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Creating A New, Safer Generation of Hyaluronic Acid for Pharmaceutical Applications
Hans Ole Klingenberg
Global Marketing Director, Novozymes Biopharma

In this sensitive application area, quality control of products is becoming subject to stricter governing from regulatory bodies, aiming at improving safety and minimalizing risk. The use of HA in such applications has also been associated with a number of challenges over recent years. The purity and consistency of its raw materials has been of particular concern as these can have a significant impact on clinical performance.

Sourcing high-quality HA has proven to be problematic for manufacturers in the past. Many commercial sources available are produced from either rooster comb extractions or various strains of Streptococcus bacteria. These can contain potentially dangerous endo- and exo-toxins or animal-derived contaminants. In an attempt to remedy this, these sources of HA are often purified using organic solvents, which themselves can pose further health issues.

New technologies have been developed in response to these challenges, one of which is Hyasis®. Developed by Novozymes Biopharma Hyasis® is pure HA manufactured with high quality, animal-free ingredients by way of an innovative Q7 cGMP biomanufacturing process. This article will discuss this method, and how the technique allows the production of a safe and consistent HA on a large scale.

New source, new techniques

The nature of the organisms used in the production of HA has been a major obstacle for manufacturers aiming to produce the material with a high degree of purity at an economically viable rate. However, a new process based on Bacillus subtilis, a non-pathogenic bacterium, has been developed. The products of this bacterium are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA), meaning it provides a viable alternative to some existing sources.

Working across three stages, including fermentation, recovery/filtration and separation, this new technique enables the delivery of high quality HA for commercial use on a large scale. These stages are examined in more detail below.

Extracellular expression

During production, traditional manufacturing techniques rely on Streptococcal HA cells to produce HA and bind it to the cell coat. In order to then recover the HA, large volumes of organic solvents are used, which in turn can cause damage to this cell coat, releasing impurities that need to be purified away. By undertaking numerous purification stages, it becomes harder and more costly to deliver the pure product that is required.

In contrast, the Bacillus subtilis organism is well adapted to growing under variable fermentation conditions. Parameters such as temperature and pH can be adjusted to optimize yield and molecular weight. As shown in Figure 1, when using an animal-free, mineral media, HA is expressed extracellularly. It is released out of the cell and into the fermentation media, allowing for a much easier recovery process and the elimination of numerous purification steps, creating a much purer end product.

Recovering a purer product

There are several recovery and filtration stages in order to extract the Bacillus-based expressed HA. The first of these is flocculation. During this stage, the HA is separated from the Bacillus cells using a centrifuge to spin down the biomass that is not needed. Once the HA is isolated, a series of exhaustive filtration steps are implemented – ultrafiltration, diafiltration and depth filtration. By using a number of different and carefully executed techniques, manufacturers have complete control over how the HA is filtered, meaning they can be confident in only extracting the pure product.

Not only does the technique of allowing HA to be released naturally from the cell make it particularly useful in achieving high levels of purity, it also plays a vital role in controlling the molecular weight of the desired end product. Whereas in previous methods, HA was stripped from a cell to extract it, resulting in a heterogeneous mix of polymers where high and low molecular weight could not be distinguished, Novozymes’ approach enables filtration to be customized to extract certain molecular weights. This holds great importance due to the fact that HA has a varying viscosity depending on its molecular weight, impacting its use in clinical applications and therapies. This is also a beneficial factor in manufacturing.

This proprietary water-based process successfully overcomes challenges by efficiently and effectively removing impurities and contaminants. The purity of Bacillus-derived HA is shown in Figure 2.

Spray dried separation technique

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 the top of a large cylinder vessel at a high temperature. This evaporates any surplus water, resulting in the final HA powder, which is then captured in containers and packaged. This also means that it is largely possible to avoid open handling steps, thereby further improving quality.

Reliable and controlled production

With this technique, for the first time, it is now possible to deliver HA material according to a desired specification, rather than having to adapt to the limitations of what is available. This is particularly valuable when producing on a manufacturing scale, as it allows the targeting and reproduction of consistent molecular weights. The production organism and process can be adjusted accordingly, producing a high or low molecular weight HA depending on the needs of a specific application. The size of the polymer produced can therefore be controlled and maintained from very early on in the fermentation process.

