Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: Method of controlling the content of selected component(s) from
polymer(s)
using molecular sieve(s).
FIELD OF THE INVENTION
The present invention relates to a method for controlling the content of
selected
component(s) in one or more polymer(s).
BACKGROUND OF THE INVENTION
Polymers and their derivatives are used in a wide range of applications. Of
particular
interest are biopolymers which can be applied in the food, cosmetic, medical
and
pharmaceutical industries. In order to meet these diverse applications it is
often necessary to
purify the polymer to remove unwanted components and, further, to control the
balance of
components in the final product.
The most abundant heteropolysaccharides of the body are the
glycosaminoglycans.
Glycosaminoglycans are unbranched carbohydrate polymers, consisting of
repeating
disaccharide units (only keratan sulphate is branched in the core region of
the carbohydrate).
The disaccharide units generally comprise, as a first saccharide unit, one of
two modified
sugars - N-acetylgalactosamine (GaINAc) or N-acetylglucosamine (GIcNAc). The
second
unit is usually an uronic acid, such as glucuronic acid (GIcUA) or iduronate.
Glycosaminoglycans are negatively charged molecules, and have an extended
conformation that imparts high viscosity when in solution. Glycosaminoglycans
are located
primarily on the surface of cells or in the extracellular matrix.
Glycosaminoglycans also have
low compressibility in solution and, as a result, are ideal as a physiological
lubricating fluid,
e.g., joints. The rigidity of glycosaminoglycans provides structural integrity
to cells and
provides passageways between cells, allowing for cell migration. The
glycosaminoglycans of
highest physiological importance are hyaluronan, chondroitin sulfate, heparin,
heparan
sulfate, dermatan sulfate, and keratan sulfate. Most glycosaminoglycans bind
covalently to a
proteoglycan core protein through specific oligosaccharide structures.
Hyaluronan forms
large aggregates with certain proteoglycans, but is exceptional as free
carbohydrate chains
form non-covalent complexes with proteoglycans.
Numerous roles of hyaluronan in the body have been identified (see, Laurent T.
C.
and Fraser J. R. E., 1992, FASEB J. 6: 2397-2404; and Toole B.P., 1991,
"Proteoglycans
and hyaluronan in morphogenesis and differentiation." In: Cell Biology of the
Extracellular
Matrix, pp. 305-341, Hay E. D., ed., Plenum, New York). Hyaluronan is present
in hyaline
cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis.
Hyaluronan is also
suspected of having a role in numerous physiological functions, such as
adhesion,
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development, cell motility, cancer, angiogenesis, and wound healing. Due to
the unique
physical and biological properties of hyaluronan, it is employed in eye and
joint surgery and
is being evaluated in other medical procedures.
HA plays an important role in the biological organism, as a mechanical support
for
the cells of many tissues, such as the skin, tendons, muscles and cartilage,
it is a main
component of the intercellular matrix. HA also plays other important parts in
the biological
processes, such as the moistening of tissues, and lubrication.
HA may be extracted from the above mentioned natural tissues, although today
it is
preferred to prepare it by microbiological methods to minimize the potential
risk of
transferring infectious agents, and to increase product uniformity, quality
and availability.
HA and its various molecular size fractions and the respective salts thereof
have
been used as medicaments, especially in treatment of arthropathies, as an
auxiliary and/or
substitute agent for natural organs and tissues, especially in ophtalmology
and cosmetic
surgery, and as agents in cosmetic preparations. Products of hyaluronan have
also been
developed for use in orthopaedics, rheumatology, and dermatology.
HA may also be used as an additive for various polymeric materials used for
sanitary and surgical articles, such as polyurethanes, polyesters etc. with
the effect of
rendering these materials biocompatible.
Due to the wide diversity of biopolymer usage particularly HA usage and
derivatives
thereof, some of which are mentioned above, and the frequent use of HA in
pharmaceutical
compositions or surgical articles as well as tailored for specific
applications, it is often
necessary to provide HA products of high purity which should be substantially
absent of other
contaminating components in the end product. The non-polymer content and ionic
composition of the polymer, particularly of HA, is also important. Often, the
sodium salt of
the HA is preferred as the most biocompatible form and other HA salts (Fe, Ca,
Cu, Zn, Al,
Mg, Mn) are avoided, especially Ca++ salts of HA. Although, in certain
applications, it can be
desirable to favour another salt of HA or create a controlled balance of
counter ions. For
example, controlled levels of calcium are actually desirable in some wound
care applications,
controlled zinc levels have been proposed for combating foot ulcers (ref
diabetologia
Croatica 30-3, 2001) and for antibacterial properties (Acta Pharm Hung.
2002;72(1):15-24);
and controlled iron levels are sometimes used to control the rheological
properties in some
types of hyaluronic acid gel (CN 1473572A; WO 95/04132), whereas, low iron
levels are
often desired to reduce HA polymer susceptibility to degradation.
