Hyaluronic acid - the biotech way

Fanny Longin, science manager, Novozymes Biopolymer A/S, reveals why a new biotech method of producing hyaluronic acid is proving to be a winner

Fanny Longin, science manager, Novozymes Biopolymer A/S, reveals why a new biotech method of producing hyaluronic acid is proving to be a winner

Hyaluronic acid (also referred to as HA or hyaluronan) is a natural and linear polysaccharide belonging to the class of the non-sulphated glycosaminoglycans. HA is a unique biopolymer in that its structure is highly conserved and identical in all species. HA is composed of alternating beta-1,3-N-acetyl glucosamine and beta-1,4-glucuronic acid disaccharide units (see figure 1). The number of repeating disaccharides can reach 10,000 or more, resulting in molecular weights of 4 MDa or more.1

Hyaluronic acid is found in the vitreous body and it is also abundant in the extracellular matrix, especially of soft connective tissue, and in the synovial fluids of articular joints. Skin tissues contain the largest amount of HA, i.e. 7-8g per average adult human.1

HA exhibits significant structural, rheological, physiological, and biological functions. Its distinctive moisturising and visco-elastic properties, coupled with its lack of immunogenicity and toxicity, have led to a wide range of proven and marketed applications within the cosmetic and biomedical industries.2 These include skin moisturisers, osteoarthritis treatment, ophthalmic surgery, eye and rewetting drops, adhesion prevention, dermal fillers and wound healing. Hyaluronic acid is also increasingly investigated as a carrier for the dermal, ophthalmic, nasal, pulmonary, parenteral, liposomal, and implantable delivery of drugs as well as for gene delivery.3

The concentration of HA in rooster combs and human umbilical cords is very high, reaching 7500mg/L and 4100mg/L, respectively.4 In the early 1980s, Balazs and coworkers developed a procedure to isolate, purify and identify hyaluronic acid from rooster combs and human umbilical cords.5 Since then, HA has been produced from rooster combs at industrial scale.

However, the rooster comb-based extraction process is faced by a growing concern due to the use of animal-derived products for technical and especially biomedical and pharmaceutical applications. Hence, microbial fermentation has emerged as a new technique for the production of HA.6 The bacterial production of HA involving a Streptococcus zooepidemicus strain was first described in 1989, giving rise to the first commercialisation of fermented HA.7 Nevertheless, streptococci are pathogenic in nature and fastidious lactic acid bacteria with demanding requirements such as media containing yeast or animal extracts, peptone and serums during the fermentation.8 The presence of bacterial endotoxins, chondroitin sulphates, proteins, nucleic acids and heavy metals in HA from streptococcal fermentation or rooster combs has also been reported.6 Finally, both extracted HA and microbial HA are purified using harsh organic solvents.

Novozymes is a global biotech specialist in enzymes and micro-organisms. Using its core competencies, the company has developed a unique method for the production of a ultra-pure sodium hyaluronate, marketed as HyaCare.9 It is produced by fermentation of a novel and non-pathogenic strain, Bacillus subtilis, from which products are Generally Regarded As Safe (GRAS).

The assembly of the two immediate HA precursor sugars, UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetyl-D-glucosamine (UDP-GlcNAc), to form HA is catalysed by the enzyme hyaluronan synthase, or HAS. In Streptococcus, the biosynthetic pathways which result in the production of these precursors also supply sugars for basic cellular processes such as cell wall biosynthesis and energy metabolism, as shown in figure 2.

To avoid potential limitations on cell growth due to HA synthesis, Streptococci incorporate HAS into an operon along with additional copies of one or more genes (‘precursor genes’), which encode key enzymes that are involved in the synthesis of the precursor sugars. For example, in addition to the HAS gene (designated hasA), the S. equisimilis HA operon includes the hasB, hasC, and hasD precursor genes encoding the enzymes UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase, respectively.

A similar strategy in developing recombinant Bacillus strains that produce HA was followed. All expression constructs utilised the hasA from S. equisimilis, in conjunction with one or more of three native B. subtilis precursor genes - tuaD (hasB homologue), gtaB (hasC), and gcaD (hasD). Gene expression was driven by a modified version of the amyQ promoter from B. amyloliquefaciens. All expression cassettes were integrated into the chromosome of B. subtilis A164D5 at the amyE locus in order to maximise genetic stability. Strains that demonstrated HA production through the appearance of a wet or slimy phenotype on agar plates were further evaluated in fermentation reactors.

During the fermentation of Bacillus subtilis for the production of hyaluronic acid, no animal derived raw materials are used, but instead, a minimal medium based on sucrose as the carbon source. Moreover, the growing HA chain is secreted into the surrounding medium and is not cell-associated. As a consequence, the Bacillus-derived HA is characterised by a vastly improved safety profile, including no risk of viral contamination or of transmittance of animal spongiform encephalopathy. It also exhibits very low protein, nucleic acid and metal ion levels. Moreover, the host strain does not produce any endotoxins or exotoxins.

The fermentation of the B. subtilis A164D5 host to produce HA is followed by a unique recovery process, during which only water-based (i.e. no organic) solvents are employed. The final recovery step consists of spray-drying, which affords the final HA powder. This remarkably energy-efficient technology, combined with the exclusive use of aqueous solvents, make the Novozymes sodium HA production process the most environment friendly developed to date.

The above biotech process leads to the production of a very fine HA powder composed of micro- and nanospheres (Figure 3). Owing to the large surface area, Bacillus-HA dissolves faster than traditional HA, which significantly reduces the time and the energy needed for batch processes and formulation manufacturing. The molecular weight of spray-dried Bacillus-derived HA is ca. 1 MDa, with a very low polydispersity (i.e. 1.3-1.4) according to SEC-MALLS-RI analysis. Moreover, the structure of Bacillus-HA is identical to that of natural HA and Streptococcus HA. This was confirmed by enzymatic hydrolysis followed by MALDI-TOF analysis, FT-IR and HPLC for monomer composition. We have also shown that HyaCare is biocompatible, non-cytotoxic, non-allergenic and non-mutagenic.

In summary, Novozymes Biopolymer has developed a new hyaluronic acid, produced by the fermentation of the safe bacterial strain Bacillus subtilis. The fermentation process is both safeand environmentally friendly: it is 100% free of animal-derived raw materials and of organic solvents. As a consequence, HyaCare is a premium hyaluronic acid with unsurpassed safety and purity. In addition, it is characterised by a well-controlled and reproducible molecular weight and exhibits advantageous formulation properties. Finally, the innovative Bacillus technology not only offers great promise in the pharmaceutical arena with demanding quality and safety requirements but has also the potential to lead to custom-tailored products with targeted molecular weights.