|Title of Invention||
"FRACTIONATION OF CHARGED POLYSACCHARIDE."
|Abstract||Polydisperse and charged polysaccharides are fractionated into low polydispersity fractions [preferably having Pd<1.1], each containing species within a narrow range of molecular weights. An aqueous solution of the polydisperse polysaccharides is contacted with an ion exchange resin in a column and the polysaccharides are subjected to selective elution by aqueous elution buffer. The selective elution consists of at least 3 sequential elution buffers having different and constant ionic strength and/or pH and in which the subsequent buffers have ionic strength and/or pH than those of the preceding step. The new preparations are particularly suitable for the production of PSA- derivatised therapeutic agents intended for use in humans and animals.|
|Full Text||FRACTIONATION OF CHARGED POLYSACCHARIDE
The present invention relates to fractionation of polydisperse and
charged polysaccharides optionally having reactive groups, into low
polydispersity fractions (preferably pd polysaccharides are useful for conjugation to substrates such as peptides,
proteins, drugs, drug delivery systems (e.g. liposomes), viruses, cells, (e.g.
animal cells, microbes, synthetic polymers etc), or for use as excipients or
diluents in pharmaceutical compositions.
Polysialic acids (PSAs) are naturally occurring unbranched polymers
of sialic acid, produced by certain bacterial strains and mammals in certain
cells [Roth et. al., 1993]. PSAs can be produced in various degrees of
polymerisation: from n = about 80 or more sialic acid residues down to n =
2 by either limited acid hydrolysis, digestion with neuraminidases or by
fractionation of the natural, bacterially or cell derived forms of the polymer.
The composition of different PSAs also varies such that there are: I.
homopolymeric forms i.e. the alpha-2,8-linked PSA comprising the capsular
polysaccharide of E. co//strain K1 and of the group-B meningococci, which
is also found on the embryonic form of the neuronal cell adhesion molecule
(N-CAM). II. Heteropolymeric forms, such as the alternating alpha-2,8 alpha-
2,9 PSA of £. co// strain K92 and the group C polysaccharides of N.
meningitidis. In addition, III. alternating copolymers containing sialic acids
monomers other than sialic acid such as group W135 or group Y of N.
meningitidis. PSAs have important biological functions including the evasion
of the immune and complement systems by pathogenic bacteria and the
regulation of glial adhesiveness of immature neurons during foetal
development (wherein the polymer has an anti-adhesive function)
[Muhlenhoff et. al., 1998; Rutishauser, 1989; Troy, 1990,1992; Cho and
Troy, 1994].There are no known receptors for PSAs in mammals. The alpha-
2,8-linked PSA of E. co// strain K1 is also known as 'colominic acid' (CA) and
is used (in various lengths) to exemplify the present invention.
SUBSTITUTE SHEET (RULE 26)
The alpha-2,8 linked form of PSA, among bacterial polysaccharides,
is uniquely non-immunogenic (eliciting neither T-cell or antibody responses
in mammalian subjects) even when conjugated to immunogenic carrier
protein, which may reflect its existence as a mammalian (as well as a
bacterial) polymer. Shorter forms of the polymer (up to n=4) are found on
cell-surface gangliosides, which are widely distributed in the body, and are
believed to effectively impose and maintain immunological tolerance to
PSA. In recent years, the biological properties of PSAs, particularly those
of the alpha-2,8 linked homopolymeric PSA, have been exploited to modify
the pharmacokinetic properties of protein and low molecular weight drug
molecules [Gregoriadis, 2001; Jain et. al., 2003; US-A-5846,951;
WO-A-0187922]. PSA derivatisation of a number of therapeutic proteins
including catalase and asparaginase [Fernandes and Gregoriadis, 1996
and 1997] gives rise to dramatic improvements in circulating half-life, and
also allows such proteins to be used in the face of pre-existing antibodies
raised as an undesirable (and sometimes inevitable) consequence of prior
exposure to the therapeutic protein [Fernandes and Gregoriadis, 2001]. In
many respects, the modified properties of polysialylated proteins are
comparable to proteins derivatised with polyethylene glycol (PEG). For
example, in each case, half-lives are increased, and proteins and peptides
are more stable to proteolytic digestion, but retention of biological activity
appears to be greater with PSA than with PEG [Hreczuk-Hirst et. al., 2002].
Also, there are questions about the use of PEG with therapeutic agents that
have to be administered chronically, as PEG is only very slowly
biodegradable [Beranovaet.al., 2000] and high molecular weight forms tend
to accumulate in the tissues [Bendele, et. al., 1998; Convers, et. al., 1997].
PEGylated proteins have been found to generate anti PEG antibodies that
could also influence the residence time of the conjugate in the blood
circulation [Cheng et. al., 1990]. Despite the established history of PEG as
a parenterally administered polymer conjugated to therapeutics, a better
understanding of its immunotoxicology, pharmacology and metabolism will
be required [Hunter and Moghimi, 2002; Brocchini, 2003]. Likewise there
are concerns about the utility of PEG in therapeutic agents that require high
dosages, (and hence ultimately high dosages of PEG), since accumulation
of PEG may lead to toxicity. The alpha 2,8 linked PSA therefore offers an
attractive alternative to PEG, being an immunologically 'invisible'
biodegradable polymer which is naturally part of the human body, and that
can degrade, via tissue neuraminidases, to the non-toxic saccharide, sialic
Our group has described, in previous scientific papers and in granted
patents, the utility of natural PSAs in improving the pharmacokinetic
properties of protein therapeutics [Gregoriadis, 2001; Femandes and
Gregoriadis, 1996, 1997, 2001; Gregoriadis et. al., 1993, 1998, 2000;
Hreczuk-Hirst et. al., 2002; Mital, 2004; Jain et. al., 2003, 2004; US-A-
05846,951; WO-A-0187922]. Now, we describe new derivatives of PSAs,
which allow new compositions and methods of production of PSA-derivatised
proteins (and other forms of therapeutic agents).
Methods have been described previously for the attachment of
polysaccharides to therapeutic agents such as proteins [Jennings and
Lugowski, 1981; US-A-5846,951; WO-A-0187922J. Some of these methods
depend upon chemical derivatisation of the 'non-reducing1 end of the
polymer to create a protein-reactive aldehyde moiety (Fig. 1,2). The
reducing end of PSA (and other polysaccharides) is only weakly reactive
with proteins under the mild conditions necessary to preserve protein
conformation and the chemical integrity of PSA during conjugation. The
non-reducing end of the sialic acid terminal unit, which contains vicinal
diols, can be readily (and selectively) oxidised with periodate to yield a
mono-aldehyde derivative. This derivative is much more reactive towards
proteins and comprises of a suitably reactive element for the attachment of
proteins via reductive amination and other chemistries. We have described
this previously in US-A-5846,951 and WO-A-0187922. The reaction is
illustrated in Fig. 1a and 2 in which;
1 a) shows the oxidation of CA (alpha-2,8 linked PSA from E. co/i)
with sodium periodate to form a protein-reactive aldehyde at the nonreducing
end of the terminal sialic acid and
2) shows the selective reduction of the Schiff s base with sodium
cyanoborohydride (NaCNBH3) to form a stable irreversible covalent bond
with the protein amino group.
The weak reactivity of the reducing end can be exploited to beneficial
effect (by destroying the non-reducing end, capping the reducing end and
derivatising with a bifunctional crosslinker), thus avoiding the product
complexity described in Fig. 1b using the established method (Fig. 1a) of
reductive amination of proteins with periodate oxidised CA.
Commercially available polymers (especially natural polymers, e.g.
colominic acid (CA); used in most of the above papers) are produced from
bacteria and are highly polydisperse. They may also contain bacterial
contaminants such as endotoxins, salts etc. Such materials might not be
suitable for the production of PSA-derivatised therapeutic agents intended
for use in humans and animals, where the chemical and molecular definition
of drug entities is of major importance because of the safety requirements of
medical ethics and of the regulatory authorities (e.g. FDA, EMEA).