Conventionally polymers, including HA, having predominantly the sodium salt
form
and low or no Ca++ and low or no other metal ion content have been provided by
carefully
avoiding the presence of calcium and other unwanted ions during the
fermentation process
step and during subsequent purification steps. The desired ion balance is
conventionally
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achieved by first creating an environment of high sodium ion concentration
from, for
example, addition of sodium salt(s) such as sodium acetate, sodium chloride,
sodium
sulphate, etc. The high sodium ion concentration is used to competitively
displace calcium
from the HA molecule. The calcium ions liberated from the HA molecule can then
be
removed from the HA molecule, or vice versa, by any of a wide range of
conventional
polymer isolation processes. Most commonly, the polymer, or particularly HA,
is precipitated
or crystallised chemically and/or by organic solvent addition, such as
ethanol, iso-
propylalcohol, acetone, chloroform, CETAB etc, thereby leaving the liberated
and unwanted
ions in the supernatant (CZ 9700350; WO 84/ 03302; EP 0694616A2 and EP 0144
019
being just some examples of this process). The liberated ions can also be
separated from the
polymer by ultra-filtration of dia-filtration techniques (WO 95/04132;
GB2249315A). It is also
possible to use aqueous extraction and other common polymer separation
techniques.
Other methods to displace undesirable components, often Ca++, from a polymer
include sequestration with sequestration agents, such as, EDTA, phosphates (CN
85103674A), etc or application ion exchange adsorbent resins (EP 0694616A2).
The disadvantages inherent in the techniques described above, depending on the
chosen method and the active agent(s) used, are that: repeated application is
often required
to achieve the desired component balance (e.g. repeated precipitation and re-
suspension or
extensive dia-filtration); and or the component selectivity is poor; and or
the capacity for the
component is poor; and or the displacement or sequestration efficiency is low;
and or
process introduces another component which is subsequently difficult to remove
and / or is
toxic; and or process stream conditions need to be manipulated to achieve the
desired
component balance.
Accordingly alternative processes for manipulating the balance of components
associated with a polymer is desirable, due to the difficulties described
above for the
conventional means of controlling the component balance in the final polymer
product. The
present invention provides such a process, which furthermore, provides several
advantages
as will be described.
SUMMARY OF THE INVENTION
The invention provides in a first aspect a method for controlling the content
of
selected component(s) in one or more polymer(s) by:
(a) contacting the polymer(s) with at least one molecular sieve; and
optionally
(b) isolating the polymer from the molecular sieve(s).
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DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for controlling the content of
undesirable or
selected components from a polymer by removing or replacing the component(s)
with a more
desirable component and or manipulating the balance of components associated
with the
polymer. The removal or manipulation of the component(s) is performed by
contacting the
polymer product with an appropriate molecular sieve(s) for a time period
sufficient to remove
or exchange or manipulate the component balance. In some applications it can
be
convenient to leave the molecular sieve, in with the polymer product; however,
if desirable,
the molecular sieve(s) may be separated from the polymer.
The term "polymer" in the present context is a substance which is made up of
many
repeating smaller chemical units or molecules. Polymers can be natural or
synthetic.
The term "polymer" also comprises liquid polymers or suspensions of polymers,
in
this context.
The "component" to be removed or manipulated comprises atoms, molecules, ions,
or
compounds.
In the present context the expression "control" means remove (completely or
partially), exchange and / or balance the content of component(s) from the
polymer or
polymer bearing liquid.
There are numerous applications where it is desirable to control, reduce or
select the
components associated with the polymer and / or polymer bearing liquid:
i) For example, for biocompatibility, hyaluronic acid is preferred in the
sodium form.
Furthermore, limitations for specific applications are often desired, for
example, calcium
containing polymers may be insoluble themselves, (e.g. calcium alginate) or
may create
problematic precipitation in common phosphate buffers, or create adverse
reactions with
active ingredients or formulation chemicals. Conversely, a controlled level of
calcium in some
applications has been specified in, for example, alginate for wound care
products. Similarly,
Fe, Ca, Cu, Zn, Al, Mg, other ions and other components
ii) Ionic content and ion type can affect the viscosity and other properties
of the
polymer; similarly for other components. Ion content has to be carefully and
accurately
controlled, for example, for the creation of hydrogels containing zinc.
iii) Polymer destabilising molecules, such as Fe, and Cu can be removed,
controlled
or replaced by another less harmful ion.
iv) Polymer products often require removal of odour or colour molecules.
The present invention provides a number of advantages over conventionally
applied
methods for manipulation of the component balance. The MOLECULAR SIEVE(S)
according
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to the invention can be applied at any point in the manufacturing process,
including to raw
materials; during manufacture or as a post-treatment of the polymer product.
Furthermore
the process conditions such as pH, temperature, polymer concentration etc. are
not as
critical as in other conventionally applied processes.
