RU2708394C2 - Method of producing immunoglobulins - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: group of inventions relates to a method of producing an immunoglobulin solution. Method for producing a solution of immunoglobulins from an initial immunoglobulin solution with a degree of purity of not less than 96 %, concentration of 1 to 10 mg/ml, containing 2 to 6 % (w/v) polyethylene glycol (PEG) or polypropylene glycol (PPG), comprises steps a) adding caprylic acid or salts thereof to the initial solution until reaching concentration of 9 to 15 mM; b) bringing pH of solution obtained at step a) to pH 5.0–5.2; c) incubating the solution obtained at step b) for a period of time and at a temperature required to inactivate envelope viruses; and d) performing the step of ultrafiltration/diafiltration of the solution obtained at step c). Also disclosed is the use of caprylic acid for virus inactivation.EFFECT: group of inventions provides using a lower concentration of caprylate for virus inactivation when producing an immunoglobulin solution, which enables to avoid or does not cause aggregation.19 cl, 11 tbl, 9 ex

Description

The present invention relates to a new method for producing immunoglobulins. The resulting immunoglobulin composition 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 which serve in the immune system to recognize and neutralize foreign objects such as bacteria, viruses or parasites. Immunoglobulins find various applications in medicine, including in the diagnosis of diseases, therapeutic methods of treatment and prenatal therapy. The most common therapeutic applications of immunoglobulins can be classified into three general groups of pathologies: primary immunodeficiencies (humoral immunodeficiency), secondary immunodeficiencies or acquired immunodeficiencies (for example, in the prevention and treatment of viral infections) and autoimmune immunodeficiencies (antibody production).

Immunoglobulins can be administered in various ways, such as intramuscular, intravenous and subcutaneous routes, among others. Of these, it is preferable to use the intravenous route, since it provides many advantages, in particular higher therapeutic efficacy.

Typically, immunoglobulins are isolated from human plasma by methods based on the Cohn fractionation method (Cohn EJ. Et al., J Am Chem Soc, 1946, 62, 459-475), the Con-Onclie 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-Nichman method (Kistler P, Nitschmann H, 1962, 7, 414-424). Accordingly, 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 were introduced to more thoroughly purify immunoglobulins (IgG) and to give them better tolerance when administered, preferably intravenously. These modifications were introduced, for example, to remove aggregates and other impurities, as well as to ensure product safety. However, adding a plurality of steps to an immunoglobulin production technique reduces yield using this technique and increases production costs. The growing demand for immunoglobulin products, mainly for intravenous administration, has made yield a key aspect in the process of their production on an industrial scale.

Of the methods described in the prior art, methods for preparing immunoglobulin compositions that are suitable for the intravenous route include those using the following steps: precipitation with polyethylene glycol (PEG), ion exchange chromatography, physical / chemical methods capable of inactivating viruses, or processing enzymes and partial chemical modification of immunoglobulin molecules.

Therefore, the need to ensure the safety of the product requires the implementation of labor-intensive stages, making it possible to eliminate pathogenic biological agents. The method that is commonly used involves the use of a solvent / detergent to inactivate lipid-coated viruses, as this does not significantly reduce the biological activity of the proteins. However, given the toxicity of solvent / detergent mixtures, this reagent should be carefully removed before the final product is obtained, and this increases the time required for this process and reduces the yield. The procedures described for removing the indicated solvent / detergent are not simple and usually require the use of chromatographic adsorption methods, either direct, based on hydrophobic interaction, or indirect capture of immunoglobulin in ion exchange resins and separation of the unrecovered solvent / detergent. In all cases, these processes are expensive and time-consuming, including significant protein loss.

However, simpler and more effective alternative treatments capable of inactivating viruses are known in the art. For example, caprylic fatty acid (also known as octanoic acid) or its salts were used.

In US Pat. No. 4,446,134, sodium caprylate was used in combination with amino acids and heat treatment as a technique for inactivating viruses in a method for producing factor VIII. Although it is believed that a virucidal agent capable of destroying lipid membranes is undissociated caprylic acid, the technique in which this agent is used is commonly known as caprylate inactivation, in accordance with the rules in biochemistry, designate a solution of an acid and its ionized form by the name of the latter, i.e. caprylate .

Caprylic acid was also used as a precipitating agent for the isolation of immunoglobulins (Steinbuch, M. et al., Arch. Biochem. Biophys., 1969, 134 (2), 279-284). The purity and yield of immunoglobulins depend mainly on the concentration of added caprylic acid and pH. Steinbuch, M. et al. also found that it is useful to add an effective amount of caprylate in two different stages with the removal of sediment between the two stages. This gives this technique the ability to remove viruses with and without shells, due to the distribution of non-immunoglobulin proteins in the sediment.

Also found in the art are descriptions of a combination of caprylate precipitation followed by ion exchange chromatography for purification of immunoglobulins (Steinbuch, M. et al., Supra).

European patent EP 0893450 discloses a method for purification of IgG (immunoglobulin G) using fraction II + III (obtained by implementing procedures based on the previously mentioned Kohn method), comprising two anion-exchange columns sequentially after the steps of adding caprylate at a concentration of 15-25 mM at the stage double precipitation and combining both effects of caprylate: a decrease in the content of non-immunoglobulin proteins by precipitation and the ability to inactivate viruses by incubation. The subsequent stages of anion exchange chromatography, in addition to removing other impurities (IgM, IgA, albumin and others), are used to remove caprylate, and for this reason double adsorption using relatively large amounts of anionic resins is required.

PCT patent application WO 2005/082937 also discloses a method for precipitating a composition that includes immunoglobulins, comprising the steps of adding caprylate and / or heptanoate to a solution or composition that contains immunoglobulins, followed by introducing the solution into an anion exchange resin column.

However, the authors of the present invention concluded that the use of caprylate in a suitable concentration and at a suitable pH value (for example, pH 5.0-5.2) in order to ensure the ability of this treatment to inactivate viruses, as described in the prior art, causes the formation of protein aggregates with a high molecular weight, which is partially irreversible when diluted and / or pH changed. Moreover, these aggregates can only be partially separated by filtration, and therefore a special subsequent separation step is required, for example, by chromatography or precipitation. The separation of these aggregates causes significant protein loss and a decrease in the yield of the industrial process for producing immunoglobulins.

In addition, the authors of the present invention concluded that the presence of aggregates formed during the treatment with caprylate, even in very small quantities, makes it difficult to correctly remove caprylate by directly performing the separation step using an ultrafiltration membrane under optimal process conditions. These aggregates impede or impede the preparation of an immunoglobulin solution in therapeutic concentrations (for example, from 5% to 20%) due to the presence of colloids (turbidity) or instability in liquid form, thereby hampering or hindering the subsequent steps of the method for producing immunoglobulins, such as nanofiltration and sterilizing filtration .

As a consequence of the foregoing, the inventors of the present invention have developed a method for preparing immunoglobulin solutions, which unexpectedly involves treatment with caprylate, capable of inactivating viruses at a lower concentration of caprylate than that described in the prior art, and which, with a suitably purified and diluted stock solution and the presence of at least one polyester or polymer glycol inhibits, warns, avoids or does not cause the appearance of aggregates.

