Use of adenoviral vectors as veterinary vaccines.

Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

Computational and experimental investigation of flow and fluid mixing in the roller bottle bioreactor.

The fully three-dimensional velocity field in a roller bottle bioreactor is simulated for two systems (creeping flow and inertial flow conditions) using a control volume-finite element method, and validated experimentally using particle imaging velocimetry. The velocity fields and flow patterns are described in detail using velocity contour plots and tracer particle pathline computations. Bulk fluid mixing in the roller bottle is then examined using a computational fluid tracer program and flow visualization experiments. It is shown that the velocity fields and flow patterns are substantially different for each of these flow cases. For creeping flow conditions the flow streamlines consist of symmetric, closed three-dimensional loops; and for inertial flow conditions, streamlines consist of asymmetric toroidal surfaces. Fluid tracers remain trapped on these streamlines and are unable to contact other regions of the flow domain. As a result, fluid mixing is greatly hindered, especially in the axial direction. The lack of efficient axial mixing is verified computationally and experimentally. Such mixing limitations, however, are readily overcome by introducing a small-amplitude vertical rocking motion that disrupts both symmetry and recirculation, leading to much faster and complete axial mixing. The frequency of such motion is shown to have a significant effect on mixing rate, which is a critical parameter in the overall performance of roller bottles.

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production.

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.

Large-scale mammalian cell culture.

Mammalian cell culture continues to draw major research efforts. A great deal of progress has recently been made in cellular physiology, especially in factors adversely affecting cell growth or viability. Through molecular genetic manipulation, cells are more readily cultivated in a medium free of animal proteins. Achieving a high cell concentration and high viability continue to be common themes in engineering research. The need to implement a control policy for fed-batch and perfusion cultures has prompted increased efforts in process monitoring and control. Integrating these advances will be beneficial for ensuring product quality and process consistency.

Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production.

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.

Two-dimensional versus three-dimensional culture systems: Effects on growth and productivity of BHK cells.

The influence of surface growth (two-dimensional microcarriers) and three-dimensional growth (aggregates and macroporous supports) in agitated, suspended batch culture systems upon growth and productivity of BHK was compared. Cultures using three porous microcarriers (CultiSpher G, Cellsnow EX, and Cytocell), one nonporous microcarrier (Cytodex 3) and natural aggregates were performed in stirred tanks using two different agitation rates (60 and 100 RPM). With the exception of Cytocell, cell growth, viability, and productivity were similar when three-dimensional structures (porous microcarriers and aggregates) were used. Nonporous microcarriers only compared well at 60 RPM as growth ceased under overagitation. These results suggest that cultures less susceptible to fluid shear are advantageous for scale-up. (c) 1996 John Wiley & Sons, Inc.

Assessment of virus infection in cultured cells using metabolic monitoring.

A rapid, in-process assessment of virus replication is disired to quickly investigate the effects of process parameters on virus infection, and to monitor consistency of process in routine manufacturing of viral vaccines. Live virus potency assays are generally based on plaque formation, cytopathic effect, or antigen production (TCID(50)) and can take days to weeks to complete. Interestingly, when infected with viruses, cultured cells undergo changes in cellular metabolism that can be easily measured. These phenomena appear to be common as they has been observed in a variety of virus-host systems, e.g., in insect cells infected with baculovirus, Vero cells infected with Rotavirus, MRC-5 cells infected with Hepatitis A virus, and MRC-5 cells infected with the Varicella Zoster Virus (VZV). In this article, changes in glycolytic metabolism of MRC-5 cells as a result of CVZ infection are described. Both glucose consumption and lactate production in VZV infected MRC-5 cells are significantly elevated in comparison to uninfected cells. Based on this result, a rapid, in-process assay to follow VZV infection has been developed. The relative increase in lactate production in infected cells (α) increases as the infection progresses and then plateaus as the infection peaks. This plateau correlates with time of peak virus titer and could be used as a harvest triggering parameter in a virus production process.X(u) = cell density of uninfected cellsX(i) = cell density of infected cellsX(T) = total cell densityL(i) = cumulative lactate production in infected culturesL(u) = cumulative lactate production in uninfected culturesq(Li) = specific lactate production of infected cellsq(Lu) = specific lactate production of uninfected cellsk(1), K(2) = constants.

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 3.5×10(9) 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.

Hydrodynamic effects on BHK cells grown as suspended natural aggregates.

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc.

Cultivation of attenuated hepatitis A virus antigen in a titanium static mixer reactor.

