, the specific monoclonal antibody production rate increased 1.6‐fold for the slower growing batch‐immobilized culture and 3‐fold for the immobilized perfusion culture.

The Production of Monoclonal Antibody in Growth-Arrested Hybridomas Cultivated in Suspension and Immobilized Modes

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG‐alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5‐fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth‐arrested cultures and nongrowth‐associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3‐fold higher antibody production rate than in growing batch cultures. These results were translated to a high‐density perfusion culture of immobilized cells in the growth‐arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate‐ and serum‐depleted batch cultures.

AMMONIA CATALYZED ORGANOSOLV DELIGNIFICATION OF POPLAR

A parametric investigation of NH4OH catalyzed solvent delignification of poplar was conducted to define pretreatment conditions which would yield an optimal separation of the biomass components and an enzymatic susceptible solid carbohydrate phase. Delignification parameters of interest included concentration of NH4OH, time and temperature of the reaction, and type of solvent. The addition of 0.82 M NH4OH to the delignification liquor increased lignin removal and decreased carbohydrate degradation, but increasing NH4OH concentration had no additional effect. At lower reaction temperatures, the extent of delignification increased with reaction time; at higher temperatures, a “relignification” of the pretreated wood was observed. The delignification and hemicellulose solubilization were modelled and rate constants reported. No major difference between three potential pulping solvents—ethanol, butanol, phenol—was observed. The enzymatic susceptibility of pretreated wood samples was approximately 6-fold great...

Growth of an adherent continuous cell line entrapped in a peg/alginate matrix

CHO-K1 cells, an anchorage-dependent line, were entrapped in beads prepared from a Na alginate/polyethylene glycol mixture and grown, through successive passages, to an average maximum density of 4.5×107 viable cells/g of bead. Cell growth and viability was unaffected by repeated alginate re-solubilization and reformation of the gel beads through five passages.

Use of Infrared Spectroscopy for the On-Line Multicomponent Analysis and Control of Bioprocesses

Current strategies for the control of bioprocesses are based on the use of material balances to indirectly estimate the status of the culture. Until recently, direct measurements of substrate and product concentrations, other than that of gaseous components, has been hampered by the lack of sensors suitable for use in a sterile environment. However, infrared spectroscopy has been shown to be useful in the quantitation of β-lactam production in the Penicillium fermentation, pyruvate utilization in the E. coli process, and simultaneous glucose uptake and ethanol formation in the Saccharomyces cerevisiae process. This technique potentially offers the unique advantage of allowing for resolution of multiple components in the reaction mixture. Its general utility as a “bioreactor sensor” will be determined by the sensitivity and stability of the instrumental analysis, the speed and accuracy of the mathematical techniques for resolution of the spectra, and the implementation of a satisfactory sampling system. This paper will address these problems and their resolution for the on-line analysis with and use of the IR spectrophotometer in the closed-loop control of the Saccharomyces cerevisiae process.