Additionally, the authors of the present invention found that the presence of at least one polyester or glycol polymer in the method of the present invention does not interfere with the activity and effectiveness of caprylate with respect to its ability to inactivate enveloped viruses.

In an additional aspect, the authors of the present invention for the first time described a method for producing immunoglobulins, which, in addition to including processing with inactivation ability under optimal conditions, suggests the ability to remove or reduce the content of caprylate and polyester or glycol polymer reagents (present earlier during the specified treatment) by using only ultrafiltration methodology. This ultrafiltration step allows purification and concentration of the product to levels that are acceptable for administration, for example, by the intravenous, intramuscular or subcutaneous route, without the formation of immunoglobulin protein aggregates in the final product. This eliminates the need for additional separation steps after treatment with caprylate, such as, for example, chromatography. Moreover, the residual levels of the polyester or polymer of glycol and caprylate after ultrafiltration provide the possibility of reaching concentrations of immunoglobulins, such as IgG, up to 20 ± 2%, which, when properly prepared in the form of the drug, do not destabilize during their storage in liquid form.

Considering the simplification of the method according to the present invention, this makes it possible to significantly improve the yield and very significantly reduce production costs compared with the previous methods described in the prior art, thereby not jeopardizing the level of safety or purity of the product.

Therefore, in a first aspect, the present invention relates to a method for preparing an immunoglobulin solution, comprising adding caprylic acid or its salts in the presence of at least one polyester or glycol polymer to a purified immunoglobulin solution and subsequently removing or reducing the content of said reagents by ultrafiltration / diafiltration.

In an additional aspect, the present invention relates to the use of caprylic acid or its salts in the presence of at least one polyester or glycol polymer for inactivating viruses in protein production processes and subsequently removing or reducing the content of these reagents by ultrafiltration / diafiltration.

In yet another aspect, the present invention relates to a single ultrafiltration / diafiltration step to remove or reduce levels of caprylic acid or its salts and / or polyester or glycol polymer, used to inactivate viruses in protein production processes.

Therefore, the present invention discloses a method for producing an immunoglobulin solution from an initial immunoglobulin solution with a purity of at least 96% in the presence of a polyester or glycol polymer, characterized in that it comprises the steps of:

a) adding caprylic acid or its salts to the initial solution;

b) adjusting the pH of the solution obtained in stage a);

c) incubating the solution obtained in stage b) for a period of time and at a temperature necessary for inactivation of enveloped viruses; and

g) performing the ultrafiltration / diafiltration step of the solution obtained in step c).

The method of the present invention may also include the stage of final preparation of the solution obtained in stage g), in the form of a preparation.

In the method of the present invention, the initial solution of immunoglobulins is obtained from fraction I + II + III, fraction II + III or fraction II obtained by the Cohn or Kon-Onclay method, or from precipitate A or I + A or GG obtained by the Kistler method Nitschman, or variants thereof, which were further purified to obtain IgG with a degree of purity of at least 96%. Preferably, the immunoglobulin stock solution is obtained from fraction II + III obtained by the method of Kohn or its modifications, which was then purified by precipitation with polyethylene glycol (PEG) and anion chromatography as described in EP 1225180 B1. According to the present invention, any of the aforementioned fractions can be subjected to a precipitation procedure using PEG followed by filtration to remove the precipitate and an additional purification step using an ion exchange column (e.g., a DEAE-Sepharose column). In all these cases, the initial solution of immunoglobulins is obtained from human plasma.

In a most preferred embodiment, the immunoglobulin stock solution is obtained from fraction II + III obtained using methods based on the Kohn method, which is further purified by any of the methods described in the prior art to achieve an adequate level of purification required by treatment with caprylate under non-precipitating conditions according to the present invention, that is, a purity index greater than 96% (w / v) IgG or equal to 96% IgG determined by electrophoresis in cellulose acetate, with an albumin content of preferably m nshim 1% or equal to 1% (wt. / vol.) on the total protein content. Thus, the indicated immunoglobulin stock solution is sufficiently purified, before and after treatment with caprylate, for the therapeutic route for which it is intended, so that no further purification is required after the virus inactivation step of the present invention.

The immunoglobulins of the stock solution of the method of the present invention can also be obtained by genetic recombination methods, for example, by expression in cell cultures; chemical synthesis methods and transgenic protein production methods.

In a most preferred embodiment, the immunoglobulins mentioned in the method of the present invention are IgG. It is contemplated that these IgGs may be monoclonal or polyclonal. In a most preferred embodiment, the IgGs are polyclonal.

It is contemplated that the polyesters or polymers of the glycol of the present invention may be alkane polyesters or polyalkane oxides, also known as polyglycols, and relate, for example, to derivatives of ethyl or ethylene and propyl or propylene, better known as polyethylene glycol (PEG) or polypropylene glycol (PPG ), or their equivalents. In addition, these reagents must be compatible with immunoglobulins in the sense that they do not impair their stability or solubility and that, due to their size, they can be easily removed using ultrafiltration techniques, or that, due to their reduced toxicity, they are consistent with therapeutic the use of immunoglobulins.

In a preferred embodiment, the polyester or glycol polymer is selected from polyethylene glycol (PEG), polypropylene glycol (PPG), or combinations thereof. Preferably, the glycol polyester or polymer is PEG, more preferably PEG with a nominal molecular weight of 3350 Da to 4000 Da, and most preferably PEG with a nominal molecular weight of 4000 Da.

The content of the aforementioned polyester or glycol polymer in the initial immunoglobulin solution is preferably from 2% to 6% (w / v) and more preferably from 3% to 5% (w / v).

It is contemplated that it may be necessary to adjust the concentration of said polyester or glycol polymer in an initial immunoglobulin solution. The specified concentration of the specified polyester or glycol polymer can be performed by diluting the initial purified solution of immunoglobulins and / or by adding it.

In accordance with the composition of the initial solution of immunoglobulins, it is assumed that before stage a) of the method of the present invention, a series of stages of purification or concentration are carried out, such as, for example:

- bringing the concentration of immunoglobulins to 1-10 mg / ml, more preferably 3-7 mg / ml. This summing up can be performed using any of the techniques known in the art, for example, by diluting or concentrating the protein in a predetermined range (determined, for example, in accordance with the total protein content by optical density at 280 nm E (1%) = 13 , 8-14.0 UA (conventional units) by the biuret method, the Bradford method or, in particular, immunonefelometry), as the case may be. Therefore, in a preferred embodiment, the immunoglobulin stock solution has an immunoglobulin concentration of preferably 1-10 mg / ml and more preferably 3-7 mg / ml; and / or

- bringing the purity of the solution of immunoglobulins, which preferably should reach at least 96% IgG relative to the total protein content. This purification can be carried out using methods completely known to a person skilled in the art, such as, for example, PEG precipitation, and filtration, and subsequent anion exchange chromatography (DEAE-Sepharose).