Therefore, we have solved the above problems by developing new
methods for the fractionation of a polydisperse polysaccharide preparation
into a series of low polydispersity ( species within a narrow range of molecular weights. These new preparations
are particularly suitable for the production of PSA-derivatised therapeutic
agents intended for use in humans and animals, where the chemical and
molecular definition of drug entities (e.g. predictable half-life) is of major
importance because of the safety requirements of medical ethics and of the
regulatory authorities (e.g. FDA, EMEA).
Ion exchange chromatography methods are well known in the art for
separation of complex mixtures. In general the prior art uses variation in
ionic strength to achieve this separation. In a solution of fully dissaciated
electrolytes the ionic strength (I) is defined as 1=0.5 E, C, Zj2, in which Cj is
the concentration of a particular ion, and Zj is the charge number of the ionic
Constantino et. al.,  have reported a chromatographic method
suitable for the isolation of the high molecular weight fraction from a
polydisperse preparation of a negatively charged polysaccharide such as the
Haemophilus influenzae type b and Neisseria meningitidis group A and group
C antigens. The material obtained still contains species with a large range of
molecular weights (i.e. it is still highly polydisperse) but it is free from small
oligosaccharides. The removal of the oligosaccharides makes this material
more suitable for use in polysaccaride based vaccines. The removal of the
low molecular weight species was accomplished, using a two step elution
process. Briefly Hib (Haemophilus influenzae type b antigen), a negatively
charged polysaccharide, was bound to an ion exchange resin and the low
molecular weight species where eluted by extensive washing with a low ionic
strength buffer. The intermediate and high molecular weight species, which
remain bound in the column were recovered by eluting with a high ionic
Zhang et al (1997) have reported use of high performance ion
exchange chromatography for the separation of colominic acid polymers.
Three stock solutions are mixed together to form a linear gradient which has
a progressively higher concentration of sodium nitrate.
The present invention may be distinguished over the disclosures of
Constantino et al and Zhang et al by its use of a step rather than a linear
gradient elution. Constantino et al use a large volume of low ionic strength
wash to remove all low molecular weight species from the column and then a
high volume high ionic strength wash to remove all other species from the
column. This does not result in separation into low polydispersity fractions.
The use of a step gradient according to the present invention, with steps that
differ only slightly in ionic strength, leads to the isolation of low
Ravenscroft et. al.,  have developed a procedure for the
molecular weight determination of low molecular weight Hib
oligosaccharides using ion exchange chromatography (IEC). Ion exchange
affords excellent resolution of small oligosaccharides with DP (degree of
polymerisation) values of 10 or lower. This was exploited to separate a
mixture of oligomers using ion exchange into individual oligomers. The DP
was measured with mass-spectroscopy and the purified oligosaccarides
were then used as standards to calibrate an ion exchange column enabling
thus the determination of the molecular weight of small oligosaccharides to
be carried out using analytical ion exchange.
The present invention generates fractions of polysaccharides of low
polydispersity in good yield which may be used to provide pharmaceutically
useful protein-polymer conjugates. None of the prior art describe techniques
which fractionate polysaccharides in this manner.
According to the invention there is provided a process for separating
polydisperse ionically charged polysaccharide into fractions of different
average molecular weight in which an aqueous solution of the polydisperse
polysaccharides is contacted with ion exchange resin in a column, the
polysaccharides are then subjected to selective elution by aqueous elution
buffer, and polysaccharide is recovered from eluted fractions, characterised
in that the selective elution involves washing the resin in the column
sequentially with at least three elution buffers each having different and
constant ionic strength and/or pH and in which the second and subsequent
buffers have higher ionic strength and/or pH than the buffers of the
immediately preceding step.
In the process, preferably the elution buffers all have the same pH,
but have successively higher ionic strength due to increasing concentrations
of ions such as ions of salts of strong mineral acids and strong mineral
bases. Preferably the salt is sodium chloride. Preferably the increase in
ionic strength is the same as that produced by increasing the NaCI
concentration by 5-100 mM, preferably 25 mM. This corresponds to an
increase in ionic strength of 0.005 to 0.1 M, preferably'0.025M.
Alternatively, the elution buffers may have successively higher pH
values. Preferably, the difference between the pH of each eluent is the
same and preferably 0.2 pH units. The pH of each buffer preferably lies in
the range 7.4-13. Typically, the first elution buffer will have a pH of around
7.4 and at least 5 elution steps will be used in the process. Ideally, the ionic
strength of all elution buffers will be less than 25M. The concentration of
hydrogen and hydrpxyl ions in the buffers is so low that their contribution to
the total ionic strength of the solution is negligible.
An increase in ionic strength and pH may be used in tandem in the
fractionation process. Alternatively, pH elution may be used initially in a
series of steps to raise the pH from 7.4 to at least 8.8, and then ionic
strength elution may begin. As an alternative, increasing the pH after a
short salt gradient may be performed, but this requires a starting pH of
greater than 7.4. A low pH range (around 4) may allow fractionation when
using a strong ion exchange column. However, since many polysaccharides
are not stable at this pH, the pH of the eluted fractions should in this case
immediately be adjusted to around 7.4.
Preferably, the elution buffers contain a base, which is preferably
It is possible for there to be an initial washing step, in which a low
ionic concentration elution buffer is washed through the column. For
instance such an initial wash step may be carried out with a buffer having a
salt concentration more than 100 mM lower in salt concentration than the
first of the essential three elution buffers used in the process. Such an initial
step may wash out low molecular weight contaminants or other contaminants
which are by-products from recovery processes for naturally occurring
materials. Such an initial wash step may involve a volume of at least 1,
preferably at least 1.5 column volumes, based on the volume of the ion
exchange resin column. It is to be noted that a column of ion exchange resin
may have a diameter of the cross-section which is greater than the height or
may, as in a conventional column, have a height which is greater than the
diameter of the cross section. The volume may be in the range 1 to 5000
ml. The height of the column may be in the range 1 cm to.5000 cm. The
cross sectional area may be in the range 1 cm to 5000 cm2. The cross
section may be of any shape but is preferably round.
We have found that it is preferred for each of the essential elution
steps to use at least 1.0, preferably at least 1.25 and most preferably at least
1.5 column volumes of the respective elution buffer. Preferably no more
than 3 column volumes of elution buffer are used. The flow rate for a 75ml
matrix is preferably 7ml/minute.
The process of the invention preferably comprises at least 5, for
instance as many as 20 or, generally in the range 6 to 12, sequential steps
of elution with the elution buffers of successively increasing ionic strength.
The ionic strength in the first of these essential steps is generally in the
range 1mM to 1M. Weaker ionic concentrations will be required for stronger
counterions, such as sulphate, than for chloride.
Preferably the ionic strength of an elution buffer is varied by varying
the level of a salt of a strong mineral acid and a strong mineral base,
preferably sodium chloride.
With regard to the recovery steps, these generally involve steps in
which the polysaccharide is isolated from the salt, for instance using
membranes, for instance ultra-filtration membranes. Such steps may allow
concentration of the polysaccharides to form more highly concentrated
solutions. Such solutions may be subjected to additional steps of membrane
treatment, for instance successive ultra-filtration or other filtration steps.
The elution buffer may contain a volatile acid or base and in this case the
recovery involves volatilisation of the volatile acid or base from the eluted
fractions. Although it is possible to recover the polysaccharide from
aqueous solutions by precipitation techniques, for instance involving non
solvents for the polysaccharides, it is preferred that no such solvents are
utilised, since this may make final isolation from the respective solvents
more difficult. Consequently the final step of recovery preferably involves
evaporation of the water and, preferably, any remaining volatile buffer
components remaining from the elution steps. In a preferred case wherein
triethanolamine is present, both the triethanolamine cation and acetate anion
are volatile and can easily be removed under vacuum.
The polysaccharide may be finally isolated from solution by drying,
preferably under reduced pressure. This is preferably performed by freezedrying.
Precipitation, preferably using a non solvent, may be carried out as a
preliminary step to fractionation to remove a portion of the population and
decrease polydispersity of the higher molecular weight fractions. Preferably,
differential ethanol precipitation is used.
The invention described herein is the use of a step gradient strategy,
which affords the stepwise removal (and subsequent collection) of
progressively higher molecular weight species from a polydisperse polymer
population bound to an ion exchange resin. The ion exchange resin may be
any strong or weak anion or cation exchange medium. Using a wash of any
given ionic strength will elute a complex population of species from the
column. By using a series of short washes in tandem, each step elutes a part
of the population that would be eluted by the next step, so at each point only
a small population is actually eluted, facilitating the fractionation of the
polydisperse preparations into fractions with narrow polydispersity. This
process may also facilitate removal of impurities e.g.salt, endotoxin etc.
In IEC, molecules do not remain absolutely motionless at low salt
concentrations. They move extremely slowly as bands. Their speed depends
upon the binding constant of a molecule to the column, which varies with the
ionic strength. When the binding constant is, for example, medium, a band
will move relatively slowly through the column and broaden to some extent.
When the binding constant is weak the band will move very quickly but with
negligible broadening. When the binding constant is strong the band will
move very slowly, mostly by broadening. A linear gradient prevents the
broadening, giving narrow bands and well-defined peaks. A step gradient on
the other hand elutes the molecules which at the particular
concentration have a low binding constant as a tight band and elutes those
which have an intermediate binding constant as a diffuse band (which is
likely to carry over into the next fraction). Molecules which have a high
binding constant will exhibit little movement and they will mostly be broad.
The result is that each step produces a peak that rises very rapidly and then
slowly declines producing a tail. Step gradients are well documented to
behave in this manner. The rise of the peak contains more of the species
with a low binding constant and the tail contains more of the species with the
intermediate binding constant (which will also carry over to the next fraction).
The longer each step runs for, the more it causes the species which have an
intermediate binding constant to broaden and eventually some are eluted,
enriching what is left in the column of higher species. So a step gradient also
elutes species, which in a linear gradient would come down at higher ionic
strength and cause some shifting of even the much higher bands. Although
this does not facilitate resolution, it is advantageous to fractionation. If two
species have a very close binding constant a step gradient can afford
purification or enrichment of the species with the higher binding constant.
The first step elutes both species, but preferentially the one with the lower
binding constant more, The second step elutes both species but since most
of the low binding constant species has already been removed in the
previous step, most of the fraction is the high binding constant species. So a
step gradient, though it cannot completely separate species with similar
binding constants, can result in significant enrichment of the one with the
higher binding constant.
Taking CA as an example, the binding constant is the result of the
average charge on the molecule. The difference in binding constants
depends on the charge difference between different species. The charge
ratio between a species with 41 charges and a species with 40 charges is a
lot lower than that between a species with 11 charges and one with 10
charges. As the amount of charges (and hence) monomers increases the
difference between the binding constants decreases. At larger molecular
weights it becomes practically negligible.
A linear gradient therefore results in good resolution for the low
molecular weight species, but as the molecular weight (and hence the
charge) increases, resolution decreases. The step gradient strategy, in
contrast, elutes more of the lower than higher molecular weight species with
each wash. Thus the step gradient produces less disperse fractions towards
the higher end of the molecular weight range and more disperse fractions in
the middle and begining of the range.
Several parameters may affect the separation.
1) pH: The charge on the polymer is pH dependent; decreasing the
pH will reduce the charge and might afford better resolution on the higher
end of the molecular weight range.
2) Step wash volume: The more washing the less of the higher
molecular weight species would be recovered, but the lower their
3) Number of steps: The number of steps is also an important
parameter. Skipping the early steps may result in a decrease on the average
size obtained in each fraction and an increase in polydispersity that
becomes less and less pronounced as the molecular weight increases. If
more steps are used it may increase the resolution of most fractions, but it
will decrease the average amount of CA in each fraction.
4) Temperature. Lower temperature increases the strength of binding
and may decrease the size of species coming down in each fraction.
In the invention the polydisperse polysaccharide may be a naturally
occurring polysaccharide, a hydrolysis product thereof or a functionalised
derivative of either of these. The invention is of particular utility in
separating naturally occurring polysaccharides such as bacterial
polysaccharides, for instance polysaccharide antigens. The invention is of
particular utility in separating sialic acid polymers and copolymers, for
instance poly(2,8-linked sialic acid), poly(2,9-linked sialic acid) or an
alternating 2,8-2,9-linked PSA. Preferably the polysaccharide is colominic
acid (CA) or an oxidised, reduced, aminated and/or hydrazide derivative
The polydispersity of molecular weight (that is the weight average
molecular weight divided by the number average molecular weight) of the
polydisperse polysaccharide should be at least 1.1, preferably at least 2.0.
We have found that the process has particular utility where the weight
average molecular weight of the polydisperse polysaccharide is at least 1
kDa, preferably at least 10 kDa, and preferably at least 100 kDa.
The process is of use for producing very low polydispersity
polysaccharide fractions. For instance the product polysaccharide
recovered from the eluted fractions preferably has polydispersity less than
1.5, most preferably less than 1.25, for instance 1.1 or even lower. We have
found it possible to achieve polydispersities down to around 1.01.
Preferably the polysaccharide starting material has at least 1, more
preferably at least 5, more preferably at least 10, for instance at least 50,
saccharide units. Preferably the polysaccharide is polysialic acid (PSA)
comprising sialic acid units linked a (2,8) or a (2,9) to one another.
There is no particular upper limit on the size of polydisperse
polysaccharides to be fractionated. In the case of PSA, however, we find
that most polymers of utility have a weight average molecular weight of up to
The PSA may be derived from any source, preferably a natural source
such as a bacterial source, e.g. E. coli K1 or K92, group B meningococci, or
even cow's milk or N-CAM. The sialic acid polymer may be a
heteropolymeric polymer such as group 135 or group V of N. meningitidis, or
may be synthesised. The PSA may be in the form of a salt or the free acid.
It may be in a hydrolysed form, such that the molecular weight has been
reduced following recovery from a bacterial source. The PSA may be
material having a wide spread of molecular weights such as having a
polydispersity of more than 1.3, for instance as much as 2 or more.
Preferably the polydispersity of molecular weight is less than 1.2, for
instance as low as 1.01.
The following description describes the preferred embodiments of the
process of the present invention carried out on the polysaccharide PSA for
which the invention is of particular utility.
A population of PSAs having a wide molecular weight distribution may
be fractionated into fractions with lower polydispersities, i.e. into fractions
with differing average molecular weights. Fractionation is preferably anion
exchange chromatography, using for elution a suitable basic buffer. When
the polysaccharide has carboxylic acid groups then anion exchange is
particularly desirable. We'have found a suitable anion exchange medium; a
preparative medium such as a strong ion exchange material based on
activated agarose, having quaternary ammonium ion pendant groups (ie
strong base). The choice of medium will be dependent upon whether pH or
ionic strength is the means for fractionation, and will be apparent to a person
skilled in the art. The elution buffer is non-reactive and is preferably volatile
so that the desired product may be recovered from the base in each fraction
by evaporation. Suitable examples are amines, such as triethanolamine.
Recovery may be by freeze-drying for instance. The fractionation method is
suitable for a PSA starting material as well as its derivatives. The technique
may thus be applied before or after the essential process steps on related
inventions described in PCT/GB04/03488, application filed even date
herewith (agent's ref: HMJ03917) and application filed even dated herewith
(agent's ref: HMJ03871), in which we describe various derivatisation
reactions carried out on polysialic acids and intermediates formed therein.
It is believed this is the first time IEC has been applied to fractionate
ionic polysaccharides with molecular weights above about 5 kDa, especially
PSA of such molecular weights.
According to a further aspect of this invention there is provided a
process for fractionating a population of ionic polysaccharide with a
molecular weight higher than 5kDa using IEC using in the elution buffer a
base or acid which is preferably volatile.
Preferably the polysaccharide has carboxylic acid groups and the ion
exchange is anion exchange. Preferably the elution buffer contains an
amine, more preferably triethanolamine. Most preferably the
polysaccharides are recovered from the fractions by drying, preferably under
reduced pressure, most preferably freeze-drying.
This method can be applied for the fractionation of CA having reactive
moieties which are stable in water (maleimide or iodoacetate etc.) and other
natural (e.g. dextran sulphate) and synthetic (e.g. polyglutamic acid,
polylysine, hyaluronic acid) charged polymers. Cation and anion exchange
chromatography can also be used for fractionation of charged polymers
using salt or pH gradient.
It is believed that it is also the first time that IEC has been used to
separate ionic polysaccharides in combination with precipitation techniques
and/or ultrafiltration methods.
The I EC method may remove by products such as endotoxins which
remain in the commercially available PSAs and CAs.
Ion pair and hydrophobic interaction chromatography can also be
used for fractionation of polymers and protein-polymer conjugates.
In the invention there is provided a new process for producing a
series of narrow polydispersity (pd polysaccharide, preferably PSA compound (native or activated ), in which
the high polydispersity, negatively charged starting material is bound to an
anion exchange resin and the fractions are eluted with a series short washes
each with a progressively higher ionic strength.
In an alternative embodiment, +ve and -ve charged broad disperse
polymers and protein-polysaccharide conjugates can be fractionated by
cation or anion exchange chromatography respectively.
According to an another aspect of the invention there is provided a
new process in which a polysialylated macromolecule, where the product is
polydisperse owing to the polydispersity of the polysialic acid starting
material conjugated to the macromolecule, is fractionated into narrow
polydispersity preparations using the same IEC and step gradient strategy
as described above.
In the process the parameters (e.g. amount of matrix used, amount of
sample loaded, temperature, flow rate, gradients etc.) are preferably
optimized for fractionation of a polysialylated macromolecule (for instance),
where the product is polydisperse owing to the polydispersity of the PSA
starting material conjugated to the macromolecule, is fractionated into
narrow polydispersity preparations using the same IEC and step gradient
strategy as described above. These steps are carried out under conditions
such that there is substantially no mid-chain cleavage of the backbone of a
long-chain (polymeric) starting material, that is no substantial molecular
weight reduction should occur.
The narrow dispersed polysaccharides can be used to generate
active groups. For instance aldehyde groups are suitable for conjugating to
amine-group containing substrates or hydrazine compounds. Processes in
which the activated product of an oxidation step is subsequently conjugated
to substrate compound are described. Preferably the conjugation reaction
(as described in our earlier publications mentioned above) involves
conjugation of PSA with an amine to form a Schiff base, preferably followed
by reduction to form a secondary amine moiety. The process is of particular
value for derivatising proteins, of which the amine group is suitably the
epsilon amine group of a lysine group or the N-terminal amino group. The
process is of particular value for derivatising protein or peptide
therapeutically active agents, such as cytokines, growth hormones,
enzymes, hormones, antibodies or fragments. Figures 1 and 2 show
reaction schemes for such reactions, wherein the polysaccharide is PSA.
Alternatively the process may be used to derivatise drug delivery
systems, such as liposomes, for instance by reacting the aldehyde with an
amine group of a liposome forming component. Other drug delivery systems
are described in our earlier case US-A-5846951. Other materials that may
be derivatised include viruses, microbes, cells, including animal cells and
Alternatively the substrate may have a hydrazine group, in which case
the product is a hydrazone. This may be reduced if desired, for additional
stability, to an alkyl hydrazide.
The derivatisation of proteins and drug delivery systems may result in
increased half life, improved stability, reduced immunogenicity, and/or
control of solubility and hence bioavailability and pharmaco-kinetic
properties, or may enhance solubility actives or viscosity of solutions
containing the derivatised active.
Preferably the polysaccharide compounds recovered from eluted
fractions comprise sialic acid units, most preferably consist of sialic acid
units. More preferably the polysaccharides have 1-1000 sialic acid units, for
instance 10-500, more preferably 10 to 50 sialic acid units. The eluted
fractions may consist of monomers, dimers, or larger polymers. Preferably,
the polydispersity of a fraction will be less than 1.26, ideally less than 1.2,
and ideally in the range 1.01 to 1.10.
It is believed that this is the first time that a polysialic aid, particularly
CA, having a polydispersity of molecular weight of less than 1.26 has been
isolated. Accordingly, a further aspect of the present invention provides a
polysialic acid having a polydispersity of molecular weight of less than 1.26,
preferably no more than 1.2, most preferably in the range 1.01 to 1.10. The
polysialic acid may be CA, or an oxidised, reduced, aminated and/or
hydrazide derivative thereof. Preferably, the polysialic acid has a molecular
weight of at least 5kDa, preferably at least 10kDa.
The fractionation process that we described here is linearly scalable
and reproducible. It is suitable for industrial scale production and for control
of polydispersity of CA. The fractionation technology, described here, allows
for fractionatjon of CA as well as other polysaccharide (preferably charged),
protein-polymer conjugates and vaccines to be prepared with a homogenous
(moriodispersed) polymer chain length.
Figure 1 a is a reaction scheme showing preparation of
Figure 1b is a reaction scheme showing preparation of products
using original conjugation methods;
Figure 2 is a reaction scheme showing preparation of protein-CA
Figure 3 shows the results of the Gel Permeation Chromatography of
Figure 4 shows % population of different CA fractions;
Figure 5 shows a typical native page of CA with molecular weights;
Figure 6 shows the native PAGE of CA (22.7KDa; pd 1.34);
Figure 7 shows a typical chromatogram for CA fractions;
Figure 8 shows the CA samples from different steps of fractionation;
Figure 9 shows the loading of different amounts of CA samples;
Figure 10 shows the fractionation of CA (150mg of CA; 5 ml matrix);
Figure 11 shows the fractionation of CA (200mg; 5ml);
Figure 12 shows the fractionation of CA-NH2;
Figure 13 shows the anion exchange Chromatography of oxidised CA
Figure 14 shows the anion exchange chromatography of
Figure 15 shows SDS PAGE for preparation of protein-polymer
conjugates with broad and narrow dispersed polymer;
Figure 16 shows native PAGE results for fractionation of CA by anion
exchange chromatography vs filteration;
Figure 17 shows the fractionation of CA by ethanol precipitation;
Figure 18 shows the fractionation of CA by ultrafilteration;
Figure 19 shows the anion exchange chromatography of GHCA
Figure 20 shows the characterization of CA (35KDa) by NMR;
Figure 21 shows % population of large scale IEC fractionation of CA
(39kDa; pd 1.4);
Figure 22 shows the native PAGE of CA (39 kDa; 12.5g) IEC
Figure 23 shows % population of small scale IEC fractionation of CA
(39kDa; pd 1.4);
Figure 24 shows the native PAGE of CA (39 kDa; 200mg) IEC
Figure 25 shows a typical GPC chromatogram for a narrow dispersed
Figure 26 shows optimisation of native-PAGE analysis of CA;
Figure 27 shows native PAGE of CA fractions from IEC;
Figure 28 shows native PAGEs of CA (22.7 kDa) IEC fractions using
increasing ionic strength of triethanolamine;
Figure 29 shows the native PAGE of Q FF fractionation of CA (22.7
kDa) using increasing ionic strength of triethanolamine acetate; and
Figure 30 shows the native PAGE of DEAE fractionation of CA (22.7
kDa) using a gradient pH system.
The invention is illustrated further in the accompanying examples.
Ammonium carbonate, ethylene glycol, PEG (8KDa), sodium
cyanoborohydride (> 98% pure), sodium meta-periodate, triethanolamine,
sodium chloride, sodium nitrate, sodium azide, PBS tablets and molecular
weight markers were obtained from Sigma Chemical Laboratory, UK. The
CA used, linear alpha-(2->8)-linked E coli K1 PSAs (22.7 kDa average, high
polydispersity 1.34; 39kDa, p.d. 1.4; 11 kDa, p.d. 1.27) was from Camida,
Ireland, Other materials included 2,4 dinitrophenyl hydrazine (2,4 DNPH)
(Aldrich Chemical Company, UK), dialysis tubing (3.5kDa and 10kDa cut off
limits; Medicell International Limited, UK), Sepharose SP HiTrap, PD-10
columns (Pharmacia, UK), Tris-glycine polyacrylamide gels (4-20%), Trisglycine
sodium dodecylsulphate running buffer and loading buffer (Novex,
UK), Sepharose Q FF and DEAE (Amersham Biosciences, UK), Tris-Borate-
EDTA (TBE) polyacrylamide gels (4-20% and 20%), TBE buffer and loading
buffer (Invitrogen, UK). Deionised water was obtained from an Elgastat
Option 4 water purification unit (Elga Limited, UK). All reagents used were of
analytical grade. A plate reader (Dynex Technologies, UK) was used for
spectrophotometric determinations in protein or CA assays.
Protein and colominic acid determination
Quantitative estimation of PSAs (as sialic acid) was carried out by the
resorcinol method [Svennerholm 1957] as described elsewhere [Gregoriadis
et. al., 1993; Fernandes and Gregoriadis, 1996, 1997]. Protein was
measured by the bicinchoninic acid (BCA) colorimetric method.
Example 1: Preparation of monofunctional PSA :
1a Activation of CA
Freshly prepared 0.02 M sodium metaperiodate (NalO4; 6 fold molar
excess over CA) solution was mixed with CA at 20°C and the reaction
mixture was stirred magnetically for 15 min in the dark. The oxidised CA was
precipitated with 70% (final concentration) ethanol and by centrifuging the
mixture at SOOOg for 20 minutes. The supernatant was removed and the
pellet was dissolved in a minimum quantity of deionised water. The CA was
again precipitated with 70% ethanol and then centrifuged at 12,000 g. The
pellet was dissolved in a minimum quantitiy of water, lyophilized and stored
at -20°C until further use (Figure 1; step 1).
1b Reduction of CA
Oxidised CA (CAO; 22.7kDa) was reduced in presence of sodium
borohydride. Freshly prepared 15mM sodium borohydride (NaBH4; in 0.1 M
NaOH diluted to pH 8-8.5 by diluting with dilute H2SO4 solution) was mixed
with CAO (100mg CA/ml) at 20°C and the reaction mixture was stirred for up
to 2h in the dark. The pH was brought down to 7 by the completion of the
reaction. The oxidised/reduced CA (CAOR) was dialysed (3.5 kDa molecular
weight cut-off for dialysis tubing) against 0.01% ammonium carbonate buffer
pH 7 at 4CC. Ultracentrifugation was used to concentrate the CAOR solution
from the dialysis tubing. The filtrate was lyophilized and stored at 4°C until
further required. The determination of any aldehyde content was determined
as described under 'determination of CA oxidation'(Figure 1; step 2).
1c Reoxidation of CA
After confirmation of no aldehyde content the CAOR was again
oxidised as reported under activation of CA except CAOR was incubated
with periodate solution for a longer time (up to 1h). The degree of oxidation
in the CAORO product was measured on lyophilized powder obtained from
this stage as well (Figure 1; step 3).
1 d Determination of the oxidation state of CA and derivatives
Qualitative estimation of the degree of colominic acid oxidation was
carried out with 2,4 DNPH, which yields sparingly soluble 2,4 dinitrophenylhydrazones
on interaction with carbonyl compounds. Non-oxidised (CA),
oxidised (CAO), reduced (CAOR) and re-oxidised (CAORO) (5mg each),
were added to the 2,4-DNPH reagent (1 .Oml), the solutions were shaken and
then allowed to stand at 37°C until a crystalline precipitate was observed
[Shriner et. al., 1980]. The degree (quantitative) of GA oxidation was
measured with a method [Park and Johnson, 1949] based on the reduction
of ferricyanide ions in alkaline solution to ferric ferrocyanide (Persian blue),
which is then measured at 630nm. In this instance, glucose was used as a
1e Preparation of CA-NH2
CAO at (10-100 mg/ml) was dissolved in 2ml of deionised water with a
300-fold molar excess of NH^CI, in a 50 ml tube and then NaCNBH4 (5 M
stock in 1 N NaOH(aq), was added at a final concentration of 5 mg/ml. The
mixture was incubated at room temperature for 3 days. A control reaction
was also set up with colominic acid instead of CAO. Product colominic acid
amine derivative was precipitated by the addition of 5 ml ice-cold ethanol.
The precipitate was recovered by centrifugation at 4000 rpm, 30 minutes,
room temperature in a benchtop centrifuge. The pellet was retained and
resuspended in 2 mi of deionised water, then precipitated again with 5 ml of
ice-cold ethanol in a 10 ml ultracentrifuge tube. The precipitate was
collected by centrifugation at 30,000 rpm for 30 minutes at room
temperature. The pellet was again resuspended in 2 ml of deionised water
1f Assay for amine content
The TNBS (picrylsulphonic acid i.e. 2, 4, 6-tri-nitro-benzene sulphonic
acid) assay was used to determine the amount of amino groups present in
the product [Satake et. al., 1960]. In the well of a microtitre plate TNBS (0.5
ul of 15 mM TNBS) was added to 90 ul of 0.1 M borate buffer pH 9.5. To
this was added 10 ul of a 50 mg/ml solution of CA-amide. The plate was
allowed to stand for 20 minutes at room temperature before reading the
absorbance at 405nm. Glycine was used as a standard, at a concentration
range of 0.1 to 1mM. TNBS trinitrophenylates primary amine groups. The
TNP adduct of the amine is detected.Testing the product purified with a
double cold-ethanol precipitation using the TNBS assay showed close to 90
1g Preparation of maleimide polymer (CA-M)
The CAORO synthesised in Example 1 c above was reacted with 5
molar equivalents of N-[(3-maleimidopropionic acid] hydrazide in 0.1 M
sodium acetate for 2h at 20°C. The product hydrazone was precipitated in
ethanol, resuspended in sodium acetate and precipitated again in ethanol,
redissolved in water and freeze-dried. The product is useful for site-specific
conjugation to the thiol groups of cysteine moieties in proteins and peptides.
1h Gel Permeation Chromatography
GA samples (CA, CAO, CAOR and CAORO) were dissolved in NaNO3
(0.2M), CH3CN (10%; 5mg/ml) and were chromatographed on over 2x
GMPWXL columns with detection by refractive index (GPC system: VE1121
GPC solvent pump, VE3580 Rl detector and collation with Trisec 3 software
(Viscotek Europe Ltd). Samples (5mg/ml) were filtered over 0.45um nylon
membrane and run at 0.7cm/min with 0.2M NaNO3and CH3CN (10%) as the
mobile phase (Figure 3).
Results of Example 1: Preparation of monofunctional PSA
The integrity of the internal alpha-2,8 linked NeuSAc residues post
periodate and borohydride treatment was analysed by GPC and the
chromatographs obtained for the oxidised (CAO), oxidised reduced (CAOR),
double oxidised (CAORO), amino CA (CA-NH2) materials were compared
with that of native CA. It was found (Fig.3) that oxidized (15 minutes) (CAO),
reduced (CAOR), double oxidised (1hr) (CAORO) and native CA exhibit
almost identical elution profiles, with no evidence that the successive
oxidation and reduction steps give rise to significant fragmentation of the
polymer chain. The small peaks are indicative of buffer salts.
The results of quantitative assay of CA intermediates in the double
oxidation process using ferricyanide were consistent with the results of
qualitative tests performed with 2,4 DNPH which gave a faint yellow precipitate
with the native CA, and intense yellow colour with the aldehyde containing
forms of the polymer, resulting in an intense yellow precipitate after ten minutes
of reaction at room temperature.
The amination content of the polymer was found to be 85% by the 2,4,6-
tri-nitro-benzene sulphonic acid (TNBS) assay. The PSA aldehyde derivative
could also be reacted with a linking compound having a hydrazide moiety and
a N-maleimide moiety to form a stable hydrazone having an active maleimide
functionally useful for reacting with a thiol group. The maleimide content of the
polymer was found to be 95% by the maleimide assay.
Example 2 - Fractionation of Colominic Acid by IEC (CA, 22.7 KDa,
2.1 Fractionation at large scale
An XK50 column (Amersham Biosciences, UK) was packed with 900 ml
Sepharose Q FF (Amersham Biosciences) and equilibrated with 3 column
volumes of wash buffer (20mM triethanolamine; pH 7.4) at a flow rate of
50ml/min. CA (25 grams in 200 ml wash buffer) was loaded on column at 50
ml per minute via a syringe port. This was followed by washing the column with
1.5 column volumes (1350ml) of washing buffer.
The bound CA was eluted with 1.5 column volumes of different elution
buffers (Triethanolamine buffer, 20 mM.pH 7.4, with OmM to 475mM NaCI in
25 mM NaCI steps) and finally with 1000mM NaCI in the same buffer to
remove all residual CA and other residues (if any). The flow rate was
The samples were concentrated to 20 ml by high pressure ultra filtration
over a 3-10kDa membrane (Vivascience, UK). These samples were buffer
exchanged into deionised water by repeated ultra filtration at 4°C. The samples
were analysed for average molecular weight and other parameters by GPC (as
reported in example 1 h) and native PAGE (stained with alcian blue) [Table 1
and 2; Figure 4, 5,6].
Example 2.2: Fractionation at medium and smaller scale
The following samples were fractionated using an identical wash and
gradient system on a smaller scale (1-75ml matrix; 0.2-3 gram of colominic
CA (CA, 22.7kDa, pd 1.34; CA, 39 KDa, pd 1.4), CAO (CAO, 22.7 kDa,
pd 1.34), monofunctional CAO (RO, 22.7kDa; pd 1.34), CA-NH2 (22.7kDa, pd
1.34), CAM (as per example 1g) produced were monitored throughout.
Narrow fractions of CA produced using above procedure were oxidised
with 20mM periodate and analysed by gel permeation chromatography (GPC)
and native PAGE for gross alteration to the polymer (Fig. 7,8).
Results of Example 2: Fractionation at large, medium and small
CA and its derivatives (22.7 kDa) were successfully fractionated into
various narrow species with a polydispersity less than 1.1 with m.w. averages
of up to 46 kDa with different % of populations (Tables 1-2 and Figures 4-8).
Table 1 shows the results of fractionating the 22.7kDa material at a scale of
75ml. Figure 7 is the GPC results and Figs.4-6 are native PAGEs of CA
(Table Removed) The 22.7kDa material is separated on a larger scale. Using GPC the
fractions from ion exchange are analysed.
All narrow fractions were successfully oxidised with 20mM periodate
and samples taken from different stages of the production process and
analysed by GPC and native PAGE showed no change in the molecular
weight and polydispersity. The data for some of the samples are shown in
Example 3 Factors affecting the fractionation of CA
The various factors affecting the fractionation of CA (e.g. washing
volume etc.) were studied [Fig. 7 -14].
Results of example 3: Factors affecting the fractionation of CA
The various factors affecting the fractionation of CA were studied. The
binding studies were done by loading 50, 100, 150 and 200mg of CA on to
the column (5ml matrix). Using 200mg of CA, more than 99% of CA was
bound to the column (Figure 9). When the column was washed with one
column volume of eluting buffer, by step gradient, the polydispersity of
polymer was found to be more than 1.1 (Fig. 11). The washing of the column
with 1.5 column volume generated CA fractions with polydispersities less
thanThe amino CA (CA-NH2; Fig. 12), oxidised CA (Fig. 13) and
monofunctional CA could successfully be fractionated in the fractions with
Example 4 - Synthesis of growth hormone (GH)-CA conjugates
(broad and narrow dispersed)
CAO (22.7 KDa) and narrow dispersed-CAO (27.7kDa pd= 1.09; 40.9
kDa. pd = 1.02) prepared in Reference example 2 were used for the
preparation of GH conjugates.
Preparation of growth hormone-CA conjugates
Growth hormone was dissolved in 0.15 M PBS (pH 7.4) and
covalently linked to different CAs (CAO and NCAO). Different CAs (22kDa,
CAO; 27.7kDa & 40.9kDa, NCA) were individually added to GH (2mg) in a
CA:GH molar ratios (12.5:1 ), sodium cyanoborohydride was added to a final
concentration of 4 mg/ml. The reaction mixtures were sealed and stirred
magnetically for 24h at 35±2°C. The mixtures were then subjected to
ammonium sulphate ((NH4)2SO4) precipitation by adding the salt slowly
whilst continuously stirring, to achieve 70% w/v saturation, stirred for 1 h at
4°C, then spun (5000xg) for 15 min and the pellets resuspended in a
saturated solution of (NH4)2SO4 and spun again for 15 min (SOOOxg). The
precipitates recovered were redissolved in 1 ml PBS pH 7.4 and dialysed
extensively (24 h) at 4°C against the same buffer. Controls included
subjecting the native protein to the conjugation procedure in the presence of
non-oxidised CA or in the absence of CA. Shaking was kept to a minimum to
avoid concomitant denaturation of the protein. Polysialylated GH was
characterised by SDS-PAGE. The polysialylated GH was passed through
anion exchange chromatography and the product fractions subjected to
SDS PAGE (Figure 15).
Results of example 4: Synthesis of GH-CA conjugates (broad
and narrow dispersed)
The GH-CA conjugates were successfully synthesised. The results of
SDS-PAGE (Fig. 15) show that in the control (with GH) migration of the
sample is similar to that for fresh GH.. In the conjugate lanes there are shifts
in the bands which typically indicates an increase in mass indicative of a
polysialylated-GH. The band width was significantly narrowed down in case
of conjugates with narrow dispersed polymer in comparison to conjugates
with broad dispersed polymers. Further, GH conjugates (with broad
dispersed polymer) were successfully separated into different species by
anion exchange chromatography (Fig. 19).
Example 5: Precipitation of CA
Differential ethanol precipitation was used to precipitate different
chain lengths of colominic acid [Fig. 1.6, 17].
Results of example 5: Precipitation of CA
The CA (22.7; pd 1.34) could not be precipitated into narrow
dispersed fractions using different strengths of ethanol (Fig. 17). However,
differential ethanol precipitation showed that smaller narrow CAs required
more ethanol (EtOH). Broad 22.7 kDa polymer was precipitated with 70%
EtOH giving a yield >80% of product polymer. A concentration of 80% EtOH
was required to precipitate > 80% of a lower MW 6.5KDa (pd process also removes part of the salt contaminating the product.
Example 6: Fractionation of CA by ultrafiltration
Samples of 22.7kDa were purified by ultrafiltration over different
molecular weight cut off membranes (5, 10, 30, 50, and 100 kDa). In all
cases retentate was examined by GPC and native PAGE [Fig. 18].
Results of example 6: Fractionation of CA by ultrafiltration
Samples of 22.7 kDa purified by ultrafiltration over different molecular
weight cut off membranes showed that there was a decrease in
polydispersity of the polymer and a shift towards higher molecular weight
with increase in membrane cut off (Fig. 18). Figure 16 shows fractionation
of CA by anion exchange chromatography (left) and filtration (right). IEC of
CA generated fractions with much narrow dispersed CAs as compared to
fractionation by filtration.
Example 7: Characterization by NMR spectroscopy
The fractionated CA polymers were characterized by 1H (400 MHz)
and 13C (100 MHz) NMR spectroscopy for impurities (if any) using D2O (Fig.
Results of example 7: Characterization by NMR spectroscopy
The 1H and 13C NMR of narrow dispersed polymer fractionated
material is free from impurities. In addition, the chemical shifts of the H-3
protons in the 1H NMR and the C-2 carbon in the 13C NMR spectra confirm
that the polymer is indeed the expected alpha-2,8 linked sialic acid material.
Example 8: Fractionation of CA (39 kDa, pd 1.4) by IEC
8.1 Fractionation at large scale
An XK50 column (Amersham Biosciences, UK) was packed with 900
ml Sepharose Q FF and equilibrated with 3 column volumes of wash buffer
(20mM triethanolamine; pH 7.4) at a flow rate of 50ml/min. CA (12.5g in 200
ml wash buffer) was loaded on column at 50 ml per minute via a syringe port
This was followed by washing the column with 1.5 column volumes (1350ml)
of washing buffer.
The bound CA was eluted with 1.5 column volumes of elution buffer
(triethanolamine, 20 mM, pH 7.4) containing different salt concentrations (0,
200, 250, 300, 350, 375, 400, 425, 450, 475, 500 and 525mM NaCI) and
finally with 1000mM NaCI in the same buffer to remove all residual CA and
other residues (if any).
The samples were concentrated to approximately 20 ml by either
high-pressure ultrafiltration over a 3 or 10 kDa membrane (Vivascience, UK)
or by Vivaflow 50 diafiltration (filtration by constantly passing the sample
through a membrane) having a 3 kDa mwco membrane (Vivascience, UK).
These samples were buffer exchanged into deionised water by either
repeated ultrafiltration or Vivaflow at 4°C. The samples were analysed for
average molecular weight and other parameters by GPC (as reported in
Example 2) and native PAGE (4-20% Tris-glycine gel stained with alcian
blue) (Figures 21 and 22).
8.2 Fractionation at small scale
CA was also fractionated on a smaller scale (200mg of CA) using
Sepharose Q FF (5ml matrix, prepacked; Amersham Biosciences, UK)
employing an identical buffer system (20mM triethanolamine; pH 7.4)
containing different salt concentrations (0, 200, 250, 300, 350, 375, 400,
425, 450, 475, 500, 525, 550 and 575mM NaCI). The bound CA was eluted
by washing the column with 1:5 column volumes (7.5ml) at a flow rate of
1ml/min with a final wash of the column using 1000mM NaCI in the usual
The samples were concentrated to approximately 0.75 ml by Vivaspin
membrane filtration (mwco 3 kDa) (Vivascience, UK), buffer exchanged into
deionised water by repeated membrane filtration at 8°C and then lyophilised.
The samples were analysed by native PAGE (4-20% tris-glycine gel stained
with alcian blue) (Figures 23 and 24).
Results of Example 8: Polydispersed CA (39 kDa; pd 1.4) was
successfully fractionated into various narrow dispersed species with
molecular weight averages ranging from 7 to 97 kDa and with different % of
populations (Figures 21-24 and Table 4).
(Table Removed) In the analysis the solvent used was 0.2M NaNO3 in MECN and PEO
and dextron were used as standards. The temperature was 22°C,3 injection
volume 100 uL and flow rate was 0.7 ml_/mn.
* Estimated approximate values by native PAGE
Figures 21 and 23 show the % population of CA in various fractions
as a result of fractionating 39 kDa CA polymer on a large scale (12.5 g CA;
900 ml matrix) and a small scale (200 mg CA; 5 ml matrix), respectively.
Figures 22 and 24 are the native PAGEs obtained as a result of the
fractionation of CA on a large and small scale. Table 4 is the GPC results of
the fractions obtained by I EC at various salt concentrations. The GPC data
shows that species up to 97 kDa are generated by the fractionation process
(see Example 9 for further details). These larger molecular weight polymers
have been shown to have a greater percentage phosphate moiety present
on the reducing end of CA compared to their lower molecular weight
counterparts by a phosphate assay, which tests for the presence of
inorganic phosphate (Rouser et. at. 1970).
Example 9: Characterisation of fractionated CA by GPC
Freshly prepared CA samples, by dissolving for example either CA,
CAO, CAOR or CAORO (4-5 mg/ml) in 0.2 M NaNO3/0.1% NaNa/10%
acetonitrile (1 ml) (or alternatively 10mM PBS, pH 7.4) and then filtering the
resulting solution over 0.2im nylon membrane (Whattman, UK), were
analysed by GPC.
Samples were chromatographed with 2x GMPWxl (250x4,6mm)
columns employing a triple detection GPC (SEC3) system (Viscotek Europe
Ltd, UK). Detectors consisted of a Viscotek Laser Refractometer (refractive
index) and a Viscotek 270 Dual Detector (right-angle light scattering detector
configured with a 4-capillary viscometer detector) collated with an OmniSEC
3.1 workstation (Viscotek Europe Ltd).
The analysis conditions used were; eluent: buffer employed to
dissolve the CA sample; flow rate: 0.7ml/min; sample loading: 100ul;
temperature: 22°C. The system was calibrated with narrow molecular weight
polyethylene glycol and broad molecular weight Dextran reference materials.
The time it takes for a polymer to be eluted from the GPC column is
converted to its molecular weight using various detectors. The light
scattering detector provides a direct measurement of absolute molecular
weight and eliminates the need for column calibration (detector gives a
proportional response to molecular weight and concentration). The radius of
gyration is not calculated when measurements are performed using a single
angle. The viscometer provides a direct measurement of intrinsic viscosity
and allows for the determination of molecular size, conformation and
structure (detector gives an inversely proportional response to molecular
density). The response from the refractive index detector is proportional to
the concentration of polymer: the constant of proportionality is dn/dc (the
same specific refractive index increment needed in light scattering).
The GPC system described above enables a number of parameters of
CA to be determined. For instance, the number average molecular weight
(Mn) and the weight average molecular weight (Mw) can be obtained and
from these numbers the polydispersity of CA can be calculated. Other
information acquired from the GPC data include the percentage recovery of
CA and the degree of branching (if any) on the polymer. The exact
concentration of the sample can also be determined from the dn/dc value (or
alternatively the dn/dc value can be calculated from the exact known
concentration of the polymer).
Results of Example 9: Table 5 shows typical data for a range of
parameters that was obtained from the analysis of a CA fraction (400mM
NaCI in 20mM triethanolamine, pH 7.4) from IEC fractionation of CA (39 kDa,
pd 1.4). In this tabe, the following definitions apply:
Mn = number average molecular weight
Mw = weight average molecular weight
Mz = Z-average molecular weight
Mp = peak average molecular weight
Mv = viscosity average molecular weight
Mw/Mn = molecular weight distribution (polydispersity)
Rh = hydrodynamic radius
IV = intrinsic viscosity
dn/dc = change in refractive index with concentration for
(Table Removed) For example, 27,956 and 26,666 Da were the values for Mw and Mn,
respectively, which gave a polydispersity of 1.048. Table 4 shows the range
of narrow dispersed molecular weights obtained by GPC analysis on the
fractionation of polydispersed CA (39 kDa; pd 1.4) with IEC, while Figure 25
shows a typical GPC chromatogram for the combined viscometer,
refratometer and light scattering curves obtained for a narrow dispersed CA
Example 10: Optimisation Studies
10.1 Optimisation of the analysis of CA by native PAGE
Fractionated and the non-fractionated CA have been analysed further
by TBE and Tris-glycine gels on native PAGE in order to optimise the
resolution of these polymers on the gel. In general, 40 ig of either narrow or
broad dispersed CA was loaded, as a 20II solution containing 10II of
loading buffer, per well on the gel. The gel was run at three different speeds
(150, 25 or 15 mV/cm) and then stained with alican blue, followed by
destaining with 2% acetic acid.
Results of Example 10.1: Figure 26 demonstrates the various
resolutions that can be obtained for different molecular weights of CA with 4-
20% Tris-glycine, 4-20% and 20% TBE gels. From the gels it can be
observed that good separation of high and low molecular weight CAs can be
observed with 4-20 and 20% TBE gels, with particularly good resolution with
the 20% TBE gel. Narrow bands are best observed when the gel is run at or 15 mV/cm compared to when the gel speed is 150mV/cm.
10.2 Concentration of CA
The filtrates of high-pressure ultrafiltration obtained from IEC
fractionation of CA (22.7 kDa, pd 1.34) were concentrated by Vivaflow
(mwco 3 kDa). These CA samples were analysed with the corresponding
filters of the high-pressure ultrafiltration by native-PAGE using a 4-20% TBE
gel (Figure 27) for any degradation of the polymer.
Results of Example 10.2: Results from the native PAGE (Figure 27)
of the CA samples obtained by Vivaflow purification of the filtrates show that
both materials had the same molecular weight. This observation of the
presence of CA in the filtrates of high-pressure ultrafiltration maybe
accounted for by a process known as reptation, whereby due to the
flexibility, deformability and its rod-like conformation of the CA polymer, the
polymer can pass through the membrane resulting in the presence of CA in
the filtrates. The gel also demonstrates that both Vivaflow and ultrafiltration
can be successfully employed to process the fractions obtained by IEC
fractionation of GA.
