Novozymes caused a stir when it developed a biofermentation process for the manufacture of hyaluronic acid. Hans Ole Klingenberg, global marketing director at Novozymes Biopharma, reveals how Hyasis is going from strength to strength.
Hyaluronic acid (HA), also known as hyaluronan, is a naturally occurring polysaccharide ubiquitous in nature, which is distributed widely throughout connective, epithelial and neural tissues in the human body. As a component of the extracellular matrix, HA provides structure to tissues, including skin and cartilage, and as a result is well suited to a wide range of biomedical applications. It functions as a binding and protecting agent in tissue hydration and lubrication.
By supplementing the natural levels of glycosaminoglycan in the human body and providing exceptional water-binding, viscoelastic and biological properties, HA serves as a major ingredient in pharmaceutical and medical device products.
Comprising a high molecular weight, HA is a linear polysaccharide composed of alternating molecules of glucosamine and glucuronic acid. From the 1980s onwards, the understanding of HA properties and functions has expanded and today HA is recognised as a high-value biomaterial with numerous proven and marketed applications within aesthetic medicine, dietary supplements and biomedical areas.1
Due to its intrinsic biocompatibility, biodegradability and diverse biological functions, HA is widely used in medical devices and pharmaceutical applications, including eye drops, viscoelastic devices and topical formulations. It can bring new and improved attributes to existing products, while also offering numerous opportunities when developing novel formulations.
Overcoming challenges
Producers of medical devices are continually faced with challenges related to abrasion, adhesion, infection and blood coagulation. When used as a biomaterial in a wide range of applications, including the manufacture of device coatings, dermal fillers, tissue engineering, wound healing as well as joint care and eye care applications, HA can help producers overcome the associated challenges and improve device efficacy.
Exhibiting significant structural, rheological, physiological and biological functions also makes HA a highly attractive carrier for drug delivery and formulation applications. As a polyanionic polymer, HA can form complexes with drugs to enhance their aqueous solubility and stability.
As a polyanionic polymer, HA can form complexes with drugs to enhance their aqueous solubility and stability
Although HA has a number of wide-ranging benefits for a variety of applications, sourcing HA for medical uses has been associated with a number of challenges over recent years. In particular, the purity and consistency of HA raw materials can have a significant impact on its clinical performance.
Many of the commercial sources of HA currently available on the market at industrial scale are produced from either rooster comb extraction or various attenuated strains of Streptococcus bacteria, which, if not treated properly, can often incorporate animal-derived contaminants or potentially dangerous endo- and exotoxins and other immunogenic components. This makes them a growing concern for regulatory bodies and has led to the development of a new technology that can deliver safer, more sustainable sources of HA with advantages for both patients and manufacturers.
Reduced contamination risks
In response to the increasing concerns surrounding the purity of many sources of HA and the introduction of more stringent quality measures for medicinal products, a Bacillus subtilis-based fermentation process has been developed by Novozymes Biopharma for the production of HA to minimise the challenges that are widely associated with traditional sources.
Bacillus subtilis is a well-established, nonpathogenic host used in the manufacture of several products with GRAS (Generally Recognized as Safe) status by the US Food and Drug Administration (FDA). Using minimal media and no animal-derived raw materials, the process eliminates the use of organic solvents at all stages of the manufacturing process, ensuring high purity and reducing contamination risks. The end product is a HA characterised by low amounts of nucleic acids, proteins, bacterial endotoxins, endotoxins and microbial contamination, which reduces the potential for adverse reactions in patients.
The resulting HA, known as Hyasis, is produced according to Q7 cGMP standards for API manufacturing, which helps manufacturers to accelerate processes and reduce testing times, and as a result, take their products to market faster and more efficiently. With stringent in-process and end-process specifications, the new generation of HA is characterised by properties that include well-controlled and reproducible molecular weight, low polydispersity and long shelf-life.
Consistent final product
Using a Bacillus-derived HA means that manufacturers are offered improved processability. Its unique recombinant fermentation and spray-drying process yields a highly consistent final product with a reproducible molecular weight and narrow polydispersity of 1.4. The narrow polydispersity allows for the delivery of highly consistent material that can translate into more consistent product formulations and improved performance in the final product.