In step a) of the process of the present invention, caprylic acid or its salts are added, preferably using a concentrated solution thereof, for example from 1.5 M to 2.5 M, to achieve a final concentration of preferably from 9 mM to 15 mM.

In a preferred embodiment, in step b), the pH of the resulting solution is adjusted to 5.0-5.2, more preferably 5.1.

In a preferred embodiment, in step c), the resulting solution is incubated for at least 10 minutes, more preferably 1-2 hours, and even more preferably 2 hours. In addition, the temperature at which the indicated incubation is carried out is from 2 ° C to 37 ° C, more preferably from 20 ° C to 30 ° C.

In a preferred embodiment, prior to step d) of the method of the present invention, the content of high molecular weight polymers or aggregates in the solution obtained in said step c) is not more than 0.2% and more preferably less than 0.1%. This percentage of polymers or molecular aggregates of immunoglobulins relative to the total protein content is determined using an exclusion HPLC gel column (high performance liquid chromatography) in accordance with the absorbance at 280 nm. The specified percentage of polymers or molecular aggregates of immunoglobulins can be estimated, for example, using the analysis method described in the monograph of the European Pharmacopoeia relating to intravenous gamma globulin.

Preferably, the immunoglobulin solution is clarified using porous filters before performing step d) ultrafiltration / diafiltration.

With respect to step d), it is contemplated that ultrafiltration / diafiltration in the method of the present invention preferably includes initial stages of diafiltration and concentration with a decrease in volume followed by diafiltration with a constant volume.

Ultrafiltration / diafiltration can be carried out on an industrial scale, preferably using the method of simultaneous dialysis and concentration, reducing the volume of the product, and diafiltration, in turn, which thus somewhat reduces the consumption of reagents and makes the process more efficient, taking into account the fact that the concentration of proteins is optimal and preferably not more than 30 mg / ml. In any case, a person skilled in the art can easily determine the most suitable and practical way to carry out this stage of ultrafiltration / diafiltration, choosing among various technological methods known in the art (for example, dilution / concentration or diafiltration / concentration, diafiltration with a constant volume or modifications and combinations of the foregoing).

The ultrafiltration / diafiltration membrane used in step d) of the method of the present invention preferably consists of polysulfone, regenerated cellulose or equivalents, such as, for example, membranes sold under the trademarks Biomax® (Millipore, USA), Omega® (Pall, USA), Kvik-flow® (General Electric, USA). However, the molecular weight cutoff threshold selected for the membrane may vary depending on various factors, such as manufacturer choice. One of skill in the art can easily determine a preferred membrane that will meet the needs in each case depending, for example, on the concentration of caprylate and polyester or glycol polymer in the solution being treated.

Preferably, step d) ultrafiltration / diafiltration is carried out using a membrane with a cutoff threshold of molecular weight of not more than 100 kDa, more preferably of 100 kDa.

In the most preferred embodiment, the ultrafiltration / diafiltration in step d) is carried out in two stages:

a first step in which the pH is adjusted to a pH of 5.0-6.0 to reduce or remove most of the caprylate, and

a second step in which the pH is adjusted to a pH of less than 5.0, preferably to a pH of 4.0-5.0, to reduce or remove most of the polyester or glycol polymer.

In a preferred embodiment, in the first step of the ultrafiltration / diafiltration step, the diafiltration is carried out using a diafiltration medium that contains alkaline salts of a carboxylic acid, for example acetic acid, at a concentration of more than 5 mM or approximately 5 mM. In a most preferred embodiment, the aforementioned diafiltration is carried out using a solution of sodium acetate at a concentration of more than 5 mM or equal to 5 mM with a pH adjusted as indicated above, i.e., to a pH of 5.0-6.0.

The number of diafiltration volumes to be performed in the first step of step d) ultrafiltration / diafiltration can be easily determined by a person skilled in the art according to the amount of caprilate taken initially and an acceptable final amount. At least three volumes of diafiltration medium are preferably used, wherein said diafiltration medium is preferably, as previously mentioned, a 5 mM sodium acetate solution with a pH of 5.0-6.0. Preferably, in this first ultrafiltration / diafiltration step, approximately 90% or more of the starting caprylate is removed, so that in this first step, the concentration of caprylate is reduced to about 1 mM or lower.

In a second step of step d) ultrafiltration / diafiltration, the immunoglobulin solution is diafiltered, preferably at a constant volume.

Preferably, the diafiltration in said second ultrafiltration / diafiltration step is carried out using a buffer solution that contains alkali metal salts formed by acetate, phosphate or their equivalents or amino acids and / or polyols, for example glycine and / or sorbitol, at the pH value indicated previously.

As is the case with the first diafiltration step, in the second step, the number of dialysis volumes used to appropriately reduce the polyester or glycol polymer used in the method of the present invention can be easily determined by one skilled in the art taking into account the desired reduction in the content or removal of the polyester or polymer glycol. In a preferred embodiment, the amount of buffer to be exchanged during diafiltration in the second step of step d) ultrafiltration / diafiltration is at least six volumes. In a most preferred embodiment, in said second step, the exchange takes place in accordance with the number of buffer volumes necessary to achieve a 100-fold or more than 100-fold reduction in the content of polyester or glycol polymer relative to the initial content of said polyester or glycol polymer before the start of step d) ultrafiltration / diafiltration.

Once the content of caprylate and polyester or glycol polymer is reduced in step d) ultrafiltration / diafiltration, at the final stage of preparation in the form of the preparation mentioned above, the solution can be adjusted to the desired final composition by adding the necessary excipients and / or stabilizers, so that to concentrate the product in order to obtain the final composition. The addition of excipients and / or stabilizers, which must be performed at the end of the preparation of the composition, can be carried out directly by adding the indicated excipients and / or stabilizers in solid form or in a concentrated solution or, even more preferably, by diafiltration using the required number of volumes of solution exchange compositions to provide an appropriate composition of the final product.

In another embodiment, the addition of excipients and / or stabilizers is carried out by completely or partially replacing the sodium acetate dialysis buffer solution used in the second step of step d) with a solution containing excipients and / or stabilizers, preferably with a pH adjusted to the same value of 4.0 -5.0, so that after final concentration, the immunoglobulin is obtained already prepared in the form of a composition.

One skilled in the art knows what types of excipients and / or stabilizers should be added to achieve the desired stability. It is believed, for example, that said excipients and / or stabilizers can be one or more amino acids, for example glycine, preferably at a concentration of 0.2-0.3 M, one or more than one carbohydrate or polyol, for example sorbitol, or a combination.

Finally, the final concentration of immunoglobulins, preferably IgG, is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use, which will be known to a person skilled in the art and may, for example, be from 5% to 22% (w / v) . The indicated concentration is influenced by any procedure known in the art, for example, concentration by ultrafiltration. It is assumed that if ultrafiltration affects the concentration of immunoglobulins, this concentration can be carried out using the same membrane as in the previous diafiltration. Obviously, the three diafiltration mentioned, as well as concentration, can also be carried out using different membranes.

