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The Science Behinu Expiession"

In iecent yeais theie has been a giowing concein iegaiuing the use of animal-ueiiveu
piouucts foi technical anu especially biomeuical anu phaimaceutical applications. Typically
extiacteu fiom ioostei combs, hyaluionic aciu, also iefeiieu to as BA oi hyaluionan, is one
example of a piouuct that coulu benefit fiom a pathogen-fiee piouuction alteinative. BA has
been useu in a wiue iange of pioven anu maiketeu applications within the cosmetic anu
biomeuical inuustiies. Its uistinctive moistuiizing anu visco-elastic piopeities, coupleu with
its lack of immunogenicity anu toxicity, have maue it populai in skin moistuiizeis,
osteoaithiitis tieatment, ophthalmic suigeiy, eye anu iewetting uiops, auhesion pievention,
wounu healing, anu ueimal filleis. BA also is investigateu incieasingly as a caiiiei foi the
ueimal, ophthalmic, nasal, pulmonaiy, paienteial, liposomal, anu implantable ueliveiy of uiugs
as well as foi gene ueliveiy.

New methous have emeigeu to make BA available as a iaw mateiial fiee fiom any animal
ueiivatives thiough a biotech piouuction methou. Enhancement Neuical is using the new
pathogen-fiee, iaw BA mateiial to uevelop anu manufactuie its Expiession" injectable fillei in
Wauwatosa, Wisconsin. By examining the chemical piopeities of BA anu compaiing the two
piouuction methous, it is eviuent that the new methou offeis numeious auvantages.

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BA is a natuial anu lineai polysacchaiiue belonging to the class non-sulphateu
glycosaminoglycans. With a stiuctuie that is highly conseiveu anu iuentical in all species, BA is
a unique biopolymei. It exhibits significant stiuctuial, iheological, physiological, anu biological
functions. BA is composeu of alteinating beta-1, S-N-acetyl glucosamine anu beta-1, 4-
glucoionic aciu uisacchaiiue units. The numbei of iepeating uisacchaiiues can ieach 1u,uuu oi
moie, iesulting in moleculai weights of 4 NBa oi moie. Figuie 1 shows BA in its powueieu
foim.


-).'$/ 01 Byaluionic aciu, with its moistuiizing anu visco-elastic piopeities

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Byaluionic aciu is founu in the vitieous bouy anu it is also abunuant in the extiacellulai matiix,
especially of soft connective tissue, anu in the synovial fluius of aiticulai joints. Skin tissues
contain the laigest amount of BA, measuiing 7-8 g pei aveiage auult human. The concentiation
of BA in ioostei combs anu human umbilical coius is veiy high, ieaching 7Suu mgL anu 41uu
mgL, iespectively. In the eaily 198us, a gioup of scientists uevelopeu a pioceuuie to isolate,
puiify anu iuentify hyaluionic aciu fiom ioostei combs anu human umbilical coius. Since then,
BA has been piouuceu fiom ioostei combs at inuustiial scale.

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Niciobial feimentation has emeigeu as a successful new technique foi the piouuction of BA.
The bacteiial piouuction of BA involving a Stieptococcus zooepiuemicus stiain was fiist
uesciibeu in 1989, giving iise to the fiist commeicialization of feimenteu BA. Neveitheless,
stieptococci aie pathogenic in natuie anu fastiuious lactic aciu bacteiia. They have uemanuing
iequiiements such as meuia containing yeast oi animal extiacts, peptone anu seiums uuiing
the feimentation. The piesence of bacteiial enuotoxins, chonuioitin sulphates, pioteins,
nucleic acius, anu heavy metals in BA fiom stieptococcal feimentation oi ioostei combs has
also been iepoiteu. Finally, both extiacteu BA anu miciobial BA aie puiifieu using haish
oiganic solvents.

As a global biotech specialist in enzymes anu micio-oiganisms, the Benmaik-baseu company
Novozymes Biopolymei useu its coie competencies to uevelop a unique methou foi the
piouuction of an ultia-puie souium hyaluionate. It is piouuceu by feimentation of a novel anu
non-pathogenic stiain, Bacillus subtilis, fiom which piouucts aie ueneially Regaiueu As Safe
(uRAS).

The enzyme hyaluionan synthase, oi BAS, catalyzes the assembly of the two immeuiate BA
piecuisoi sugais, 0BP-glucoionic aciu (0BP-ulc0A) anu 0BP-N-acetyl-B-glucosamine (0BP-
ulcNAc), to foim BA. In Stieptococcus, the biosynthetic pathways that iesult in the piouuction
of these piecuisois also supply sugais foi basic cellulai piocesses such as cell wall
biosynthesis anu eneigy metabolism. This ielationship is illustiateu in Figuie 2.


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-).'$/ 41 Bacteiial pathway foi the piouuction of hyaluionic aciu in iecombinant Bacillus
subtilis stiains

To avoiu potential limitation on cell giowth uue to BA synthesis, Stieptococci incoipoiate BAS
into an opeion along with auuitional copies of one oi moie genes that encoue key enzymes
involveu in the synthesis of the piecuisoi sugais. Foi example, in auuition to the BAS gene
(uesignateu hasA), the S. equisimilis BA opeion incluues the hasB, hasC, anu hasB piecuisoi
genes encouing the enzymes 0BP-glucose uehyuiogenase, 0BP-glucose pyiophosphoiylase,
anu 0BP-N-acetylglucosamine pyiophosphoiylase, iespectively.