The titanium static mixer reactor, demonstrated for a variety of vaccine processes during the late 197s, was investigated for the production of attenuated hepatitis A virus antigen from anchorage-dependent MRC-5 cells. This reactor system used Charles River Biotechnological Services cabinets for monitoring and process control. Cell inoculation protocols, using 6000-10,000 cells/cm(2), resulted on over 95% attachment at both the laboratory and pilot scales. Indirect monitoring techniques using oxygen, glucose, L-serine, and L-glutamine uptake rates were indicative of cell growth prior to virus inoculation as well as environmental and/or nutrient limitations. Seven laboratory-scale (3900 cm(2)) runs and one pilotscale (265,000 cm(2)) run were conducted to investigate refeeding regiments, parallel versus perpendicular element orientation, increased element surface area per unit volume, and scale-up performance. In general, lysate antigen yields achieved were similar to those of parallel T-flasks cultivated under similar conditions.

Studies of baby hamster kidney natural cell aggregation in suspended batch cultures.

Microcarrier cultures of animal cells of industrial relevance are known to shed aggregates into the suspension phase. For a BHK cell line, which is known to be prone to aggregate naturally, microcarrier and aggregate forms of culture are compared in spinner culture. In microcarrier cultures, it is shown that increasing initial microcarrier concentration yields decreasing concentration of smaller aggregates in suspension; roughly equivalent concentrations of total cells and single cells in suspension are obtained. In the absence of Cytodex 3, aggregate final size is hydrodynamically controlled in batch and semicontinuous suspension culture. Rate of agitation is the main variable controlling aggregate size in batch cultures. The range of agitation rates studied (20 to 70 rpm in 250 mL spinner flasks) produced aggregates with maximum sizes of 200 microns. Necrotic centers were not observed; this was confirmed by Trypan blue viability measurements after mechanical dissociation of aggregates and also by the constant productivity obtained from different aggregate sizes. Comparing aggregate and microcarrier culture conditions, it is shown that at 100 rpm maximum total cell concentration is larger in the absence of microcarriers; dead cell concentrations, most of which exist in suspension, are slightly larger in microcarrier culture. Total viable cell concentrations in aggregate, hydrodynamically controlled culture, are almost one order of magnitude higher than in microcarrier cultures. These results suggest that there might be advantages in using aggregate cultures under hydrodynamic control of aggregate size in lieu of microcarrier cultures for naturally aggregating cell lines.

Changes in animal cell natural aggregates in suspended batch cultures.

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical dissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing in size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1.2 x 10(6) cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 x 10(8) cells/ml, with a porosity of 55%. Viability was consistently in the range 85-90%, both for aggregate and suspended cells.

Repeated-batch cultures of baby hamster kidney cell aggregates in stirred vessels.

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate characteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this 'selection' procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.

Monoclonal antibody process development using medium concentrates.

A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

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 degrees C for one to two weeks, while virus accumulation was performed at 32 degrees 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 x 10(6) cells/ml at 5-10 milligrams beads were readily attained and could be maintained in the absence of infection at either 37 degrees C or 32 degrees 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.

Experimental collision efficiencies of polymer-flocculated animal cells.

The determination of particle collision kinetics is useful to decouple the effects of process parameters on individual events in flocculation. This paper discusses the effects of flocculation conditions on the collision efficiency of ATCC strain CRL 1606 hybridomas flocculated with poly-L-histidine. Experimental determinations of the collision efficiency of cells in Couette flow are presented over a range of experimental conditions. The collision efficiency correlates with the cell zeta potential to the -2.4 power at high surface coverage, consistent with literature results in latex systems. At low coverage, accounting for the distribution of polymer on the cells corrects for deviation from the high-coverage behavior. Collision is dependent on the hydrodynamic environment as well. At high surface coverage, collision efficiency is weakly dependent on hydrodynamic conditions and follows a dependency on the shear rate and viscosity to the -0.32 power. This is consistent with ionic coagulation theory. At low surface coverage, the collision efficiency is strongly dependent on the viscous fluid forces. The results versus both dose and shear rate over the entire range of surface coverages are consistent with weak intercell bonding. Collision kinetics in the presence of high molecular weight dextrans show steric hindrance to cell collision.

Effects of paddle impeller geometry on power input and mass transfer in small-scale animal cell culture vessels.

Process scaleup for stirred-tank animal cell cultures such as suspension and microcarrier cultures often begins at the bench scale in small spinner vessels. In order to initiate process development under the proper conditions, it is essential to know the physical conditions under which the cells are grown. In this article, power inputs and surface oxygen transfer rates to culture medium in 500-mL Corning spinner vessels were determined as a function of the impeller geometry, impeller height, and agitation speed. The results obtained indicate that power dissipation dependency differs from literature correlations and may compromise scale up at constant power input from these vessels. These results are of general utility to researchers using small-scale vessels.