Molecular sieves can be highly selective for particular components or groups
of
components. Reaction equilibrium is normally achieved rapidly meaning faster
processing
and closer control of the product characteristics in terms of the component
balance
associated with the polymer. The molecular sieves demonstrate a high capacity
for the
component(s) to be manipulated and therefore there is generally only the need
for a single
MOLECULAR SIEVE(S) treatment. The simple addition, contact and subsequent
removal of
the MOLECULAR SIEVE(S) by conventional solid-liquid separation methods, means
there is
no need for dedicated equipment and no need to subsequently remove soluble
reactants or
additions. It is possible to leave the MOLECULAR SIEVE(S) in the polymer
solution which
further simplifies the process. Process costs can also be reduced since the
present method
allows raw materials containing components which are undesirable in the end
product, to be
used in upstream steps, e.g. tap water (Ca++ containing) for fermentation and
dilution instead
of de-ionized water. Furthermore the MOLECULAR SIEVE(S) can often be added
directly
and without the requirement for pre-equilibrium or pre-treatment. Molecular
sieves are
generally low in toxicity.
High molecular weight contaminants / impurities
Since the method according to the invention is applied for the removal of
component(s) from a polymer product, component(s) may even be added in an
upstream
process step without causing any problems for the downstream purification
steps.
Since the method according to the invention is conveniently applied for the
removal
of calcium from a polymer product, Ca++ may even be added in an upstream
process step
without causing any problems for the downstream purification steps.
The addition of calcium, or other divalent salts, makes it possible to remove
high
molecular weight contaminants/impurities by flocculation in an early
purification step; it is
also possible to remove these impurities from cell free preparations of the
glycosaminoglycan
of interest. This is further described in WO2004/001054. The possibility of
adding
component(s), particularly calcium during upstream process steps constitutes a
further
advantage of the present invention compared to traditional methods.
The above advantages are provided by the method according to the invention for
controlling the content of selected (often undesirable) components from one or
more
polymers comprising the steps of:
(a) contacting the polymer(s) with a molecular sieve; and optionally
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(b) isolating the polymer product from the molecular sieve(s).
In the present context "controlling the content of selected components" means
removing (completely or partially) the component(s) and or replacing
(exchanging) the
component(s) with a more desirable component and or manipulating the balance
of
components associated with the polymer or polymer bearing liquid.
A"molecular sieve" in the present context means materials having molecule-
sized
pores that can be used in separating larger molecules from smaller ones. They
include, but
are not limited to, zeolites, carbon molecular sieves, silica gels, activated
alumina. Typically,
molecular sieves have a lattice structure creating a cage like structure with
windows which
admit only molecules of less than a certain size. By using different source
materials and
different conditions of manufacture, it is possible to produce a range of
molecular sieves of
differing access dimensions. The dimensions can often be precise for a
particular molecular
sieve(s) because they derive from the crystal structure of that sieve.
Examples of some of the molecules admitted by different molecular sieves are
given
in Table 17.3 "Classification of Some Molecular Sieves" from Chemical
Engineering, Volume
2, 4th Edition ; JM Coulson and JF Richardson; Pergamon Press, 1991 together
with further
details on molecular sieves. A more comprehensive database of relevant
structures and the
properties of molecular sieves is hosted by the International Zeolite
Association
(http://www.iza-online.org/) at (http://www.iza-structure.org/databases/).
Relevant basic texts
for molecular sieves include: DW Breck: Zeolite Molecular Sieves, Wiley, New
York, 1974;
RM Barrer: Hydrothermal Chemistry of Zeolites, Academic Press, 1982; Intro to
Zeolite
Science and Practice, H van Bekkum, EM Flannigen, PA Jacobs and JC Jansen,
Studies in
Surface Science, Vol 137, 1-1060, (2001) Elsevier, Amsterdam.
In a particular embodiment the molecular sieve(s) is a zeolite. The classical
definition
of a zeolite is a crystalline, porous aluminosilicate. However, some
relatively recent
discoveries of materials virtually identical to the classical zeolite, but
consisting of oxide
structures with elements other than silicon and aluminium have stretched the
definition. Most
researchers now include virtually all types of porous oxide structures that
have well-defined
pore structures due to a high degree of crystallinity in their definition of a
zeolite. In the
present context all of the above are comprised in the term "zeolite". In these
crystalline
materials we call zeolites, the metal atoms (classically, silicon or aluminum)
are surrounded
by four oxygen anions to form an approximate tetrahedron consisting of a metal
cation at the
centre and oxygen anions at the four apexes. The tetrahedral metals are called
T-atoms for
short, and these tetrahedra then stack in, regular arrays such that channels
form. The
possible ways for the stacking to occur is virtually limitless, and hundreds
of unique
structures are known.
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The zeolitic channels (or pores) are microscopically small, and in fact, have
molecular
size dimensions such that they are often termed "molecular sieves". The size
and shape of
the channels have extraordinary effects on the properties of these materials
for adsorption
processes, and this property leads to their use in separation processes.
Components can be
separated via shape and size effects related to their possible orientation in
the pore, and or
by differences in strength of adsorption. Therefore, components can be
selectively removed.
