Addressing Shear Stress in Bioreactors

We have developed our new Poloxamer 188 EMPROVE® EXPERT to achieve reliable quality and consistency providing shear stress protection for large scale cell culture processes in biopharmaceutical productions. We have mainly worked together with mAb manufacturers and performed several tests to identify correlations between physical and chemical properties and cell culture performance. It has been decided to implement a cell based assay with different CHO cells with well-characterized shear sensitivity to assess and classify the shear protective effect. First vaccine manufacturers are currently testing our new product and we are curious to see the results. But so far the new poloxamer has not yet been used in vaccine production.

Poloxamer 188 is commonly used in the biopharmaceutical industry as a cell culture media additive to protect cells from the turbulent environment of sparged bioreactors. It has been demonstrated that Poloxamer 188 can function as highly effective shear protectant as it decreases physical damage to suspensions of animal cell cultures by physical and biochemical means. Managing mixing time and speed, impeller choice, agitation, gas entrance velocities etc. in typical culture conditions can support sparging-associated cell damage. Nevertheless achievable cell densities and thereby process productivity are limited without a shear protectant especially with regard to the industry trend towards higher cell densities e.g. perfusion processes. From our view there is currently no real alternative to the addition of a shear protectant.

During the early development phase we tested the product quality with various cell lines, mainly CHO cells (e.g. CHOZN, CHO-K1, CHO-S) and a few others in our R&D labs. During the later development and implementation phase of a cell culture assay we decided to use a CHO cell line for regular product release in our QC lab as this is commonly used by our customers in the biopharmaceutical industry.

To develop a product with consistent quality and proven functionality in upstream processes was our main objective when starting the project as this was a real pain point in the industry. Nevertheless we see our reliable supply (large manufacturing capacities in Europe) and the transparency we offer with our Emprove® dossiers as other benefits beyond protection from shear. The Emprove® Program is known for outstanding quality with comprehensive documentation. Each product in the portfolio is complemented with three types of dossiers to help facilitate qualification, risk assessment and process optimization efforts: Material Qualification Dossier, Quality Management Dossier and Operational Excellence Dossier. They provide information on the manufacturing process, stability data, elemental impurity information, product quality reports, analytical procedures, supply chain,  and much more.

Yes, Poloxamer 188 in seed trains is needed to adapt the cells to the media composition and to protect the cells from shear. Especially after thaw, cells are very sensitive.

We have not investigated this in detail. Due to cell specific sensitivity differences, one cannot make general comments to this question. And due to the effectiveness of Poloxamer 188 this is an unnecessary risk.

This is mainly cell line dependent - we saw effective protection between 0,5-2 g/L in our stress test, which is of course a simplified model. According to literature, concentrations up to 5 g/l have been used for very stressful conditions (high VCD Perfusion), providing protection without any negative side effects. As a minimum we have also seen very few customers using <0,5g/L Poloxamer 188 in their processes.

Modeling the turbulent flow in bioreactors is very complex from a fluid mechanic point of view. There is turbulent kinetic energy dissipation (TKE) from the agitators, but also stress due to bubble formation and bubble burst. These effects cannot be modeled exactly in shaken systems. We have developed a shaken system that does induce turbulent flow in baffled shake flasks, however, the types of shear might be present in different proportions, compared to a bioreactor. We will be working with academic partners to answer these questions.

We have tested 100+ customer samples and have built an extensive sample library and compared our results. We could see that our conclusions are quite representative to the results our customers had with the respective material. For sure total cell densities and viabilities can differ but we are able to distinguish between good and poor performing lots. Several customers have an internal quality requirement to confirm CoA results from suppliers and have to test the incoming material with their proprietary cell line. Nevertheless it might be possible to perform reduced testing after an internal risk assessment as Merck will perform full spec testing for every batch.

The typical concentration in chemically defined cell culture media is usually 1-2g/L but this is always dependent on the cell line, the media composition, the process, etc. We also had some applications where we could reduce the concentration to 0,5g/L thanks to our improved Poloxamer 188 and still had full shear protection in our model system and with our cell line. It may be worth trying to reduce Poloxamer concentration as it contributes to foaming and impacts oxygen transfer. However, the main function in the media is the shear protection effect and this should not be put at risk.