AU2016231646B2 - Method for the preparation of immunoglobulins - Google Patents

Abstract

Method for the preparation of immunoglobulins The present invention relates to 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 a temperature necessary for the inactivation of enveloped viruses; d) performing a step of ultrafiltration/diafiltration on the solution obtained in step c).

Description

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 Soc, 1946, 62, 459-475), the

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 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.

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 EP0893450 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.

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

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 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 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, 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.

According to a first aspect, the present invention provides 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, comprising the steps of:

(22894369_1):GGG

7a

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 a temperature necessary for the inactivation of enveloped

viruses; and

d) performing a step of ultrafiltration/diafiltration on the

solution obtained in step c).

According to a second aspect, the present invention provides a

solution of immunoglobulins prepared according to the method of

the first aspect.

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.

(22894369_1):GGG

In a further aspect, the present invention relates to the implementation of a single step 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 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 an IgG purity greater than or equal to 96%. Preferably, 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, as described in the document EP1225180B1. According to the present patent, any of the above fractions could be subjected to a precipitation procedure 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 Sepharose). 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.

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

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

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.

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/ml; and/or

- 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.

In 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 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).

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,

on 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 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 sodium acetate at a concentration greater than or equal to

5 mM adjusted to the pH mentioned above, i.e. between 5.0

and 6.0.

The number of diafiltration volumes to be performed in the

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

diafiltration medium preferably being, as mentioned

previously, a 5 mM solution of sodium acetate at pH 5.0

6.0. 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.

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 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 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 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 preferably for between 1 and 2 hours.

Clarification is then performed using depth filters (for

example, Cuno 90LA, 50LA, Seitz EK, EK-1, EKS, or

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 6.0.

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 between 4.0 and 5.0.

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 pm, 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 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.

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.

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 II+III, 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 PEG-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.

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 80 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

ultrafiltration 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).

Finally, the said solution was filtered using a depth

filter (CUNO*, Ultrafilter, Denmark) and absolute filters

or membranes with a pore size of 0.22 pm (CVGL*, Millipore, USA; or DFL*, 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

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

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

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

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.

Taibl l ResI~ults ctaM ned for the startingg aea; T m3t

SS am.. a. .a" rf ,t

to on :C "..en

effluet

t d,.1S.

2'

99 1

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 according to the state of the art and as described in patent application PCT W02005/073252 (70% recovery, based on a yield of 4.8 g/l 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

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 Sepharosa

chromatography. Thus, material B was obtained after the

precipitation with PEG of the extraction suspension of

fraction II+III 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.

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.

42'1

44..;Q - A

' -14 0) 4

10,' -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

.0.............................................. ..... _ _ __ __ _.......................

........- ...................2.... ... .. -------

................................................... ......................... ...............

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 90% IgG, see group B), causes the

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

1%, 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 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/ml 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 DEAE 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 generated in the step following the treatment with caprylate and in the diafiltered and concentrated final product, for each experimental group.

I/ V/ V ----- -(----)----- ------- --------------------

00

0~~~ q!: J 4-0-

V

>~0

() l ,

H 10 '0 I

4-14-z

5 -- -- -- - -- -- -- - - -- - -- -- - -- -- -- -- - -- -- - - -- -- -- -- - -- -- -- -- - -- -- -- - - -- -- -- -- - -- -- --

As an e sen n Tble3, he esuts ut ntoevienc

tht teamn wt apyae ne te secfe conditions, on a purified solution of immunoglobulin, at a concentration of 5±2 mg/ml 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 (<1%) (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 B), which was separated into four distinct experimental groups: groups B1, B2, B3 and B4.

The material of group B1 was processed at an approximate protein concentration of 4% and an approximate PEG concentration of 0.6%.

The material of group B2 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 B1, 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 (%)

B1 ~ 40 ~ 6 1.6 < 0.1 0.5

B2 ~ 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 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 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

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

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

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.

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.

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 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 5.0.

The results obtained put into evidence that when the purified solution was subjected to a treatment with 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±2%, an approximate protein concentration of 40 mg/ml

and an approximate PEG content of 0.6%, 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.

The first of the ultrafiltration/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(2

) buffer (mM) (mM))

0 20 2.3 13.2

2 13 0.6 19.3 5 13 <0.2 32.8 20 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

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, applying 8 dialysis volumes of a buffer solution with acetate, 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 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

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%.

- 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

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

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

ultrafiltration/diafiltration and at different pH values:

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 1 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 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 (O.D. Caprylate Caprylate

(mg/ml) 280 nm) (mM) (O.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

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 be

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.

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 40-60 nm, with a lipid envelope 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 15°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 material divided by the quantity of virus detected in the resulting sample at the end of the treatment, expressed in logio.

Table 9 details the characteristics of the starting

material of each test, as well as the RF obtained.

Table 9. Viral inactivation results observed in the

caprylate treatment tests with

viral inoculum, in the presence or absence of PEG.