Since silicon typically exits in a 4+ oxidation state, the silicon-oxygen
tetrahedra are
electrically neutral. However, in zeolites, aluminium typically exists in the
3+ oxidation state;
so that aluminium-oxygen tetrahedra form centres that are electrically
deficient one electron.
Thus, zeolite frameworks are typically anionic, and charge compensating
cations populate
the pores to maintain electrical neutrality.
In a particular embodiment of the invention the molecular sieve is chosen from
the
group of zeolites where the porosity of the material is compatible with the
component to be
removed, in a further embodiment the porosity of the material is compatible
with Ca++; in a
further embodiment ions populating the zeolite pores to maintain electrical
neutrality are
those desired in the polymer product; in a further embodiment sodium ions are
the ions
populating the zeolite pores to maintain electrical neutrality.
Those molecular sieves classically grouped as "Type 4" have molecular sieve(s)
dimensions appropriate for the sequestration of calcium ions having Linde
sieve 4A
dimensions of approximately 0.4 nm. A number of these molecular sieves have
sodium ions
populating the zeolite pores to maintain electrical neutrality.
In a particular embodiment of the invention the zeolite is therefore a Type 4
zeolite. In
a further embodiment the pores of the molecular sieve contains sodium ions.
In one embodiment of the invention the polymer is a biopolymer. A "biopolymer"
is
any polymeric substance (examples being, but not limited to, polysaccharides,
proteins
nucleic acids, etc,) formed in a biological system. Many examples of common
biopolymers
exist, including, but not limited to: Chitosan, glucan, keratin, cellulose,
gelatine,
glycosaminoglycans and derivatives of all these polymers.
In a particular embodiment the biopolymer is a polysaccharide, and in a
further
particular embodiment the polysaccharide is a glycosaminoglycan.
Glycosaminoal c~
According to the invention a glycosaminoglycan may be any carbohydrate polymer
having a molecular weight of at least 700 Daltons; preferably a molecular
weight of at least
10,000 Daltons; more preferably a molecular weight of at least 20,000 Daltons,
even more
preferably a molecular weight of at least 30,000 Daltons.
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Preferred glycosaminoglycans are hyaluronic acid, chondroitin sulphate,
chondroitin
(non-sulphated), heparin, heparin sulphate, dermatan sulphate, and keratin
sulphate.
Hyaluronic acid is constituted by alternating and repeating units of D-
glucoronic acid and N-
acetyl-D-glucosamine, to form a linear chain having a molecular weight of up
to 15,000,000
Daltons.
Preferred Glycosaminoglycans according to the invention are Glycosaminoglycans
having a molecular weight of from 700 Daltons to 15,000,000 Daltons.
It is to be noted that the term "hyaluronic acid" in the present application
and claims
may mean indifferently hyaluronic acid in its acidic form or in its salt form
such as for
example sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate,
calcium
hyaluronate, or others.
The terms "hyaluronan" or "hyaluronic acid" are used in literature to mean
acidic
polysaccharides with different molecular weights constituted by residues of D-
glucuronic and
N-acetyl-D-glucosamine acids, which occur naturally in cell surfaces, in the
basic
extracellular substances of the connective tissue of vertebrates, in the
synovial fluid of the
joints, in the endobulbar fluid of the eye, in human umbilical cord tissue and
in cocks' combs.
The term "hyaluronic acid" is in fact usually used as meaning a whole series
of
polysaccharides with alternating residues of D-glucuronic and N-acetyl-D-
glucosamine acids
with varying molecular weights or even the degraded fractions of the same, and
it would
therefore seem more correct to use the plural term of "hyaluronic acids". The
singular term
will, however, be used all the same in this description; in addition, the
abbreviation "HA" will
frequently be used in place of this collective term.
The biopolymer, e.g. a glucosaminoglucan, can be provided from animal tissues
or
more preferably by culturing a host cell expressing the biopolymer.
Fermentation broth
The glycosaminoglycan may be obtained from any fermentation broth. The
glycosaminoglycan may furthermore be one which is producible by a method
comprising
cultivating a host cell.
The host cell may preferably be a micro-organism. The micro-organism may
be a unicellular micro-organism, e.g., a prokaryote, or a non-unicellular
micro-organism, e.g.,
a eukaryote. Useful unicellular cells are bacterial cells such as gram
positive bacteria
including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus,
Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii,
Bacillus coagulans,
Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,
Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus
thuringiensis; or
a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or
gram negative
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bacteria such as E. coli and Pseudomonas sp. In a particular embodiment, the
bacterial host
cell is a Bacillus lentus cell, a Bacillus licheniformis cell, a Bacillus
stearothermophilus cell or
a Bacillus subtilis cell. Mutant Bacillus subtilis cells particularly adapted
for recombinant
expression are described in WO 98/22598.
The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a
plant
cell or a fungal cell. Useful mammalian cells include Chinese hamster ovary
(CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other
immortalized cell lines available, e.g., from the American Type Culture
Collection. The
transformation method, selectable marker gene and any other parts of the
expression
construct may be chosen from those well known and available to one skilled in
the art.