The method of the present invention also contemplates the possibility of introducing a nanofiltration step to increase the product safety spectrum. There are many steps in the procedure whereby a product can be nanofiltered using commercially available filters (e.g. Planova® and Bioex® manufactured by Asahi-Kasei, DV® and SV4® manufactured by Pall, Virosart® manufactured by Sartorius, Vpro®, manufactured by Millipore or equivalents) with pore sizes of 20 nm or less and up to 50 nm, preferably with pore sizes of 20 nm or less, or even 15 nm nanofilters can be used. The intermediate stages at which the nanofiltration stage can be carried out can be carried out, for example, on the initial solution of immunoglobulins, or on the material treated with caprilate after the stage of ultrafiltration / diafiltration (as soon as the content of caprilate and polyester or glycol polymer is reduced), or in the material after concentration and preparing a solution of immunoglobulins, preferably IgG, (final product) in the form of a preparation. The person skilled in the art will choose the best option depending, inter alia, on the pore size of the membrane, the filtration area required in accordance with the procedure time, the volume of the product to be nanofiltered, and the yield of protein.

The final product obtained by the method of the present invention fully meets the criteria of the European Pharmacopoeia with respect to the content of isohemagglutinins. However, the method of the present invention also contemplates the possibility of incorporating a selective and specific capture step of anti-A and / or anti-B blood antibodies to achieve the maximum reduction in their content. This step is preferably carried out using biospecific affinity resins, as described in the prior 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 possibly be included at the discretion of a person skilled in the art at any stage of the method of the present invention or may be performed before or after the implementation of the method of the present invention.

Therefore, with respect to the method for preparing the immunoglobulin solution of the present invention, in the most preferred embodiment, an immunoglobulin stock solution with a purity of at least 96% IgG is used. The concentration of IgG in this solution is preferably adjusted to a concentration of from 1 mg / ml to 10 mg / ml, and preferably from 3 mg / ml to 7 mg / ml, and it contains (due to the addition at the previous stages) PEG at a concentration of 4 ± 1% (w / v) or PEG is added to it to a concentration of 4 ± 1% (w / v). Then, the pH of the solution is adjusted to 5.0-5.2 with acetic acid and sodium caprylate is added (for example, using a concentrated solution of said sodium caprylate). In a preferred embodiment, the concentrated caprylate solution is added slowly and with stirring to the purified IgG solution. After the addition of all caprylate, calculated to bring the product to a final concentration of caprylate 9-15 mm, the pH is finally adjusted to 5.0-5.2 if necessary, and the solution is incubated, preferably at a temperature of 2-37 ° C and more preferably at a temperature of 25 ± 5 ° C for at least 10 minutes and preferably for 1-2 hours.

Purification is then performed using porous filters (e.g. Cuno 90LA, 50LA, Seitz EK, EK-1, EKS or equivalents).

The solution thus obtained is then treated with ultrafiltration / diafiltration equipment formed by polysulfone-containing membranes, for example Biomax® manufactured by Millipore or Omega® from Pall, preferably in the form of a composite cartridge. The solution is recycled through each ultrafiltration / diafiltration element, preferably in a volume of about 100-500 l / h and at a temperature of 5 ± 3 ° C. The pressure difference between the inlet and outlet pressures (atmospheric pressure) is preferably from 1 bar (100 kPa) to 3 bar (300 kPa). Then, the first diafiltration step of the ultrafiltration / diafiltration step is started to remove caprylate, preferably using an exchange of at least three volumes of a buffer, preferably formed by a solution of sodium acetate at a concentration of at least 5 mM and a pH of 5.0-6.0. Preferably, with each volume of buffer added or consumed, the volume of the product solution is reduced by half from the original volume, with the exception of the last addition.

After the first step of diafiltration (by dilution and concentration, or in an equivalent way), the pH of the resulting solution is adjusted to 4.0-5.0 with, for example, acetic acid. Then, diafiltration is started at a constant volume, preferably using six or more volumes of a buffer solution formed by sodium acetate at a concentration of not less than 5 mM and at a pH of 4.0-5.0.

The aforementioned sodium acetate dialysis buffer solution may possibly be completely or partially replaced with an amino acid solution, for example, glycine at a concentration of 0.2-0.3 M, possibly in combination with carbohydrates and polyols, for example sorbitol, whose pH is preferably adjusted to such the same values are 4.0-5.0, so that after final concentration the immunoglobulin is obtained already prepared in the form of a preparation.

After applying preferably at least six volumes (more preferably six to ten volumes) of the aforementioned dialysis solutions at pH 4.0-5.0, the product can be prepared in the form of a preparation, if it is not already in the form of a preparation, by direct addition to the resulting solution of excipient (s) and / or stabilizer (s), such as, for example, glycine or other amino acids, as well as carbohydrates, for example sorbitol, or a combination thereof, in the solid state or in the form of a concentrated solution of the specified excipient (s) ) and / or and stabilizer (s). Then the IgG solution obtained by reducing the volume is concentrated to achieve an IgG concentration suitable for intravenous, intramuscular or subcutaneous use.

The specified concentrated solution, with suitably adjusted values of the concentration of excipient (s) and / or stabilizer (s) and pH, is subjected to absolute filtration using filters with a pore size of 0.2 μm and possibly nanofiltration. Finally, an IgG solution under aseptic conditions is dosed in the form of injectable preparations in ampoules, test tubes, vials or other glass containers, which are then hermetically closed. Another option is dosing into compatible rigid or flexible plastic containers, such as bags or vials.

The dosed product passes through quarantine and visual inspection before it is stored at a temperature of 2-30 ° C to preserve up to at least 2 years.

Moreover, as previously mentioned, the present invention also for the first time discloses the use of caprylic acid or its salts in the presence of at least one polyester or glycol polymer to inactivate viruses in the process of producing a protein in which said polyester or glycol polymer and caprylic acid or its salt subsequently removed by ultrafiltration.

Preferably, said proteins are selected from the group of proteins that includes immunoglobulins, albumin, coagulation factors such as factor VII, factor VIII and factor IX, and von Willebrand factor. Even more preferably, said proteins are immunoglobulins. In a most preferred embodiment, said proteins are IgG.

The present invention will now be described in more detail with reference to various examples of embodiments. However, these examples are not intended to limit the scope of the present invention, but are intended only to illustrate its description.

EXAMPLES

Example 1. The method of the present invention for obtaining from plasma a solution of immunoglobulins, which is virologically safe, does not contain aggregates and has a sufficient yield for industrial use

The starting material was 16 liters of an immunoglobulin solution that contained IgG as the main protein component, obtained by the method described in European patent EP 1225180 B1. Briefly, said solution was obtained by extracting gammaglobulin from fraction II + III using the Cohn method. To carry out this extraction of gammaglobulin from fraction II + III, this fraction was previously isolated by fractionation of human plasma using ethanol. Then it was suspended in the presence of carbohydrate and the content of most of the accompanying proteins was reduced by precipitation using PEG-4000. Finally, the final purification of the fraction was carried out by adsorption on an ion exchange resin column (DEAE-Sepharose). The column effluent thus obtained (the fraction not adsorbed on the resin, i.e. DEAE) had an electrophoretic purity in cellulose acetate (ACE) of 98 ± 2% immunoglobulins, pH 6.0, a turbidity of 2.6 nephelometric turbidity units (NTU), and an IgG concentration approximately 5 mg / ml.

The pH of the resulting solution was brought to pH 5.1 by adding acetic acid, and its temperature to 2-8 ° C. Then, the concentration of this immunoglobulin solution was adjusted to a final concentration of 13 mM by adding a concentrated sodium caprylate solution.

The caprylate immunoglobulin solution was heated to 25 ° C and incubated at this temperature for 2 hours with slow stirring. During the incubation procedure, the pH was maintained at 5.10 ± 0.05. The turbidity of the resulting solution was 17.3 NTU.

The caprylate-treated solution was cooled to approximately 8 ° C for subsequent clarification using a porous filter (CUNO®, Ultrafilter, Denmark). Approximately 20 liters of filtered liquid was obtained after the indicated clarification (including washing) with an IgG concentration of approximately 4 mg / ml and a turbidity of less than 3 NTU.

The aforementioned clarified solution was dialyzed by ultrafiltration using membranes with a nominal cutoff threshold of molecular weight of 100 kDa (Biomax®, Millipore, USA). Ultrafiltration was carried out in two differentiated stages: in the first stage, the material, which had a pH of 5.1, was subjected to three stages of sequential dialysis and concentration by diafiltration using a 5 mM acetate solution, whose pH was adjusted to 5.1, and by concentration to approximately 30 UA. In the second stage, the pH of the solution, which had a sufficient concentration of proteins, caprylate and PEG, was adjusted to 4.5 ± 0.1 and then dialysis was started using eight volumes of a 5 mM acetate solution with a pH of 4.5. The product was then prepared in the form of a preparation by dialysis using approximately 20 liters of a 200 mM glycine solution with a pH of 4.2 and concentrated in the same ultrafiltration element to a value of 140.5 UA in order to obtain an IgG solution with a concentration of 10% (w / v) .).

Finally, this solution was filtered using a porous filter (CUNO®, Ultrafilter, Denmark) and absolute filters or membranes with a pore size of 0.22 μm (CVGL®, Millipore, USA or DFL®, PALL, USA).

Table 1 presents the characteristics of the starting material, several intermediate products and the final product in accordance with the method described above. Regarding the results included in this table, it should be noted that turbidity was measured by nephelometry; the percentage of polymer or immunoglobulin molecular aggregates relative to the total content of the detected proteins was determined on a gel column for size exclusion HPLC in accordance with the optical density at 280 nm; the concentration of caprylate was determined by the enzymatic method by quantitative determination of the colorimetric substrate; PEG concentration was determined using an HPLC gel filtration column using a refractometric detector; and the percent yield of this process was calculated according to the concentration of IgG quantified by nephelometry.

The results obtained in this example show that treating the aforementioned purified solution with caprylate does not cause any formation of immunoglobulin aggregates or other precipitates, maintaining the molecular distribution unchanged in the product. Therefore, after treatment with caprylate, no purification steps were required to remove aggregates and / or sediments. This fact greatly facilitated the production process and made it possible to directly apply the material to the ultrafiltration membrane.

Thus, the subsequent ultrafiltration process satisfactorily achieved the goal of effectively reducing the chemical content of the production process (i.e., PEG and caprylate), and also provided the possibility of subsequent preparation in the form of a preparation and concentration of the purified immunoglobulin solution to obtain a suitable composition for its therapeutic use.

As can be seen from table 1, the yield of protein obtained in this case, from the initial effluent to 10% of the concentrated product was 89.4%, demonstrating the effectiveness of this process on an industrial scale. This yield was higher than the value obtained by traditional methods in accordance with the prior art and as described in PCT patent application WO 2005/073252 (70% yield based on 4.8 g / L yield compared to the original 6.8 g / l).

Example 2. The influence of the purity of the initial solution of immunoglobulins during treatment with caprylate

In this example, the effect of the purity of the initial immunoglobulin solution and the presence of concomitant proteins in the starting material subjected to the method of the present invention was evaluated.

Two independent experimental test groups were formed:

- in group A, the starting material was an effluent of a DEAE-Sepharose column with electrophoretic purity (ACE) of 98 ± 2% IgG, that is, the starting material described in Example 1;

- in group B, the starting material, designated as 4% PEG filtrate, was obtained by the same process described in Example 1, up to the stage before chromatography on DEAE-Sepharose. Thus, material B was obtained after precipitation using a PEG extraction suspension of fraction II + III and had an electrophoretic purity (ACE) of approximately 90% IgG.

Both starting materials (group A and group B) with an equivalent PEG content of approximately 4% were subjected to treatment with caprylate at a concentration of 13 mM and with a pH of 5.0-5.2 and purified as shown 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 the characteristics of the material obtained in the steps following processing with caprylate.

The results obtained and summarized in Table 2 show that the addition of caprylate in an effective concentration for inactivation (13 mM) to a low-purity material (approximately 90% IgG, see group B) precipitates the components of the solution, resulting in a sharp increase in turbidity ( above 500 NTU). Thus, the molecular distribution results for this solution showed the precipitation of a portion of the high molecular weight concomitant proteins.

The addition of caprylate in quantities and under the conditions described previously (13 mM caprylate, pH 5.0-5.2) to a low-purity material led to a precipitated suspension, which made it necessary to include additional separation and purification steps to separate high molecular weight proteins and precipitated aggregates. Consequently, the molecular composition of the product of group A, treated with caprylate, i.e. with an aggregate content exceeding 1%, demonstrated the inefficiency of processing this product into a purified final product without additional purification or separation steps, such as PEG precipitation, chromatography or equivalent methods. Finally, this fact demonstrates the effectiveness of using caprylate as an agent with the ability to inactivate viruses under non-precipitating conditions, only when it is added to the material of a sufficient degree of purity.

Example 3. The effect of the composition of the starting material on the formation of aggregates

The objective of this experiment was to assess the effect of the composition of the initial immunoglobulin solution, for which caprylate treatment was used.

Two independent experimental test groups A and B were formed based on materials with an equivalent degree of purity (97.9 ± 1.5%), but of different composition.

In group A, the starting material was a column effluent (obtained in accordance with the starting 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 as concentrated and dialyzed effluent, was the same column effluent mentioned for group A, but after concentration and dialysis. Therefore, the DEAE column effluent (mentioned above in Example 1 and corresponding to group A of the present example) was subjected to an additional stage of dialysis and concentration by ultrafiltration, so that the PEG content was reduced by approximately 6 times, and the protein was concentrated to approximately 4%, then there are 40 mg / ml.

The material obtained in both experimental groups A and B was subjected to treatment with caprylate at a concentration of 13 mM and a pH of 5.0-5.2 and ultrafiltration under the conditions described in Example 1 to obtain a product with an IgG concentration of 10%.