The narrow polydispersity allows for the delivery of highly consistent material that can translate into more consistent product formulations and improved performance in the final product
As a result of the porosity and reduced size of the HA’s spray-dried particles, the product also dissolves four times faster than HAs of Streptococcal origin. This saves significant time and cost in production and ensures increased facility throughput, while the high purity of the material permits sterilisation by autoclaving while mitigating molecular weight loss.
In addition, individual batch processing times can be reduced, resulting in higher yields and lower cost per production batch, while unparalleled product consistency also eliminates the need for batch selection.
The unique recombinant fermentation and spray-drying process yields a highly consistent final product
As one of the most versatile human biomaterials, novel sources of HA can be used in applications across several medical fields. In the ophthalmology arena one of the major problems with contact lenses is dryness that causes discomfort in the eye. This is why millions of people each year stop using contact lenses completely. A number of re-wetters are available on the market to combat this problem, and a key ingredient in modern formulations is HA.
HA is essentially composed of sugar chains that combine to form a sponge-like structure with water trapped between the chains. This gives re-wetting drops the ability to function as an effective lubricant and mechanical cushion in the eye. HA’s unique physical properties enable it to maintain high viscosity and bind to the surface of the eye without causing blurry vision or undue friction. These properties also make HA an ideal ingredient for buffer solutions used to store contact lenses.
Individual batch processing times can be reduced, resulting in higher yields and lower cost per production batch
For the treatment of osteoarthritis HA can be directly injected into the space between the joints of the knee. As HA is part of the synovial fluid found in the joint space, replenishing HA can give patients sustained pain relief for 6–9 months. The main benefits of HA injections are lubrication and cushioning of the joint, which decreases a patient’s pain and thereby prolongs the time before a knee joint replacement is necessary.
Many injectable dermal fillers contain HA to fill out wrinkles, lips, fine lines, dimples, and some scarring, such as that from acne. It is an alternative to Botox, a toxin that numbs the nerves. In contrast to Botox, hyaluronic acid is naturally found in the skin and therefore no skin test is usually required prior to treatment. A few hours after an injection, a noticeable lift and increase in volume of the skin can be seen.
Tailoring for specific properties
To develop unique end user products, manufacturers sometimes require specific HA properties. The properties of emerging sources of HA can be tailored or adjusted by chemical modification through functionalisation and crosslinking to meet individual manufacturer needs. Newly introduced technology for preparing crosslinked HA hydrogels is based on a simple, reproducible and safe process that does not employ any organic solvents. Owing to an effective purification step, the resulting transparent and homogenous hydrogels do not contain any detectable residual crosslinking agent.
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 the tailoring of gel viscoelastic properties, thereby making the new technology versatile and adaptable to the needs of a specific application.
HA can be customised to achieve a specified viscosity, enabling the product to be adapted for a wide range of drug delivery and medical device applications
The resulting hydrogels can withstand both autoclaving and extrusion through clinical needles of different gauge sizes without significant alteration of their rheological properties. This means that HA can be customised 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 and aesthetic medicine. It also presents manufacturers with the capability to design novel products with a competitive edge, improve product quality and drive efficiency.
Non-animal origins
Recent years have seen the importance of using non-animal derived ingredients in manufacturing processes rise dramatically. With regulatory authorities beginning to clamp down on the safety of medical products, manufacturers are actively looking for ways to produce safe, nonpathogenic products. Bacillus-derived HA represents a safe, consistent source of hyaluronic acid that is animal-free and Q7 cGMP compliant. Offering a high degree of processability, while at the same time decreasing regulatory burden, the new generation of HA enables manufacturers to optimise the efficacy, safety and usability of their products, holding significant benefits for patients and the industry.
References
1. Balaz EA, Laurent TC. Round table discussion: New applications for hyaluronan. In: Laurent TC, editor. The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives. London: Portland Press Ltd; 1998. P 325-336.