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The Science Behind Expression™
In recent years there has been a growing concern regarding the use of animal-‐derived products for technical and especially biomedical and pharmaceutical applications. Typically extracted from rooster combs, hyaluronic acid, also referred to as HA or hyaluronan, is one example of a product that could benefit from a pathogen-‐free production alternative. HA has been used in a wide range of proven and marketed applications within the cosmetic and biomedical industries. Its distinctive moisturizing and visco-‐elastic properties, coupled with its lack of immunogenicity and toxicity, have made it popular in skin moisturizers, osteoarthritis treatment, ophthalmic surgery, eye and rewetting drops, adhesion prevention, wound healing, and dermal fillers. HA also is investigated increasingly as a carrier for the dermal, ophthalmic, nasal, pulmonary, parenteral, liposomal, and implantable delivery of drugs as well as for gene delivery. New methods have emerged to make HA available as a raw material free from any animal derivatives through a biotech production method. Enhancement Medical is using the new pathogen-‐free, raw HA material to develop and manufacture its Expression™ injectable gel in Wauwatosa, Wisconsin. By examining the chemical properties of HA and comparing the two production methods, it is evident that the new method offers numerous advantages. Introduction to HA HA is a natural and linear polysaccharide belonging to the class non-‐sulphated glycosaminoglycans. With a structure that is highly conserved and identical in all species, HA is a unique biopolymer. It exhibits significant structural, rheological, physiological, and biological functions. HA is composed of alternating beta-‐1, 3-‐N-‐acetyl glucosamine and beta-‐1, 4-‐glucoronic acid disaccharide units. The number of repeating disaccharides can reach 10,000 or more, resulting in molecular weights of 4 MDa or more. Figure 1 shows HA in its powdered form.
Figure 1: Hyaluronic acid, with its moisturizing and visco-‐elastic properties
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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, measuring 7-‐8 g per average adult human. The concentration of HA in rooster combs and human umbilical cords is very high, reaching 7500 mg/L and 4100 mg/L, respectively. In the early 1980s, a group of scientists developed a procedure to isolate, purify and identify hyaluronic acid from rooster combs and human umbilical cords. Since then, HA has been produced from rooster combs at industrial scale.
Production of HA Microbial fermentation has emerged as a successful new technique for the production of HA. The bacterial production of HA involving a Streptococcus zooepidemicus strain was first described in 1989, giving rise to the first commercialization of fermented HA. Nevertheless, streptococci are pathogenic in nature and fastidious lactic acid bacteria. They have demanding requirements such as media containing yeast or animal extracts, peptone and serums during the fermentation. 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. Finally, both extracted HA and microbial HA are purified using harsh organic solvents. As a global biotech specialist in enzymes and micro-‐organisms, the Denmark-‐based company Novozymes Biopolymer used its core competencies to develop a unique method for the production of an ultra-‐pure sodium hyaluronate. It is produced by fermentation of a novel and non-‐pathogenic strain, Bacillus subtilis, from which products are Generally Regarded As Safe (GRAS).
The enzyme hyaluronan synthase, or HAS, catalyzes the assembly of the two immediate HA precursor sugars, UDP-‐glucoronic acid (UDP-‐GlcUA) and UDP-‐N-‐acetyl-‐D-‐glucosamine (UDP-‐GlcNAc), to form HA. In Streptococcus, the biosynthetic pathways that result in the production of these precursors also supply sugars for basic cellular processes such as cell wall biosynthesis and energy metabolism. This relationship is illustrated in Figure 2.
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Figure 2: Bacterial pathway for the production of hyaluronic acid in recombinant Bacillus
subtilis strains To avoid potential limitation on cell growth due to HA synthesis, Streptococci incorporate HAS into an operon along with additional copies of one or more genes that encode key enzymes 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 New Approach A similar strategy was followed to develop recombinant Bacillus strains that produce HA. All expression constructs utilized 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 maximize genetic stability. Strains that demonstrated HA production through the appearance of a wet or slimy phenotype on agar plates were evaluated further in fermentation reactors. During the fermentation of Bacillus subtilis for the production of HA, no animal derived raw material are used. Instead, the carbon source is a minimal medium based on sucrose. The growing HA chain is secreted into the surrounding medium and is not cell-‐associated. As a consequence, the Bacillus-‐derived HA is characterized by a vastly improved safety profile. There is no risk of viral contamination or of transmittance of animal spongiform encephalopathy. It also exhibits very low levels of protein, nucleic acid and metal ion. Moreover, the host strain does not produce any endotoxins or exotoxins.
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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. Benefits of the New Method 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 new biotech process leads to the production of a very fine HA powder composed of micro-‐ and nanospheres, as shown in Figure 3. Owing to the large surface area, Bacillus-‐HA dissolves faster than traditional HA. This significantly reduces the time and energy needed for batch processes and formulation manufacturing.
Figure 3: Scanning Electron Microscope picture of Bacillus-‐derived hyaluronic acid
The molecular weight of spray-‐dried Bacillus-‐derived HA is ca. 1 MDa, with a very low polydispersity of 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. It has been shown that this ultra-‐pure sodium hyaluronate from is biocompatible, non-‐cytotoxic, non-‐allergenic and non-‐mutagenic. Enhancement Medical uses the raw HA product from Novozymes as a non-‐toxic and non-‐pathogen delivery medium for its Expression™ injectable gel. In formulating Expression™, the HA molecules are cross-‐linked with divinyl sulfone (DVS) and the gel is swelled to equilibrium.
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In Summary This new hyaluronic acid produced by the fermentation of the safe bacterial strain Bacillus subtilis is characterized by a well-‐controlled and reproducible molecular weight and exhibits advantageous formulation properties. This innovative Bacillus technology not only offers great promise in the pharmaceutical arena with demanding quality and safety requirements but also has the potential to lead to custom-‐tailored products with targeted molecular weights. The fermentation process is both safe and environmentally friendly. It is 100% free of animal-‐derived raw materials and of organic solvents. As a consequence, Expression™ by Enhancement Medical contains a premium hyaluronic acid with unsurpassed safety and purity. Further Reading Additional information regarding this new HA production method is available from Enhancement Medical and from Novozymes. Additionally, the following article and the references contained therein: “Hyaluronic Acid -‐ The Biotech Way”, Manufacturing Chemist, 22 Dec 2008, www.manufacturingchemist.com/technical/article_page/Hyaluronic acid the biotech way/41631
Figure 4: Novozymes Biopolymer A/S was one fo the winning entries in the 2008 Innovatin Awards at CPhI in Frankfurt. The company won Gold for the innovative process of producing
hyaluronic acid via biotechnology
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