Development and Applications of a Novel, First-in-Class Hyaluronic

Chapter 22

Downloaded by UNIV OF OKLAHOMA on August 21, 2013 | http://pubs.acs.org Publication Date (Web): August 11, 2010 | doi: 10.1021/bk-2010-1043.ch022

Development and Applications of a Novel, First-in-Class Hyaluronic Acid from Bacillus Khadija Schwach-Abdellaoui,* Birgit Lundskov Fuhlendorff, Fanny Longin, and Jens Lichtenberg Novozymes Biopolymer A/S, Kroghshoejvej 36, DK-2880 Bagsvaerd, Denmark *[email protected]

Since 1934, hyaluronic acid (HA) has been extracted from rooster combs and employed for various applications such as ophthalmic surgery, orthopedic and wound healing. A breakthrough in HA production occurred in 1985, when the more sophisticated streptococcal fermentation process was implemented and considered the ‘gold standard’ since then. Novozymes Biopolymer radically shifted the paradigm by employing a unique and non-pathogenic strain, Bacillus subtilis, as an “industry factory” to produce a new biosynthetic HA product: HyaCare®. HyaCare® powder is composed of microparticles with enhanced surface properties that improve solubilization time compared to conventional processes. This new technology combined with the molecular and physico-chemical properties of HA provides improved safety, purity, consistency, stability and ease of filtering. HyaCare® is produced using an advanced fermentation process without ingredients derived from animal sources or organic solvents. It is an extremely pure source of HA in which there are no exotoxins and low level of proteins. These properties make it suitable for use in biomedical and pharmaceutical applications.

Introduction Hyaluronic acid (HA) is a natural linear polysaccharide consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked through β-1,3 glycosidic © 2010 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


bonds while consecutive disaccharide repeating units are linked through β-1,4 bonds (Figure 1). In vertebrates HA is ubiquitous in all organs and fluids and in the extracellular matrix of soft connective tissues where it provides a backbone for the distribution and organization of proteoglycans, fibrin, fibronectin and collagen. Since HA was discovered in 1934, it has been extracted from rooster combs and employed for various applications such as ophthalmic surgery, orthopedic and wound healing. Recovery of HA from rooster combs necessitates extensive purification using harsh organic solvents to remove antigenic avian proteins. A breakthrough in HA production occurred in 1985, when the more sophisticated streptococcal fermentation process was implemented and since then considered as the ‘gold standard.’ Streptococci are fastidious organisms to grow, are natively pathogenic, and have the potential to produce exotoxins. Moreover it is difficult to control HA molecular weight from streptococcal fermentation. Novozymes Biopolymer radically shifted the paradigm by employing a unique and non-pathogenic strain, Bacillus subtilis, as an ‘industry factory’ to produce a new biosynthetic HA product: HyaCare® (1). B. subtilis is one of the most well-characterized gram-positive microorganisms that can be genetically manipulated using a wide array of tools available. It does not produce, nor does the genome sequence encode, a hyaluronidase which could degrade HA. Thus, these organisms offer several advantages as possible hosts for producing HA of well defined molecular weight and narrow polydispersity (2). Recombinant Bacillus species have been used for several decades to produce industrial enzymes and small molecules such as riboflavin and amino acids and many of these products have achieved GRAS (Generally Recognized As Safe) status. The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been introduced into Bacillus subtilis and expressed, resulting in the production of authentic HA. HyaCare® is produced using an advanced fermentation process during which no ingredients derived from animal sources or organic solvents are used. It is an extremely pure source of HA containing no exotoxins and very low level of proteins. Here we report the physicochemical properties, thermal stability and filterability of this new source of HA compared to streptococcal HA (sHA) as well as its interaction with ingredients and drugs. Preliminary toxicity evaluation is also reported here.

Experimental HA powder was dissolved into PBS buffer to mimic the most common formulations used in Eye Care at concentrations ranging from 0.1 to 2%. Samples were autoclaved under various temperatures and times (110°C/20 min, 121°C/16 min and 134°C/3 min). Molecular weights (MW) were determined using SEC/MALLS/RI (Size-Exclusion-Chromatography combined with Multi-Angle-Laser-Light-Scattering and Refractive Index detector). HA solutions were prepared in PBS buffer at concentrations ranging from 0.025% to 0.5% (w/v), preserved with Polyhexamethylene Biguanide (PHMB, 306 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. Molecular structure of the HA disaccharide repeating unit. Cosmocil® CQ) and were filtered under house vacuum through a Durapore® membrane filter (0.22 µm GV) adapted on a Büchner funnel. The filtration time was recorded with t=0 when the solution had just been loaded on the filter. PBS solutions were prepared with HyaCare concentrations ranging from 0.1 to 0.5% and containing 0.3 or 0.5% gentamicin. Clearance was evaluated by visual inspection and dynamic viscosity was measured using Brookfield Viscometer LVDV-II +Pro, (E-246-99) equipped with a small sample adapter. HyaCare® was evaluated in two separate in vivo toxicological studies where the animals were treated either by the intravenous (IV) or the subcutaneous (SC) route of administration. In each of the two studies twenty-four male rats of the Wistar strain were randomly divided into four groups each comprising of 6 rats per group. The animals were treated with HyaCare® at the dose levels of 25 (low dose), 50 (mid dose) and 100 (high dose) mg/kg b.wt/day, respectively for a period of 14 consecutive days. All animals were closely monitored for body weight changes, clinical signs and at termination they were subjected to a full gross necropsy.

