Dermal fillers

Sodium hyaluronate

Bacillus-derived hyaluronic acid for dermal filler applications
Dermal filling is a nonsurgical aesthetic procedure using injections of soft tissue substitutes as a means to correct contour defects such as those resulting from intrinsic and extrinsic aging, disease, trauma, and scarification1. Existing dermal filler products can be distinguished according to their constitutive material (synthetic or natural), their indication, and the duration of the correction provided (nonpermanent, semipermanent, or permanent fillers). Due to the lack of long-term clinical safety data with semipermanent and permanent fillers and the risk of unaesthetic filler migration after injection, nonpermanent fillers are currently the most favored class of products. Within this class, fillers based on natural substances have become popular due to their safety and resorbability. In particular, hyaluronic acid (HA) dermal fillers are used in a considerable number of procedures each year due to their unequalled ease of administration, effectiveness, and safety profile2. differentiate the currently available commercial products. However, regardless of these characteristics, the purity of the HA raw material and the innocuousness of the crosslinking technology are crucial elements in achieving a safe dermal filler product.

Bacillus-derived sodium hyaluronate, an ingredient of choice for HA-based dermal fillers
Novozymes’ sodium hyaluronate material (sodium salt of hyaluronic acid) is derived from the world’s first and only recombinant manufacturing process which is based on fermentation of the safe and nonpathogenic bacterial strain Bacillus subtilis. This process uses minimal medium, no animal-derived raw materials, and a proprietary water-based technology, which eliminates the use of organic solvents at any stage of the manufacturing process. The resulting sodium hyaluronate material is characterized by an unmatched purity and particularly low amounts of nucleic acids, proteins, bacterial endotoxins, and microbial contamination* which are believed to reduce hypersensitivity reactions when the material is injected either as linear or crosslinked HA. Furthermore, the controlled production process affords a HA material with a reproducible molecular weight and narrow size distribution. These properties lead to homogeneous crosslinked HA hydrogels with a welldefined mesh size and reproducible biodegradation profile. All these features are advantageous when sourcing HA as an ingredient for dermal fillers: the ultrapurity and reproducible molecular properties of Novozymes’ HA can indeed contribute to the safety, consistency, and robustness of final products.
Benefits of Novozymes’ Bacillus-derived HA • • Ingredient produced without animal-derived raw materials or organic solvents Ultrapure material with particularly low amounts of nucleic acids, proteins, bacterial endotoxins, and microbial contamination Reproducible molecular weight and narrow size distribution Bioactive molecule known for its rejuvenation properties Superior heat stability permitting autoclaving without significant loss of product properties Ingredient of choice for the preparation of safe, consistent, and robust dermal filler products

Hyaluronic acid and aging of the skin
While HA is ubiquitous in the human body, it is most abundant in the skin, where it is found in both the dermis and the epidermis. HA’s role in the skin relates to shaping the extracellular space, maintaining tissue hydration, and mediating cellular events such as cell proliferation, migration, and differentiation. With age, the content of HA in the skin decreases3. This has direct consequences on the water content and texture of the skin which becomes dry and less turgid and elastic4. Furthermore, aging skin displays wrinkles and an altered pigmentation due to a less effective microvascularization and fewer nutrient/waste cellular exchanges. Injections of dermal fillers based on linear HA (mesotherapy) or crosslinked HA help restore the elemental structure of the skin and boost skin rejuvenation due to the natural bioactivity of HA molecules.
Hyaluronic acid dermal fillers are used in a considerable number of procedures each year due to their unequalled ease of adminstration, effectiveness, and safety profile.

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Hyaluronic acid dermal fillers
According to recent statistics, the majority of aesthetic procedures carried out in the US currently occur with HA dermal fillers5. This trend is expected to increase since there presently is no other class of filling agent that rivals the properties and advantages of HA fillers2. HA fillers offer high biocompatibility and a low risk of adverse reaction in patients. In addition, they do not require skin testing 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 HA hydrogel solution all constitute variables used to

Versatile and proven crosslinking technology to support dermal filler development
Novozymes’ proprietary technology for preparing crosslinked HA is based on a simple, reproducible, and safe process. The resulting transparent and homogeneous hydrogels do not contain any detectable residual crosslinking agent and can withstand both autoclaving and 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 making Novozymes’ crosslinking technology versatile and adaptable to customers’ specific requirements. 5 6 Figure 1 presents the storage (G’) and loss (G’’) moduli of a crosslinked HA hydrogel formulation as a function of the frequency of deformation. The 5 G’ values of formulations characterized by different ratios of crosslinked 4 HA hydrogel to HA hydrogel solution show that the elastic properties of 3 these formulations can be fine tuned (Fig. 2). Finally, the average force required to extrude different hydrogel formulations through a 30G needle 2 was below 15 N which shows that these could easily be injected in vivo 1 (Fig. 3). The well-defined rheological properties and excellent injectability of 0 Novozymes’ crosslinked HA formulations can offer maximum0 convenience to practitioners and comfort to patients for enhanced precision and efficacy. 1.0
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6 5
Extrusion force (N)

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4 3 2 1 0



2.0Formulation 1

Formulation 1

Formulation 2

Formulation 3


3.0 Formulation 2


Storage (G') and loss (G'') moduli (Pa)

HA concentration (% w/v) Fig. 3. Average extrusion force Formulation inject 2 Formulation 3 HA concentration (% w/v) needed1 to Formulation different Novozymes crosslinked HA hydrogel formulations, differing by the ratio of crosslinked HA hydrogel to HA hydrogel solution, through a 30G needle (according to texture analysis). Formulation G Formulation 2 Formulation11 30Formulation 2 22 G 27 G Formulation 3 30 G 22 G 27 G



Benefits of Novozymes’ crosslinked HA technology • • • • Proven and versatile technology affording gels with customized viscoelastic properties No detectable residual crosslinking agent Ability to withstand autoclaving and extrusion without significant alteration of rheological properties Optimal injection mechanics for increased practitioner convenience and patient comfort Well tolerated, safe, and biocompatible







Frequency (Hz)

Fig. 1. Storage (G’) and loss (G’’) moduli as a function of the frequency of deformation of a typical Novozymes crosslinked HA hydrogel formulation (according to rheological analysis). — Storage modulus — Loss modulus

Supporting future dermal filler developments
Dermal fillers are becoming more and more engineered in order to enhance the precision and flexibility of the correction provided. With expertise in HA, crosslinked HA, and the interaction of these biomaterials with active ingredients, Novozymes is well-placed to provide support for the development of your innovative solutions.
* For the specifications of Novozymes’ Bacillus-derived sodium hyaluronate, feel free to contact us.


Storage (G') modulus (Pa)



Sturm et al. A systematic review of dermal fillers for age-related lines and wrinkles. ANZ

Journal of Surgery (2011) 81, 9–17.

Beasley et al. Hyaluronic acid fillers: a comprehensive review. Facial Plastic Surgery (2009)
© Novozymes A/S · No. 2011-15533-01

1 0.01 0.1 1 10

25, 86–94.

Frequency (Hz)

Ghersetich and Lotti. Hyaluronic acid in cutaneous intrinsic aging. International Journal of

Dermatology (1994) 33 2, 119–122.
Fig. 2. Storage (G’) modulus as a function of the frequency of deformation of different Novozymes crosslinked HA hydrogel formulations differing by the ratio of crosslinked HA to HA hydrogel solution (according to rheological analysis). The proportion of HA hydrogel solution decreases from Formulation 1 to 3. — Formulation 3 — Formulation 1 — Formulation 2

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.

The American Society for Aesthetic Plastic Surgery:

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