Industrial choices for protein production by large-scale cell culture.

Traditional barriers to large-scale mammalian culture have now been addressed, with the standard stirred-tank reactor emerging as industry's technology of choice. The issues of adapting cells to suspension culture, shear sensitivity and oxygen supply have been largely resolved. But for many low-volume and specialty applications, such as the production of viral vaccines and gene therapies, reactor technology remains diversified, with reactor types ranging from roller bottles to stacked plates and hollow fibers.

Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

Influence of metam sodium on the dissipation and residual biological activity of the herbicides EPTC and pebulate in surface soil under black plastic mulch.

Metam sodium is a potential replacement for methyl bromide, which is used to control soil pests. Metam sodium rapidly breaks down in the soil to form methylisothiocyanate (MITC). Dissipation of the herbicides EPTC and pebulate in a silt loam soil under plastic mulch in the absence and presence of metam sodium was examined in field experiments in 1998 and 1999 at Knoxville, Tennessee. EPTC half-life (DT(50)) was 9 d, but when applied in conjunction with metam sodium DT(50) increased to 22 d. Similarly, average pebulate DT(50) was 8 d and increased to 23 d when applied in conjunction with metam sodium. This increase in herbicide DT(50) with the addition of metam sodium is thought to be due to a reduction in soil microorganisms that degrade EPTC and pebulate. EPTC applied with metam sodium injured tomato plants and reduced total crop yield more than EPTC, pebulate, or pebulate with metam sodium. The increased tomato injury may have been related to the greater and prolonged activity of EPTC and slower EPTC dissipation in the presence of metam sodium or MITC.

Biocatalysis for pharmaceuticals--status and prospects for a key technology.

In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.

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.

Usefulness of the transcendental meditation program in the treatment of patients with coronary artery disease.

Twenty-one patients with documented coronary artery disease were tested at baseline by exercise tolerance testing, and assigned to either stress reduction using the Transcendental Meditation (TM) program or to a wait-list control, After 8 months, the TM group had a 14.7% increase in exercise tolerance, an 11.7% increase in maximal workload, an 18% delay in onset of ST-segment depression, and significant reductions in rate-pressure product at 3 and 6 minutes, and at maximal exercise compared with the control group.

Advancing observational quality methods for the measurement of forensic psychiatric care.

Discusses some of the current problems associated with observational quality measurement. Uses Lerninger's conceptual theory-generating model as the basis of discussion. Puts forward a qualitative approach which breaks away from the traditional methods.

Titration of human-bovine rotavirus reassortants using a tetrazolium-based colorimetric end-point dilution assay.

A colorimetric end-point dilution assay was developed for the titration of rotavirus-containing samples that uses commercially available tetrazolium dyes as an indicator of virus infection. This assay offers several advantages over both plaque assays and traditional end-point dilution methods. The latter assays require manual counting of plaques or the scoring of wells for the presence of virus based on observed cytopathic effects. The colorimetric end-point dilution assay enables the scoring of wells based upon absorbance readings alone, thereby eliminating time-consuming and subjective manual screenings. This method also has the potential for automating the analysis of large numbers of samples. Virus titers of human-bovine rotavirus reassortants obtained using this method are comparable to those determined by plaque assay. The scoring of wells based on absorbance readings was also found to agree with manual scoring of cytopathic effects and with the production of viral antigen.

Inside looking out: innovations in community health nursing.

The Portland Veterans Affairs Medical Center provides a range of health care services for an aging population. The chronic, multiple health problems of veterans and the complexity of the health care delivery system often lead to fragmented and discontinuous care. The innovative model of advanced practice nursing described here involves using hospital-based community health CNSs in the role of community health coordinators. The three CNSs described here each have unique and overlapping responsibilities. Based primarily in the medical center, they are able to facilitate a smooth patient transition from the hospital to the community using knowledge and experience to assess, plan, implement, and evaluate care needs of three defined populations. In this article, responsibilities of the community health CNS are defined and the Portland VA Medical Center and population served are described. The history of the community health coordinator role is outlined, followed by the current role description and targeted populations, and role impacts to date.

In pursuit of the optimal fed-batch process for monoclonal antibody production.

Fed-batch culture currently represents the most attractive choice for large scale production for monoclonal antibodies (MAbs), due to its operational simplicity, reliability, and flexibility for implementation in multipurpose facilities. Development of highly productive cell lines, maximization of cell culture longevity, and maintenance of high specific antibody secretion rates through genetic engineering techniques, nutrition supplementation, waste product minimization, and control of environmental conditions are important for the design of high-yield fed-batch processes. Initially simple supplementation protocols have evolved into sophisticated serum-free multi-nutrient feeds that result in MAb titers on the order of 1-2 g/L. Limited research has been published to date on the effect of various culture parameters on potentially important quality issues, such as MAb glycosylation and stability. Although most fed-batch protocols to date have relied on relatively simple control schemes, increasingly sophisticated algorithms must be applied in order to take full advantage of the potentially additive effects of manipulating nutrient and environmental parameters to maximize fed-batch process productivity.

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process.

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed.

Effect of specific growth rates on productivity in continuous open and partial cell retention animal cell bioreactors.

A clonal derivative of a transfectant of the SP2/0 myeloma cell line producing a chimeric monoclonal antibody was cultivated in both continuous open and continuous partially-closed bioreactors. Using an open system for the determination of kinetic parameters, we showed that the production of this chimeric mAb was growth associated. As such, the volumetric productivity increased linearly with increasing dilution rate up to the maximum dilution rate. Three continuous cultivations employing partial cell retention were conducted. In agreement with mathematical predictions, the product titer and volumetric productivity were independent of the degree of cell retention when the total dilution was held constant. When cells were maintained at a low specific growth rate, the product titer was independent of dilution rate and the volumetric productivity increased with increasing dilution rate, again in agreement with mathematical predictions. Since the partially-closed bioreactor could be operated at dilution rates in excess of the maximum specific cellular growth rate, volumetric productivities were greater than those achievable in the open bioreactor. However, when cells were maintained at a high specific growth rate, cell accumulation was limited and product titers decreased at high dilution rates. Therefore, the volumetric productivity in this latter case did not increase at higher dilution rates.

Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture.

A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day(-1). The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day(-1). Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.

Influence of a Maharishi Ayur-vedic herbal preparation on age-related visual discrimination.

An ancient system of natural medicine--Maharishi Ayur-Vedic--prescribes certain herbal formulas to enhance cognitive functioning, prevent illness, and alleviate the detrimental effects of the aging process. A double-blind study was conducted to test the effect of an Ayurvedic herbal preparation, Maharishi Amrit Kalash (MAK), on an age-related alertness task. Forty-eight men over 35 years of age were randomly assigned to receive MAK tablets or a closely matched placebo twice daily for six weeks. A visual discrimination task consisted of the identification of the exact location of a stimulus "v" within an array of x symbols in tachistoscopic presentations. The MAK group improved significantly more in their performance of this task after three and six weeks of treatment relative to the placebo group. Performance was highly correlated with age, and because successful performance apparently requires an unrestricted flow of homogeneous attention as well as focalized concentration, it is concluded that MAK may enhance attentional capacity or alertness, and thus reverse some of the detrimental cognitive effects of aging.