Photos courtesy of Novozymes Biopharma

The field of aesthetic technology has grown rapidly during the past 10 years, with cosmetic procedures taking advantage of the most recent innovations in biotechnology. In a market driven by individuals’ desire to achieve a more youthful appearance, growth has been as a result of the rising demand for non-surgical dermal filler procedures, performed primarily to correct scarring, wrinkles and other depressions in the skin.

Dermal fillers are soft tissue substitutes, designed in such a way that enables injection into the dermis for purposes of improving the appearance of contour defects. They are made up of several types of materials of synthetic, man-made and natural origin, with the most popular ones being collagen-based fillers, poly lactic-based fillers, calcium hydroxylapatite fillers and hyaluronic acid (HA) fillers. As a result of the lack of long-term clinical safety data with permanent fillers and the risk of unaesthetic filler migration after injection, non-permanent fillers are currently the most favored class of products. They work chiefly as a substitute for lost collagen and fat under the skin, offering varying results and duration of effect.

Fillers based on natural substances are becoming increasingly popular due to their safety and resorbability. In particular, HA dermal fillers are being used in a considerable number of procedures each year due to their unequalled ease of administration, effectiveness and safety profile.¹

The Effect of HA on the Skin

HA, also known as hyaluronan, is a naturally occurring polysaccharide distributed widely throughout connective, epithelial and neural tissues in the human body. By providing structure to tissues including skin, HA contributes to the optimal functioning of the body, which makes it well suited to a wide-range of dermal applications. Comprising a high molecular weight, HA is a linear polysaccharide composed of alternating molecules of n-acetyl glucosamine and glucuronic acid.

Since the 1980s, the understanding of HA properties and functions has blossomed into a truly scientific enterprise, meaning the biopolymer is recognized as a high-value product with numerous proven and marketed applications within cosmetic and biomedical areas.²

Historically HA first distinguished itself in the cosmetic industry due to its incomparable moisturizing properties. Indeed, HA is one of the most hygroscopic molecules found in nature, a property that confers its ability to attract and retain moisture from its immediate environment. In addition to maintaining hydration, HA’s role in the skin also relates to shaping the extracellular space and mediating cellular events such as cell proliferation, migration and differentiation.

As the amount of HA in the skin declines with age, this has an impact upon water content and texture, with skin becoming dry and less turgid and elastic.³ In addition, aging skin show signs of wrinkles or folds in the skin, as well as an altered pigmentation as a consequence of less effective microvascularization and a reduction in nutrient/waste celluar exchanges. Dermal fillers based on linear HA or crosslinked HA can be injected to help restore the elemental structure of the skin and boost skin rejuvenation due to the natural bioactivity of HA molecules.

Dermal Filling Using HA

As HA occurs naturally in the skin, it is widely accepted that temporary fillers containing HA offer the lowest risk and are less likely to cause any adverse reactions in patients. Recent statistics show that the majority of aesthetic procedures carried out in the U.S. use HA dermal fillers.⁴ This trend is expected to increase as currently there is no other class of filling agent that rivals the properties and advantages of HA fillers.¹ As well as offering high biocompatibility, HA fillers do not require a pre-treatment skin test and they can be degraded naturally in vivo by endogenous hyaluronidase. Parameters such as HA source, HA concentration, nature of crosslinking agent, crosslinking process (single or multiple crosslinking), particle size and ratio of crosslinked HA hydrogel to uncrosslinked HA solution, all constitute variables used to differentiate commercial products currently on the market. However despite the undisputed benefits of HA, purity of the HA raw material and the innocuousness of the crosslinking technology must be assured by manufacturers in order to achieve a safe product.

Current Challenges for Manufacturers

Until recently, the source of a specific HA product has not been considered to be a clinically important point of differentiation amongst competitive products available to the market. However, with its growing use as an injectable, there is increasing demand for a source that can offer the advantages of existing products, while also ensuring an enhanced level of purity and a higher degree of safety.