The host cell may be a fungal cell. "Fungi" as used herein includes the phyla
Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the
Oomycota
and all mitosporic fungi. Representative groups of Ascomycota include, e.g.,
Neurospora,
Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium
(=Aspergillus), and the true
yeasts listed below. Examples of Basidiomycota include mushrooms, rusts, and
smuts.
Representative groups of Chytridiomycota include, e.g., Allomyces,
Blastocladiella,
Coelomomyces, and aquatic fungi. Representative groups of Oomycota include,
e.g.,
Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of
mitosporic
fungi include Aspergillus, Penicillium, Candida, and Alternaria.
Representative groups of
Zygomycota include, e.g., Rhizopus and Mucor.
The fungal host cell may be a yeast cell. "Yeast" as used herein includes
ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast
belonging to
the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided
into the
families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of
four
subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),
Nadsonioideae,
Lipomycoideae, and Saccharomycoideae (e.g., genera Kluyveromyces, Pichia, and
Saccharomyces). The basidiosporogenous yeasts include the genera
Leucosporidim,
Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeast
belonging to the
Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g.,
genera
Sporobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida).
In another embodiment, the fungal host cell is a filamentous fungal cell.
"Filamentous fungi" include all filamentous forms of the subdivision Eumycota
and
Oomycota. The filamentous fungi are characterized by a mycelial wall composed
of chitin,
cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
Vegetative growth
is by hyphal elongation and carbon catabolism is obligately aerobic. In
contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of a
unicellular thallus
and carbon catabolism may be fermentative. In a more particular embodiment,
the
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filamentous fungal host cell is a cell of a species of, but not limited to,
Acremonium,
Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,
Penicillium, Thielavia,
Tolypocladium, and Trichoderma.
Fungal cells may be transformed by a process involving protoplast formation,
transformation of the protoplasts, and regeneration of the cell wall in a
manner known per se.
The micro-organism producing the glycosaminoglycan of interest is cultivated
in a
nutrient medium suitable for production of the glycosaminoglycan using methods
known in
the art. For example, the micro-organism may be cultivated by shake flask
cultivation, small-
scale or large-scale fermentation (including but not limited to continuous,
batch, fed-batch, or
solid state fermentations) in laboratory or industrial fermentors. The
cultivation takes place in
a suitable nutrient medium comprising carbon and nitrogen sources and
inorganic salts,
using procedures known in the art. Suitable media are available from
commercial suppliers
or may be prepared according to published compositions (e.g., in catalogues of
the American
Type Culture Collection). As an example the production of a glycosaminoglycan
produced in
a micro-organism W02003/054163 describes the production of hyaluronic acid in
a Bacillus
host cell.
As mentioned above, traditional methods or processes for the production of
purified
glycosaminoglycans with a desired component balance, such as e.g. hyaluronic
acid in the
sodium salt form, include:
-Precipitation of the polymer in a sodium ion rich environment;
-Crystallisation in a sodium ion rich environment;
-Precipitation of the unwanted component, for example, with phosphate and
application of a
sodium ion rich environment;
-Dialysis or Dia-filtration in a sodium ion rich environment;
-Sequestration and application of a sodium rich environment; and
-Other conventional means for polymer purification.
One problem associated with the known methods for removing calcium is that
they
almost always introduce a soluble contaminant that needs to be removed by
further
processing steps. Furthermore these methods often utilises chemical
precipitants or other
aids which are then subsequently difficult to remove from the product and may
be toxic or
detrimental to the final application. This is true of e.g. competitive
substitution of the calcium
in the presence of an excess of sodium ions. The displaced calcium and excess
of sodium
counter ions need to be removed. It is also the case when using sequestration
agents such
as EDTA. The EDTA must be subsequently removed. Also precipitation of the
calcium ion,
with say phosphates requires the excess of precipitant to be removed as well
as the
precipitate formed.
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Such processing steps are less easy to control because the reaction
equilibrium
changes as the concentration of the soluble(s) components change, achievement
of reaction
equilibrium is often relatively slow, capacity for the component to be
manipulated is often
relatively low, the techniques only work for a narrow range of process
conditions (e.g. pH,
ionic strength, polymer concentration, etc). Furthermore they are often time
consuming and
require more than one application, which leaves the product susceptible to
degradation and
are not selective or efficient means to control the component balance in the
product.
According to the present invention a method/process is provided comprising the
steps
of contacting the polymer or polymer bearing liquid to be modified with the
appropriate
type(s) of molecular sieve(s) under appropriate conditions; and separating the
MOLECULAR
SIEVE(s) from the polymer, if necessary.
Contact, between the polymer bearing liquid to be modified with the MOLECULAR
SIEVE(S),
can be achieved by any of a number of means including, but not limited to:
= Simple suspension or mixing together
= Passage of the polymer bearing liquid through a: packed, settled, expanded,
fluidised
bed of molecular sieves
= Contact with molecular sieves used as a body feed, pre-coat or aid to
filtration.
