The rapidly growing interest for cell and gene therapies demands the development of robust, scalable, and cost-effective bioprocesses for viral vector production. For the production of lentiviral vector (LVV) at high titers, we have developed an inducible packaging system in suspension HEK293 cells from which we can also generate stable producer cell lines, in serum-free conditions. To evaluate the potential of this platform, we have generated a stable cell line that produces an LVV encoding a green fluorescent protein (GFP) and obtains 10E+07 to 10E+08 transduction units (TU)/mL at the 4 L, 10 L and 50 L scales. Functional LVV titers were maintained across all scales in bioreactors with different configurations and geometries indicating process robustness. Further, the addition of 10% feed increased the volumetric productivity by 3.5-fold in comparison to batch production, making our platform suitable for large-scale LVV production and showing a real potential for commercial manufacturing.
Tag: <span>fed-batch</span>
A rapid increase in the number of gene therapy trials and products has led to a comparable increase in the need for industrial production of viral gene therapy vectors such as lentiviral, adeno-associated, and adenoviral vectors. Current production systems are limited with respect to scalability and robustness. With our CAP® and CAP-T™ cell lines, we have developed a novel system for high-density suspension culture, efficient and reproducible transfection, and highly efficient production of viral vectors. By upstream process optimization, we have obtained a robust and high-density fed-batch culture system which can be scaled in any current bioreactor format. A design-of-experiments approach has been employed to optimize transient production of lentiviral vectors with significantly higher titers than can be obtained with adherent HEK293T cells…
A basic engineering study has been performed to evaluate three different strategies for the production of monoclonal antibodies (MAbs) from Chinese hamster ovary (CHO) cells. Cells are expanded in suspension culture and are then inoculated into either fed batch or perfusion culture for MAb production. The first strategy, which is also the current industry standard, uses fed batch culture with the cells in suspension in a stirred tank fermenter. The second strategy uses perfusion culture with the cells immobilized on Cytopore™ microcarriers in a stirred tank fermenter. The third and final strategy is perfusion culture with Cytoline™ microcarriers in a fluidized bed fermenter. Perfusion cultures, while leading to a somewhat lower product titer, were characterized by a much smaller equipment footprint. This in turn led to a >30% reduction in investment costs and a 12% reduction in MAb production costs calculated over five years of depreciation and ten years of production time…
Building a MAb bioprocessing plant is a process which normally takes three years. Before starting the engineering work, a “locked” process is necessary. This means that all the steps have to be defined by volume, time, material balances and product yield. These calculations are based on the results obtained during process development. The titer and yield of functional, recoverable product determines the plant size. Optimal volumetric productivity [g/(liter reactor volume * day)] is of utmost importance. The main difference between fed batch and perfusion culture is that in the fed batch, a centrifuge is required for cell removal, whereas in perfusion culture, cell removal is performed by dead end filtration. This is possible because the majority of the cells are immobilised on the microcarriers, thus minimizing the burden on the clarification unit…
Bioreactor productivities are highly dependent on the process used to cultivate mammalian cells. These productivities directly affect the manufacturing plant capacity, and thereby the economics of production of monoclonal antibodies (MAbs). Historically, companies have chosen bioreactor process strategies that emphasize simplicity of scale-up at the expense of productivity, and conducted manufacturing using well-characterized and relatively straightforward batch processes. Such processes have successfully produced small or moderate quantities (ranging from ~100 g to ~ 1 kg per lot) of the desired antibody. Given the anticipated demand for large-scale quantities of MAbs (and the high stakes for the companies investing in these new biological entities), it is worthwhile to revisit these past selection strategies and see if — and under what conditions — they remain optimal today…
The Sf-9 insect cell/baculovirus expression system is one of the most commonly used protein expression systems. It is the preferred system for generating large amounts of protein in a short period of time, and it has been successfully used to express several hundreds of different proteins. A representative list of the different proteins made in our laboratory over the past decade with the Sf-9 insect cell/BEVS system is given in Table 1. These proteins are often used in drug screening studies and structure function analysis. Proteins intended for therapeutic purposes are not normally produced using this technology, although a few examples do exist. There is also an unexplored potential for the cells to be used for the production of recombinant viral vectors. Recent reports demonstrating the ability of baculoviruses to express proteins in mammalian cells, with mammalian promoters, indicate that BEVS technology might soon have a major role to play in the field of gene delivery…