Experimental Starting Treatment Caprylate Viral group material temperature concentration reduction (mM) factor

IgG PEG (°C) (RF) (mg/ml) (mg/ml)

Dialysed and 10 1.5 25 9 > 4.19 concentrated > 4.36 effluent 13 > 3. 95

> 4.13

9 > 4. 62

> 5.10 25 13 > 4.71 Column 5 40 effluent > 4.57

> 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

concentration of caprylate, after treatment at different 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 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 pm), 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 PARAMETER THE METHOD OF SEIFCATON THE INVENTION (Eur. Ph.)

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 (%) IgG2 66.7 equivalent to IgG2 27.6 plasma IgG 3 3 IgG 4 2.7 Integrity of 93 Fc fragment

PRODUCT OBTAINED OBTAIED BY BY SPECIFICATIONS PARAMETER THE METHOD OF SEIFCATON THE INVENTION (Eur. Ph.)

Purity profile:

Ig purity (ACE) (%) 99.6 > 95

(mg/ml) < 0.002 IgM

NAPTT (Dil. 1/10) (s) 308 Activated factor XI (ng/ml) Not detected

TGT FXI (nM thrombin) < 53

Isoagglutinin titre Agglutination Agglutination Anti-A 1:16 Aguiao < 1:64 Agglutination Anti-B 1:16

Proteolytic activity < 2 < 35

PKA (UI/ml) PKA(UIml)0.6 ±0.07 < 1 ACA (CH 5 o/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

isohemagglutinins, concentration of IgM, procoagulant 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.

This intermediate was obtained by the same plasma

fractionation method described in the present invention up

to Fraction II+III. The procedure then continued with the

alcoholic reprecipitation of the extraction suspension of

fraction II+III, 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

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

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.

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

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

after the treatment with caprylate.

I

A~' - e -----

('A T A N ' N

"-1 C 1'

4-1'

3---- ----- -'----------- 3~z C '', 711 ) (

its (9 AcIV

>4 4

() Th PE nUarlt aus orsodt h au

obaiedbymen of anltia dtermintio)

5' n

The results show he iblt f te iatvto

traten wit carl nerteseiie odtos

using imuolo an i solutio sufiietl puifedb 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.

Claims (22)

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, comprising 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 a temperature necessary for the inactivation of enveloped

viruses; and

d) performing a step of ultrafiltration/diafiltration on the

solution obtained in step c).

2. The method according to claim 1, wherein the said method also

comprises a step of final formulation of the solution of

immunoglobulins obtained in step d).

3. The method according to claim 1 or 2, 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.

4. The method according to claim 3, 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

(22894418_1):GGG anionic chromatography.

5. The method according to any one of the preceding claims,

wherein the initial solution of immunoglobulins has a

concentration of immunoglobulins between 1 and 10 mg/ml.

6. The method according to claim 4, wherein the initial solution

of immunoglobulins has a concentration of immunoglobulins between

3 and 7 mg/ml.

7. The method according to any one of the preceding claims,

wherein the polyether or polymer of glycol is selected from

polyethylene glycol (PEG), polypropylene glycol (PPG) or

combinations of the same.

8. The method according to claim 7, wherein the concentration of

PEG in the initial solution is between 2% and 6% (w/v).

9. The method according to claim 7, wherein the concentration of

PEG in the initial solution is between 3% and 5% (w/v).

10. The method according to any one of claims 7 to 9, wherein the

PEG is PEG with a nominal molecular weight of 4000 Da.

11. The method according to any one of the preceding claims,

wherein in step b), the solution obtained is adjusted to a pH of

5.1.

12. The method according to any one of the preceding claims,

wherein in step c), the solution is incubated for at least 10

minutes at a temperature between 20C and 370C.

(22894418_1):GGG

13. The method according to claim 12, wherein in step c), the

solution is incubated for 2 hours at a temperature between 200C

and 30°C.

14. The method according to any one of the preceding claims,

wherein the initial solution of immunoglobulins has an albumin

content less than or equal to 1% (w/v) with respect to the total

proteins.

15. The method according to any one of the preceding claims,

wherein the initial solution of immunoglobulins is derived from

human plasma.

16. The method according to claims 1 to 17, wherein the

immunoglobulins of the initial solution of immunoglobulins are

obtained by genetic recombination techniques, chemical synthesis

techniques or transgenic protein production techniques, or in cell

cultures.

17. The method according to any one of the preceding claims,

wherein step d) of ultrafiltration/diafiltration is carried out

using a membrane of 100 kDa.

18. The method according to any one of the preceding claims,

wherein step d) of ultrafiltration/diafiltration 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

(22894418_1):GGG less than or equal to 5.0, in order to reduce or eliminate most of the polyether or polymer of glycol.

19. The method according to claim 20, wherein in the second phase

of step d) of ultrafiltration/diafiltration, the pH is adjusted to

between 4.0 and 5.0.

20. The method according to claim 2, wherein in the step of final

formulation, excipients and/or stabilisers are added, selected

from one or more amino acids, one or more carbohydrates or polyols,

or combinations of the same.

21. The method according to any one of the preceding claims,

wherein the final concentration of immunoglobulins is adjusted to

a concentration suitable for its intravenous, intramuscular or

subcutaneous use.

22. A solution of immunoglobulins prepared according to the

method of any one of the preceding claims.

Instituto Grifols, S.A.

Patent Attorneys for the Applicant/Nominated Person

SPRUSON&FERGUSON

(22894418_1):GGG