Computational fluid dynamics analysis of mixing and gas-liquid mass transfer in wave bag bioreactor.

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (kL a) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector.

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

Targeting lipopolyplexes using bifunctional peptides incorporating hydrophobic spacer amino acids: synthesis, transfection, and biophysical studies.

We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif.

The fractal structure of polycation-DNA complexes.

We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.

Computational-fluid-dynamics (CFD) analysis of mixing and gas-liquid mass transfer in shake flasks.

CFD (computational fluid dynamics) techniques were used to predict mixing and gas-liquid mass transfer in a 250 ml shake flask operating over a range of shaking frequencies between 100 and 300 rev./min, shaking diameters between 20 and 60 mm, and fill volumes between 25 and 100 ml. Interfacial area, a, volumetric mass-transfer coeffcient, kLa, and the power input per unit volume, epsilonv, of the liquid were predicted to be 300

Rapid characterization of outer-membrane proteins in Neisseria lactamica by SELDI-TOF-MS (surface-enhanced laser desorption ionization-time-of-flight MS) for use in a meningococcal vaccine.

Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal Neisseria species, particularly with Neisseria lactamica. We have reported previously that immunization with N. lactamica outer-membrane vesicles containing the major OMPs (outer-membrane proteins) protected mice against lethal challenge with meningococci of diverse serogroups and serotypes and has the potential to form the basis of a vaccine against meningococcal diseases [Oliver, Reddin, Bracegirdle et al. (2002) Infect. Immun. 70, 3621-3626]. In the present study, we have shown that biomass production and the profile of outer-membrane vesicle proteins may be affected by fermentation conditions and, in particular, media composition. Ciphergen SELDI-TOF Protein Chips were used as a rapid and sensitive new method in comparison with conventional SDS/PAGE. SELDI-TOF-MS (surface-enhanced laser-desorption ionization-time-of-flight MS) reproducibly identified three major OMPs (NspA, RmpM and PorB) and detected the changes in the protein profile when the growth medium was altered. The findings of this work indicate that SELDI-TOF-MS is a useful tool for the rapid optimization of OMP production in industrial fermentation processes and can be adapted as a Process Analytical Technology.

Separation of immunoglobulin G precipitate from contaminating proteins using microfiltration.

A small-scale stirred-cell device was used to separate an antivenom antibody precipitate from a suspension containing contaminating soluble proteins. The device has a total volume of 200 ml and is equipped with a filter membrane with a cut-off size of 3 microm. About 90% of the antibody-precipitate particles in the feed were 29 microm or smaller, and the concentration of solids was 12% (w/w). The microfiltration cell was operated in a constant-volume (continuous/diafiltration) mode, and its performance was compared with an industrial disc-stack centrifuge currently used in the manufacture of antibody precipitate. In terms of product purity, the separation performance of the microfiltration operation was found to be comparable with the disc-stack centrifuge, whereas the overall yield was 10% better than that obtained from the centrifuge. This was attributed to the ability of microfiltration to reduce material losses by integrating a number of operations in a single piece of equipment. These included separation, concentration and buffer exchange, as well as dissolution and final recovery of the antibody in an appropriate buffer. The results obtained from the stirred cell are potentially scaleable, and dynamic microfiltration is shown to be an attractive process option.

Shear-induced release of disabled herpes simplex virus from baby-hamster kidney cells.

A new process route is proposed to increase the production yield of disabled herpes simplex virus type 1 (HSV-1 DIS). Infected baby-hamster kidney (BHK) cells were subjected to a range of shear rates between 3.69 x 10(3) s(-1) and 51.3 x 10(3) s(-1) in the gap between a pair of co-axial cylinders. Analysis of the supernatant fractions of sheared material established that optimal virus release was achieved by exposing the infected cells to a shear rate of 42.7 x 10(3) s(-1) for a period of 1 min. Compared with the current laboratory process, the titre of HSV-1 DIS was increased over 30-fold, from about 1 x 10(6) to 30 x 10(6) pfu (plaque-forming units)/ml. Evaluation of the supernatant fractions by flow cytometry, total protein assay, PAGE and dot-blot assays showed no evidence of cell disruption, supporting the hypothesis that shear-induced release of the cell-membrane-bound virus was achieved without compromising downstream purification. The proposed method is scalable, and since no additional chemicals are required, it provides an attractive option for enhanced recovery of virus particles for therapeutic applications.