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A similai stiategy was followeu to uevelop iecombinant Bacillus stiains that piouuce BA. All
expiession constiucts utilizeu the hasA fiom S. equisimilis, in conjunction with one oi moie of
thiee native B. subtilis piecuisoi genes: tuaB (hasB homologue), gtaB (hasC), anu gcaB (hasB).
uene expiession was uiiven by a mouifieu veision of the amyQ piomotei fiom B.
amyloliquefaciens. All expiession cassettes weie integiateu into the chiomosome of B. subtilis
A164BS at the amyE locus in oiuei to maximize genetic stability. Stiains that uemonstiateu BA
piouuction thiough the appeaiance of a wet oi slimy phenotype on agai plates weie evaluateu
fuithei in feimentation ieactois.

Buiing the feimentation of Bacillus subtilis foi the piouuction of BA, no animal ueiiveu iaw
mateiial aie useu. Insteau, the caibon souice is a minimal meuium baseu on suciose. The
giowing BA chain is secieteu into the suiiounuing meuium anu is not cell-associateu. As a
consequence, the Bacillus-ueiiveu BA is chaiacteiizeu by a vastly impioveu safety piofile.
Theie is no iisk of viial contamination oi of tiansmittance of animal spongifoim
encephalopathy. It also exhibits veiy low levels of piotein, nucleic aciu anu metal ion.
Noieovei, the host stiain uoes not piouuce any enuotoxins oi exotoxins.

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The feimentation of the B. subtilis A164BS host to piouuce BA is followeu by a unique
iecoveiy piocess, uuiing which only watei-baseu (i.e. no oiganic) solvents aie employeu. The
final iecoveiy step consists of spiay-uiying, which affoius the final BA powuei.


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This iemaikably eneigy-efficient technology, combineu with the exclusive use of aqueous
solvents, make the Novozymes souium BA piouuction piocess the most enviionment fiienuly
uevelopeu to uate. The new biotech piocess leaus to the piouuction of a veiy fine BA powuei
composeu of micio- anu nanospheies, as shown in Figuie S. 0wing to the laige suiface aiea,
Bacillus-BA uissolves fastei than tiauitional BA. This significantly ieuuces the time anu eneigy
neeueu foi batch piocesses anu foimulation manufactuiing.


-).'$/ =1 Scanning Election Nicioscope pictuie of Bacillus-ueiiveu hyaluionic aciu

The moleculai weight of spiay-uiieu Bacillus-ueiiveu BA is ca. 1 NBa, with a veiy low
polyuispeisity of 1.S-1.4 accoiuing to SEC-NALLS-RI analysis. Noieovei, the stiuctuie of
Bacillus-BA is iuentical to that of natuial BA anu Stieptococcus BA. This was confiimeu by
enzymatic hyuiolysis followeu by NALBI-T0F analysis, FT-IR anu BPLC foi monomei
composition. It has been shown that this ultia-puie souium hyaluionate fiom is biocompatible,
non-cytotoxic, non-alleigenic anu non-mutagenic.

Enhancement Neuical uses the iaw BA piouuct fiom Novozymes as a non-toxic anu non-
pathogen ueliveiy meuium foi its Expiession" injectable fillei. In foimulating Expiession",
the BA molecules aie cioss-linkeu with uivinyl sulfone (BvS) anu the gel is swelleu to
equilibiium. This pioviues the Expiession" injectable fillei piouuct with an 8u2u gel to fluiu
iatio with up to 26 mgmL of BA. Thus the foimulation combineu with the highly iobust BA
molecules iesult in a veiy iich anu potent fillei compaieu to otheis. Clinicians finu

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Expiession" uoes not have the hyuiophilic effect when placeu into tissue, eliminating
guesswoik fiom coiiections anu pioviuing gieatei utility foi multi-puipose use.

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This new hyaluionic aciu piouuceu by the feimentation of the safe bacteiial stiain Bacillus
subtilis is chaiacteiizeu by a well-contiolleu anu iepiouucible moleculai weight anu exhibits
auvantageous foimulation piopeities. This innovative Bacillus technology not only offeis gieat
piomise in the phaimaceutical aiena with uemanuing quality anu safety iequiiements but also
has the potential to leau to custom-tailoieu piouucts with taigeteu moleculai weights. The
feimentation piocess is both safe anu enviionmentally fiienuly. It is 1uu% fiee of animal-
ueiiveu iaw mateiials anu of oiganic solvents. As a consequence, Expiession" by
Enhancement Neuical contains a piemium hyaluionic aciu with unsuipasseu safety anu puiity.

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Auuitional infoimation iegaiuing this new BA piouuction methou is available fiom
Enhancement Neuical anu fiom Novozymes. Auuitionally, the following aiticle anu the
iefeiences containeu theiein:

"Byaluionic Aciu - The Biotech Way", Nanufactuiing Chemist, 22 Bec 2uu8,
www.manufactuiingchemist.comtechnicalaiticle_pageByaluionic aciu the biotech
way416S1

-).'$/ B1 Novozymes Biopolymei AS was one fo the winning entiies in the 2uu8 Innovatin
Awaius at CPhI in Fiankfuit. The company won uolu foi the innovative piocess of piouucing
hyaluionic aciu via biotechnology

1u2u1 Innovation Biive, Ste 4Su
Wauwatosa, WI SS226
(414) 918 - 428u
infoenhancementmeuical.com
www.enhancementmeuical.com