Results and Discussions HyaCare® MW was investigated for several batches using SEC-MALLS and showed very good consistency in both MW and polydispersities (I) (Figure 2). This is due to the fact that bacillus-derived HA is excreted extracellularly and extracted using a gentle aqueous process mainly comprising ultrafiltration and ions exchange steps and that has minor effect on HA degradation. Control of the molecular weight and polydispersity of HA is very important for the functionality of the HA-based products, especially in the biomedical and pharmaceutical applications. In case of HyaCare®, detailed study of the MW decrease upon autoclavation was allowed by plotting the relative retained MW as a function of the HA concentration (Figure 3). Results showed that the higher the HA concentration, the higher the retained HyaCare® molecular weight after autoclavation, regardless of the autoclavation conditions. For most concentrations, the autoclavation at 134°C for 3 min was milder or significantly milder than the other selected autoclavation conditions. It is noteworthy that the purity of HA is of outmost importance when carrying out an autoclavation step. It is well-established that cations such as Cu+, Fe2+ and Sn2+ enhance the depolymerization of HA as they are involved in redox reactions that may lead to the formation of radicals, which in turn degrade the HA chains. 307 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 2. Consistency in MW (■) and polydispersities (—) for several HyaCare® batches

Figure 3. HA retained molecular weight after autoclavation of HyaCare® solutions at 110°C/20 min (white), 121°C/16 min (gray) and 134°C/3 min (black) Results show that high MW HA is more sensitive to autoclavation than medium MW HA at 0.1% and 1% concentration (Figure 4). A possible explanation could be the increased number of available sites to thermal degradation in high MW HA compared to medium MW HA. HyaCare® was significantly more stable than sHA high MW and sHA medium MW. Topical ophthalmic formulations containing thermolabile drugs are typically sterilized by filtration at production scale. However, those containing HA may be difficult to filter due to the relatively high viscosity of this ingredient even at low concentrations. The molecular weight does not affect the filtration time up to 0.1%. However, at 0.2% concentration, it takes up to three times longer to filter 308 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 4. HA retained molecular weight after autoclavation at 134°C/3 min comparing HyaCare® (white) with medium MW sHA (HA2, gray) and high MW sHA (HA1, black). The MW was normalized

Figure 5. Filtration time of HA solutions at various concentrations; (white) medium MW HyaCare®, (0.89 MDa); (black) high MW s-HA (2.25 MDa). a high MW HA solution than a medium MW HA solution (Figure 5). The effect is even more pronounced when the concentration is increased to 0.3%. Today, commercial eye drops recommended for the treatment of dry eye contain from 0.1% to 0.3% HA. Sterile filtration of these ophthalmic solutions is a crucial step in the manufacturing process in that its optimization can not only reduce the number of adverse events linked to the handling of highly viscous solutions, but also lower the production costs (3). When stored with the commonly used preservatives in pharmaceutical formulations namely, benzalkonium chloride and polyhexamethylene biguanide, HyaCare® did not show any degradation at 25 and 40°C or interaction with the preservatives (results not shown). 309 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 6. Dynamic viscosity of HyaCare formulations with (---) and without gentamicin (—) Gentamicin is one of the most commonly used antibiotics in ophthalmology and dermatology and other biomedical applications due to its wide antibacterial spectrum and because, unlike many antibiotics, it is stable at the high temperatures used during sterilization. Sustained high local concentrations of antibacterial compounds such as gentamicin are required to minimize and even to cure some diseases such as chronic osteomyelitis. Results of the combination of Hyacare® and gentamicin show that the drug did not interfere with HA at any concentration studied. The clearance of the solution was maintained even at high concentrations of HA and the dynamic viscosity was unchanged with or without gentamicin (Figure 6). We believe that HyaCare® will represent the ideal carrier for sustained released of gentamicin as HA is already used and well tolerated for ophthalmic, dermatological and other biomedical applications. The toxicological findings following IV administration to rats comprised of clinical signs such as mild lethargy observed immediately after dosing in some of the rats from high dose treated group, which however recovered within 10 to 20 minutes after dosing. No significant difference was observed in the body weight gain in any of the treatment groups compared to the control. The gross pathological examination of the HyaCare® treated groups did not reveal any treatment related changes compared to the control group. Rats treated by SC administration for 14 days showed no clinical signs of toxicity or body weight changes in any groups during the treatment period. The gross pathological examination of the HyaCare® treated groups did not reveal any treatment related changes except marginal reddening and/or hardening at the injection site. The present findings are supported by several in vitro studies previously conducted including cytotoxicity, dermal and eye irritancy tests (data not shown). The low toxicity of HyaCare® is mainly due to its high purity and to the fact that HA is a natural component of the extracellular matrix (4). 310 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

In conclusion, based on the present in vivo toxicological studies and the previously conducted in vitro tests HyaCare® is considered exhibiting no or a very limited toxicological potential at all end points.


Conclusion Several HA-based products in the biomedical space are currently using high molecular weight HA. We believe that in the majority of the cases this is due to the fact that mainly high MW was available from streptococcal fermentation when these products were developed. Our studies demonstrate that medium molecular weight from Bacillus fermentation has superior properties in term of consistency in molecular weight and polydispersity and the ease of sterile filtration. Moreover the higher purity of HyaCare® compared to the available sources of HA offers the possibility of heat sterilization with minor degradation under given conditions and allows its use with various ingredients, preservatives and drugs without degradation, precipitation or decrease in viscosity. Last but not least the encouraging preliminary toxicity studies conducted on rats are very promising and show that even IV injection of HA will now be possible when safe and high purity source is used. We believe that high MW is still needed for applications such as ophthalmic surgery where strong cohesive properties are needed and could not be obtained by using medium MW even at very high concentrations. High MW HyaCare from Bacillus subtilis fermentation is thus under current development. Finally, these unique properties will undoubtedly make HyaCare® the product of choice for biomedical and pharmaceutical applications, especially for drug delivery.

References 1.

2. 3.

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311 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Development and Applications of a Novel, First-in-Class Hyaluronic

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