Table 3 describes in detail the main characteristics of the material processed in the aforementioned experimental groups A and B, as well as the characteristics of the material obtained in the stage following the treatment with caprylate and diafiltered and concentrated final product for each experimental group.

As can be seen from Table 3, the results confirm that caprylate treatment of the purified immunoglobulin solution at a concentration of 5 ± 2 mg / ml and in the presence of PEG at a concentration of 40 ± 10 mg / ml (4 ± 1%) (group A, column effluent) in certain conditions does not cause any change or aggregation of the solution of immunoglobulins, maintaining the molecular distribution of the product unchanged during and after the addition of caprylate with an undetectable fraction of aggregates of less than 0.1%.

However, when the same caprylate treatment conditions were applied to a material with a low PEG content (less than 1%) (group B), a significant increase in the number of immunoglobulin aggregates was observed after caprylate was added. Moreover, it was not possible to remove this aggregated content by ultrafiltration under the applicable conditions, and comparable polymer levels were found in the final product.

Considering that the main distinguishing characteristics of the starting materials used in experimental groups A and B were protein concentration and PEG concentration, an additional test was conducted to verify the effect of each of these parameters on subsequent treatment with caprilat.

In this experiment, the starting point was one batch of concentrated and dialyzed effluent (starting material of the previous group B), which was divided into four separate experimental groups: groups B1, B2, B3 and B4.

Group B1 material was processed at a protein concentration of approximately 4% and a PEG concentration of approximately 0.6%.

The material of group B2 was processed at the same protein concentration of about 4%, but the PEG content was changed to 4 ± 1% (w / w).

In groups B3 and B4, the material was diluted to 0.5 ± 0.2% protein. Regarding the PEG content in group B3, it was adjusted to a concentration of approximately 0.6% (w / w), while in group B4, the PEG content was changed to 4 ± 1% (w / w).

The pH of the resulting material in these four experimental groups was adjusted to 5.10 ± 0.05 and the caprylate concentration in it was up to 15 mM, and then incubated at 25 ° C for 2 hours. The results obtained are shown in Table 4.

The results presented in Table 4 show that during the treatment with caprylate under the specified conditions, the protective effect of PEG was observed in combination with a sufficient dilution of the protein. Noteworthy is the fact that when the starting material had an approximate protein concentration of 5 ± 2 mg / ml and a PEG concentration of 4%, after treatment with caprylate, the levels of aggregates obtained were non-detectable (less than 0.1%).

Example 4. The effect of pH on the solubility of a solution of immunoglobulins treated with caprylate

It is known that the removal of PEG from solutions of immunoglobulins, as well as the concentration of these immunoglobulins to suitable concentrations for their intravenous administration, should preferably take place at pH values of about 4.5.

Moreover, taking into account the insolubility of caprylic acid at pH values below its pKa (4.89), in this experiment, we evaluated the effect of pH on the solubility of a caprylate-treated immunoglobulin solution in order to determine the appropriate pH value to start its ultrafiltration.

To this end, a batch of column effluent obtained in accordance with the original method described in detail in Example 1 was processed to obtain an immunoglobulin solution treated with 13 mM caprylate and clarified.

This intermediate product, which constitutes the material before the ultrafiltration stage, was acidified by the addition of acetic acid to bring its pH value when treated with caprylate (5.1) to pH values of about 4.5. Then, the appearance and solubility of the solution for each of the estimated pH values were evaluated, and the formation of colloidal particles was quantified by nephelometric measurement of turbidity.

Table 5 shows the results of the assessment of appearance and turbidity obtained for each of the estimated pH values.

The results obtained, as can be seen from Table 5, demonstrate that when an immunoglobulin solution treated with 13 mM caprylate was acidified to pH 5.0 or lower, a whitish precipitate appeared along with a clear increase in turbidity. Most likely, this effect arose due to the formation of insoluble caprylic acid in colloidal form, which made it impossible to start the ultrafiltration process at pH values below 5.0.

The obtained results prove that when the purified solution was subjected to treatment with caprylate in a concentration range effective for inactivation of viruses (9-15 mm caprylate), and under the conditions described earlier, it is preferable to start the subsequent stage of ultrafiltration at a pH value of not less than the pH of the treatment for inactivation of viruses , i.e. 5.1, in order to increase the concentration of the ionized and soluble form of caprylate and, therefore, facilitate its penetration through the ultrafiltration membrane.

Example 5. The influence of the acetate content in the dialysis solution on the reduction of caprylate by ultrafiltration / diafiltration A series of independent ultrafiltration / diafiltration processes was carried out in the presence of various concentrations of acetate in the buffer solution used for dialysis of the product.

The starting material used, designated as concentrated and dialyzed effluent, was the same as the group B of Example 3. The specified 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 processed caprylate and then ultrafiltration / diafiltration using membranes with a nominal molecular weight cut-off threshold of approximately 100 kDa.

The ultrafiltration / diafiltration step used included the first step of concentrating to about 4% (w / v) IgG, the second step of dialysis using eight volumes of dialysis solution, and finally concentrating to about 9-10% (w / v) IgG

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 acetate, respectively, and with a pH adjusted to 5.0-5.5 in all cases.

The number of dialysis volumes = In (Cf / Co) / (R-1); where Cf is the concentration after dialysis with the number of dialysis volumes in question, Co is the concentration before dialysis, and R is the retention coefficient.

The results in Table 6 show that the ultrafiltration / diafiltration method using membranes with a cutoff threshold of molecular weight of approximately 100 kDa using 8 dialysis volumes of buffer solution with acetate at pH 5.0-5.5 and with a minimum concentration of acetate of about 5 mm and at least 50 mm allows you to satisfactorily achieve the goal of effectively reducing the content of caprylate to suitable levels in the final concentrated product.

Conversely, when the solution used for dialysis was water for injection or a buffer solution with acetate levels of 2 mM, caprylate was not effectively removed from the filtrate.

This proves that the ultrafiltration / diafiltration method using a membrane with a molecular weight cut-off threshold of about 100 kDA under the conditions described above is effective in reducing the caprylate content from the previous treatment, given that the correct levels of the indicated reagent were determined in the final concentrated product.

Example 6. Simultaneous removal of chemicals (PEG and caprylate) through a single ultrafiltration step

A batch of IgG was processed in accordance with the method described in Example 1 to obtain a solution inactivated by caprylate and clarified. The specified solution with an approximate protein concentration of 0.5% and a pH of 5.1 was processed using ultrafiltration / diafiltration equipment formed by polysulfone membranes of the Biomax® type (Millipore, USA) with a molecular weight cut-off threshold of 100 kDa. Ultrafiltration / diafiltration was carried out in two separate stages as described in Example 5:

- at the first stage, carried out at a pH of 5.1, 5.6 or 5.8, the material was subjected to sequential dialysis and concentration stages by diafiltration with at least three volumes of a 5 mM acetate buffer solution with a pH adjusted to 5.1, 5, 6 or 5.8, and concentrating the protein to an approximate value of 2%;

- at the second stage, as soon as the caprylate content was reduced to approximately one tenth, the pH of the solution was adjusted to 4.5 ± 0.1 or 5.1. The product was then adjusted to adequate protein concentrations to begin dialysis, and dialysis was started with eight volumes of 5 mM acetate buffer pH 4.5 or 5.1.

