Barbara Jardin

On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures

Respiration rates in Spodoptera frugiperda (Sf‐9) cell bioreactor cultures were successfully measured on‐line using two methods: The O2 uptake rate (OUR) was determined using gas phase pO2 values imposed by a dissolved oxygen controller and the CO2 evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf‐9 cultures. Infection led to increases in volumetric and per‐cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant β‐galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5–100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant β‐galactosidase.

On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process.

On‐line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (ϵ′) and CO2 evolution rates (CER) were measured on‐line using the biomass monitor and the infrared CO2 analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf‐9) and Trichoplusia ni (High‐5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off‐line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The ϵ′, CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The ϵ′ profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf‐9 cultures under conditions of cell densities from 3 to 50 × 106 cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High‐5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off‐line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.

Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells.

Production of a TGFβ receptor with high density baculovirus infected Sf-9 cells (7×106cells ml-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGFβ receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO2 partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO2 at this concentration has been assessed at smaller scale for TGFβ receptor and β-gal expression, in controlled atmosphere incubators.

Insect cell density in bioreactor cultures can be estimated from on-line measurements of optical density

Total cell density in growing insect cell suspension cultures was accurately estimated from both off-line and on-line measurements of optical density (OD). The latter measurements were done with an in-situ autoclavable OD sensor. The ability to continuously monitor cell density in insect cell cultures may be useful for the development of a large-scale process for recombinant protein production using baculovirus expression vectors.

Recombinant Baculovirus as a Highly Potent Vector for Gene Therapy of Human Colorectal Carcinoma: Molecular Cloning, Expression, and In Vitro Characterization

Present therapeutic strategies for most cancers are restricted mainly to the primary tumors and are also not very effective in controlling metastatic states. Alternatively, gene therapy can be a potential option for treating such cancers. Currently mammalian viral-based cancer gene therapy is the most popular approach, but the efficacy has been shown to be quite low in clinical trials. In this study, for the first time, the insect cell-specific baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been evaluated as a vector for gene delivery to colorectal cancer cells. Experiments involving factorial design were employed to study the individual and combined effects of different parameters such as multiplicity of infection (MOI), viral incubation time and epigenetic factors on transduction efficiency. The results demonstrate that baculovirus gene delivery system holds immense potential for development of a new generation of highly effective virotherapy for colorectal, as well as other major carcinomas (breast, pancreas, and brain), and offers significant benefits to traditional animal virus-based vectors with respect to safety concerns.

Recombinant Protein Production in Large-Scale Agitated Bioreactors Using the Baculovirus Expression Vector System.

The production of recombinant proteins using the baculovirus expression vector system (BEVS) in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantification, bioreactor preparation and operation, the infection process, final harvesting, and primary separation steps for recovery of the product are presented. Furthermore, methods involved with advanced on-line monitoring and bioreactor control, which have a significant impact on the overall process success, are briefly discussed.

Topical application of complement C3 in collagen formulation increases early wound healing.

Abstract Background: The complement system is composed of bactericidal and hemolytic proteins that increase capillary leakage and inflammatory cell migration. The role of complement C3 to augment wound healing has not yet been studied. Methods: We examined the effects of topical complement C3 formulation at two concentrations (10 and 100 nM) on the rat surgical skin incision model. Skin was examined for maximal breaking strength and sectioned for histological examination. Fibronectin and collagen I content were measured using western blot analysis. Results: There was a statistically significant 74% increase in maximum wound strength with the topical application of 100 nM of C3 at day 3 (850 ± 138 g) when compared to the control rats (490 ± 57 g). Histological correlation was seen with an increased inflammatory cell and fibroblast infiltration in treated wounds as compared to control rats as early as 3 days post-wounding. Western blots revealed increased fibronectin and collagen I levels in C3 treated wounds. Conclusions: Topical application of complement C3 in collagen formulation to skin wounds significantly increases wound healing as early as 3 days after wounding. This is correlated with increased inflammatory cell recruitment and the subsequent early fibroblast migration and increased collagen deposition and organization in wounds.

Accelerated wound healing with topical application of complement C5.

Background: Delayed-healing traumatic, surgical, and chronic wounds can be detrimental to patients and the health care system. The authors set out to investigate the effects of complement C5, a naturally occurring chemotactic cytokine, on wounds. Methods: The authors examined the effects of complement C5 on the rat paired skin incision model. Each rat served as its own control where topical collagen was applied to one incision and 100 nM of C5 in collagen vehicle was applied to the other incision. Rats were killed on days 3 (n = 6), 7 (n = 6), and 28 (n = 5) after wounding. Results: There was a statistically significant, 65 percent increase in maximum wound breaking strength with the topical application of C5 at day 3 (p < 0.01). The increase persisted to 14 percent at 7 days after wounding (p < 0.05). When compared with the sham group, the C5-treated wound strength increased by 83 percent at day 3 and 64 percent at day 7. There was no change in breaking strength at 28 days. Western blot analysis demonstrated a significant increase in collagen and fibronectin content in the C5-treated wounds. Conclusions: Topical application of C5 to skin wounds significantly increases wound healing maximum breaking strength as early as 3 days and up to 7 days after wounding. C5 accelerated wound healing by at least 4 days in the first week of wounding. This was correlated with an increase in vascular permeability, increased inflammatory cell recruitment, subsequent fibroblast migration, and increased collagen deposition.

Expression and Production of Human Interleukin-7 in Insect Cells Using Baculovirus Expression Vector System (BEVS)

Interleukin-7 (IL-7) is a glycoprotein cytokine with significant clinical and biomedical potential, such as cancer therapy and HIV infections. Earlier it has been cloned and expressed in various protein expression systems; however, they are not efficient for large-scale production. To address this inadequacy, we report in this paper the production of recombinant human interleukin-7 (hIL-7) in insect cells. A recombinant bacmid containing hIL-7 was constructed, purified, and characterized. It was used to infect Trichoplusia ni (BT1-TN-5B1/High Five™) insect cells. Result shows that T. ni cells successfully produce hIL-7 in shake flask cultures. A scale up to 2.5-L laboratory batch bioreactor showed the efficacy of this system for large-scale production. Our results offer a highly efficient, inexpensive, and convenient system for the large-scale expression and production of recombinant hIL-7.

Expression of a Secreted Protein in Mammalian Cells Using Baculovirus Particles

There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.