All current commercial sources of HA are produced from either rooster comb extraction or various attenuated strains of Streptococcus bacteria, which may potentially result in contamination risks from animal proteins, viruses or endotoxins. Streptococci are inherently pathogenic to human beings due to their secretion of toxins and resulting hemolytic properties. This makes them a concern to regulatory bodies such as the U.S. Food and Drug Administration and European Medicines Agency (EMA). Moreover, both extracted HA and microbial HA are purified using harsh organic solvents, which poses further health issues to patients. Consequentially, there is a need to move away from the use of HA produced by these methods, in favor of new technology that is able to deliver safer, more sustainable sources.

The Introduction of Bacillus-Derived HA

The popularity of animal-free ingredients is continuously increasing, with regulatory authorities beginning to enforce tighter quality controls on cosmetic products to improve safety and minimize risk to patients. A new Bacillus subtilis-based fermentation process has been developed for the production of HA to overcome the challenges and provide an alternative to animal-derived sources. Bacillus subtilis is a non-pathogenic host whose products are Generally Recognized as Safe (GRAS) by the FDA. The process uses minimal medium, no animal-derived raw materials, and a water-based technique, which removes the use of organic solvents at any stage during the manufacturing process. The resulting HA is characterized by low amounts of nucleic acids, proteins, bacterial endotoxins, exotoxins and microbial contamination which reduces hypersensitivity reactions when the material is injected either as linear or crosslinked HA.

The controlled Bacillus-derived production process affords a HA material with a reproducible molecular weight and narrow size distribution. These properties lead to homogenous crosslinked HA hydrogels with a well-defined mesh size and reproducible biodegradation profile. In addition, this source of HA offers improved processibility due to the porosity and reduced size of its spray-dried particles and can dissolve much faster than HAs of streptococcal origin. This reduces filtration time at large scale and as a result, reduces manufacturing costs. A high degree of purity of the material also permits sterilization by autoclaving without significant loss of product properties.

Versatile Crosslinking Technology To Support Dermal Filler Development

Using a Bacillus-derived fermentation process for preparing crosslinked HA is a simple and safe process. The resulting transparent and homogenous hydrogels do not contain traceable levels of residual crosslinking agent and can tolerate extrusion through needles of different gauge sizes without significant alteration of their rheological properties. Control of parameters such as starting HA concentration and HA-to-crosslinking-agent ratio permits tailoring gel viscoelastic properties, thereby enabling the product to be adjusted to suit application-specific needs. This capability presents manufacturers with the ability to tailor their products, or design them with a competitive edge. Figure 1 shows the storage (G’) and loss (G”) moduli of a crosslinked HA hydrogel formulation as a function of the frequency of a deformation. The G’ values of formulations characterized by different ratios of crosslinked HA hydrogel to HA hydrogel solution demonstrate that the elastic properties of these formulations can be fine tuned (Figure 2). The average force required to extrude different hydrogel formulations through a 30G needle was below 15 N (see Figure 3), demonstrating that these could easily be injected in vivo.

Demand for dermal fillers based on natural substances by the cosmetics industry is on the increase. Products are also becoming increasingly engineered in order to enhance precision and flexibility of the correction provided. Bacillus-derived HA offers new possibilities to the industry by providing a pure and biocompatible source which confers numerous advantages to both manufacturers and patients. The innovative technology offers unequalled ease of administration, effectiveness and an unmatched safety profile which make it suitable for the preparation of safe, consistent and robust dermal filler products.  



















References
1    Beasley et al. Hyaluronic acid fillers: a comprehensive review. Facial Plastic Surgery (2009) 25, 86–94.
2    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.
3    Oh et al. Intrinsic aging- and photoaging-dependent level changes of glycoaminoglycans and their correlation with water content in human skin. Journal of Dermatological Science (2011) doi:10.1016/j.dermsci.2011.02.007.
4    The American Society for Aesthetic Plastic Surgery: http://www.surgery.org.

Hans Ole Klingenberg is Product Director at Novozymes Biopharma. He can be reached at [email protected].