Finally, the product was prepared in the form of a preparation by dialysis with six volumes of a 200 mM glycine solution and a pH of 4.2 and concentrated to obtain a 10% IgG solution.

Table 7 shows the percent passage of PEG and caprylate obtained at the beginning of each ultrafiltration / diafiltration step and at different pH values.

The results in Table 7 show that at the beginning of the ultrafiltration / diafiltration step in step I, caprylate showed very high transmission values at pH 5.1-5.8. These values led to a very strong decrease in the content of caprylate during the indicated stage I of the ultrafiltration / diafiltration step (a decrease in the content of caprylate by more than 10 times relative to the initial content was obtained). On the contrary, the passage of PEG was very low (less than 20%) at the indicated stage I, and its complete removal was almost impossible at a pH of more than 5 in the presence of caprylate.

On the other hand, in stage II, as can be seen from Table 7, the passage of PEG was very high at pH 4.5, with a value of 82%. Additionally, it was found that during this stage II, the content of caprylate also decreased, taking into account the fact that at the beginning of this stage it was present at a residual level of less than or equal to 1 mm, which provides almost 100% passage.

Table 8 describes in detail the change in the concentration of protein, PEG and caprylate at each stage of the ultrafiltration / diafiltration stage and at the final stage of preparation in the form of a preparation.

In accordance with the PEG and caprylate values recorded at each stage and each stage, and taking into account the protein concentration at each stage, the PEG reduction coefficient was 4 in stage I (at pH 5.1) of ultrafiltration / diafiltration and 90 in stage II (at pH 4.5 of the ultrafiltration / diafiltration step, giving a total reduction factor (step I and step II) of about 350 times (initial absorption of 6.9 compared to the absorption of 0.02 obtained at the end of the ultrafiltration / diafiltration step).

In the case of caprylate, the reduction coefficient was 55 in stage I (at pH 5.1) of the ultrafiltration / diafiltration stage and 13 in stage II (at pH 4.5) of the ultrafiltration / diafiltration stage, giving the overall reduction coefficient (stage I and stage II) approximately 700 times (the initial absorption of 2.2 compared with the absorption of 0.003 obtained at the end of the ultrafiltration / diafiltration stage).

The results showed that the content of a reagent with the ability to inactivate viruses (caprylic acid or caprylate), as well as a precipitant (PEG) can be effectively reduced by a single ultrafiltration step using a membrane with a molecular weight cut-off threshold of approximately 100 kDa, choosing physical and chemical conditions that will be used at each stage of the ultrafiltration / diafiltration stage (including pH, protein concentration, number of dialysis volumes, dialysis buffer) and leading to the final production IgG product, concentrated to 10%, with small residual concentrations of both reagents suitable for intravenous use.

Example 7. Evaluation of the virus-inactivating ability of caprylate in the presence of PEG

Various independent experiments were performed in which column effluent or Analyzed and concentrated effluent (obtained in accordance with Examples 1 and 3, respectively) were taken as starting material to evaluate the ability of caprylic acid or caprylate in the presence of PEG to eliminate or inactivate lipid-coated viruses.

Both materials had an immunological purity of 98 ± 2% and a protein concentration of 5-10 mg / ml, while the PEG content in them was different, being 40 mg / ml and 1.5 mg / ml, respectively.

Virus inactivation tests were performed using the bovine diarrhea virus (BVDV) of the flavivirus family (Flaviviridae), 40-60 nm, with a lipid membrane and medium resistance to physical and chemical agents.

In each test, the corresponding starting material was inoculated with the virus to a value of not more than 0.5% and subjected to a virus inactivating treatment for two hours at a temperature of 15 ° C or 25 ° C using caprylate concentrations of 9 mM or 13 mM.

Quantification of the BVDV viral load in the various examples obtained was performed using the TCID50 test (50% dose of tissue culture infection) using the MBDK cell line. Virus attenuation factor (RF) of the virus inactivation stage was determined as an indicator of the viral load determined in the inoculated starting material divided by the amount of virus detected in the resulting sample at the end of the treatment, expressed in log ₁₀ .

Table 9 describes in detail the characteristics of the source material of each test, as well as the obtained RF value.

The results of the attenuation of the virus obtained in all tests (see Table 9) showed a high ability to inactivate BVDV in both starting materials even for a minimum 9 mM concentration of caprylate after treatment at different temperatures (15 and 25 ° C). Moreover, these tests showed that for each of the analyzed PEG concentrations, no interference with the ability of caprylate to inactivate the virus was detected by PEG, given that equivalent results were obtained with both analyzed materials.

Example 8. Characterization of an intravenous immunoglobulin solution obtained in accordance with the production method of the present invention

This example is intended to establish the biochemical and functional properties of a solution of immunoglobulins with 10% (w / v) proteins obtained by the method of the present invention.

Two batches of effluent from the DEAE column were processed in accordance with the method described in detail in Example 1 to obtain an inactivated viral solution with caprylate in approximately 200 liters of plasma.

The specified solution with caprylate after clarification was subjected to dialysis and concentration by ultrafiltration at separate stages, as described in Example 6, in order to achieve the removal of basic production residues (PEG and caprylate). Then the specified purified solution with an approximate concentration of protein of 2.5% was prepared in the form of a preparation by dialysis at a constant volume of approximately 6 volumes of a buffer solution consisting of 1% sorbitol and 240 mm glycine, with a pH adjusted to 4.5 ± 0, one. Finally, this solution was concentrated by ultrafiltration and the optical density was adjusted to 140 ± 5 UA (280 nm), equivalent to 10% (w / v) of the proteins, and its pH was adjusted to a final pH of 5.25 ± 0.25.

The resulting product (IGIV 10% (w / v)), stabilized with sorbitol and glycine and once clarified and filtered using sterilizing membranes (0.22 μm), was dosed in glass bottles with chlorobutyl stoppers to determine the most relevant analytical quality parameters, the immutability and stability of the immunoglobulin solution for intravenous administration. The average analytical values obtained for two batches, as well as standard indicators for the European Pharmacopoeia are shown in Table 10.

The above results confirm that the obtained product remains essentially unchanged as a result of the purification process of the present invention with respect to such parameters as the absence of polymer, undesirable biological activity, such as the activity of PKA or ACA, among others, keeping several functional properties intact with respect to plasma, such as the ratio of IgG subclasses and the integrity of the Fc fragment, while demonstrating an excellent purity profile (low titer of anti-A / anti-B isohemagglutinins, IgM concentration, procoagulant activity, and so on).

It was concluded that, in general, the method of the present invention for producing 10% (w / v) IGIV, comprising the step of inactivating viruses using caprylate in the presence of PEG and its subsequent separation, as well as the final preparation in the form of a preparation, is completely feasible and scalable in relation to the final product, prepared in the form of a preparation and concentrated in the form of a solution of 10% IGIV protein (w / v), giving a final product that is fully consistent with the indicators established in the European Pharmacopoeia.