Prediction of size distribution of lipid-peptide-DNA vector particles using Monte Carlo simulation techniques.

Concerns with insertional mutagenesis for retrovirus and immunogenicity for adenovirus have motivated research into development of non-viral vectors that can safely deliver desired gene constructs to target cells in tissues and organs. Many non-viral vectors suffer from unacceptably poor in vivo cell transfection and low transgene expression. Evidence suggests that cell transfection is linked to particle size - vector particles below about 200 nm are considered desirable. Experimental measurements indicate, however, that vector particles are susceptible to significant aggregation under most conditions of pH and ionic strength, including physiological conditions, although there are currently no means of predicting the kinetics of aggregation. The present paper addresses this challenge by presenting a mathematical framework based on the Monte Carlo simulation techniques for modelling the dynamics of aggregation. The approach is used to simulate the evolution of particle-size distribution for an integrin-targeting lipid-peptide-DNA vector system in buffers of different pH and ionic strength. The simulations required two input parameters, including the initial-size distribution of the particles and a fitting parameter (alpha). Comparison of simulations with experimental data showed that alpha was closely related to the zeta potential of the particles in the buffer medium, making simulations fully predictive. The modelling approach may be used in other vector systems.

An automated microplate-based method for monitoring DNA strand breaks in plasmids and bacterial artificial chromosomes.

A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r2 > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of <5% coefficient of variation. Automation of the method showed equivalence to the manual procedure with high reproducibility and low variability within wells. The method described requires as little as 0.5 ng of DNA per well and a 96-well microplate can be analysed in 12 min providing an attractive option for analysis of high molecular weight vectors. A preparation of a 116 kb bacterial artificial chromosome was subjected to chemical and shear degradation and DNA integrity was tested using the method. Good correlation was obtained between time of chemical degradation and shear rate with fluorescence response. Results obtained from pulsed- field electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method.

The influence of physico-chemical and process conditions on the physical stability of plasmid DNA complexes using response surface methodology.

Research is progressing fast to find safe and effective methods of delivering therapeutic genes to patients afflicted with a range of genetic and acquired diseases that either do not respond at all, or respond poorly, to treatment with small-molecule drugs or protein-replacement therapy. A technical barrier that remains relates to the need for scalable operations that can consistently and reproducibly make large quantities of the therapeutic gene vectors under the current Good Manufacturing Practice ('cGMP'). The present investigation focuses on these issues and introduces a new method of assessing the engineering effects of process and material factors on the colloidal properties of plasmid-DNA delivery systems based on response surface methodology (RSM) and experimental techniques. Previously, experiments have shown that several factors can reduce the physical stability of non-viral delivery systems. Specifically, it has been demonstrated that the mean size and charge of plasmid DNA condensed by cationic agents are affected by many factors, including the pH and ionic strength of the buffer, and the method of preparation. For example, the method and intensity of mixing of the DNA with condensing and conjugating agents have been shown to be important. Using RSM to analyse new experimental data in the present paper, we report on the impact of these factors and, more crucially, the effects of interaction between the factors on the colloidal properties of the DNA-vector complexes. Specifically, for plasmid DNA condensed by poly-L-lysine, interactions between ionic strength, pH and DNA concentrations play a critical role. Whether poly-L-lysine should be used as a condensing agent in the final delivery system remains to be demonstrated. However, the use of RSM combined with the scaleable experimental approach described in this paper may be applied to other delivery systems.

Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA.

The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment of early clinical results. pDNA is a large molecule and has properties that are similar to those of the contaminating chromosomal DNA. These, combined with the low initial concentration of plasmids in the host cell, provide unique process challenges that require significant upfront design to establish robust manufacturing processes that can also comply with current Good Manufacturing Practice ('cGMP') and produce milligram-to-kilogram quantities of pDNA product. This review describes promising scaleable processes that are currently being assessed for production of therapeutic supercoiled pDNA. Fermentation strategies for improving supercoiled plasmid yield and reducing contaminant concentrations are reviewed, and downstream processes are assessed for their ability to efficiently remove cellular contaminants, separate the supercoiled form of the pDNA from its open circular and linear forms, and prepare the purified drug substance for formulation. Current strategies are presented for developing stable delivery systems, and approaches to quality assurance and quality control are discussed.

A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells.

Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5alpha/pSVbeta was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s(-1), with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed.