Note: Descriptions are shown in the official language in which they were submitted.
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Method for the preparation of immunoglobulins
DESCRIPTION
The present invention relates to a new method for the
preparation of immunoglobulins. The immunoglobulin
composition obtained is suitable, for example, for
parenteral administration.
Immunoglobulins are glycoproteins that can be found in
soluble form in the blood and other body fluids of
vertebrates, and are used by the immune system to identify
and neutralise foreign bodies such as bacteria, viruses or
parasites. Immunoglobulins have various medical
applications such as the diagnosis of diseases,
therapeutic treatments and prenatal therapy. The most
common therapeutic applications of immunoglobulins can be
classed in three general groups of pathologies: primary
immunodeficiencies (humoral immune deficiency), secondary
immunodeficiencies or acquired immunodeficiencies (for
example, in the prevention and treatment of virus
infections) and autoimmune immunodeficiencies (development
of antibodies).
Immunoglobulins can be administered by various routes such
as the intramuscular, intravenous and subcutaneous routes,
among others. Of these, it is preferable to use the
intravenous route, since it offers numerous advantages,
particularly greater therapeutic efficacy.
Immunoglobulins are usually purified from human plasma by
using procedures based on the Cohn fractionation method
(Cohn EJ. et al., J Am Chem Soo, 1946, 62, 459-475), the
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Cohn-Oncley method (Oncley JL. et al., J Am Chem Soc,
1949, 71, 541-550) or other equivalent methods based on
cold ethanol fractionation, for example the Kistler-
Nitschmann method (Kistler P, Nitschmann H, 1962, 7, 414-
424). Thus, using fractions rich in immunoglobulins (such
as fraction II+III, or fraction II, or precipitate A, or
gamma globulin GG precipitate) obtained by any of the
above methods. Modifications have been introduced in order
to purify the immunoglobulins more exhaustively (IgG) and
make them tolerable for administration, preferably
intravenously. The said modifications have been
introduced, for example, in order to remove aggregates and
other impurities, as well as to ensure the safety of the
product. However, the addition of multiple steps to the
procedure for the preparation of immunoglobulins reduces
the yield of the procedure and increases manufacturing
costs. Growing demand for immunoglobulin products, mainly
for intravenous administration, has made yield a critical
aspect in the process of producing them on an industrial
scale.
Of the methods described in the prior art, the procedures
for obtaining immunoglobulin compositions that are
tolerable via the intravenous route include those that use
the following steps: precipitation with polyethylene
glycol (PEG), ion-exchange
chromatography,
physical/chemical methods with the capacity for viral
inactivation, or treatment with enzymes and partial
chemical modification of the immunoglobulin molecules.
Thus, it is necessary to ensure the safety of the product
by implementing robust steps with the ability to eliminate
pathogenic biological agents. The method generally used
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involves the use of a solvent/detergent to inactivate
viruses with a lipid envelope, since this does not
severely reduce the biological activity of the proteins.
However, given the toxicity of solvent/detergent mixtures,
this reagent must be extensively eliminated before
obtaining the final product, and this increases the time
required for the process and reduces the yield. The
procedures described for the elimination of the said
solvent/detergent are not simple and usually require the
use of chromatography adsorption techniques, either
directly by hydrophobic interaction or by indirect capture
of the immunoglobulin in ion-exchange resins and
separation of the untrapped solvent/detergent. In all
cases, the processes are costly and laborious, involving
significant losses of protein.
However, simpler and more efficient alternative treatments
with the ability to inactivate viruses are known in the
state of the art. For example, caprylic fatty acid (also
known as octanoic acid) or salts of the same have been
used.
In patent US4446134, sodium caprylate is used in
combination with amino acids and heat treatment as a viral
inactivation procedure in a method for the preparation of
factor VIII. Although it is believed that the virucidal
agent capable of disintegrating the lipid membranes is
undissociated caprylic acid, the procedure that uses the
said agent is commonly known as inactivation by caprylate,
in accordance with the biochemical convention of denoting
a solution of an acid and its ionised form with the name
of the latter, i.e. caprylate.
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Caprylic acid has also been used as a precipitation agent
for purifying immunoglobulins (Steinbuch, M. et al., Arch.
Biochem. Biophys., 1969, 134(2), 279-284). The purity of
the immunoglobulins and the yield depend mainly on the
concentration of caprylic acid added and the pH.
Steinbuch, M. et al. also state that it is advantageous to
add an effective quantity of caprylate in two different
steps, with elimination of the precipitate between the two
steps. This would give the procedure the ability to
eliminate viruses both with and without envelopes, thanks
to the distribution of non-immunoglobulin proteins in the
precipitate.
Descriptions are also found in the state of the art of the
combination of precipitation with caprylate followed by
ion-exchange chromatography for the purification of
immunoglobulins (Steinbuch, M. et al., v. supra).
European patent EP08934.50 discloses a method for the
purification of IgG using fraction II+III (obtained by
means of procedures based on the Cohn method mentioned
previously), including two anionic exchange columns in
series after the steps of adding caprylate at a
concentration of 15-25 mM in a double precipitation step
and combining both effects of the caprylate: the reduction
of non-immunoglobulin proteins by precipitation, and the
capacity for viral inactivation by means of incubation.
The subsequent anionic exchange steps, in addition to
removing other impurities (IgM, IgA, albumin and others),
are used to eliminate the caprylate, and for this reason
double adsorption is required, using relatively large
quantities of anionic resins.
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Patent application PCT W02005/082937 also discloses a
method for the preparation of a composition that includes
immunoglobulins and that comprises the steps of adding
caprylate and/or heptanoate to the solution or composition
5 that comprises immunoglobulins and, subsequently, applying
the said solution in a column with anionic exchange resin.
However, the present inventors have realized that the use
of caprylate at an appropriate concentration and pH (for
example, pH 5.0-5.2) in order to provide the treatment
with viral inactivation capacity, as has been described in
the prior art, causes the formation of protein aggregates
with a high molecular weight, which are partially
irreversible by dilution and/or change of pH. Furthermore,
these aggregates are only partially separable by
filtration, and therefore require a specific subsequent
step of separation, for example by means of chromatography
or precipitation. The separation of these aggregates
causes significant losses of protein and a reduction in
the yield of the industrial process of immunoglobulin
production.
In addition, the present inventors have realized that the
presence of aggregates formed during the treatment with
caprylate, even at very low levels, hinders the correct
elimination of the caprylate by the direct application of
a step of separation using an ultrafiltration membrane
under optimal process conditions. These aggregates hinder
Or prevent the preparation of a solution of
immunoglobulins at therapeutic concentrations (for
example, between 5% and 20%) due to the presence of
colloids (turbidity) or instability in the liquid form,
thus hindering or preventing subsequent steps of the
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method for the preparation of immunoglobulins, such as
nanofiltration and sterilising filtration.
As a consequence of the above, the present inventors have
developed a method for the preparation of immunoglobulin
solutions which, surprisingly, includes a caprylate
treatment with the capacity for viral inactivation at a
lower concentration of caprylate than that described in
the prior art and which, the initial solution being
suitably purified and diluted, and in the presence of at
least one polyether or polymer of glycol, inhibits,
prevents, avoids or does not promote the appearance of
aggregates.
In addition, the present inventors have discovered that
the presence of at least one polyether or polymer of
glycol in the method according to the present invention
does not interfere with the activity and efficacy of the
caprylate in terms of its capacity for inactivating
enveloped viruses.
In an additional aspect, the present inventors describe
for the first time a method for obtaining immunoglobulins
which, as well as including the treatment with
inactivation capacity under optimal conditions,
contemplates the possibility of eliminating or reducing
the caprylate and polyether or polymer of glycol reagents
(previously present during the said treatment) by using
only the ultrafiltration technique. This ultrafiltration
step makes it possible to purify and concentrate the
product to levels that are tolerable for its
administration, for example via the intravenous,
intramuscular or subcutaneous route, without producing
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immunoglobulin protein aggregates in the final product.
This eliminates the need to introduce additional
separation steps after the treatment with caprylate, such
as, for example, chromatography. Moreover, the remnant
levels of polyether or polymer of glycol and caprylate
after the ultrafiltration make it possible to achieve
concentrations of immunoglobulins, for example IgGs, of up
to 20 + 2%, which, if correctly formulated, do not
destabilise during their conservation in liquid form.
Given the simplification of the method according to the
present invention, this makes it possible to substantially
improve the yield and very significantly reduce the
production costs compared with the previous methods
described in the prior art, without thereby compromising
the level of safety or purity of the product.
Therefore, in a first aspect, the present invention
relates to a method for the preparation of a solution of
immunoglobulins that comprises the addition of caprylic
acid or salts of the same, in the presence of at least one
polyether or polymer of glycol, to the purified solution
of immunoglobulins, and the subsequent elimination or
reduction of the said reagents by means of
ultrafiltration/diafiltration.
In an additional aspect, the present invention relates to
the use of caprylic acid or salts of the same, in the
presence of at least one polyether or polymer of glycol,
for viral inactivation in protein production processes,
and the subsequent elimination or reduction of the said
reagents by means of ultrafiltration/diafiltration.
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In a further aspect, the present invention relates to the
implementation of a single stop of
ultrafiltration/diafiltration for the elimination or
reduction of the levels of caprylic acid or salts of the
same and/or the polyether or polymer of glycol used for
viral inactivation in protein production processes.
Therefore, the present invention discloses a method for
the preparation of a solution of immunoglobulins based on
an initial solution of immunoglobulins with a purity
greater than or equal to 96% in the presence of a
polyether or polymer of glycol, characterised in that it
comprises the steps of:
a) adding caprylic acid or salts of the same to the
initial solution;
b) adjusting the pH of the solution obtained in step a);
C) incubating the solution obtained in step b) for the
time and at the temperature necessary for the inactivation
of enveloped viruses; and
d) performing a step of ultrafiltration/diafiltration on
the solution obtained in step c).
The method according to the present invention may also
comprise a step of final formulation of the solution
obtained in step d).
In the method according to the present invention, the
initial solution of immunoglobulins is derived from
fraction I+II+III, fraction or fraction II,
obtained according to the Cohn or Cohn-Oncley method, or
from precipitate A or I+A or GG, obtained according to the
Kistler-Nitschmann method, or variations on the same,
9
which have been additionally purified to obtain an IgG purity
greater than or equal to 96%. Preferably, the initial solution of
immunoglobulins is derived from fraction obtained according to
the Cohn method or variations on the same, which has been
subsequently purified by means of precipitation with PEG and
anionic chromatography, as described in the document EP1225180B1.
According to the present patent, any of the above fractions could
be subjected to a precipitation procedure 10 using PEG, followed
by filtration in order to eliminate the precipitate and an
additional purification step using an ionic exchange column (for
example, a column with DEAE SepharoseTm). In all of these cases,
the initial solution of immunoglobulins is derived from human
plasma.
In the most preferred embodiment, the initial solution of
immunoglobulins is derived from fraction II + III obtained by
procedures based on the Cohn method, which is additionally purified
by any one of the methods described in the prior art to achieve
an adequate level of purification to be subjected to the treatment
with caprylate under the non-precipitating conditions of the
present invention, i.e. a purity value greater than or equal to
96% (w/v) of IgG determined by electrophoresis in cellulose
acetate, with an albumin content preferably less than or equal to
1% (w/v) with respect to the total proteins. Thus, the said initial
solution of immunoglobulins is sufficiently purified, before and
after the treatment with caprylate, for the route of therapeutic
administration for which it is intended, so that no additional
purification is required after the step with viral inactivation
capacity of the present invention.
Date Recue/Date Received 2020-09-01
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The immunoglobulins of the initial solution of the method
according to the present invention can also be obtained by
genetic recombination techniques, for example by
expression in cell cultures; chemical synthesis
5 techniques; or transgenic protein production techniques.
In the most preferred embodiment, the immunoglobulins
mentioned in the method according to the present invention
are IgGs. It is contemplated that the said IgGs may be
10 monoclonal or polyclonal. In the most preferred
embodiment, the IgGs are polyclonal.
It is contemplated that the polyethers or polymers of
glycol of the present invention may be polyethers of
alkane or oxides of polyalkane, also known as polyglycols,
and refer, for example, to derivatives of ethyl or
ethylene and propyl or propylene, better known as
polyethylene glycol (PEG) or polypropylene glycol (PPG),
or equivalents of the same. In addition, the said reagents
must be compatible with the immunoglobulins in the sense
that they do not compromise their stability or solubility
and that, due to their size, they can be favourably
eliminated by ultrafiltration techniques, or that, due to
their lower toxicity, they are compatible with therapeutic
use of the immunoglobulins.
In a preferred embodiment, the polyether or polymer of
glycol is selected from polyethylene glycol (PEG),
polypropylene glycol (PPG) or combinations of the same.
Preferably, the polyether or polymer of glycol is PEG,
more preferably a PEG with a nominal molecular weight of
between 3350 Da and 4000 Da, and most preferably a PEG
with a nominal molecular weight of 4000 Da.
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The content of the above-mentioned polyether or polymer of
glycol in the initial solution of immunoglobulins is
preferably between 2% and 6% (w/v), and more preferably
between 3% and 5% (w/v).
It is contemplated that it may possibly be necessary to
adjust the concentration of the said polyether or polymer
of glycol in the initial solution of immunoglobulins. The
said adjustment of the said polyether or polymer of glycol
can be effected by diluting the initial purified solution
of immunoglobulins and/or by adding the same.
According to the composition of the initial solution of
immunoglobulins, it is contemplated that, before step a)
of the method according to the present invention, a series
of steps of purification or adjustment of concentrations
are carried out, such as, for example:
- adjustment of the concentration of immunoglobulins to
between 1 and 10 mg/ml, more preferably between 3 and 7
mg/ml. This adjustment can be effected by any of the
procedures known in the state of the art, for example by
dilution or concentration of the protein to the
established range (determined, for example, according to
total protein by optical density at 280 nm E(1%) = 13.8 -
14.0 UA, by the Biuret method, by the Bradford method, or
specifically by immunonephelometry), as the case may be.
Therefore, in a preferred embodiment, the initial solution
of immunoglobulins has a concentration of immunoglobulins
preferably between 1 and 10 mg/ml, and more preferably
between 3 and 7 mg/m1; and/or
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- adjustment of the purity of the solution of
immunoglobulins, which should preferably reach at least
96% of IgG with respect to the total proteins. This
purification can be effected by techniques fully known to
a person skilled in the art, such as, for example, by
precipitation with PEG, and filtration and subsequent
anionic exchange chromatography (DEAE Sepharose).
In step a) of the method according to the present
invention, caprylic acid or salts of the same are added,
preferably using a concentrated solution of the same, for
example between 1.5M and 2.5M, to achieve a final
concentration preferably between 9 mM and 15 mM.
In a preferred embodiment, in step b), the solution
obtained is adjusted to a pH between 5.0 and 5.2, more
preferably to 5.1.
Iii a preferred embodiment, in step c), the solution
obtained is incubated for at least 10 minutes, more
preferably between 1 and 2 hours, and still more
preferably 2 hours. In addition, the temperature at which
the said incubation is carried out is between 2 C and
37 C, more preferably between 20 C and 30 C.
In a preferred embodiment, before step d) of the method
according to the present invention, the content of
polymers or aggregates with a high molecular weight in the
solution obtained in the said step c) is less than or
equal to 0.2%, and more preferably less than 0.1%. This
percentage of polymers or molecular aggregates of
immunoglobulins with respect to the total proteins is
determined by exclusion HPLC gel column according to the
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optical density value at 280 nm. The said percentage of
polymers or molecular aggregates of immunoglobulins can be
evaluated, for example, using the analysis method
described in the monograph on intravenous gammaglobulin of
the European Pharmacopoeia.
Preferably, the solution of immunoglobulins is clarified
using depth filters before performing step d) of
ultrafiltration/diafiltration.
With respect to step d), it is contemplated, preferably,
that the ultrafiltration/diafiltration in the method
according to the present invention has initial steps of
diafiltration and concentration by reduction of volume,
followed by the application of diafiltration at constant
volume.
The ultrafiltration/diafiltration can be carried out on an
industrial scale preferably by the method of simultaneous
dialysis and concentration, reducing the volume of product
and diafiltering in turn, so that the consumption of
reagents is somewhat lower and the process more efficient,
taking account of the fact that the concentration of
proteins is optimal and preferably less than or equal to
30 mg/ml. In any event, a person skilled in the art can
easily determine the most appropriate and practical way of
performing this step of ultrafiltration/diafiltration,
choosing from among the various operating procedures known
in the state of the art (for example,
dilution/concentration or diafiltration/concentration,
diafiltration at constant volume, or modifications and
combinations of the above).
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The ultrafiltration/diafiltration membrane used in step d)
of the method according to the present invention
preferably consists of polysulphone, regenerated cellulose
or equivalents, such as, for example, the membranes
marketed under the brands Biomax (Millipore, USA), Omega
(Pall, USA), Kvik-flow (General Electric, USA). However,
the molecular weight cut-off chosen for the membrane may
vary depending on various factors, for example the
manufacturer of choice. A person skilled in the art can
easily determine the membrane of choice, which will be
adjusted to the needs of each case depending, for example,
an the concentration of caprylate and of the polyether or
polymer of glycol in the solution to be processed.
Preferably, step d) of ultrafiltration/diafiltration is
effected by means of a membrane with a molecular weight
cut-off of less than or equal to 100kDa, more preferably
of 100 kDa.
In the most preferable embodiment, the
ultrafiltration/diafiltration of step d) is performed in
two phases:
a first phase in which the pH is adjusted to between 5.0
and 6.0 in order to reduce or eliminate most of the
caprylate, and a second phase in which the pH is adjusted
to less than 5.0, preferably to a pH of between 4.0 and
5.0, in order to reduce or eliminate most of the polyether
or polymer of glycol.
In a preferred embodiment, in the first phase of the step
of ultrafiltration/diafiltration, the diafiltration is
performed using a diafiltration medium that comprises
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alkaline salts of carboxylic acid, for example acetic
acid, at a concentration greater than or equal to 5 mM
approximately. In the most preferable embodiment, the
aforesaid diafiltration is performed using a solution of
5 sodium acetate at a concentration greater than or equal to
5 mM adjusted to the pH mentioned above, i.e. between 5.0
and 6Ø
The number of diafiltration volumes to be performed in the
10 first phase of step d) of ultrafiltration/diafiltration
can be easily determined by a person skilled in the art
according to the quantity of caprylate used initially and
the acceptable final quantity. Preferably, at least three
volumes of the diafiltration medium are used, the said
15 diafiltration medium preferably being, as mentioned
previously, a 5 mM solution of sodium acetate at pH 5.0-
6Ø Preferably, in this first phase of the
ultrafiltration/diafiltration, approximately 90% or more
of the initial caprylate is eliminated, so that in this
first phase the concentration of caprylate is reduced to
approximately 1 mM or less.
In the second phase of step d) of
ultrafiltration/diafiltration, the solution of
immunoglobulins is diafiltered, preferably at constant
volume.
Preferably, the diafiltration in the said second phase of
the ultrafiltration/diafiltration is performed using a
buffered solution that contains alkaline metal salts
formed by acetate, phosphate or equivalents, or amino
acids and/or polyols, for example glycine and/or sorbitol
at the pH value indicated previously.
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As in the case of the first phase of the diafiltration, in
the second phase the number of dialysis volumes used to
suitably reduce the polyether or polymer of glycol used in
the method according to the present invention can be
easily determined by a person skilled in the art taking
account of the required reduction or elimination of the
polyether or polymer of glycol. In a preferred embodiment,
the quantity of buffer to be exchanged in the
diafiltration of the second phase of step d) of
ultrafiltration/diafiltration is equal to or greater than
six volumes. In the most preferred embodiment, in the said
second phase, the exchange is carried out according to the
number of volumes of buffer necessary to obtain a
reduction in the polyether or polymer of glycol equal to
or greater than 100 times the initial content of the said
polyether or polymer of glycol before beginning step d) of
ultrafiltration/diafiltration.
Once the caprylate and the polyether or polymer of glycol
have been reduced in step d) of
ultrafiltration/diafiltration, in the final formulation
step mentioned previously the solution can be adjusted to
the desired final composition by adding the necessary
excipients and/or stabilisers, so as to concentrate the
product in order to achieve the final formulation. The
addition of the excipients and/or stabilisers to be
carried out after the final formulation can be effected
directly by adding the said excipients and/or stabilisers
in solid form or in a concentrated solution or, still more
preferably, by means of diafiltration employing the
necessary number of exchange volumes of a formulation
solution to ensure the appropriate composition of the
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final product.
In another embodiment, the addition of the excipients
and/or stabilisers is carried out by wholly or partially
replacing the dialysis buffer solution of sodium acetate
used in the second phase of step d) with a solution
comprising the excipients and/or stabilisers, adjusted
preferably to the same pH value of between 4.0 and 5.0 so
that after the final concentration the immunoglobulin is
already formulated.
A person skilled in the art knows which types of
excipients and/or stabilisers must be added in order to
achieve a desired stability. It is contemplated, for
example, that the said excipients and/or stabilisers may
be one or more amino acids, for example glycine,
preferably at a concentration of between 0.2 and 0.3 M;
one or more carbohydrates or polyols, for example
sorbitol; or combinations of the same
Finally, the final concentration of immunoglobulins,
preferably IgGs, is adjusted to a concentration suitable
for its intravenous, intramuscular or subcutaneous use,
which will be known to a person skilled in the art and
may, for example, be between 5% and 22% (w/v). The said
concentration is effected by any procedure known in the
state of the art, for example concentration by
ultrafiltration. It is contemplated that if the
concentration of the immunoglobulins is effected by
ultrafiltration, the said concentration may be carried out
using the same membrane as in the previous diafiltration.
Obviously, the three diafiltrations mentioned, as well as
the concentration, may also be carried out using different
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membranes.
The method according to the present invention also
contemplates the possibility of introducing a step of
nanofiltration in order to increase the safety margin of
the product. There are multiple phases in the procedure in
which the product can be nanofiltered with commercially
available filters (for example, Planova and Bioex made
by Asahi-Kasei, DV and SV4 made by Pall, Virosart made
by Sartorius, Vpro made by Millipore, or equivalents)
with pore sizes from 20 nm or less and up to 50 nm,
preferably with pore sizes of 20 nm or less, or even
nanofilters of 15 nm can be used. The intermediate steps
in which a nanofiltratiion step can be carried out are,
for example, in the initial solution of immunoglobulins;
or in the material treated with caprylate after the step
of ultrafiltration/diafiltration (once the caprylate and
the polyether or polymer of glycol have been reduced); or
in the material after concentrating and formulating the
solution of immunoglobulins, preferably IgGs (final
product). A person skilled in the art will select the best
option depending on, among other things, the pore size of
the membrane, the filtration area required according to
the time of the procedure, the volume of product to be
nanofiltered, and the protein recovery.
The final product obtained by the method according to the
present invention complies in full with the criteria of
the European Pharmacopoeia in relation to the content of
isohemagglutinins. However, the method according to the
present invention also contemplates the option of
including a step of selective and specific capture of
anti-A and/or anti-B blood antibodies in order to maximise
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their reduction. This step is preferably carried out using
biospecific affinity resins, as has been described in the
state of the art. For example, by using biospecific
affinity resins with ligands formed by trisaccharides, a
significant reduction in the level of isohemagglutinins
can be achieved (Spalter et al., Blood, 1999, 93, 4418-
4424). This additional capture may optionally be
incorporated, at the discretion of the person skilled in
the art, in any step of the method of the present
invention, or may be done before or after carrying out the
method of the present invention.
Therefore, with respect to the method for the preparation
of a solution of immunoglobulins according to the present
invention, in the most preferred embodiment an initial
solution of immunoglobulins with a purity greater than or
equal to 96% of IgGs is used. This solution is adjusted to
a concentration of IgGs preferably between 1 mg/ml and 10
mg/ml, and preferably between 3 mg/ml and 7 mg/ml, which
contains (by addition in previous steps) or to which is
added PEG to a concentration of 4 + 1%. (w/v). The pH of
the solution is then adjusted to between 5.0 and 5.2 with
acetic acid, and sodium caprylate is added (for example,
using a concentrated solution of the said sodium
caprylate). In the preferred embodiment, the concentrated
solution of caprylate is added to the purified solution of
IgGs, slowly and with agitation. After adding all the
caprylate calculated to bring the product to the final
concentration of between 9 and 15 mM of caprylate, the
final pH is then adjusted, if necessary, to between 5.0
and 5.2, and the solution is incubated preferably at a
temperature of between 2-37 C, and more preferably at a
temperature of 25 + 5 C, for at least 10 minutes, and
20
preferably for between 1 and 2 hours.
Clarification is then performed using depth filters (for example,
Cuno'm 90LA, 50LA, Seitz'm EK, EK-1, EKS, or 5 equivalents).
The solution thus obtained is then processed by means of an
ultrafiltration/diafiltration equipment formed by membranes
comprising polysulphone, for example Biomax made by Millipore
or Omega from Pall, preferably in the form of a stackable cassette.
The solution is recirculated through
each
ultrafiltration/diafiltration unit, preferably at a volume of
between 100-500 L/h approximately and at a temperature of 5 3 C.
The pressure drop between the inlet and outlet pressures
(atmospheric pressure) is preferably between 1 and 3 bar.
Next, the first diafiltration phase of the step of
ultrafiltration/diafiltration is begun in order to eliminate
the caprylate, preferably applying an exchange of at least
three volumes of buffer formed preferably by a solution of sodium
acetate at a concentration equal to or greater than 5 mM and at a
pH of between 5.0 and 6Ø Preferably, with each volume of buffer
added or consumed, the volume of the solution of product is reduced
to half of the initial volume, except for the last addition.
After the first phase of diafiltration (by dilution and
concentration or equivalent), the pH of the solution obtained
is adjusted to between 4.0 and 5.0 using, for example, acetic
acid. Diafiltration at constant volume is then begun, preferably
using six or more volumes of a buffer solution formed by sodium
acetate at a concentration equal to or greater than 5 mM and at
a pH of
Date Recue/Date Received 2020-09-01
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between 4.0 and 5Ø
The above-mentioned dialysis buffer solution formed by
sodium acetate may optionally be wholly or partially
replaced by a solution of amino acids, for example glycine
at a concentration of 0.2-0.3 M, optionally combined with
carbohydrates and polyols, for example sorbitol, adjusted
preferably to the same pH value of between 4.0 and 5.0 so
that after the final concentration the immunoglobulin is
already formulated.
After applying preferably at least six volumes (more
preferably between six and ten volumes) of the above-
mentioned dialysis solutions at a pH of between 4.0 and
5.0, the product can be formulated, if this is not already
the case, by directly adding excipient/s and/or
stabiliser/s to the solution obtained, such as, for
example, glycine or other amino acids, as well as
carbohydrates, for example sorbitol, or a combination of
the same, in the solid state or in the form of a
concentrated solution of the said excipient/s and/or
stabiliser/s. Next, the solution of IgGs obtained by
volume reduction is concentrated to achieve the
appropriate IgG concentration for intravenous,
intramuscular or subcutaneous use.
The said concentrated solution, suitably adjusted with
respect to the concentration of excipient/s and/or
stabiliser/s and the pH, is applied by absolute filtration
using filters with a pore size of 0.2 pim, and is
optionally nanofiltered. Finally, the solution of IgGs is
aseptically dosified in injectable preparations, ampoules,
vials, bottles or other glass containers, which are then
CA 2943328 2019-08-28
22
hermetically sealed. Another option is dosification in
compatible rigid or flexible plastic containers, for
example bags or bottles.
The dosified product goes through quarantine and visual
inspection before being put into storage at a temperature
between 2 and 30 C for conservation up to at least 2
years.
Moreover, as mentioned previously, the present invention
also discloses for the first time the use of caprylic acid
or salts of the same in the presence of at least one
polyether or polymer of glycol for viral inactivation in
protein production processes, in which the said polyether
or polymer of glycol and the caprylic acid or salts of the
same are subsequently eliminated by means of
ultrafiltration.
Preferably, the said proteins are selected from the group
of proteins that comprises immunoglobulins; albumin;
coagulation factors such as factor VII, factor VIII and
factor IX; and von Willebrand factor. Still more
preferably, the said proteins are immunoglobulins. In the
most preferred embodiment, the said proteins are IgGs.
Described herein are one or more of the following items:
1. A method for the preparation of a solution of
immunoglobulins based on an initial solution of
immunoglobulins with a purity greater than or equal
to 96% in the presence of a polyether or polymer of
glycol, the method comprising:
(a) adding caprylic acid or a salt thereof to the
initial solution at a concentration between 9 mM
22a
and 15 mM;
(b) adjusting the pH of the solution obtained in (a)
to a pH between 5.0 and 5.2;
(c) incubating the solution obtained in (b) for the
time and at a temperature necessary for the
inactivation of enveloped viruses; and
(d) performing a step of
ultrafiltration/diafiltration on the solution
obtained in (c).
2. The method according to item 1, wherein the initial
solution of immunoglobulins is derived from fraction
I+II+III, fraction II+III, or fraction II, obtained
according to the Cohn or Cohn-Oncley method, or from
precipitate A or I+A or GG, obtained according to the
Kistler-Nitschmann method, or variations on the same,
which have been additionally purified to obtain a
purity greater than or equal to 96% of IgG.
3. The method according to item 2, wherein the initial
solution of immunoglobulins is derived from fraction
II+III obtained according to the Cohn method or
variations on the same, which has been subsequently
purified by means of precipitation with PEG and
anionic chromatography.
4. The method according to any one of items 1 to 3,
wherein the initial solution of immunoglobulins has a
concentration of immunoglobulins between 1 and 10
mg/ml.
5. The method according to any one of items 1 to 3,
wherein the initial solution of immunoglobulins has a
concentration of immunoglobulins between 3 and 7
mg/ml.
6. The method according to any one of items 1 to 5,
CA 2943328 2019-08-09
22b
wherein the polyether or polymer of glycol is
polyethylene glycol (PEG), polypropylene glycol
(PPG), or a combination thereof.
7. The method according to item 6, wherein the
concentration of PEG in the initial solution is
between 2% and 6% (w/v).
8. The method according to item 6, wherein the
concentration of PEG in the initial solution is
between 3% and 5% (w/v).
9. The method according to any one of items 6 to 8,
wherein the PEG is PEG with a nominal molecular
weight of 4000 Da.
10. The method according to any one of items 1 to 9,
wherein the solution obtained in (b) is adjusted to a
pH of 5.1.
11. The method according to any one of items 1 to 10,
wherein the solution in (c) is incubated for at least
10 minutes at a temperature between 2 C and 37 C.
12. The method according to any one of items 1 to 10,
wherein the solution in (c) is incubated for 2 hours
at a temperature between 20 C and 30 C.
13. The method according to any one of items 1 to 12,
wherein the initial solution of immunoglobulins has
an albumin content less than or equal to 1% (w/v)
with respect to the total proteins.
14. The method according to any one of items 1 to 13,
wherein the initial solution of immunoglobulins is
derived from human plasma.
15. The method according to any one of items 1 to 14,
wherein the immunoglobulins of the initial solution
of immunoglobulins are obtained by genetic
recombination techniques, chemical synthesis
techniques or transgenic protein production
CA 2943328 2019-08-09
22c
techniques, or in cell cultures.
16. The method according to any one of items 1 to 15, wherein the
step of ultrafiltration/diafiltration in (d) is carried out
using a membrane of 100 kDa.
17. The method according to any one of items 1 to 16, wherein the
ultrafiltration/diafiltration in (d) is carried out in two
phases:
- a first phase in which the pH is adjusted to between 5.0
and 6.0 in order to reduce or eliminate most of the
caprylate; and
- a second phase in which the pH is adjusted to a value less
than or equal to 5.0, in order to reduce or eliminate most
of the polyether or polymer of glycol.
18. The method according to item 17, wherein in the second phase,
the pH is adjusted to between 4.0 and 5Ø
19. The method according to any one of items 1 to 18, wherein
said method further comprises a step of final formulation of
the solution of immunoglobulins obtained in (d).
20. The method according to item 19, wherein in the step of final
formulation, excipients and/or stabilizers are added.
21. The method according to item 20, wherein the excipients and/or
stabilizers comprise: one or more amino acids, one or more
carbohydrates or polyols, or any combination thereof.
22. The method according to any one of items 1 to 21, wherein the
solution of immunoglobulins is adjusted to a final
concentration suitable for intravenous, intramuscular, or
subcutaneous use.
Date Recue/Date Received 2020-09-01
22d
The present invention will now be described in greater
detail with reference to various examples of embodiment.
However, these examples are not intended to limit the
scope of the present invention, but only to illustrate its
description. ____________________________________________________
CA 2943328 2019-08-09
CA 02943328 2016-09-26
23
EXAMPLES
Example 1. Method according to the present invention for
obtaining, from plasma, a solution of immunoglobulins that
is virally safe, free from aggregates and with an adequate
yield for industrial application.
The starting material was 16 litres of a solution of
immunoglobulins, which contained IgGs as the majority
protein component, obtained by the method described in
European Patent EP1225180B1. In summary, the said solution
was obtained by extracting gammaglobulin from fraction
II+III using the Cohn method. In order to perform this
extraction of gammaglobulin from fraction 11+111, the said
fraction was previously isolated by fractionation of human
plasma using ethanol. It was then suspended in the
presence of a carbohydrate, and the content of the
accompanying majority proteins was reduced by
precipitation with DEC-4000. Lastly, final purification of
the fraction was performed by adsorption in an ion-
exchange resin column (DEAE Sepharose). The column
effluent thus obtained (fraction not adsorbed in the
resin, i.e. the DEAE) had an electrophoretic purity in
cellulose acetate (ACE) of immunoglobulins of 98 + 2%, a
pH of 6.0, a turbidity of 2.6 Nephelometric Turbidity
Units (NTU) and an IgG concentration of approximately 5
mg/ml.
The solution obtained was adjusted to a pH of 5.1 by
adding acetic acid, and to a temperature of between 2 and
8 C. This solution of immunoglobulin was then brought to a
final concentration of 13 mM by adding a concentrated
solution of sodium caprylate.
CA 02943328 2016-09-26
24
The solution of immunoglobulins with caprylate was heated
to 25 C and incubated at this temperature for 2 hours
under slow agitation. During the incubation procedure, the
pH was maintained at 5.10 + 0.05. The turbidity of the
resulting solution was 17.3 NTU.
The solution treated with caprylate was cooled to an
approximate temperature of 8 C for subsequent
clarification using a depth filter (CUNO, Ultrafilter,
Denmark). Some 20 litres of filtered liquid were obtained
from the said clarification (including rinsing), with an
IgG concentration of approximately 4 mg/ml and a turbidity
of less than 3 NTU.
The above-mentioned clarified solution was dialysed by
ultrafiltration using membranes with a nominal molecular
weight cut-off of 100kDa (Biomax% Millipore, USA). The
ulLrafiltration was carried out in two differentiated
phases: in the first phase, the material, which had a pH
of 5.1, was subjected to three steps of sequential
dialysis and concentration, by means of diafiltration
using a 5 mM solution of acetate adjusted to pH 5.1 and by
means of concentration to approximately 30 UA. In the
second phase, the solution, which had an adequate
concentration of proteins, caprylate and PEG, Was brought
to pH 4.5 + 0.1 and dialysis was then begun using eight
volumes of 5mM solution of acetate at pH 4.5. Next, the
product was formulated by means of dialysis using
approximately 20 litres of 200 mM glycine solution at pH
4.2, and was concentrated in the same ultrafiltration unit
to a value of 140.5 UA with the aim of obtaining a
solution of IgGs with a concentration of 10% (w/v).
CA 02943328 2016-09-26
Finally, the said solution was filtered using a depth
filter (CtJNO , Ultrafilter, Denmark) and absolute filters
or membranes with a pore size of 0.22 um (CVGL ,
5 Millipore, USA; or DEL , PALL, USA).
Table 1 shows the characterisation of the starting
material, multiple intermediate products and the final
product, according to the method described above. With
10 respect to the results included in the said table, it
should be noted that the turbidity was measured by
nephelometry; the percentage of polymer or molecular
aggregates of immunoglobulins, with respect to the total
proteins detected, was determined by exclusion HPLC gel
15 column according to the optical density value at 280 nm;
the concentration of caprylate was determined using an
enzymatic method by quantification of colorimetric
substrate; the concentration of PEG was determined by
means of an HPLC filtration gel column using a refractive
20 index detector; and the percentage recovery of the process
was calculated according to the concentration of IgG
quantified by nephelometry.
The results of this example show that the treatment with
25 caprylate of the above-mentioned purified solution does
not induce any formation of immunoglobulin aggregates or
other precipitates, maintaining unchanged the molecular
distribution of the product. Consequently, after the
treatment with caprylate, no purification steps were
necessary in order to eliminate aggregates and/or
precipitates. This fact greatly facilitated the production
process and allowed the direct application of the material
to the ultrafiltration membrane.
CA 02943328 2016-09-26
26
Table 1. Results obtained for the starting material, multiple
intermediate products and
the final product in the method of Example 1.
sample ' IgG Turbidity Aggregates Caprylate PEG
Recoveryl
Concentration (NTU) (Polymer/Dimer)Concentration oncentration
(%)
(mg/mi) (%) (mm) (mg/m1)
Starting 4.6 2.6 <0.1/3.0 0 38.4 100
material
(ion-
exchange
column
effluent,
already
adjusted
to pH
3.1)
Material 4.3 17.3 <0.1/3.0 12.1 38.2 97.8
treated
with
,:.aprylate
Clarified 3.7 2.2 <0.1/2.8 11.1 38.0 95.2
dacerial
Final 99.1 3.5 <0.1/2.7 0.1 0.1 89.4
product
Thus, the subsequent ultrafiltration process
satisfactorily achieved the objective of efficiently
reducing the chemical reagents of the manufacturing
process (i.e. PEG and caprylate), as well as allowing the
subsequent formulation and concentration of the purified
solution of immunoglobulin to obtain the appropriate
composition for its therapeutic use.
As can be seen in Table 1, the protein recovery obtained
in this case, from the starting effluent to the 10%
concentrated product, was 89.4%, showing the viability of
this process on an industrial scale. This recovery was
greater than the value obtained by conventional methods
27
according to the state of the art and as described in PCT patent
application publication No. W02005/073252 (70% recovery, based
on a yield of 4.8 g/1 compared with an initial 6.8 g/l).
Example 2. Influence of the purity of the initial solution of
immunoglobulins in the treatment with caprylate.
In this example, an evaluation was made of the impact of the purity
of the initial solution of immunoglobulins and 10 the presence of
accompanying proteins in the starting material subjected to the
method of the present invention.
Two independent experimental test groups were created:
In group A, the starting material was the DEAE Sepharose column
effluent, with an electrophoretic purity (ACE) of 98+2% IgG,
i.e. the starting material described in Example 1.
In group B, the starting material, designated 4% PEG Filtrate, was
obtained by the same process described in Example 1 up to the step
before the DEAE Sepharose chromatography. Thus, material B was
obtained after the precipitation with PEG of the extraction
suspension of fraction
and had an approximate
electrophoretic purity (ACE) of 90% IgG.
Both starting materials (group A and group B), with an equivalent
PEG content of approximately 4%, were subjected to a treatment
with caprylate at a concentration of 13 mM and a pH of between
5.0 and 5.2, and were purified as indicated in Example 1.
Date Recue/Date Received 2020-09-01
CA 02943328 2016-09-26
28
Table 2 details the main characteristics of the starting
material used in both test groups (A and B, respectively),
as well as those of the material produced in steps
subsequent to the treatment with caprylate.
Table 2. Turbidity and aggregation percentage results obtained
in the multiple steps of the
method of the present invention for groups A and B described in
Example 2.
! Starting PuritylAlbumin Solution before Solutior. after Solution
after
material (%) ,content treatment with addition of
incubation (2 hours
1
(group) (mg caprylate caprylate at 25 C)
n
, Alb/g
0
I TgG) ____________________________________ ____ __________________________
t..)
kc,
0.D.TurbidityAggregates,TurbidityAggregates,TurbidityAggregates,
.N
w
w
280 (NTU) polymer (NTU) polymer (NTU) polymer
t.)
i
o
nm , (%) (%)
(%)
o
w
1-,
I (UA)1 1
m
1
o
Column 97.9 t <0.4 6.7. 2.9 <0.1 15. 1 <0.1
17 <0.1 kr)
1
t.)
m
effluent 1.5
(A)
1
Fil.trate 90.2 36 8.3 8 1.9 517 0.9 591
1.0
after 2.9
treatment
with PEG
(B) ,
,
CA 02943328 2016-09-26
The results obtained and collected in Table 2 showed that
the addition of caprylate at an effective concentration
for inactivation (13 mM), to a material of lower purity
(approximately 909,5 IgG, see group B), causes the
5 precipitation of components of the solution, giving rise
to a drastic increase in turbidity (superior a 500 NTU).
Thus, the molecular distribution results for the solution
showed the precipitation of part of the accompanying
proteins with a high molecular weight.
The addition of caprylate, in the quantities and under the
conditions described previously (13 mM of caprylate, pH
between 5.0 and 5.2), to a material of low purity gave
rise to a precipitated suspension that made it necessary
to include additional steps of separation and purification
in order to separate the proteins with a high molecular
weight and precipitated aggregates. Therefore, the
molecular composition of the product of group A treated
with caprylate, i.e. with an aggregate content exceeding
196, shows the non-viability of processing this product
into a purified final product unless additional steps of
purification or separation are included, such as steps of
precipitation with PEG, chromatography or equivalent
methods. Finally, this fact shows the viability of the use
of caprylate as an agent with viral inactivation capacity,
under non-precipitating conditions, only when it is added
to a material of sufficient purity.
Example 3. Effect of the composition of the starting
material on the generation of aggregates.
The objective of this experiment was to evaluate the
impact of the composition of the initial solution of
CA 02943328 2016-09-26
31
immunoglobulins to which the treatment with caprylate is
applied.
Two independent experimental test groups were created, A
and B, starting from materials of equivalent purity (97.9
+ 1.5%) but of different composition.
In group A, the starting material was the column effluent
(obtained according to the initial method described in
Example 1), with a protein concentration of 5+2 mg/m1 and
a PEG-4000 concentration of 4+1%.
In group B, the starting material, designated Concentrated
and Dialysed Effluent, was the same column effluent
mentioned for group A, but after being concentrated and
dialysed. Therefore, the DEAF column effluent (mentioned
in Example 1 above and corresponding to group A of the
present example) was subjected to an additional step of
dialysis and concentration by ultrafiltration so that the
PEG content was reduced by an order of approximately 6
times and the protein was concentrated to an approximate
value of 4%, i.e. 40 mg/ml.
The material obtained in both experimental groups, A and
B, was subjected to a treatment with caprylate at a
concentration of 13 mM and a pH of between 5.0 and 5.2,
and was ultrafiltered under the conditions described in
Example 1 in order to obtain a product with an IgGs
concentration of 10%.
Table 3 details the main characteristics of the material
processed in the above-mentioned experimental groups A and
B, as well as the characteristics of the material
tri
cr >
'cs n LQ
= Cl)
Table 3. Results obtained for the multiple steps of the
method 11 P 0
P)
o ,b b
cr n
0, 1-1 m
gi of the present
P 0 1-, gi
rt invention in groups A and B of Example 3.
pr W pr
rt M
O = 0 1 Starting Starting material Nominal
Solution after Concentrated final 1-t) 0 0,
P 0
ct m
P
= m material caprylate
addition of product h b 1-
O
m o 0,
b b (group) (mM) caprylate
P
(1-
n e-
IgG, PEG Purity
TurbidityAggregates,CaprylateAggregates, m n
0 cr
H- IA
cr P (mg/ml) (mg/ml) (%) (NTU) polymer (mM)
polymer '0 o
0 Cl)N
m
r r ko
1-, (96)
(96) 11 IA
n
P - 0-. M W
'0 w
n
t.)
P w Column 5 2 40 1097.9 13 7.0 <0.1 <0.1
<0.1 Hi m
cr
1--
11
p w o
`-< 0- effluent 1.5 9
p, 0
1-. .8 <0.1 0.1 <0.1 rr Hm 1-,
1--, ':3-
M H m
P 0 (A)
. 11 0 1
rr 11.9 <0.1 0.1
<0.1 i- 0 o
ko
0 1-1
m
t.)
m Concentrated - 40 - 6 98 2 13 11.9 ' 0.5 0.3
0.3 m
F__,
=
rt and dialysed 13.4 C.4 0.5
0.5 0,
rr
O
effluent b-'
h 10.0 C.3
0.5 0.4 n 0
,-m
o
(B)
b
(i
n Er
ct
M Fi
=
H- m
O ct p
rt
hi rr
o P
m
cr m
hc1 m
M
O 0, rr
n H-
r- 0,
ill
t-Th (I)
H-
H-P
P 1-"
O
n P Cl
=
M H
CA 02943328 2016-09-26
33
conditions, on a purified solution of immunoglobulin, at a
concentration of 5+2 my/m1 and in the presence of a PEG
concentration of 40+10 mg/ml (4+1%) (group A, column
effluent), does not induce any alteration or aggregation
of the solution of immunoglobulins, maintaining unchanged
the molecular distribution of the product during and after
the addition of the caprylate, with an undetectable
proportion of aggregates of less than 0.1%.
However, when these same conditions for the treatment with
caprylate were applied to a material with a low PEG
content (<190 (group B), a substantial increase in
immunoglobulin aggregates was observed after the addition
of caprylate. Moreover, it was not possible to eliminate
this aggregate content by ultrafiltration under the
conditions used, and comparable levels of polymer were
measured in the final product.
Given that the main differential characteristics between
the starting materials used in experimental groups A and B
were the protein concentration and the PEG concentration,
an additional test was performed with the aim of
ascertaining the influence of each of these parameters on
the subsequent treatment with caprylate.
In this experiment, the starting point was a single batch
of Concentrated and Dialysed Effluent (initial material of
the previous group Eh which was separated into four
distinct experimental groups: groups El, B2, B3 and B4.
The material of group Bl was processed at an approximate
protein concentration of 4% and an approximate PEG
concentration of 0.6%.
CA 02943328 2016-09-26
34
The material of group 02 was processed at the same protein
concentration of approximately 4%, but the PEG content was
readjusted to a value of 4+1% (w/w).
In groups B3 and B4, the material was diluted to 0.5 +
0.2% of protein. With regard to the PEG content, in group
B3 this was brought to a concentration of approximately
0.6% (w/w), while in group B4 the PEG content was
readjusted to 4+1% (w/w).
The resulting material obtained in the four experimental
groups was brought to a pH of 5.10 + 0.05 and a caprylate
concentration of 15 mM, and was then incubated at 25 C for
2 hours. The results obtained are shown in Table 4.
Table 4. Results obtained for the initial material and
after incubation with caprylate for groups El, B2, B3 and
B4 of Example 3.
Experimental Starting Material Solution
group treated
with 15mM
caprylate
at 25 C for
2 hours
IgG PEG Turbidity Aggregates, Aggregates,
(mg/ml) (mg/ml) (NTU) Polymer (%) Polymer (%)
Bit - 40 - 6 1.6 < 0.1 0.5
32 - 40 40 + 6 2.5 < 0.1 0.3
B3 5 + 2 - 6 1.3 < 0.1 0.5
B4 5 + 2 40 + 6 1.3 < 0.1 < 0.1
The results shown in Table 4 show that during the
CA 02943328 2016-09-26
treatment with caprylate under the established conditions,
a PEG protective effect was observed in combination with a
sufficient protein dilution. It is remarkable that when
the starting material was at an approximate protein
5 concentration of 5+2 mg/ml and a PEG concentration of 4%,
undetectable values of aggregates were obtained after the
treatment with caprylate (<0.1%).
Example 4. Effect of pH on the solubility of the solution
10 of immunoglobulins treated with caprylate.
It is known that the elimination of PEG in solutions of
immunoglobulins, as well as the concentration of the said
immunoglobulins to appropriate concentrations for their
15 intravenous use, must take place preferably at pH values
around 4.5.
Moreover, given the insolubility of caprylic acid at pH
values below its pKa (4.89), in the present experiment the
20 effect of pH on the solubility of the solution of
immunoglobulins treated with caprylate was evaluated, with
the aim of establishing an appropriate pH value for
beginning its ultrafiltration.
25 To this end, a batch of column effluent obtained in
accordance with the initial method detailed in Example 1
was processed to obtain the solution of immunoglobulins
treated with 13 mM caprylate and clarified.
30 This intermediate, which constitutes the material before
the ultrafiltration step, was acidified by the addition of
acetic acid to take it from the pH of the treatment with
caprylate (5.1) to pH values around 4.5. Subsequently, the
CA 02943328 2016-09-26
36
appearance and solubility of the solution was evaluated
for each of the evaluated pH values, and the generation of
colloidal particles was quantified by nephelometric
measurement of turbidity.
Table 5 shows the appearance and turbidity results
obtained for each of the evaluated pH values.
Table 5. Turbidity and visual appearance results obtained
for the different pH values analysed in Example 4.
pH Turbidity (NTU) Visual appearance
5.1 5.6 Transparent
5.0 10.0 Transparent, small
crystals
4.8 32.5 White, precipitated
crystals
4.6 53.0 White, precipitated
crystals
4.4 57.1 White, precipitated
crystals
The results obtained, as seen in Table 5, showed that when
the solution of immunoglobulins treated with 13 mM
caprylate was acidified to a pH of below pH 5.0, the
appearance of a whitish precipitation was observed, along
with a distinct increase in turbidity. This effect was
very probably due to the formation of insoluble caprylic
acid in colloidal form, which made it non-viable to begin
the process of ultrafiltration at pH values below 5Ø
The results obtained put into evidence that when the
purified solution was subjected to a treatment with
CA 02943328 2016-09-26
37
caprylate, in the effective concentration range for viral
inactivation (between 9-15 mM of caprylate) and under the
conditions described previously, it is preferable to begin
the subsequent ultrafiltration step at a pH greater than
or equal to the pH of the viral inactivation treatment,
i.e. 5.1, with the aim of increasing the concentration of
the ionised and soluble form of caprylate and therefore
facilitating its permeability through the ultrafiltration
membrane.
Example 5. Effect of the acetate content in the dialysis
solution on the reduction of caprylate by
ultrafiltration/diafiltration.
A series of independent ultrafiltration/diafiltration
processes were carried out in the presence of different
concentrations of acetate in the buffer solution used for
the dialysis of the product.
The starting material used, designated Concentrated and
Dialysed Effluent, was the same as that of group B of
Example 3. The said starting material, with an IgG purity
of 98+296-, an approximate protein concentration of 40 mg/ml
and an approximate PEG content of 0.69,1, was subjected to a
treatment with caprylate and subsequently to
ultrafiltration/diafiltration using membranes with a
nominal molecular weight cut-off of approximately 100 kDa.
The applied ultrafiltration/diafiltration step comprised a
first phase of concentration to approximately 4% (w/v) of
IgG, a second phase of dialysis using eight volumes of
dialysis solution, and finally a concentration to an
approximate value of 9-10% (w/v) of IgGs.
CA 02943328 2016-09-26
38
The first of the ultrafiltratisn/diafiltration tests was
performed using water for injection, while the subsequent
tests were carried out using buffer solutions with
increasing concentrations of acetate, more specifically 2,
5, 20 or 50 mM of acetate respectively, and with an
adjusted pH of between 5.0 and 5.5 in all cases.
Table 6. Results obtained for the
ultrafiltration/diafiltration step using different
concentrations of acetate in the dialysis buffer.
Concentration Nominal Dialysed product
of acetate addition of Caprylate in Permeability
present in the caprylate the dialysed of the
dialysis (mM) product caprylate(
buffer (mM) (mM)(1)
0 20 2.3 13.2
2 13 0.6 19.3
5 13 <0.2 32.8
13 <0.2 43.3
50 13 0.2 45.3
(1) Values determined after dialysing with 8 dialysis
volumes
(2) Permeability calculated by means of the following
15 formula:
Number of Dialysis Volumes - ln (Cf/Co)/(R-1); where Cf is
the concentration after dialysing with the number of
dialysis volumes in question, Co is the concentration
before dialysis, and R is the retention coefficient.
The results of Table 6 show that the procedure of
ultrafiltration/diafiltration using membranes with a
molecular weight cut-off of approximately 100 kDa,
CA 02943328 2016-09-26
39
applying 8 dialysis volumes of a buffer solution with
acetatc, at a pH between 5.0 and 5.5 and with a minimum
concentration of acetate around 5 mM and at least 50 mM,
satisfactorily achieves the objective of efficiently
reducing the caprylate to appropriate levels in the final
concentrated product.
On the contrary, when the solution used for the dialysis
was water for injection or a buffer solution with acetate
levels of 2 mM, the caprylate was not effectively
eliminated in the filtrate.
This puts into evidence that the method of
ultrafiltration/diafiltration using a membrane with a
molecular weight cut-off of approximately 100 kDa, under
the conditions described previously, is effective in
reducing the caprylate deriving from the previous
treatment, given that correct levels of the said reagent
were detected in the final concentrated product.
Example 6. Simultaneous elimination of the chemical
reagents (PEG and caprylate) by means of a single step of
ultrafiltration.
A batch of IgGs was processed in accordance with the
method described in Example 1 to obtain the solution
inactivated with caprylate and clarified. The said
solution, with an approximate protein concentration of
0.5% and a pH of 5.1, was processed using an
ultrafiltration/diafiltration equipment formed by
polysulphone membranes of the Biomax type (Millipore,
USA) with a molecular weight cut-off of 100 kDa. The
ultrafiltration/diafiltration was carried out in two
CA 02943328 2016-09-26
differentiated phases, as described in Example 5:
- In the first phase, carried out at pH 5.1, 5.6 or 5.8,
the material was subjected to steps of sequential dialysis
5 and concentration by means of diafiltration with not fewer
than three volumes of buffer solution of acetate 5 mM
adjusted to pH 5.1, 5.6 or 5.8, and concentrating the
protein to an approximate value of 2%.
10 - In the second phase, once the content of caprylate had
been reduced to approximately one tenth, the solution was
brought to a pH of 4.5 + 0.1 or 5.1. The product was then
brought to an adequate concentration of protein and PEG to
begin dialysis, and the dialysis was begun with eight
15 volumes of buffer solution of acetate 5 mM at a pH of 4.5
or 5,1.
Finally, the product was formulated by means of dialysis
with six volumes of glycine solution at a concentration of
20 200 mM and a pH of 4.2, and was concentrated in order to
obtain a 10% solution of IgGs.
Table 7 shows the percentage of passage of PEG and
caprylate obtained at the start of each of the phases of
25 ultrafiltration/diafiltration and at different pH values:
CA 02943328 2016-09-26
41
Table 7. Passage of PEG and caprylate in the two phases of
the ultrafiltration/diafiltration step at the different pH
values analysed.
Phase pH PEG passage Caprylate
(caprylate (%) passage (%)
concentration)
Start of Phase 5.1 17 74
I (13 mM) 5.6 15 98
5.8 15 100
Start of Phase 5.1 40 100
II (1 mM) 4.5 82 97
The results of Table 7 show that at the start of the
ultrafiltration/diafiltration step, in Phase I, the
caprylate showed very high passage values between pH 5.1
and pH 5.8. These values resulted in a very high reduction
of caprylate during the said Phase I of the
ultrafiltration/diafiltration step (a caprylate reduction
of more than 10 times was obtained with respect to the
initial content). On the contrary, the passage of PEG was
very low (<20%) in the said Phase I, and its total
elimination was practically non-viable at pH >5 in the
presence of caprylate.
On the other hand, in Phase II, as can be seen in Table 7,
the passage of PEG was very high at pH 4.5, with a value
of 82%. In addition, it was found that during this Phase
II the caprylate is also reduced, given that at the start
of the phase it is present at a residual level of i mM,
which allows the passage to be practically 100%.
Table 8 details the evolution in the concentration of
protein, PEG and caprylate in each of the phases of the
CA 02943328 2016-09-26
42
ultrafiltration/diafiltration step and in the final
formulation step.
Table 8. Quantity of PEG and caprylate (measured by
concentration and optical density) in the solution after
the viral inactivation step, Phases I and II of the
ultrafiltration/diafiltration step, formulation at pH 4.2,
and in the final solution.
Phase/Step PEG PEG
(0.D.Caprylate Caprylate
(mg/ml) 280 nm) (mM) (0.D. 280
nm)
Inactivated and 38 6.9 12 2.2
clarified
solution
Solution at the 45 1.8 0.9 0.04
end of Phase I of
the
ultrafiltration/
diafiltration
step
Solution at the 0.7 0.02 0.1 0.003
end of Phase II
of the
ultrafiltration/
diafiltration
step
Formulated 0.1 0.003 <0.1 <0.003
solution at pH
4.2
Final 0.1 0.001 <0.1 <0.001
concentrated
solution
CA 02943328 2016-09-26
43
In accordance with the PEG and caprylate values recorded
at each step and phase, and considering the protein
concentration at each step, the PEG reduction factor was 4
in Phase I (at pH 5.1) of the
ultrafiltration/diafiltration step and 90 in Phase II (at
pH 4.5) of the ultrafiltration/diafiltration step, giving
a total reduction factor (Phase I and Phase II) of
approximately 350 times (an initial absorbance of 6.9
compared with an absorbance of 0.02 obtained at the end of
the ultrafiltration/diafiltration step).
In the case of the caprylate, the reduction factor was 55
in Phase I (at pH 5.1) of the
ultrafiltration/diafiltration step and 13 in Phase II (at
pH 4.5) of the ultrafiltration/diafiltration step, giving
a total reduction factor (Phase I and Phase II) of
approximately 700 times (an initial absorbance of 2.2
compared with an absorbance of 0.003 obtained at the end
of the ultrafiltration/diafiltration step).
The results showed that the reagent with viral
inactivation capacity (caprylic acid or caprylate), as
well as the precipitation reagent (PEG), could he
efficiently reduced by means of a single step of
ultrafiltration using a membrane with a molecular weight
cut-off of approximately 100 kDa, selecting the physical
and chemical conditions to be applied in each phase of the
ultrafiltration/diafiltration step (among others pH,
protein concentration, number of dialysis volumes,
dialysis buffer) and giving rise to a final product of
IgGs concentrated to 10% with some remaining
concentrations of both reagents suitable for intravenous
use.
CA 02943328 2016-09-26
44
Example 7. Evaluation of viral inactivation capacity with
caprylate in the presence of PEG.
Various independent experiments were performed, taking as
the starting material the column effluent or the dialysed
and concentrated effluent (obtained in accordance with
Examples 1 and 3, respectively), to evaluate the capacity
of caprylic acid or caprylate in the presence of PEG for
eliminating or inactivating viruses with a lipid envelope.
Both materials had an immunoglobulin purity of 98+2% and a
protein concentration of between 5 and 10 mg/ml, while
their PEG content differed, at 40 mg/ml and 1.5 mg/ml
respectively.
Viral inactivation tests were performed using the Bovine
Viral Diarrhoea virus (BVDV) of the Flaviviridae family,
of 40-Go nm, with a lipid cnvolope and an average
resistance to physical and chemical agents.
In each test, the corresponding starting material was
inoculated with the virus to a value less than or equal to
0.5% and subjected to a viral inactivation treatment for
two hours at a temperature of 1.5 C or 25 C, applying
caprylate concentrations of 9 mM or 13 mm.
The quantification of the viral load of BVDV in the
different samples produced was carried out by means of the
TCID50 test (50% Tissue Culture Infectious Dose) using the
MBDK cell line. The viral reduction factor (RF) of the
viral inactivation step was determined as the quotient of
the viral load detected in the inoculated starting
CA 02943328 2016-09-26
material divided by the quantity of virus detected in the
resulting sample at the end of the treatment, expressed in
logio
5 Table 9 details the characteristics of the starting
material of each test, as well as the RE obtained.
Table 9. Viral inactivation results observed in the
caprylate treatment tests with
10 viral inoculum, in the presence or absence of PEG.
Experimental Starting Treatment Caprylate Viral
group material temperature concentration reduction
(mm) factor
IgG PEG ( C) (RE)
(mg/m1) (mg/m1)
Dialysed and 10 1.5 25 9 L- 4.19
concentrated
> 4.36
effluent
13 > 3. 95
> 4.13
9 14. 62
5.10
13 4.71
Column 5 40
4.57
effluent
'2 4.32
15 13 4.27
The viral reduction results obtained in all the tests (see
Table 9) show a high capacity for inactivation of BVDV in
both starting materials, even for a minimal 9 mM
15 concentration of caprylate, after treatment at different
CA 02943328 2016-09-26
46
temperatures (15 and 25 C). Furthermore, these tests
showed that at each of the PEG concentrations analysed,
there is no observed interference by the PEG in the viral
inactivation capacity of the caprylate, given that
equivalent results were obtained with both evaluated
materials.
Example 8. Characterisation of the intravenous
immunoglobulin solution obtained according to the
production method of the present invention.
It is intended to establish the biochemical and functional
characteristics of the solution of immunoglobulins with
10% (w/v) proteins obtained by the method of the present
invention.
Two batches of DEAE column effluent were processed
according to the method detailed in Example 1 in order to
obtain the inactivated virus solution with caprylate, at
an approximate scale of 200 litres of plasma.
The said solution with caprylate, once clarified, was
dialysed and concentrated by ultrafiltration in
differentiated phases, as described in Example 6, with the
aim of achieving the elimination of the main process
remnants (PEG and caprylate). Subsequently, the said
purified solution, at an approximate protein concentration
of 2.5%, was formulated by dialysis at constant volume for
approximately 6 volumes of a buffer solution consisting of
sorbitol 1% and glycine 240 mM, adjusted to pH 4.5 + 0.1.
Finally, the said solution was concentrated by
ultrafiltration and adjusted to an optical density of 140
+ 5 UA (280 nm), equivalent to 10% (w/v) proteins, and was
CA 02943328 2016-09-26
47
adjusted to a final pH of 5.25 + 0.25.
The product obtained (IGIV 10% (w/v)), stabilised with
sorbitol and glycine, and once clarified and filtered
using sterilising-grade membranes (0.22 lam), was dosified
in glass bottles with chlorobutyl stoppers by determining
the most relevant analytical parameters of the quality,
unalterability and stability of a solution of
immunoglobulin for intravenous administration. The average
analytical values obtained for the two batches, as well as
the specification values of the European Pharmacopoeia,
are shown in Table 10.
Table 10. Characterisation of the solution of intravenous
immunoglobulins at 10% (w/v)
PRODUCT
OBTAINED BY
SPECIFICATIONS
PARAMETER THE METHOD OF
(Fur. Ph.)
THE INVENTION
PH 5.25 4.0-7.4
Osmolality (mOsm/kg) 306 > 240
Sodium (mM) < 3.2 n.e.
Turbidity (NTU) 4.4 n.e.
Molecular Distribution
(%)
Polymer
< 0.1 < 3.0
Dimer 7.6 Mon.+Dim. > 90
Monomer 92.5
Fractions < 0.3
IgG subclasses (%)
IgG, 66.7
IgG2 27.6 equivalent to
plasma
IgC3 3
IgG4 2.7
Integrity of
93
Fc fragment
CA 02943328 2016-09-26
48
PRODUCT
OBTAINED BY
PARAMETER THE METHOD OF SPECIFICATIONS
.
THE INVENTION (Eur Ph.)
Purity profile:
Ig purity (ACE) (%) 99.6 > 95
< 0.002
IgM (mg/ml)
NAPTT (Dil. 1/10) (s) 308
Activated factor XI
(ng/m1) Not detected
TGT FXI (nM thrombin) < 53
Isoagglutinin titre
Agglutination
Anti-A 1:16 Agglutination
Agglutination
Anti-B 1:16
Proteolytic activity
< 2 < 35
PKA (UI/ml)
0.6 + 0.07 < 1
ACA (CH50/mg)
Eur. Ph.: European Pharmacopoeia; n.e.: Not Established; TGT
FXI: Thrombin Generation Test (using plasma deficient in Factor
IX); NAPTT PKA: Prekallikrein Activator; ACA: Anti-Complementary
Activity.
The above results enhance that the product obtained is
essentially unaltered as a result of the purification
process of the present invention in terms of parameters
such as the absence of polymer, undesirable biological
activity such as PKA or ACA activity, among others,
preserving some functionality characteristics intact with
respect to plasma, such as proportion of IgG subclasses
and Fc fragment integrity, and simultaneously showing an
excellent purity profile (low titre of anti-A/anti-B
isonemagglutinins, concentration of IgM, procoagulant
CA 02943328 2016-09-26
49
activity, etc.).
It is concluded that the overall method of the present
invention for obtaining IGIv 10% (w/v), incorporating the
step of viral inactivation with caprylate in the presence
of PEG and its subsequent separation, as well as the final
formulation, is totally viable and scalable to the final
product formulated and concentrated as IGIV 10% (w/v)
protein solution, giving a final product that complies
perfectly with the values established in the European
Pharmacopoeia.
The stability studies carried out, which are essential for
commercial viability of the product, showed the
suitability of the formulations with sorbitol (to 5%),
glycine (to isotonia) or a combination of both, in the pH
range between 4.2 and 6.0, for stabilising 10% (w/v)
solutions of intravenous immunoglobulin at ambient
temperature (25 C 30 C) for two years.
Example 9. Applicability of the treatment with caprylate
to a fraction rich in IgG obtained by alternative
methods.
An evaluation was made of the validity of the application
with caprylate under the conditions described in the
present invention, using other process intermediates
obtained using alternative purification methods.
Two independent experiments were performed using as the
starting material a plasma intermediate rich in IgG, the
designated Suspension of Fraction II, from the Cohn-Oncley
ethanol fractionation.
CA 02943328 2016-09-26
This intermediate was obtained by the same plasma
fractionation method described in the present invention up
to Fraction 11+111. The procedure then continued with the
5 alcoholic reprecipitation of the extraction suspension of
fraction 11+111, followed by separation of fraction III,
finally obtaining fraction II with a purity greater than
96%. The suspension of the said fraction II, once purified
with bentonite and dialysed with water to eliminate the
10 alcohol content, served as the starting material for these
experiments.
In the two experiments performed, the material derived
from two plasma batches was separated into two different
15 groups, A and B, according to their PEG content. In group
B, the starting material was brought to a nominal PEG
concentration of 40 mg/ml by adding a concentrated
solution of PEG-4000.
20 Subsequently, both materials derived from both groups (A y
B) were diluted to an approximate protein concentration of
5 mg/ml, adjusted to a pH value of 5.1, and subjected to
treatment with caprylate until reaching a nominal
concentration of 13 mM and a pH of between 5.0 and 5.2, as
25 described in the method of the invention.
Table 11 details the main characteristics of the starting
material used in both test groups (A and B, respectively),
as well as the characteristics of the material generated
30 after the treatment with caprylate.
0-1
O Fr IA 0 - Table 11. Main characteristLcs of the starting
material used in
ul ti 0- r
P- CD (D (-t -----
= P P groups A and B and of
LQ c-r H- H
= hl --'
P (1) 0
0 (-D the final product obtained in the same. The purity of the
b 0 0,
(1- initial solution of
1--
H- m immunoglobulins is 99.6 0.6% (n = 5 batches)
= 0-
m P
n" PP
Experimental Solution before treatment with Solution treated with
caprylate
O 0 a
I-' p 0 group caprylate (2 hours at
25 C)
n
0 1-0 0 P)
n
Cl hl hri'0
= ,.<
f-1 o
PEG 0.D.TurbidityAggregates,0.D.TurbidityAggrega7,es,Caprylate
t..)
H. p P 1-'
W,
= ct m P P (mg/ml) 280 (1NITU)
polymer 280 (NTU) polymer (mM) (1) ,N
w
CD 1-, (1-
w
m k< CD
t.)
o < Cr (1) no (%)
no (%) m
H P.
P c) po
lii 0
rt cl,
(I) Cl Pi F--, (UA) (UA)
o M
i = I-1 1-,
H- P CD <0.01 6.9 3.3 <0.1 7.0
10.1 0.5 11.2 c,
, m
ko
= 0- rr
m 1
U) Cl'< rt A <0.01 6.9 3.0 <0.1
7.1 16.5 1.2 11.2 N.)
n (1)
a) n m
rh 1-1 0 _
H) Li) 0 11 34.1 7.1 2.5 <0.1 7.2
14.5 0.1 11.9
P. 'CS I-h P- 1-1
O 0 P0
1--- n W m
,O B 33.6 6.9 3.0 <0.1 7.2
14.4 <0.1 12.4
M H- rt Cr 0
t-t1 H-
rt P- (T) 0
P. 0 aa
a
Cr
H-
= o a)
[I b n rt
P- a rrCl
1-h H- P- CD
H. ri- <
(I) H- 51)
Cl 0 rr PO
= P- I--,
0-' (I) 0
k< -P a)
CA 02943328 2016-09-26
52
different methods, there being no induced formation of
immunoglobulin aggregates or other irreversible
precipitates, which greatly facilitates the subsequent
purification process.
The results show that in combination with a sufficient
dilution of the protein and a sufficient degree of purity,
the protective effect of PEG on the generation of
immunoglobulin polymers is apparent.
This experimental example demonstrates the viability of
the use of caprylate only as a reagent with viral
inactivation capacity under non-precipitating conditions,
and/or aggregation promoting conditions, when it is added
to a material with sufficient purity and complying with
the specified conditions relating to the protein and PEG
concentration.
Although the invention has been presented and described
with reference to embodiments of the same, it will be
understood that these embodiments are not limitative of
the invention, since there could be multiple variables in
terms of manufacturing or other details that will be
evident to a person skilled in the art after interpreting
the subject matter disclosed in the present description
and claims. Consequently, all variants or equivalents will
be included in the scope of the present invention if they
can be considered to fall within the broadest scope of the
following claims.
