Animal-cell culture in aqueous two-phase systems

In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in this thesis focuses on exploring the opportunities for physical integration of fermentation and part of the downstream processing by means of extractive fermentations in Aqueous Two-Phase Systems (ATPSs). This in situ recovery concept, where the product is extracted from the bioreactor during fermentation, combines fermentation, primary recovery and part of the purification in a single step. The subsequent reduction of process steps and of processing time may provide an opportunity to improve the process economy or even the product quality. ATPSs are thought to be appropriate, because they form a relatively mild environment to cells and they are suitable for protein extractions. ATPSs can be formed by dissolving two polymers, e.g. PEG and dextran in an aqueous solution. Above certain concentrations two aqueous phases will form, each enriched in one of the polymers.

In chapter 2 of this thesis, first the influence of the individual ATPS-forming polymers PEG and dextran on the relevant physical culture medium parameters and on the growth of a model (hybridoma) cell line were characterized. It was found that the polymers raised the osmotic pressure. This could be compensated for, however, by reducing the Na-chloride concentration of the culture medium. Subsequently, it was found that cell growth was possible in culture media containing up to 0.025 g/g of PEG or up to 0.15 g/g of dextran. Using these findings, ATPSs of PEG and dextran were selected. In ATPSs of PEG 35.000, dextran 40.000 and culture medium, the hybridoma cells partitioned to the dextran-rich phase and could be cultured over prolonged periods of time. The IgG product, however, partitioned along with the cells in the lower phase.

In chapter 3, first the partitioning of the model (mouse/mouse) hybridoma cell line, was investigated systematically using a statistical experimental design. It was found that the ionic composition had the largest effect on cell partitioning. Only at a low phosphate- to chloride-ion ratio cells partitioned into the dextran-rich phase, otherwise they were present in the interface. The cell partitioning could be optimized by choosing a low dextran Mw (40 kD), a high PEG Mw (35 kD) and a low tie-line length (10 g/g). In the second place, the cell partitioning and cell growth of other cell lines (a mouse/rat hybridoma and a CHO cell line) were studied in the ATPS culture media described in chapter 2. It was found that both cell lines, partitioned almost completely into the lower phase. Moreover the hybridoma cell line was able to grow well in the ATPS (hybridoma) culture medium. This medium therefore appears to be suitable for extractive bioconversions with a wide range of hybridoma cells.

In chapter 4, first the partitioning of product (IgG) was studied systematically. In this study the same variables were used as in the cell partition study. In all of the ATPSs the IgG partitioned predominantly into the lower phase. The partition coefficient varied between 0.78 and 0.0002, in none of the ATPSs IgG concentrated in the top-phase. The tie-line length, the dextran molecular weight and the PEG molecular weight had the most pronounced effect on IgG partitioning. In non of the ATPSs tested good separation between the cells and their product was achieved. In the second place, therefore, the opportunities to manipulate the product partitioning with a ligand coupled to PEG were explored. A number of dye-resins was screened for their ability to bind the IgG antibody. The mimetic green 1 A6XL dye-resin was found to bind IgG. The dye-ligand coupled to PEG improved the IgG-partition coefficient by three orders of magnitude (in the presence of a low-salt buffer and at 1% PEG-ligand), resulting in a partition coefficient of 25.

In chapter 5, the effect of the PEG-dye-ligand on both IgG and hybridoma partitioning was characterized more extensively. It was found that the binding of IgG to the PEG-ligand was affected severely by the Na-chloride concentration. The tie-line length and pH affected IgG partitioning to a lesser extent. The desired partitioning of IgG into the top phase (with 1% PEG replaced by PEG-ligand), was only obtained when, in addition to the K-phosphate buffer, no Na-chloride was present. In ATPS culture medium increasing the percentage of PEG replaced by PEG-ligand up to 100%, did increase the IgG partition coefficient up to 0.7, but was not effective in concentrating the IgG in the top phase. Moreover, addition of the PEG-ligand to ATPS culture medium changed the hybridoma cell partitioning from the bottom phase to the interface.

This thesis has shown the feasibility of animal cell cultivation in ATPSs and it has shown the usefulness of statistical experimental design in characterizing partitioning in ATPSs. Furthermore it has pointed out the pitfalls and possibilities for the separation of animal cells from their protein products. Therefore, it presents an important step towards proof of principle for the technical feasibility of extractive fermentations with animal cells in ATPSs. Further research remains necessary, however, to amongst others improve the separation between hybridoma cells and their IgG product, for designing and implementing fermentation processes, for scale up and for establishing the economical feasibility.

In chapter 6, the recent developments in the field of extractive bioconversions in ATPSs in general are reviewed. A number of recent developments may give a new impetus to this technology and lead to a more widespread use in industry. First of all the use of extractive bioconversions in ATPSs for high value protein products, in combination with the ongoing development of low cost ATPSs, is promising. Furthermore, the application of novel analytical techniques, in combination with statistical experimental designs may lead to improved design of and control over extractive bioconversions in ATPSs.