The type, amount and contacting conditions of the Molecular sieve(s) required
to
achieve the desired component balance in the polymer product can be simply
determined by
someone skilled in the art.
The component to be removed or manipulated according to the invention
comprises
atoms, molecules, ions, or compounds. Examples of components which can be
removed by
particular molecular sieve types include, but are not limited to, those given
in Table 17.3
"Classification of Some Molecular Sieves" from Chemical Engineering, Volume 2,
4 th Edition ;
JM Coulson and JF Richardson; Pergamon Press, 1991. A more comprehensive
database
of relevant structures and the properties of molecular sieves is hosted by the
International
Zeolite Association (http://www.iza-online.org/) at (http://www.iza-
sfiructure.org/databases/).
Relevant basic texts for molecular sieves include:
= DW Breck: Zeolite Molecular Sieves, Wiley, New York, 1974
= RM Barrer: Hydrothermal Chemistry of Zeolites, Academic Press, 1982
= Intro to Zeolite Science and Practice, H van Bekkum, EM Flannigen, PA Jacobs
and
JC Jansen, Studies in Surface Science, Vol 137, 1-1060, (2001) Elsevier,
Amsterdam
Particularly, zeolites can be used to control the content of (remove or
manipulate)
organic solvents (including but not limited to: alcohols, aldehydes, ketones,
etc.), metal ions,
anions, quaternary ammonium compounds, SDS, EDTA, CETAB, TCA, cetyl pyrimidine
chloride, etc.
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Particularly the MOLECULAR SIEVE(S) can be used to remove or manipulate the
balance of ion, more particularly cations.
In one embodiment the ion is a divalent ion, more particularly Ca++
In a particular embodiment the invention relates to a method for controlling
the
content of calcium ions from a polymer using a MOLECULAR SIEVE(s).
In a particular embodiment the invention relates to a method for controlling
the
content of calcium and sodium ions from a polymer using a Molecular sieve(s).
The component(s) to be removed or partly removed can be suspended in the
liquid
comprising the polymer or the component(s) can be associated or be present on
the
polymer.
In a further embodiment the component(s) is exchanged for another component.
Ca-
ions can e.g. be exchanged for Na-ions.
In a still further embodiment the component(s) balance in the product is
controlled.
In a particular embodiment the molecular sieve(s) is a zeolite.
"Appropriate conditions" in this context mean those of the process stream and
those
conditions which can be easily determined by one skilled in the art for
effecting the
component removal or manipulation. The appropriate conditions can include but
are not
limited to; molecular sieve type, molecular sieve dosing, temperature, pH,
polymer
concentration, ionic strength, solvent concentration, mixing, incubation time,
etc.
In a more particular embodiment the polymer is a glycosaminoglycan.
The Molecular sieve(s) can be removed from the polymer by any of a number of
means, for example, but not limited to: filtration, centrifugation,
floatation, sedimentation,
phase exclusion, etc. In some cases, it may not be necessary to remove, or may
be
desirable to leave, the Molecular sieve(s) in the polymer bearing liquid.
The particular component removal or manipulation process according to the
invention
can be optimised or improved through, for example, but not limited to,
manipulation of: pH,
temperature, viscosity, concentration, mixing, ionic strength, additives,
ingredients, etc. More
than one polymer may be treated in the same polymer bearing liquid. More than
one type of
Molecular sieve(s) may be contacted with the polymer bearing liquid. More than
one
treatment with Molecular sieve(s) may be used to manipulate the polymer(s)
bearing liquid.
In a particular embodiment the method of the invention provides control
(removal,
exchange and/ or balancing) of ions in the polymer bearing liquid using an
appropriate
zeolite. Particularly the polymer is a glycosaminoglycan.
In a more particular embodiment the polymer is hyaluronic acid.
In another particular embodiment the process of the invention provides control
of the content
of ions on the polymer itself using an appropriate zeolite. Particularly the
polymer is
hyaluronic acid.
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In still another embodiment the process of the invention provides control of
the
content of calcium and sodium ions on the polymer itself using an appropriate
molecular
sieve. Particularly the polymer is hyaluronic acid.
EXAMPLES
Example 1. Hyaluronic acid purification without a calcium removal stage.
In this experiment a 5 g/I solution of hyalyronic acid, obtained from
fermentation of a
recombinant Bacillus subtilis, was diluted with ordinary tap water and
filtered to remove host
cells. The filtrate was then extensively dialysed against deionised water to
remove the bulk
of free calcium not on the HA molecule itself. The resulting dialysed product
contained 4.4
wt% calcium relative to the mass of hyaluronic acid.
Example 2. Hyaluronic acid purification with a conventional calcium removal
stage by dia-
filtration against a sodium salt.
In this experiment hyaluronic acid was obtained as described in Experiment 1.
