Barry C. Buckland

Metabolic engineering and directed evolution for the production of pharmaceuticals.

The tools of metabolic and enzyme engineering have been well developed in academic laboratories and are now being applied for the optimization of biocatalysts used in the production of a wide range of pharmaceutically important molecules. Engineered microorganisms with a diverse set of modified or non-native enzyme activities are being used both to generate novel products and to provide improved processes for the manufacture of established products, such as in the production of precursors, intermediates, and complete compounds of importance to the pharmaceutical industry, including polyketides, nonribosomal peptides, steroids, vitamins, and unnatural amino acids. The use of directed evolution has rapidly emerged to be the method of choice for the development and selection of mutated enzymes with improved properties. A variety of such methods have been used to alter the activity, stability and availability of an array of enzymes. The industrial practice of these technologies at large scale is, however, in its infancy and stands as an exciting challenge for process scientists today.

The process development challenge for a new vaccine

The challenges of vaccine development are not limited to identification of suitable antigens, adjuvants and delivery methods, but include regulatory, technical and manufacturing hurdles in translating a vaccine candidate to the clinic. Process development is the technological foundation that underlies the manufacture of new vaccines and is central to successful commercialization.

Asymmetric bioreductions: application to the synthesis of pharmaceuticals

Selected examples of asymmetric bioreductions of pharmaceutically relevant prochiral ketones are reviewed. These data show that microbial screens lead to the identification of appropriate biocatalysts, and that the use of miniaturized and semi-automated technology can greatly reduce both labor and lead times. The same data also highlight the need to evaluate a . relatively large andror diverse microbial population highlighting biodiversity . We also found that in many instances the luxury of producing either enantiomers with high optical purity, enantiocomplementarity, can be achieved when employing different microbial strains. Process development studies reviewed here demonstrate that it is possible in some cases to understand and control the production of an unwanted enantiomer or by-product. Finally, a specific example, the asymmetric bioreduction of a ketone by Candida sorbophila, shows that process development studies which optimized, the bioreduction . environmental conditions pH, temperature . . . , the addition of ketone, and the implementation of a nutrient feeding strategy in conjunction with the use of a defined cultivation medium were key in achieving increased bioreduction rates and product titers. When scaled-up in pilot plant bioreactors, the bioreduction process supported the production of several kilograms of . . . R -alcohol enantiomeric excess e.e. ) 98% , with an isolated product yield of about 80%. q 2001 Elsevier Science B.V. All rights reserved.

Bioconversion of indene to cis (1S,2R) indandiol and trans (1R,2R) indandiol by Rhodococcus species

Abstract cis (1 S ,2 R ) indandiol or trans (1 R ,2 R ) indandiol are both potential precursors to (−)- cis (1 S ,2 R )-1-aminoindan-2-ol, a key chiral synthon for Crixivan ® (Indinavir), a leading HIV protease inhibitor. Enrichment and isolation studies yielded two Rhodococcus sp. strain B 264-1 (MB 5655) and strain I-24 (MA 7205) capable of biotransforming indene to cis (1 S ,2 R ) indandiol and trans (1 R ,2 R ) indandiol respectively. Isolate MB 5655 was found to have a toluene dioxygenase, while isolate MA 7205 was found to harbor both toluene and naphthalene dioxygenases as well as a naphthalene monooxygenase. When scaled up in a 14- l bioreactor, MB 5655 produced up to 2.0 g/ l of cis (1 S ,2 R ) indandiol with an enantiometric excess greater than 99%. MA 7205 cultivated under similar conditions produced up to 1.4 g/ l of trans (1 R ,2 R ) indandiol with an enantiomeric excess greater than 98%. Process development studies yielded titers greater that 4.0 g/ l of cis indandiol for MB 5655. Due to their resistance to indene toxicity and easy cultivation in bioreactors, both Rhodococcus sp. strains appeared as good candidates for future strain engineering and process development work.

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimers disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.

Pilot-scale harvest of recombinant yeast employing microfiltration: a case study

In order to develop a cost-effective recovery process for an intracellular product, crossflow microfiltration was studied for the harvest of a recombinant yeast under severe time constraint. It was required to process yeast broth in a short period of time to minimize the risk for product degradation. Preliminary microfiltration studies employing flat sheet membranes showed high throughout with initial fluxes on the order of water fluxes (> 1000 LMH, regime I, < 2 min), followed by a rapid decay towards a low pseudo-steady state flux (20 LMH, regime II, > 2 min). Exploitation of these high fluxes and control of their eventual decline were crucial in establishing a rapid crossflow filtration process. The effect of several parameters, such as initial cell concentration, shear rate, transmembrane pressure, membrane pore size and medium composition on filtration performance were investigated to better understand the flux decline mechanisms. We found that the major contributor to flux decay was reversible fouling by the cake formation on the membrane surface. Within the operating boundaries of our microfiltration system, large-pore membrane (0.65 micron) was much more desirable for harvesting our yeast (10 microns size) without cell leakage than smaller pore ones (0.22 micron and 0.45 micron). Among adjustable operating parameters, feed flow rate (i.e., shear rate) exerted significant impact on average flux, whereas manipulation of transmembrane pressure afforded little improvement. Although initial cell concentration affected adversely the permeation rates, growth medium components, especially soy-peptone, was deemed pivotal in determining the characteristics of cell cake, thus controlling yeast microfiltration.

Enhancement of Lipase Production during Fed-Batch Cultivation of Pseudomonas aeruginosa MB 5001

Abstract Lipase production by Pseudomonas aeruginosa (strain MB 5001) was enhanced 6.6-fold by developing a fed-batch fermentation process. In batch culturing, lipase synthesis occurred during decelerated cell growth. Building on this information, lipase production was increased by feeding a balanced solution of glucose and ammonium chloride (carbon to nitrogen ratio of 4.13). Best performance was achieved when feeding was initiated during mid growth phase, prior to lipase biosynthesis. This lipase fermentation process was found to be unusually sensitive to dissolved oxygen tension, requiring cultivation of the microorganisms initially under dissolved oxygen limiting conditions followed by non-limiting dissolved oxygen conditions (lipase production phase). These process improvements yielded rapidly to scale up from laboratory bioreactors (23-l) to pilot plant bioreactors (1,900-l).

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and kLa). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.

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.

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×109 cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.