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Novel bioreactor design and its characterization

Mammalian cells have been in use for the last three decades and have gained paramount importance for the production of biopharmaceutical products (Vaccines and recombinant protein products). Various cell lines can be exploited to produce biologicals products, obtained from different sources such as insect, fish, duck, mammals and humans. Animal cells have semi-permeable outer cell membranes instead of a cell wall. Because of this, they are shear-sensitive. Their fragility, large size, slow growth rates and low oxygen consumption rates make them difficult to grow. Cells tend to form aggregates in adverse conditions and may end up with heterogeneity and undesired protein formation during fermentation. Liquid shear forces from stirring, bubble formation and direct sparging of the bioreactor have drastic effects on the overall growth, optimum cell density and productivity of the fermentation batch, owing to shear sensitivity. Small changes in the micro-environment during fermentation can produce proteins with heterogeneity in structure and varying biological activity. Hence, there is a need to develop a methodology to protect the growing cells in a bioreactor from the shear condition. The methodology should also exhibit optimum mass transfer and low shear mixing and easy purification of the product. Therefore, there is a need for a comprehensive vessel design which is suitable for a variety of mammalian cells. A novel vessel has been designed and characterization of its engineering aspects initiated. Preliminary results showed that this design has less damage to mammalian cells resulting in high cell concentration and extended longevity for economical production of biologicals.


Comparison of Spectroscopy Technologies for Improved Monitoring of Cell Culture Processes in Miniature Bioreactors.

Abstract: Cell culture process development requires the screening of large numbers of cell lines and process conditions. The development of miniature bioreactor systems has increased the through-put of such studies; however, there are limitations with their use. One important constraint is the limited number of offline samples that can be taken compared to those taken for monitoring cultures in larger-scale bioreactors. The small volume of miniature bioreactor cultures (15mL) is incompatible with the large sample volume (600µL) required for bioanalysers routinely used. Spectroscopy technologies may be used to resolve this limitation. The purpose of this study was to compare the use of NIR, Raman and 2D-fluorescence to measure multiple analytes simultaneously in volumes suitable for daily monitoring of a miniature bioreactor system. A novel design-of-experiment approach is described that utilizes previously analysed cell culture supernatant to assess metabolite concentrations under various conditions whilst providing optimal coverage of the desired design space. Multivariate data analysis techniques were used to develop predictive models. Model performance was compared to determine which technology is more suitable for this application. 2D-fluorescence could more accurately measure ammonium concentration (RMSECV 0.031g.L(-1) ) than Raman and NIR. Raman spectroscopy however, was more robust at measuring lactate and glucose concentrations (RMSECV 1.11 and 0.92g.L(-1) respectively) than the other two techniques. The findings suggest that Raman spectroscopy is more suited for this application than NIR and 2D-fluorescence. The implementation of Raman spectroscopy increases at-line measuring capabilities, enabling daily monitoring of key cell culture components within miniature bioreactor cultures. This article is protected by copyright. All rights reserved.

Pub.: 09 Mar '17, Pinned: 29 Aug '17

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed-batch media.

Abstract: Perfusion medium was successfully developed based on our fed-batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed-batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2x to increase medium depth. The medium osmolality was also optimized where we found ∼360 mOsm/kg was desirable resulting in a residual culture osmolality of ∼300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N and D were rebalanced to reduce lactate and ammonium levels, and increase the cell specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell-day yielding 1.2 g/L-day at steady-state. This perfusion process was then successfully scaled up to a 100L single-use bioreactor with an ATF6 demonstrating similar performance as a 2L bioreactor with an ATF2. Large volume handling challenges in our fed-batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug-and-play use with the bioreactor. This article is protected by copyright. All rights reserved.

Pub.: 04 Apr '17, Pinned: 29 Aug '17

Microcarrier choice and bead-to-bead transfer for human mesenchymal stem cells in serum-containing and chemically defined media

Abstract: The production of stem cells for clinical applications requires a suitable mass expansion and harvest process, which is implemented in a microcarrier-based bioreactor. Important parameters to consider include the choice of microcarriers for cell expansion, the growth medium and the scale-up strategy. We investigated six different microcarriers to determine whether they can support the growth and harvest of primary human mesenchymal stromal/stem cells (hMSCs) derived from bone marrow, and the immortalized cell line hMSC-TERT. A serum-containing medium (SCM) and a new chemically defined medium (CDM) were compared under dynamic culture conditions. We also investigated bead-to-bead transfer from spinner flasks to a stirred tank reactor as a scale-up strategy. We observed cell type-dependent differences in growth rate and attachment behavior on each microcarrier. Missing serum components in the CDM led to slower cell attachment and growth, whereas microcarriers suitable for the SCM were also suitable for CDM. Glass-coated microcarriers supported hMSC-TERT growth and bead-to-bead transfer in SCM, whereas plasma-treated plastic surfaces promoted cell growth in CDM. We demonstrated that stem cell cultures can be scaled up by bead-to-bead transfer, avoiding the need for pre-cultures in tissue flasks. The replacement of pre-cultures with a monitored bioprocess could therefore facilitate the development of conditions suitable for large-scale stem cell production.

Pub.: 22 Mar '17, Pinned: 29 Aug '17

Compact cell settlers for perfusion cultures of microbial (and mammalian) cells.

Abstract: As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 liter perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. This article is protected by copyright. All rights reserved.

Pub.: 28 Jul '17, Pinned: 29 Aug '17