Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.     

Quantification of the HIV-1 virus-like particle production process by super-resolution imaging: From VLP budding to nanoparticle analysis

Virus-like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super-resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293-transient gene expression (TGE), High Five-baculovirus expression vector system (BEVS) and Sf9-BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293-TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non-assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM-based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.     

Galactogen, a virus-like particle from slugs

Virus-like particles were isolated from three species of slugs. Further investigation showed that the preparations contained very little nucleic acid and not more than 2% protein. Colorimetric and chromatographic analyses showed that they were composed almost entirely of galactogen and glycogen.     

GFP expression in Hydra: lessons from the particle gun

The cnidarian Hydra is an important model organism to study pattern formation and tem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have hot been accessible to gene transfection studies, we have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.     

Succinic acid production from Bacteroides fragilis: process optimization and scale up in a bioreactor

We report the effect of different physiological and nutritional parameters on succinic acid production from Bacteroides fragilis. This strain initially produced 0.70gL(-1) of succinic acid in 60h. However, when process optimization was employed, 5.4gL(-1) of succinic acid was produced in medium consisting of glucose (1.5%); tryptone (2.5%); Na(2)CO(3) (1.5%), at pH 7.0, when inoculated with 4% inoculum and incubated at 37 degrees C, 100rpm for 48h. A marked enhancement in succinic acid production was observed when the optimized conditions were employed in a 10L bioreactor. A total of 12.5gL(-1) of succinic acid was produced in 30h. This is approximately 12-fold increase in succinic acid production when compared to the initial un-optimized medium production. This enhancement in succinic acid production may be due to the control of CO(2) supply and the impeller speed. This is also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.     

Development and optimization of an adenovirus production process

Adenoviral vectors have a number of advantages such as their ability to infect post-mitotic tissues. They are produced at high titers and are currently used in 28% of clinical protocols targeting mainly cancer diseases through different strategies. The major disadvantages of the first generation of recombinant adenoviruses are addressed by developing new recombinant adenovirus vectors with improved capacity and safety and reduced inflammatory response. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. HEK-293 complementing cell line physiology, metabolism and viral infection kinetics were studied at small scale to identify optimal culture conditions. Batch, fed-batch and perfusion culture modes were evaluated. Development of new monitoring tools (in situ GFP probe) and quantification techniques (HPLC determination of total viral particles) contributed to acceleration of process development. On-line monitoring of physiological parameters such as respiration and biovolume of the culture allowed real-time supervision and control of critical phases of the process. Use of column chromatographic steps instead of CsCl gradient purification greatly eased process scale-up. The implementation of the findings at large scale led to the development of an optimized and robust integrated process for adenovirus production using HEK-293 cells cultured in suspension and serum-free medium. The two-step column-chromatography purification was optimized targeting compliance with clinical material specifications. The complete process is routinely operated at a 20-L scale and has been scaled-up to 100 L. Scale-up of adenoviral vector production in suspension and serum-free medium, and purification according to regulatory requirements, are achievable. To overcome metabolic limitations at high cell densities, use of perfusion mode with low-shear cell retention devices is now a common trend in adenovirus manufacturing. Further process improvements will rely on better understanding of the mechanisms of virus replication and maturation in complementing host cells.     

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

Background

Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly.

Results

SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production.

Conclusions

The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.     

Laccase production from oil palm industry solid waste: Statistical optimization of selected process parameters

Oil palm frond parenchyma tissue was used as a solid substrate for the production of laccase via solid‐state fermentation using the white rot fungus Pycnoporus sanguineus. With a rectangular aluminium tray as solid‐state fermentation bioreactor, process parameters such as bed height, moisture and supplemented nitrogen (as urea solution) levels were studied and optimized using a statistical design of experiment. The moisture level exerted a significant effect on the process. The interaction effect observed between bed height and supplemented nitrogen level suggested that uniform distribution of supplemented nitrogen into the substrate bed was important. The proposed regression model sufficiently predicted the process response over the experimental range tested. The optimum parameter combination for laccase production was a 3‐cm bed height, 72% w/w moisture and 0.21% w/v supplemented nitrogen. Laccase productivity remained constant when the tray size was increased from 1.4 to 3.4‐fold.     

A monolith purification process for virus-like particles from yeast homogenate

Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. The monolith structure provides a more open pore structure to improve accessibility for these materials and better mass transport from convective flow and reduced pressure drops. To examine the performance of this format for bioprocessing we selected the challenging capture of a VLP from clarified yeast homogenate. Using a recombinant Saccharomyces cerevisiae host it was found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity was shown to be three-fold superior for the monolith with equivalent 90% recovery of the VLP. To understand the impact of the crude feed material confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. It was seen that the lipid formed a layer on top of the column, even after regeneration of the column with isopropanol, resulting in increasing pressure drops with the number of operational cycles. Removal of the lipid pre-column significantly reduces the amount and rate of this fouling process. Using Amberlite/XAD-4 beads around 70% of the lipid was removed, with a loss of VLP around 20%. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25 mg/mL column.     

