Beth Junker

Scale-up methodologies for Escherichia coli and yeast fermentation processes

Scale-up techniques from the literature have been compiled and reviewed for applicability to Escherichia coli and yeast processes. The consistency of design and operating parameters for the pilot scale vessels in an existing fermentation pilot plant, ranging in nominal volume from 100 l to 19,000 l, was established and compared favorably with approaches found in the literature. Differences were noted as a function of parameters such as fermentor scale, vessel geometry, agitator type/size and ungassed/gassed power input. Further analysis was conducted using actual fermentation data for historical and recent development processes collected over a 10-year-period, focussing on operating conditions at peak culture oxygen uptake rates. Scale-up estimates were performed based on geometric similarity, agitator tip speed, gassed power per unit volume and mixing time. Generally, scale-up calculations from the 280 l scale were most similar to the parameters of installed equipment. Scale-up from the 30 l laboratory scale typically underpredicted parameters with scale-up from the 280 l scale being most appropriate. The 19,000 l fermentor installation was notably different in geometric similarity from the 280 l-1900 l scales since its design was meant to accommodate a wide range of operating volumes. Analysis of historical and recent processing performance was conducted for single cell bacterial or yeast fermentations which challenged peak operating conditions of the fermentors. Identification of key issues associated with scale-up for these specific pilot plant vessels was believed to be critical to efficient process development, clinical material production, and expected process transfer to a manufacturing facility.

Foam and Its Mitigation in Fermentation Systems

Key aspects of foaming and its mitigation in fermentation systems are presented. Foam properties and behavior, conditions that affect foaming, and consequences of foaming are discussed, followed by methods to detect and prevent foam, both without and with the use of antifoam, and their implications. Antifoams were catalogued according to their class (e.g., polyalkylene glycols, silicone emulsions, etc.) to facilitate recognition of antifoams possessing similar base compositions. Relatively few published studies directly comparing antifoams experimentally are available, but those reports found only partially identify clear benefits/disadvantages of any one antifoam type. Consequently, desired characteristics, trends in antifoam application, and chemical types of antifoams are evaluated on the basis of a thorough review of available literature reports describing a specific antifoamapos;s usage. Finally, examples of specific foaming situations taken from both the literature and from actual experience in an industrial fermentation pilot plant are examined for their agreement with expected behavior.

Development of a highly productive and scalable plasmid DNA production platform.

With the applications of DNA vaccines extending from infectious diseases to cancer, achieving the most efficient, reproducible, robust, scalable, and economical production of clinical grade plasmid DNA is paramount to the medical and commercial success of this novel vaccination paradigm. A first generation production process based on the cultivation of Escherichia coli in a chemically defined medium, employing a fed‐batch strategy, delivered reasonable volumetric productivities (500–750 mg/L) and proved to perform very well across a wide range of E. coli constructs upon scale‐up at industrial scale. However, the presence of monosodium glutamate (MSG) in the formulation of the cultivation and feed solution was found to be a potential cause of process variability. The development of a second generation process, based on a defined cultivation medium and feed solution excluding MSG, was undertaken. Optimization studies, employing a plasmid coding for the HIV gag protein, resulted in cultivation conditions that supported volumetric plasmid titers in excess of 1.2 g/L, while achieving specific yields ranging from 25 to 32 μg plasmid DNA/mg of dry cell weight. When used for the production of clinical supplies, this novel process demonstrated applicability to two other constructs upon scale‐up in 2,000‐L bioreactors. This second generation process proved to be scalable, robust, and highly productive.

On-line and in-situ monitoring technology for cell density measurement in microbial and animal cell cultures

Commercially available on-line and in-situ devices for monitoring cell density are reviewed in this article. Principles of operation are described as well as capabilities of these probes in specific measurement applications based on literature reports. Pilot-scale experimental observations from three optical density probes, the Cerex, Monitek and Wedgewood designs, have been included for Escherichia coli fermentations. Requirements for future on-line and in-situ instruments are discussed as well as the impact of current limitations on widespread application.

Isolation and Structure Elucidation of Thiazomycin —A Potent Thiazolyl Peptide Antibiotic from Amycolatopsis fastidiosa

Thiazolyl peptides are a class of rigid macrocyclic compounds richly populated with thiazole rings. They are highly potent antibiotics but none have been advanced to clinic due to poor aqueous solubility. Recent progress in this field prompted a reinvestigation leading to the isolation of a new thiazolyl peptide, thiazomycin, a congener of nocathiacins. Thiazomycin possesses an oxazolidine ring as part of the amino-sugar moiety in contrast to the dimethyl amino group present in nocathiacin I. The presence of the oxazolidine ring provides additional opportunities for chemical modifications that are not possible with other nocathiacins. Thiazomycin is extremely potent against Gram-positive bacteria both in vitro and in vivo. The titer of thiazomycin in the fermentation broth was very low compared to the nocathiacins I and III. The lower titer together with its sandwiched order of elution presented significant challenges in large scale purification of thiazomycin. This problem was resolved by the development of an innovative preferential protonation based one- and/or two-step chromatographic method, which was used for pilot plant scale purifications of thiazomycin. The isolation and structure elucidation of thiazomycin is herein described.

Use of soybean oil and ammonium sulfate additions to optimize secondary metabolite production

A valine‐overproducing mutant (MA7040, Streptomyces hygroscopicus) was found to produce 1.5 to 2.0 g/L of the immunoregulant, L‐683,590, at the 0.6 m3 fermentation scale in a simple batch process using soybean oil and ammonium sulfate‐based GYG5 medium. Levels of both lower (L‐683,795) and higher (HH1 and HH2) undesirable homolog levels were controlled adequately. This batch process was utilized to produce broth economically at the 19 m3 fermentation scale. Material of acceptable purity was obtained without the multiple pure crystallizations previously required for an earlier culture, MA6678, requiring valine supplementation for impurity control.

Evaluation of a microcarrier process for large-scale cultivation of attenuated hepatitis A

Microcarrier culture was investigated for the propagation of attenuated hepatitis A vaccine in the anchorage-dependent human fibroblast cell line, MRC-5. Cells were cultivated at 37°C for one to two weeks, while virus accumulation was performed at 32°C over 21 to 28 days. The major development focus for the microcarrier process was the difference between the cell and virus growth phases. Virus antigen yields, growth kinetics, and cell layer/bead morphology were each examined and compared for both the microcarrier and stationary T-flask cultures. Overall, cell densities of 4–5×106 cells/ml at 5–10 g/l beads were readily attained and could be maintained in the absence of infection at either 37°C or 32°C. Upon virus inoculation, however, substantial cell density decreases were observed as well as 2.5 to 10-fold lower per cell and per unit surface area antigen yields as compared to stationary cultures. The advantages as well as the problems presented by the microcarrier approach will be discussed.

Retrofit of CD-6 (Smith) impeller in fermentation vessels.

We extended prior studies on the influence of impeller type on fermentation performance to include a novel low-power-number, high-efficiency radial flow impeller, the CD-6, possessing six curved blades on a disk turbine. Dual impeller combinations of CD-6/CD-6, CD-6/Maxflo T, and CD-6/HE-3 were compared with Rushton/Rushton and Maxflo T/Maxflo T base cases. Qualitative comparisons of unaerated and aerated power draw in both water and glycerol were conducted. These suggested minimal power drops with aeration for dual CD-6 impellers and hybrids containing the CD-6 impeller design. We also examined fermentation performance for Streptomyces and Glarea secondary metabolite fermentations. A qualitative comparison of the data suggested that dual CD-6 impellers and hybrids containing the CD-6 impeller design resulted in reasonable power draws, improved mass transfer rates with airflow increases, and acceptable peak titers. These arrangements may warrant further study under a wider range of production conditions.

Indirect methods for characterization of carbon dioxide levels in fermentation broth

Various factors which influence dissolved carbon dioxide levels were indirectly evaluated in pilot scale and laboratory studies. For pilot scale studies, off-gas carbon dioxide (percentage in exit air) was measured using a mass spectrometer and then its potential impact on dissolved carbon dioxide concentrations qualitatively examined. Greater volumetric air flowrates reduced off-gas carbon dioxide levels more effectively at lower airflow ranges and thus lowered expected dissolved carbon dioxide levels through gas stripping. Lower broth pH values decreased off-gas carbon dioxide levels but increased expected dissolved carbon dioxide levels due to the pH-dependence of the gas/liquid carbon dioxide equilibrium. While back-pressure increases had an insignificant effect on off-gas carbon dioxide levels, they directly affected expected dissolved carbon dioxide levels according to Henrys law. Laboratory studies, conducted using both uninoculated and inoculated fermentation media, quantified the response of the media to pH changes with bicarbonate addition, specifically its buffering capacity. This effect then was related qualitatively to expected dissolved carbon dioxide levels. Higher dissolved carbon dioxide levels, as demonstrated by reduced pH changes with bicarbonate addition, thus would be expected for salt solutions of increased ionic strength and higher protein content media. In addition, pH changes with greater bicarbonate additions declined for fermentation samples taken over the course of a one week cultivation, most likely due to the higher protein content associated with biomass growth. The presence of weak acids/bases initially in the media or formed as metabolic by products, as well as the concentration of buffering ions such as phosphate, also were believed to be important contributing elements to the buffering capacity of the solution.

A next generation, pilot-scale continuous sterilization system for fermentation media

A new continuous sterilization system was designed, constructed, started up, and qualified for media sterilization for secondary metabolite cultivations, bioconversions, and enzyme production. An existing Honeywell Total Distributed Control 3000-based control system was extended using redundant High performance Process Manager controllers for 98 I/O (input/output) points. This new equipment was retrofitted into an industrial research fermentation pilot plant, designed and constructed in the early 1980s. Design strategies of this new continuous sterilizer system and the expanded control system are described and compared with the literature (including dairy and bio-waste inactivation applications) and the weaknesses of the prior installation for expected effectiveness. In addition, the reasoning behind selection of some of these improved features has been incorporated. Examples of enhancements adopted include sanitary heat exchanger (HEX) design, incorporation of a “flash” cooling HEX, on-line calculation of Fo and Ro, and use of field I/O modules located near the vessel to permit low-cost addition of new instrumentation. Sterilizer performance also was characterized over the expected range of operating conditions. Differences between design and observed temperature, pressure, and other profiles were quantified and investigated.