August 1, 2013 (Vol. 33, No. 14)

Mikael Bundgaard-Nielsen Novozymes Biopharma

Enhancing Consistency and Safety Using an Innovative Fermentation Process

Hyaluronic acid (HA) is a naturally occurring polysaccharide providing structure to tissues including skin and cartilage.

By supplementing the body’s levels of glycosaminoglycan and providing water-binding, viscoelastic, and biological properties, HA serves as a major ingredient in pharmaceutical and medical devices.

The challenge for manufacturers has been sourcing high-quality HA, as those widely available are produced from either rooster comb extraction or various attenuated strains of Streptococcus bacteria. These production methods can be problematic as they can contain potentially dangerous endo- and exotoxins, or animal-derived contaminants.

In addition, these sources of HA are purified using organic solvents, which pose further health issues to patients and make them a concern for regulatory agencies. Until recently, the source of a specific HA product has not been considered to be clinically important. However, with its growing use in health technologies and interventions, and the spotlight on potential contamination risks, demand is increasing for safer alternatives.

Recognizing these issues, Novozymes Biopharma developed Hyasis® HA using a Bacillus subtilis fermentation process that offers manufacturers a high degree of purity and biocompatibility, as well as heat stability and targeted molecular weight. By implementing a three-stage process, the technology provisions a high-quality HA for use on a large scale.

Production Methodology

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.

Figure 1. Extracellular expression provides better control of molecular weight and higher purity.

The final step incorporates a spray-drying method to separate water from the HA product. The method works by spraying fine droplets of the HA solution through a large cylinder vessel at a high temperature. The surplus water is evaporated, resulting in the final HA powder, which is then captured and packaged. This also means that it is possible to avoid open handling steps, further improving quality.

Figure 2. Bacillus-derived HA enables the targeting of molecular weight at scale.

Improved Development Strategies

The method allows increased control over manufacturing processes with regard to both consistency and the purity of the final material. In terms of consistency, using a process that excretes HA into the fermentation media enables the targeting and reproduction of molecular weight at scale. This means that from very early on in the fermentation process the size of the polymer produced can be controlled and maintained (Figure 2).

By doing this the production organism and process can be adjusted to manufacture a high molecular weight or low molecular weight product depending on the needs of a specific application. This means that rather than manufacturers having to adapt to the limits of traditional methods, the process can be adjusted to fit their requirements.

Using a water-based filtration process and a bacterial host without endotoxins results in a high-grade HA material that is compliant with ICH Q7 and cGMP guidelines, offering manufacturers improved consistency and purity (Figure 3).

Figure 3. The HA presents very low endotoxin and microbial contamination levels.

An additional benefit of Bacillus HA is its better processability. Due to the porosity and reduced size of the product’s spray-dried particles, it dissolves up to four times faster than Streptococcus-derived HAs, and filters substantially faster, saving significant time and cost in production. The high degree of purity of the material also permits sterilization by autoclaving without significant loss of product viscosity (Figure 4).

When developing unique end user products, manufacturers often require specific HA properties. The characteristics of the new HA can be adjusted during biomanufacturing by chemical modification through crosslinking to meet individual needs.

Owing to the effective purification steps, the resulting transparent and homogenous hydrogels do not contain any detectable residual crosslinking agent. HA can therefore be customized during manufacture to achieve a specified viscosity, enabling the product to be adapted for many applications including ophthalmology, joint care, aesthetic medicine, and animal care.

Figure 4. Bacillus-derived HA demonstrates improved performance and viscosity when autoclaved.

Mikael Bundgaard-Nielsen ( is vp of biopharma operations at Novozymes Biopharma.