Selective flocculation and precipitation for the improvement of virus-like particle recovery from yeast homogenate

The purification of an intracellular product from a complex mixture of contaminants after cell disruption is a common problem in processes downstream of fermentation systems. This is particularly challenging for the recovery of particulate (80 nm in diameter) multimeric protein products, named virus-like particles (VLPs), from cell debris and other intracellular components. Selective flocculation for debris removal followed by selective precipitation of the target protein can be used as a preclarification step to aid purification. In this paper, selective borax flocculation of cell debris in yeast homogenate, followed by selective poly(ethylene glycol) precipitation of VLPs are defined with a view to demonstrating their potential in aiding the initial clarification stages of the purification sequence. The translation from laboratory scale to pilot scale operation is addressed, demonstrating the challenge of scale-up of solid-liquid separation stages for biological particle processing.     

Biochemical and physiological studies of the yeast virus-like particle.

A study was made of the virus-like particle (VLP) of Saccharomyces cerevisiae S7. This strain contains elevated amounts of P1 double-stranded ribonucleic acid (dsRNA) but no P2 dsRNA. The amount of dsRNA contained in cells grown on a fermentable carbon source (glucose) was compared with that in cells grown on a nonfermentable carbon source (ethanol). It was found that ethanol-grown cells contain higher levels of dsRNA than glucose-grown cells. In the former, the amount of dsRNA increased during the logarithmic phase of growth, whereas in the latter it increased during the transition from the logarithmic to the stationary phase. A method was devised to isolate VLPs from these cells by using CsCl gradients, and the yield was assessed by monitoring the recovery of dsRNA. Three proteins were found to be tightly associated with these particles. They have molecular weights of 75,000, 53,000, and 37,000. Together they account for almost all of the coding capacity of the P1 dsRNA that the VLP contains.     

Characterization and localization of a virus-like particle in aDrechslera species

A virus-like particle (VLP) has been found in a species of the fungusDrechslera. Four double-stranded RNA species with sizes of 3.8, 2.8, 2.7, and 2.2 kbp were isolated using CF-11 cellulose. These dsRNAs are associated with a 35-nm particle at a density of 1.37 g/cm³ in CsCl. The particle contains a major protein of 117 kDa and a minor protein of 89 kDa. In cellular fractionations of 2-week-old cultures, the VLPs are found in the mitochondrial pellet, but do not band with mitochondria in sucrose step gradients. In 1-week-old cultures, however, the VLP dsRNA is found in the cytoplasmic fraction. VLPs have not been reported previously in this genus.     

Kinetics of retrovirus production and decay.

There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271-278; Mulligan RC. 1993. Science 260:926-932; Orkin SH, Motulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C. Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed.     

Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization

A triphasic process was developed for the production of beta dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphE(al)) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter(-1) citrate, pH 6.5, and 37 degrees C are ideal parameters for CphE(al) production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter(-1) CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter(-1) induced CphE(al) production with only about 30% efficiency in comparison to that with CGP. CphE(al) was purified utilizing its affinity for the substrate and its specific binding to CGP. CphE(al) turned out to be a serine protease with maximum activity at 50 degrees C and at pH 7 to 8.5. Phase III comprises degradation of CGP to beta-aspartate-arginine and beta-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphE(al) preparation. Optimum degradation parameters were 100 g liter(-1) CGP, 10 g liter(-1) crude CphE(al) powder, and 4 h of incubation at 50 degrees C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphE(al) powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.     

Statistical analysis and optimization of biodiesel production from waste coffee grounds by a two-step process

Biodiesel was produced using waste coffee grounds (WCGs) via a two-step process comprising lipid extraction and subsequent transesterification steps. Each step was statistically analyzed, and optimum conditions for each step were suggested. WCGs were found to have 16.4% lipid content with 1.9% free fatty acid (FFA) content. The liquid-solid ratio (LSR) significantly influenced lipid extraction from WCGs, while extraction time and temperature did not; 92.7% of lipid extraction efficiency was achieved at 13.7 mL-hexane/g-WCGs, 30 min of extraction time, and 25°C. Owing to the relatively low FFA content, an alkaline catalyst (NaOH) reaction was used that requires less amount of catalyst, methanol, and shorter reaction time compared to an acid catalyst reaction. Reaction time and temperature were the major factors affecting biodiesel conversion, and 94.0% of biodiesel conversion was obtained at optimum conditions for transesterification: 0.5% catalyst, 1.5 mL-methanol/g-lipid, 45°C, and 9 h of reaction time. With the use of statistical analysis tools, high lipid extraction efficiency and biodiesel conversion were achieved at relatively mild conditions, which would reduce biodiesel production cost substantially.     

The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction

Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.     

HBc-based virus-like particle assembly from inclusion bodies using 2-methyl-2, 4-pentanediol

Hepatitis B virus core antigen (HBc) has recently been used as carriers to develop recombinant vaccines. However, not virus-like particles (VLPs) but inactive inclusion bodies are often formed for the chimeric proteins when expressed in Escherichia coli. A novel method for in vitro assembly of chimeric HBc-MAGE3 II from inclusion bodies to VLPs was established in this study. The method utilized 2-methyl-2, 4-pentanediol (MPD), an amphipathic di-alcohol, to dissociate sodium dodecyl sulfate (SDS) from the solubilized chimeric protein to initiate VLP assembly. The HBc-MAGE3 II could assemble into VLPs only when the molar ratio of SDS/protein subunit was less than 0.14. After removing SDS/MPD by desalting and further purification, VLPs with similar morphology to the natural virus were obtained. This method could be used for preparation of other VLPs expressed as inclusion bodies.