A variety of complex glycoproteins, such as antibodies, fusion proteins, growth factors, and enzymes, are increasingly found in the biopharmaceutical industry product pipeline. This has created a need for the expansion of mammalian cell-based manufacturing capacities.
Currently, the market supply of glycoproteins totals several tons. This supply covers the demand for over 50 approved mammalian cell-based biologics. Several trains of stainless-steel stirred tank bioreactors in fed-batch modes whose final manufacturing scales go up to 20,000 L are the industrial gold standard for this supply.
Cost pressures from the healthcare sector and mounting competition among biologics, as well as less expensive manufacturing in Asia and Eastern Europe, have been driving forces in the design of disposable-based facilities. For these designs, a savings of up to 40% on capital expenditures is expected, whereas operating costs for such facilities are at least equal to conventional ones.
At the same time, a number of disposable bioreactors with sizes of up to 1,000 L are under development. The challenge for these systems is to show equivalent fed-batch performance in terms of process characteristics, productivity, quality, and reproducibility, as well as offering proper safety and sturdiness in operation.
Historically, continuous processes were developed for products prone to decomposition and therefore not feasible in fed batches. Short residence time in bioreactors is mandatory for these products. However, such continuous processes are complex, logistically problematic, and costly. ProBioGen(www.probiogen.de) has designed and prototyped a disposable bioreactor system to address the expected needs.
The rationale behind bioreactor design has been to support a continuous protein-production process. This maintains a permanent cell-free product that is capable of harvesting at high cell densities. It includes a requested combination of reliable mammalian cell retention and a highly efficient oxygen supply, as well as minimized shear stress.
For reliable cell retention, a polyethersulfone microfiltration membrane was selected and qualified. The crucial selection criteria were sharp exclusion rates with pore sizes up to 1.4-µm maximum (measured by the bubble-point method) and surface properties preventing membrane clogging over cell culture periods of 60 days.
The prerequisite for a highly efficient oxygen supply is an effective transfer rate of oxygen to every single cell in the system, even at exceptionally high cell densities. Due to low solubility of oxygen in culture media, different concepts for adequate gas supply were developed (sparger, gassing membranes). ProBioGen designed a solution that ensures a maximum distance of less than 1 mm between each cell in the system and the gas phase. Thus the ideal shape for the cell culture space is a tube.
A biomathematical model was developed at the Technical University of Hamburg-Harburg describing cell survival in such tubes. The basic assumptions of the model are an oxygen utilization rate of 1.6 pg/c*h for low-consuming cell lines (OURlow) and 6.4 pg/c*h for high-consuming ones (OURhigh). The model assumes that: the membranes are tubes filled with medium and cells, the tubes are placed in oxygen-containing gas phase, the oxygen transfer is realized only by passive diffusion.
A membrane-wall thickness of 200 µm is sufficient to support a cell density of 108 cells/mL (Figure 1) for high-consuming cell lines and a theoretical density of 109 cells/mL for low consumers inside the tube. The latter one is the cell density similar to dense human tissue.
Shear stress caused by microturbulences at the dimension of single cells is a major obstacle at high gassing and mixing rates in stirred tanks. However, the mixing of suspension cell cultures is necessary to avoid nutrient gradients. ProBioGen’s design minimizes shear stress by gentle mixing rates and sheltering the cells inside the membrane tube. In contrast to other systems, the oxygen transfer efficiency is completely independent of mixing parameters.
Identical cell culture tubes are fixed horizontally in a disposable, rotating cylindrical bioreactor vessel. Placing the cell culture tubes alternatively in oxygen or culture medium allows a sufficient nutrient supply, waste removal, and product harvest (Figure 2).
With a given length, different volumetric scales of bioreactors can be manufactured just by increasing the vessel diameter. The packaging density of the tubes stays constant.
Family, Facility, and Feasibility
An efficient bioreactor system for producing biopharmaceuticals needs at least three scales: A small one for process development, a middle-sized one for preclinical and investigational clinical material production, and a large-scale one for market supply. The characterizing size parameter of the bioreactor family is the volume of the cylindrical vessel.
The Process Development Device with a vessel volume of 100 mL will operate eight modules in parallel. Therefore, an efficient process development strategy with a speedy evaluation of process parameters is possible.
The pilot-scale bioreactor has a vessel volume of 10 L. The commercial-scale bioreactor allows a market supply with a vessel volume of 100 L, ten-times higher than the pilot. Remaining true to the basic mobility, a commercial-scale bioreactor with a 400-L vessel volume is envisaged.
Proper layout of the supporting bioreactor hardware (pumps, control unit, power supply) allows the running of pilot- and commercial-scale disposable vessels with the same bioreactor hardware (Figure 3). If feasible, this may substantially speed up technical transfer from pilot to commercial plants.
In a recent facility design it can be shown that the well-known design parameters and the foreseeable savings for fully disposable plants are applicable. The foundation of this facility design is the single manufacturing unit (SMU). Each SMU contains four areas (growing inoculum, upstream, downstream, final filtration and filling), separated by a passage-cross for material flow and surrounded by a corridor for staff flow.
Lab-scale feasibility was shown using special prototypes each with 100-mL cell culture space. Processes of different durations were run with a CHO-cell line secreting a recombinant protein under serum-free conditions. By varying process parameters, the daily productivity of the bioreactor could be increased significantly (Figure 4).
Summary and Outlook
The first experiments with lab-scale prototypes of the new bioreactor system confirmed a proper cell retention, robust process operations of up to 60 days, andprocess improvements due to growing expertise in system operation. A productspecific process development program is expected to be completed within the next ten months. Thereafter, the performance of the pilot-scale bioreactor with specific emphasis on biopharmaceutical requirements will be tested in a feasibility study.
When the eightfold minibioreactor device supports reliable fast optimization of process parameters and the pilot-scale bioreactor specifications meet regulatory requirements, the bioreactor family is well positioned to be tested for clinical-grade manufacturing of glycoproteins. This would naturally involve working together with partners and regulatory bodies on the issues of disposable cultureware qualification (leachable compounds, manufacturing validation, and cultureware release testing).
ProBioGen is confident that in the future this technology might enable the development and approval of highly complex fragile glycoproteins at reasonable economic efforts. The technology also supports both future disposable-based green-field manufacturing plants and efficient retrofitting of brown-field facilities in any part of the world.