A
calcium controlling step (not involving MOLECULAR SIEVE(S) according to the
invention)
was introduced involving competitive substitution of the calcium in the
presence of an excess
of sodium ions as follows:
The hyaluronic acid solution was dia-filtered against an excess of sodium
ions. The
liberated calcium ions were thereby removed from the solution by passage
through the dia-
filtration membrane. The liberated calcium ions from the hyaluronic acid could
equally have
been removed by polymer precipitation. In the present case the hyaluronic acid
solution was
dia-filtered against 3 x volumes of a 10 wt% sodium sulphate solution at
constant volume
followed by extensive dialysis against deionised water to remove excess
sulphate and
sodium ions. The resulting product contained 1.2 wt% calcium relative to the
hyaluronic acid.
Example 3. Control of calcium content of hyaluronic acid using a cation
exchange resin.
Hyaluronic acid was produced as described in Example 1 above. A strong cation
exchange resin with SO3 (> 2 eq/I) in the sodium ion form was used to
manipulate the
calcium ion content of the polymer.
The performance of the exchange resin was characterised for the range of
conditions likely to be met throughout hyaluronic acid manufacturing and
purification
processes described earlier.
In one example, 1.0 wt% exchange resin was added to a 0.5 wt% solution of
hyaluronic acid containing 2wt% calcium relative to the hyaluronic acid. After
120 minutes of
incubation with stirring the resin was filtered from the solution. The
resulting filtrate
contained hyaluronic acid with a calcium content of 0.5 wt% calcium relative
to the hyaluronic
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acid. After an incubation of 240 minutes under the same conditions the calcium
content
relative to the hyaluronic acid was below detection.
Example 4. Control of calcium content of Hyaluronic acid using a cation
exchange gel.
Hyaluronic acid was produced as described in Example 1 above. A strong cation
exchange gel having a sulphonated functional group (2.05 eq/I) was used to
manipulate the
calcium ion content of the polymer.
The performance of the exchange resin was characterised for the range of
conditions likely to be met throughout hyaluronic acid manufacturing and
purification
processes described earlier.
In one example, 0.25 wt% exchange gel was added to a 0.5 wt% solution of
hyaluronic acid containing 2 wt% calcium relative to the hyaluronic acid. The
mixture was
incubated with stirring and allowed to come to equilibrium which was achieved
in 1 hour. The
gel was filtered from the solution. The resulting filtrate contained
hyaluronic acid with a
calcium content of 0.3 wt% calcium relative to the hyaluronic acid. Under the
same
conditions, 1wt% of the exchange gel in the sodium form reduced the calcium
content
relative to the hyaluronic acid to below detection.
In another pilot scale example, 0.5 wt% exchange gel in the sodium form was
added, without pre-treatment, to a 0.7 wt% solution of hyaluronic acid
containing 211 ppm
calcium. The mixture was incubated with stirring for 2 hours before the gel
was filtered from
the solution. The resulting filtrate contained 14ppm calcium relative to the
hyaluronic acid
corresponding closely to bench scale experiments under the same conditions.
Under the
same conditions, 1wt% of the exchange gel reduced the calcium content relative
to the
hyaluronic acid to below detection.
The starting material and the filtrate treated with 1wt% of the exchange gel
in the
sodium form were dialysed against deionised water to remove free ions. From
analysis of
the resulting hyaluronic acid, it was found that the ions on the hyaluronic
acid, following
treatment with 1wt% of the exchange gel in the sodium form, had been replaced
by sodium
ions.
Example 5. Reduction of iron in fermentation broth using a cation exchange
gel.
Gel type was a strong cation exchanger having functional groups: sulphonates
(2.05
eq/I).
An excess of the ion exchange gel was used to reduce the level of iron in the
fermentation broth. Ions such as iron, copper and others have been
demonstrated to reduce
the stability of hyaluronic acid towards degradation.
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Raw fermentation broth containing hyaluronic acid was clarified by dilution
with
ordinary tap water and filtration to remove the microorganisms. This clarified
broth was then
incubated and stirred with an excess (10 wt%) of exchange gel for 1 hour. The
exchange gel
was then filtered from the solution. The iron content of the filtrate was
found to be below
detection (< 1 ppm relative to the hyaluronic acid) compared to 0.4 wt% in the
clarified broth
(relative to the hyaluronic acid)
The original clarified broth and that treated to remove iron were subsequently
heat
treated. The exchange gel treated material was found to be substantially more
heat stable
with regard to molecular weight than the untreated clarified broth material
under the same
conditions.
Treatment of the clarified broth did not change the molecular weight profile
or
concentration of the hyaluronic acid contained.
It was found that other process fluids and components could be controlled with
similar application and procedures to those demonstrated in experiments 3 and
4. Removal
of components from the clarified fermentation broth stabilised the hyaluronic
acid towards
thermal degradation.
