1. Field of the Invention
The present invention provides methods of increasing yield in the protein production by cultured cells, especially mammalian cells. Specifically, the present invention relates to methods of preparing protein product(s), e.g., a glycoprotein product(s), wherein the protein product characteristics are controlled by manipulating the cell culture environment to increase stress applied to the cells.
2. Related Background Art
A large proportion of biotechnology products, whether commercially available or in development, are protein therapeutics. There is a large and increasing demand for production of proteins in mammalian cell cultures and for improved methods related to such production. Such improved methods are needed especially when large glyco-proteins with low cellular expression levels are produced. One such protein, FVIII, has an expression level at least two to three orders lower than other recombinant proteins produced in mammalian cells. A common problem encountered in late-phase development of large-scale therapeutic protein production is increasing demand due to larger clinical trials and contaminations in the cell culture production plant which decrease capacity. To meet the increased demand the total production level can be increased by several ways. However, most of them such as finding a better cell clone or improving the culture medium are very tedious tasks and therefore not often quick enough options. Other ways to increase the productivity is to increase the production scale or increase the density of cells in fed-batch or perfusion mode culture. Also these process changes are accompanied with large investment costs and for the case of high density cultures oxygen limitation in the culture tank will generally set a limit for the maximum cell density that can be used for production. Therefore, there is a need in art for new methods of increasing productivity.
Keane J. T. et al. Effect of shear stress on expression of a recombinant proetine by chinese hamster ovary cells; Biotechnology and Bioengineering, 81:211-220, 2003, subjected attached CHO cells to shear force for 32 h and monitored recombinant human growth hormone production and glucose metabolism. They observed that when shear force was increased from 0.005 N/m2 (0.02 W/m3) to 0.80 N/m2 (6.4×102 W/m3), recombinant protein production rate was reduced by 51%, glucose uptake rate was increased by 42%, and lactate production was decreased by 50%.
Godoy-Silva R et al. Physiological responses of CHO cells to repetitive hydrodynamic stress; Biotechnology and Bioengineering, Vol. 103, No. 6, Aug. 15, 2009, examined the effect of repetitive hydrodynamic stress on CHO cells and came to the conclusion that energy dissipation rate up to 6.4×106 W/m3 did not affect cell growth, death, and productivity.
J. A. Frangos et al. Shear stress induced stimulation of mammalian cell metabolism; Biotechnology and Bioengineering, Vol. 32, Pp. 1053-1060(1988) discloses a flow apparatus for the study of the metabolic response of anchorage-dependent cells to a wide range of steady and pulsatile shear stresses under well-controlled conditions. The data demonstrate that physiological levels of steady shear stress and the onset of shear stress dramatically stimulate prostacyclin production in cultured human endothelial cells.
Giard and co-workers observed that human fibroblasts secrete up to 30-fold greater amounts of interferon when maintained on microcarrier in spinner flasks compared to cells in roller bottles (D. J. Giard, D. H. Loeb, W. G. Thilly, D. 1. C. Wang, and D. W. Levine, Biotechnol. Bioeng., 21, 433(1979)). Since the shear stresses that cells are exposed to in the spinner flasks are much higher than those in roller bottles, the increased production may be attributable to shear-induced stimulation of interferon synthesis.
Timm Tanzeglock et al, Induction of mammalian cell death by simple shear and extensional flows; Biotechnology and Bioengineering, Vol. 104, No. 2, Oct. 1, 2009 discloses whether the type of shear flow, to which cells are exposed, influences the initiation of cell death. It is shown that mammalian cells, indeed, distinguish between discrete types of flow and respond differently. Two flow devices were employed to impose accurate hydrodynamic flow fields: uniform steady simple shear flow and oscillating extensional flow. To distinguish between necrotic and apoptotic cell death, fluorescensce activated cell sorting and the release of DNA in the culture supernatant was used. Results show that chinese hamster ovaries and human embryonic kidney cells will enter the apoptotic pathway when subjected to low levels of hydrodynamic stress (around 2 Pa) in oscillating, extensional flow. In contrast, necrotic death prevails when the cells are exposed to hydrodynamic stresses around 1 Pa in simple shear flow or around 500 Pa in extensional flow. These threshold values at which cells enter the respective death pathway should be avoided when culturing cells for recombinant protein production to enhance culture longevity and productivity.