For example, if a target molecular weight for a batch is specified at 850 kilodalton, then it is possible to meet that target with a high degree of accuracy, regardless of the amount of product needed by the customer. This is demonstrated in Figure 3.

Due to the limits of traditional methods, manufacturers may have had to adapt their formulations to suit the materials available. A defined and reproducible molecular weight ensures that HA batches can be delivered to requirements without any off-specification product.

Undeniable advantages

A major challenge in using HA of Streptococcal origin is that the human body is adept at recognizing Streptococcus and many of its cell components. The commonality between Streptococcal production systems and Streptococcal infections has meant that a proportion of a population's immune systems will automatically recognize impurities from Streptococcal derived HA following an injection. As a result, their bodies will launch an inflammatory response which may lead to unwanted side-effects. By using Bacillus subtilis as the production organism, such toxins are not produced. It is therefore a much safer starting point for the production of a standard HA.

The high-grade HA material developed in this way is compliant with ICH Q7 and cGMP guidelines, offering manufacturers a level of consistency and purity that was not previously possible. Importantly, working with raw materials that are already Q7 compliant has a significant impact on the speed of the regulatory process and greatly reduces testing time.

On top of all this, Bacillus HA has some surprising additional benefits. One of these is its improved processability, which is due to the porosity and reduced size of its spray-dried particles. Manufacturers can save significant time and cost during production, as the product dissolves up to four times faster than Streptococcal-derived HAs and filters substantially faster.

Creating specific therapies

The characteristics of this new HA can be adjusted during biomanufacturing through chemical modification so to tailor to individual production needs. This is very valuable to manufacturers who often require specific HA properties. In addition to techniques mentioned here, a newly introduced technology for preparing crosslinked HA hydrogels has been developed. This is based on a reproducible process that does not employ any organic solvents. The resulting transparent and homogenous hydrogels do not contain any detectable residual crosslinking agent, keeping purity levels up.

The variation of parameters, such as starting HA concentration and crosslinking agent to HA weight ratio, as well as subsequent processing of the hydrogels, such as extrusion and combination with native HA solutions, permit tailoring gel viscoelastic properties. As a result, HA can be customised during manufacture to achieve a specified viscosity, enabling the product to be adapted for a wide-range of drug delivery and medical device applications including ophthalmology, joint care, aesthetic medicine and animal care.


Bacillus subtilis is a non-pathogenic, animal-free production organism, and its use in the production of HA presents minimal risks of contamination and achieves a highly pure final product.

These latest technologies and developments ensure high levels of safety and consistency in the production of HA. Importantly, the flexibility of the process also allows molecular weight, and as a result viscosity, to be accurately defined, meaning that the final HA can be designed to fit the specific needs of manufacturers, while still meeting the stringent regulations of today. The combination of these features translates into clear benefits for drug manufacturers and patients alike.

About the Author

Hans Ole Klingenberg is Director for the Global Marketing group in Novozymes Biopharma. Hans Ole has been with Novozymes for more than 10 years and has, through his time at Novozymes, held various positions in the area of corporate business development, with a focus on establishing new business entities for Novozymes in the biopharmaceutical industry. Hans Ole has, since 2007, been working in Novozymes’ Biopharma business with a focus on marketing, and is commercially responsible for the establishment of Novozymes’ new Hyaluronic Acid business franchise. Hans Ole has, with a bachelor in Chemistry and master in Economics from Copenhagen University. He is based in Denmark.

Novozymes is the world leader in bioinnovation. Together with customers across a broad array of industries, we create tomorrow's industrial biosolutions; improving our customers’ business and the use of our planet's resources.

About Novozymes

With over 700 products used in 130 countries, Novozymes’ bioinnovations improve industrial performance and safeguard the world's resources by offering superior and sustainable solutions for tomorrow's ever-changing marketplace.

Novozymes’ natural solutions enhance and promote everything from removing trans fats in cooking, to advancing biofuels to power the world tomorrow. Our never-ending exploration of nature's potential is evidenced by over 6,000 patents, showing what is possible when nature and technology join forces.

Our 6,000+ employees working in research, production and sales around the world are committed to shaping businesses today and our world tomorrow.


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