Example 6. Control of calcium content of Hyaluronic acid and replacement of
ions on the
hyaluronic acid with sodium ions using the sodium form of powdered aluminium
silicate Type
4A Zeolite
Hyaluronic acid was produced as described in Example 1 above. A powdered Type
4A zeolite in the sodium form was used to manipulate the calcium ion content
of the polymer.
The performance of the exchange resin was characterised for the range of
conditions likely to be met throughout hyaluronic acid manufacturing and
purification
processes described earlier. The characteristics were reproduced at both bench
and pilot
scale and diverse hyaluronic acid batches, produced as in Experiment 1, were
tested.
The zeolite was found to be able to remove around 0.06mg calcium from the
hyaluronic acid
bearing solutions per mg zeolite when the two were contacted. This ratio was
found to be
largely independent of the process conditions used (eg pH, temperature, HA
concentration,
etc). This made it very simple to control the calcium level in the hyaluronic
acid bearing
solution by simple dosed addition of zeolite. Equilibrium was achieved in less
than 15
minutes in all cases.
In a typical example only 0.2 wt% of powdered zeolite was contacted with a
hyaluronic acid solution containing 142 ppm calcium by direct addition without
pre-
equilibriation or pre-treatment. After 20 minutes incubation with stirring,
the zeolite was
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filtered from the solution. The resulting filtrate contained 14 ppm calcium.
Contact with 0.3
wt% zeolite under the same procedure reduced the calcium to below detection
limits.
Hyaluronic acid starting material and after treatment with 0.3wt% of the
powdered
zeolite were analysed. It was found that the ions on the hyaluronic acid,
following treatment
with 0.3wt% of the zeolite, had been replaced by sodium ions.
Treatment of the hyaluronic acid did not change the molecular weight profile
or
concentration and a detailed characterisation of the treated hyaluronic acid
revealed no
detrimental changes.
It was found that the degree of calcium removal from the hyaluronic acid
containing
solution could be reproducibly predicted and controlled. The ions on the
hyaluronic acid were
replaced by sodium ions. There were no adverse effects on the hyaluronic acid.
Example 7. Reduction of iron in fermentation broth using the sodium form of
powdered
aluminium silicate Type 4A Zeolite
Ions such as iron, copper and others have been demonstrated to reduce the
stability
of hyaluronic acid towards degradation. An excess of the sodium form of a
powdered
aluminium silicate Type 4A Zeolite was used to reduce the level of iron in
fermentation broth.
Raw fermentation broth obtained from fermentation of a recombinant Bacillus
subtilis, was diluted with ordinary tap water and filtered to remove host
cells to give a 3.5 g/I
hyaluronic acid solution containing 24 ppm iron ion. This clarified broth was
then incubated
and stirred with an excess (3 wt%) of powdered zeolite for 1 hour. The zeolite
was then
filtered from the solution. The iron content of the filtrate was found to be
below detection (<
1 ppm relative to the hyaluronic acid).
The original clarified broth and that treated to remove iron were subsequently
heat
treated. The exchange gel treated material was found to be substantially more
heat stable
with regard to molecular weight than the untreated clarified broth material
under the same
conditions.
Treatment of the clarified broth did not change the molecular weight profile
or
concentration of the hyaluronic acid contained.
Example 8. Control of calcium content of Hyaluronic acid and replacement of
ions on the
hyaluronic acid with sodium ions using a sodium form of a granular Type 4A
zeolite
The performance of the granular zeolite for calcium ion removal and
replacement
with sodium ion was characterised for the range of conditions likely to be met
throughout
hyaluronic acid manufacturing and purification processes described earlier.
The
characteristics were reproduced at both bench and pilot scale and diverse
hyaluronic acid
batches, produced as in Experiment 1, were tested.
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Typically, this granular Type 4A zeolite was able to remove around 0.1 mg
calcium
from the hyaluronic acid bearing solutions per mg zeolite when the two were
contacted. This
ratio was found to be largely independent of the process conditions used. This
made it very
simple to control the calcium level in the hyaluronic acid bearing solution.
Equilibrium was
achieved in less than 10 minutes in all cases.
In a typical example 0.2 wt% of the granular zeolite was contacted with a
hyaluronic
acid solution containing 220 ppm calcium. After 20 minutes incubation with
stirring, the
zeolite was filtered from the solution. The resulting filtrate contained 20
ppm calcium.
Contact with 0.25 wt% zeolite (II) under the same conditions reduced the
calcium to below
detection limits.
Hyaluronic acid from the starting material and from the filtrate treated with
0.25 wt%
of the granular zeolite was analysed. It was found that the ions on the
hyaluronic acid,
following treatment with 0.25 wt% of the zeolite had been replaced by sodium
ions.
Treatment of the hyaluronic acid did not change the molecular weight profile
or
concentration and a detailed characterisation of the treated hyaluronic acid
revealed no
detrimental changes.
It was found that the degree of calcium removal from the hyaluronic acid
containing
solution could be reproducibly predicted and controlled. The ions on the
hyaluronic acid were
replaced by sodium ions. There were no adverse effects on the hyaluronic acid.
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