WO 2006/103258A1 discloses a method for increasing the yield of a protein produced by cultivating eukaryotic cells and adding an ionic substance to the culture medium prior to harvest of the protein. Suitable ionic substances are the salts of the Hofmeister series and amino acids.
WO 2008/006494A1 discloses a process for the culturing of cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, preferably an antibody, wherein at least one cell culture medium component is fed to the cell culture and wherein the cell culture comprising the cells, the biological substance and cell culture medium is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor. Preferably part of the substances of lower molecular weight is continuously removed from the cell culture.
Zhang, Hu et al report in Current Pharmaceutical Biotechnology, Volume 11, Number 1, January 2010, pp. 103-112(10) that mammalian cell cultivation plays a great role in producing protein therapeutics in the last decades. Many engineering parameters are considered for optimization during process development in mammalian cell cultivation, only shear and mixing are especially highlighted in this paper. It is believed that shear stress due to agitation has been over-estimated to damage cells, but shear may result in nonlethal physiological responses. There is no cell damage in the regions where bubbles form, break up and coalescence, but shear stress becomes significant in the wake of rising bubbles and causes great damage to cells in bubble burst regions. Mixing is not sufficient to provide homogeneous dissolved oxygen tension, pH, CO2 and nutrients in large-scale bioreactors, which can bring severe problems for cell growth, product formation and process control. Scale-down reactors have been developed to address mixing and shear problems for parallel operations. Engineering characterization in conventional and recently developed scale-down bioreactors has been briefly introduced. Process challenges for cultivation of industrial cell lines in high cell densities as well as cultivation of stem cells and other human cells for regenerative medicine, tissue engineering and gene therapy are prospected. Important techniques, such as micromanipulation and nanomanipulation (optical tweezers) for single cell analysis, computational fluid dynamics (CFD) for shear and mixing characterization, and miniaturized bioreactors, are being developed to address those challenges.
Timothy A. Barrett et al. in Biotechnology and Bioengineering, Vol. 105, No. 2, pages 260-275 report about experimentation in shaken microplate formats offering a potential platform technology for the rapid evaluation and optimization of cell culture conditions. There is described a detailed engineering characterization of liquid mixing and gas-liquid mass transfer in microwell systems and their impact on suspension cell cultures.
Provided that cell growth and antibody production kinetics are comparable to those found in currently used shake flask systems then the microwell approach offers the possibility to obtain early process design data more cost effectively and with reduced material requirements. This work describes a detailed engineering characterization of liquid mixing and gas-liquid mass transfer in microwell systems and their impact on suspension cell cultures. For growth of murine hybridomas cells productizing IgGl, 24-well plates have been characterized in terms of energy dissipation (P/V) (via Computational Fluid Dynamics, CFD), fluid flow, mixing and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted kLa values varied between 1.3 and 29 h−1; liquid-phase mixing time, quantified using iodine decolorization experiments, varied from 1.7 s to 3.5 h; while the predicted P/V ranged from 5 to 35 W m−3. CFD simultations of the shear rate predicted hydrodynamic forces will not be detrimental to cells. For hybridomas cultures however, high shaking speeds (>250 rpm) were shown to have a negative impact on cell growth, while a combination of low shaking speed and high well fill volume (120 rpm; 2,000 μL) resulted in oxygen limited conditions. Based on these findings a first engineering comparison of cell culture kinetics in microwell and shake flask formats was made at matched average energy dissipation rates. Cell growth kinetics and antibody titer were found to be similar in 24-well microtiter plates and 250 mL shake flasks. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproductible and comparable to currently used shake flask systems while offering at least a 30-fold decrease in scale of operation and material requirements. Linked with automation this provides a route towards the high through-put evaluation of robust cell lines under realistic suspension culture conditions.
William G. Whitford and John S. Cadwell in BioProcess International 2009, Vol. 7, No. 9, pages 54-64 report about growing interest in hollow-fiber perfusion bioreactors.
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