Example 11: Fractionation of CA (22.7 kDa, pd1.34) by IEC using
increasing ionic strength of triethanolamine/HCI
Polydispersed CA was also fractionated by Sepharose Q FF (1ml
matrix, prepacked) using a range of triethanolamine concentrations at pH 7.4
in the absence of any salt such as NaCI. Thus, CA (40mg; 1ml) (22.7kDa; pd
1.34) was loaded on to a Q FF column (1ml matrix; prepacked; Amersham
Biosciences). The bound CA was eluted by passing 1 ml of each
triethanolamine buffer (50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1100 and 1200mM triethanolamine) through the column at a flow rate
of 1ml/min with a final wash of the column using 2000mM triethanolamine.
The samples were directly lyophilised and then analysed by native PAGE
(TBE gel stained with alcian blue).
Results of Example 11: Figure 8 demonstrates that fractionation of
CA can be achieved in the presence of a varying concentration of
triethanolamine at pH 7.4.
Example 12: Fractionation of CA (22.7 kDa, pd 1.34) by (EC using
increasing ionic strength of triethanolamine acetate
Polydispersed CA was also fractionated by Sepharose Q FF (1ml
matrix, prepacked) using a range of triethanolamine acetate concentrations
at pH 7.4 in the absence of any salt such as NaCI. The triethanolamine
acetate buffer was prepared using triethanolamine and adjusting the pH to
7.4 using acetic acid. Polydispersed CA (40mg; 1 ml) (22.7kDa; pd 1.34) was
loaded on to a Q FF column (1ml matrix; prepacked). The bound CA was
eluted by passing 1 ml of each triethanolamine acetate buffer (300, 400, 500,
600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 and 1500mM
triethanolamine acetate) through the column at a flow rate of 1 ml/min with a
final wash of the column using 2000mM triethanolamine acetate. A small
sample of each fraction (2011) was then buffer exchanged with water using a
micro membrane dialysis system, lyophilised and then analysed by native
PAGE (20% TBE gel stained with alcian blue).
Results of Example 12: Figure 29 demonstrates that fractionation of
CA can be successfully achieved in the presence of a varying concentration
of triethanolamine at pH 7.4.
Example 13: Fractionation of CA (22.7 kDa, pd 1.34) using a pH
HEPES and ethanolamine buffer system was used to create a pH
step gradient. Buffers at pH 7.6, 7.8 and 8.0 were set up using increasing
concentrations of HEPES from 10 to 50mM and setting the pH to the
appropriate value with NaOH. The concentration of sodium ions did not
exceed 36mM, Buffers at pH 8.2, 8.3, 8.5, 8.7, 8.9, 9.1, 9.3, 9.5 and 9.7 were
created by mixing appropriate amounts of 1M ethanolamine (20 to 70mM
final concentration) with HEPES 50mM (10 to 50mM final concentration) and
setting the pH with NaOH, making sure that the sodium ion concentration did
not exceed 30mM. The final buffer was a 70mM ethanolamine pH 11.
30 mg of polydispersed CA (22.7 kDa; pd 1.34) was dissolved in pH
7.6 buffer (1ml_) and oaded onto a DEAE sepharose column (1ml matrix,
prepacked) also with the pH 7.6 buffer. The column was washed with 5ml of
the pH 7.6 buffer (flow rate 1ml/min) followed by passing 2ml of each buffer
through the column, collecting 1ml fractions. SOOil of each eluted fraction
was lyophilised and then re-dissolved in 50il of deionized water for analysis
by native PAGE (20% TBE gel stained with alcian blue).
Results of Example 12: Figure 30 demonstrates that fractionation of
CA can be successfully achieved in the presence of an increasing pH
strength. DEAE sepharose is an anion exchange matrix with a tertiary amino
group, /V-diethyl-amino-ethyl, which looses its charge at high pH. Thus, high
pH is used to deprotonate the matrix, change its charge and elute any
species bound by ionic interactions on the column. A mixed gradient can
also be used where a pH gradient is first used to fractionate the low
molecular weight species followed by an ionic strength gradient to elute the
higher m.w. species.
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1. A process for separating polydisperse ionically charged polysaccharide into fractions, in which an aqueous solution of the polydisperse polysaccharide is contacted with ion-exchange resin in a column, subjected to selective elution by aqueous elution buffer, and polysaccharide is recovered from eluted fractions, characterised in that the selective elution involves washing the resin in the column sequentially with at least three elution buffers each having different and constant ionic strength or pH or both ionic strength and pH in which the second and subsequent buffers have higher ionic strength or pH, or both ionic strength and pH than the buffers of the immediately preceding step, and in that eluted fractions each have different average molecular weight and a polydispersity of less than 1.5.
2. The process as claimed in claim 1, wherein each fraction has a polydispersity of less than 1.25, optionally wherein each fraction has a polydispersity of 1.1 or less.
3. The process as claimed in claim 1 wherein the elution buffers all have the same pH.
4. The process as claimed in claim 1 wherein the difference between the ionic strength of successive steps is substantially the same.
5. The process as claimed in claim 4 wherein the said difference is 0.005 to 0.1M.
6. The process as claimed in any preceding claim wherein the ionic strength is varied by varying the level of a salt of a strong mineral acid and a strong mineral base.
7. The process as claimed in claim 1 wherein the elution buffers have successively higher pH values.
8. The process as claimed in claim 7 wherein the difference between the pH of successive steps is substantially the same.
9. The process as claimed in claim 8 wherein the said difference is 0.2 pH units.
10. The process as claimed in any preceding claim wherein the pH of each buffer is in the range 7.4-13.
11. The process as claimed in any of claims 7 to 10 wherein the second and subsequent buffers have higher ionic strength and pH than the buffers of the immediately proceeding step.
12. The process as claimed in any preceding claim wherein each elution buffer is used in an amount of at least 1.0 column volumes.
13. The process as claimed in any preceding claim wherein the elution buffer optionally contains a volatile acid or base and in which the recovery involves volatilisation of the volatile acid or base from the eluted fractions.
14. The process as claimed in claim 13 wherein the buffer contains a base which is an amine.
15. The process as claimed in any preceding claim wherein the weight average molecular weight of the polydisperse polysaccharide is at least 5 kDa.
16. The process as claimed in any preceding claim wherein the selective elution involves at least five steps, optionally six to twelve steps.
17. The process as claimed in any preceding claim optionally comprising a preliminary step before contact with the ion-exchange resin of non-solvent precipitation.
18. The process as claimed in any preceding claim wherein the polysaccharide is a naturally occurring polysaccharide, a hydrolysis product thereof or a functionalized derivative of either.
19. The process as claimed in claim 18 wherein the naturally occurring polysaccharide is a bacterial polysaccharide.
20. The process as claimed in claim 18 or 19 wherein the polysaccharide is a sialic acid polymer or copolymer, wherein optionally the sialic acid polymer or copolymer is in a hydrolysed form.
21. The process as claimed in claim 20 wherein the polysaccharide is a poly (2,8-linked sialic acid), a poly(2,9-linked sialic acid) or an alternating 2,8-2,9- linked PSA.
22. The process as claimed in claim 20 wherein the polysaccharide is CA or an oxidised, reduced, aminated and/or hydrazide derivative thereof.
23. The process as claimed in any of claims 20 to 22 wherein each fraction has a polydispersity of less than 1.2.
24. The process as claimed in any preceding claim wherein the polydispersity of the polydisperse polysaccharide is at least 2.0.
25. The process as claimed in any preceding claim optionally comprising conjugating fractionated polysaccharide to a protein.
26. The process as claimed in any preceding claim for separating polydisperse ionically charged protein-polysaccharide conjugates.
27. A process as claimed in any preceding claim as and when used for obtaining a polysialic acid with a polydispersity of less than 1.26.
|Indian Patent Application Number||1099/DELNP/2007|
|PG Journal Number||38/2012|
|Date of Filing||09-Feb-2007|
|Name of Patentee||LIPOXEN TECHNOLOGIES LIMITED|
|Applicant Address||LONDON BIOSCIENCE INNOVATION CENTRE, 2 ROYAL COLLEGE STREET, LONDON NW1 0NH, UNITED KINGDOM|
|PCT International Classification Number||C08B 37/00|
|PCT International Application Number||PCT/GB2005/003149|
|PCT International Filing date||2005-08-12|