While a number of producers have succeeded in creating commercial HAs using traditional methods, producing the material with a high degree of purity at an economically viable rate has been challenging due to the nature of the production organisms used. These techniques rely on Streptococcus HA cells to produce HA and bind it on to the cell coat rather than releasing it.
For a Streptococcal producer to then recover the HA, large volumes of organic solvents are used. This creates additional challenges when the cell coat is disrupted, as further impurities are released that need to be purified away.
In contrast, B. subtilis is well adapted to growing under variable fermentation conditions, where parameters such as temperature and pH can be adjusted to optimize yield and molecular weight. Using an animal-free, mineral media, the process is unique in that it expresses HA extracellularly, which means it will essentially extrude HA out of the cell and into the fermentation media allowing for an easier recovery process (Figure 1).
The bioprocessing method includes several recovery and filtration stages with the first being flocculation. The HA is separated from the Bacillus cells using a centrifuge to spin down the biomass. A series of exhaustive filtration steps are then performed including ultrafiltration, diafiltration, and depth filtration. Using a number of different and carefully executed techniques enables complete control over how the HA is filtered.
The Bacillus method’s technique of allowing HA to be released from the cell naturally makes it useful in achieving high levels of purity, as well as for controlling the molecular weight of the end product. When HA is torn from a cell to extract it, as with the Streptococcus model, it results in a heterogeneous mix of polymers without the ability to distinguish between high and low molecular weight.
Using the new approach enables filtration to be customized to manage the fact that HA has a varying viscosity depending on its molecular weight. From the outset, it was essential that a technique was found to modify all elements of filtration including flow, process times, and temperature, to enable a highly viscous polymer such as HA to be moved through a system, while at the same time removing impurities. This proprietary water-based process successfully overcomes challenges by eliminating impurities to a very high degree.