Stability studies, which are essential for the commercial viability of the product, demonstrated the suitability of compositions with sorbitol (up to 5%), glycine (until isotonic), or a combination of them in the pH range 4.2–6.0 to stabilize 10% (w / v). ) intravenous immunoglobulin solutions at ambient temperature (25 ° C-30 ° C) for two years.

Example 9. The applicability of treatment with caprylate to the enriched IgG fraction obtained by alternative methods

The validity of the use of caprylate treatment under the conditions described in the present invention was evaluated using other process intermediates obtained using alternative purification methods.

Two independent experiments were performed using an IgG enriched plasma intermediate as the starting material, designated as a suspension of fraction II from Cohn-Onclie fractionation with ethanol.

This intermediate was obtained by the same plasma fractionation method described in the present invention up to fraction II + III. Then this procedure was continued using alcohol re-precipitation of the extraction suspension of fraction II + III, followed by separation of fraction III, finally obtaining fraction II with a degree of purity of more than 96%. A suspension of the indicated fraction II, being purified by bentonite and dialyzed with water to remove alcohol contents, served as the starting material for these experiments.

In two experiments, the material obtained from two lots of plasma was divided into two different groups A and B, in accordance with the content of PEG in them. In group B, the concentration of PEG in the starting material was adjusted to a nominal concentration of 40 mg / ml by adding a concentrated solution of PEG-4000.

Then, both materials obtained from both groups (A and B) were diluted to a protein concentration of approximately 5 mg / ml, the pH was adjusted to 5.1, and subjected to caprylate treatment to achieve a nominal concentration of 13 mM and a pH of 5.0-5.2 as described in the method of this invention.

Table 11 describes in detail the main characteristics of the starting material used in both experimental groups (A and B, respectively), as well as the characteristics of the material obtained after treatment with caprylate.

The results show the effectiveness of inactivating treatment with caprylate under certain conditions using an immunoglobulin solution sufficiently purified by various methods, where the formation of immunoglobulin aggregates or other irreversible precipitation does not occur, which greatly facilitates the subsequent purification process.

The results demonstrate that in combination with a sufficient dilution of the protein and a sufficient degree of purity, the protective effect of PEG in relation to the formation of immunoglobulin polymers is clearly visible.

This experimental example demonstrates the effectiveness of caprylate only as a reagent with the ability to inactivate viruses under non-precipitating conditions and / or conditions that stimulate aggregation when it is added to the material with a sufficient degree of purity and corresponding to certain conditions related to protein and PEG concentration.

Although the invention has been presented and described with reference to its embodiments, it should be understood that these embodiments do not limit the invention, since there may be many variables from the point of view of production or other details that will be obvious to a person skilled in the art after covering the subject matter disclosed in the present description and claims. Therefore, all variants or equivalents will be included in the scope of the present invention, if they can be considered falling within the broadest scope of the following claims.

Claims (25)

1. A method of obtaining an immunoglobulin solution from an initial immunoglobulin solution with a purity of at least 96%, a concentration of 1 to 10 mg / ml and containing 2 to 6% (w / v) polyethylene glycol (PEG) or polypropylene glycol (PPG), characterized in that it comprises the steps of: a) adding caprylic acid or its salts to the initial solution to achieve a concentration of from 9 to 15 mm; b) adjusting the pH of the solution obtained in stage a) to a pH of 5.0-5.2; c) incubating the solution obtained in stage b) for a period of time and at a temperature necessary for inactivation of enveloped viruses; and g) performing the ultrafiltration / diafiltration step of the solution obtained in step c). 2. The method according to p. 1, characterized in that the method also includes the stage of final preparation of the solution of immunoglobulins obtained in stage g), in the form of a preparation. 3. The method according to p. 1 or 2, characterized in that the initial solution of immunoglobulins is obtained from fraction I + II + III, fraction II + III or fraction II obtained by the method of Cohn or Kon-Onclay, or from precipitate A, or I + A, or GG obtained by the Kistler-Nichman method, or variants thereof, which were further purified to achieve a purity of at least 96% IgG. 4. The method according to p. 3, characterized in that the initial solution of immunoglobulins is obtained from fraction II + III obtained by the method of Kohn or its modifications, which was then purified by precipitation with polyethylene glycol (PEG) and anion chromatography. 5. The method according to p. 4, characterized in that the concentration of immunoglobulins in the initial solution of immunoglobulins is 3-7 mg / ml 6. The method according to p. 1, characterized in that the concentration of PEG in the initial solution is from 3 to 5% (wt./about.). 7. The method according to p. 1 or 6, characterized in that the PEG is a PEG with a nominal molecular weight of 4000 Da. 8. The method according to p. 1, characterized in that in stage b) the pH of the resulting solution is adjusted to pH 5.1. 9. The method according to any one of paragraphs. 1-8, characterized in that at stage c) the solution is incubated for at least 10 minutes at a temperature of from 2 to 37ºC. 10. The method according to p. 8, characterized in that at stage c) the solution is incubated for 2 hours at a temperature of from 20 to 30ºC. 11. The method according to any one of paragraphs. 1-10, characterized in that the albumin content in the initial solution of immunoglobulins is not more than 1% (wt./about.) Relative to the total protein content. 12. The method according to any one of paragraphs. 1-11, characterized in that the initial solution of immunoglobulins is obtained from human plasma. 13. The method according to PP. 1-12, characterized in that the immunoglobulins of the initial solution of immunoglobulins are obtained by genetic recombination methods, chemical synthesis methods or methods for producing transgenic proteins, or in cell cultures. 14. The method according to any one of paragraphs. 1-13, characterized in that stage d) ultrafiltration / diafiltration is carried out using a membrane of 100 kDa. 15. The method according to any one of paragraphs. 1-14, characterized in that stage d) ultrafiltration / diafiltration is carried out in two stages: - the first stage, in which the pH value is adjusted to a pH of 5.0-6.0 to reduce the content or remove most of the caprylate; - and the second stage, in which the pH value is brought to a value not higher than 5.0 to reduce the content or remove most of the specified glycol. 16. The method according to p. 15, characterized in that in the second stage of step d) ultrafiltration / diafiltration, the pH value is adjusted to a pH of 4.0-5.0. 17. The method according to p. 2, characterized in that at the stage of final preparation in the form of a preparation, excipients and / or stabilizers selected from one or more than one amino acid, one or more than one carbohydrate or polyol or combinations thereof are added. 18. The method according to any one of paragraphs. 1-17, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use. 19. The use of caprylic acid or its salts with a concentration of from 9 to 15 mm in the presence of from 2 to 6% (w / v) polyethylene glycol (PEG) or polypropylene glycol (PPG) for inactivation of viruses in the method for producing an immunoglobulin solution according to claim 1 wherein said PEG or PPG and caprylic acid or its salts are subsequently removed by ultrafiltration.