HK1246812B

HK1246812B - Method for the preparation of immunoglobulins

Info

Publication number: HK1246812B
Application number: HK18106311.9A
Authority: HK
Inventors: 佩雷‧里斯托尔德巴尔特; 萨尔瓦多尔‧格兰沙加蒙; 胡安‧伊格纳西奥‧霍尔克拉捏托; 玛丽亚‧默西迪丝‧法罗托玛斯; 纽莉亚‧乔巴基立福
Original assignee: 盖立复集团公司
Filing date: 2018-05-15
Publication date: 2023-08-25

Description

Method for producing immunoglobulins

Technical Field

The present invention relates to a novel process for the preparation of immunoglobulins. The obtained immunoglobulin composition is suitable for, e.g., parenteral administration.

Background

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 recognize and neutralize foreign bodies such as bacteria, viruses, or parasites. Immunoglobulins have a variety of medical applications, such as diagnosis of disease, therapeutic treatment (therapeutic treatment) and prenatal treatment. The most common therapeutic applications of immunoglobulins can be divided into three major groups of pathologies: primary immunodeficiency (humoral immunodeficiency), secondary immunodeficiency or acquired immunodeficiency (e.g., in the prevention and treatment of viral infections) and autoimmune deficiency (development of antibodies).

The immunoglobulin may be administered by a variety of routes, such as intramuscular, intravenous, and subcutaneous routes, among others. Among them, the intravenous route is preferably used because it provides many benefits, in particular greater therapeutic efficacy.

Immunoglobulins are typically purified from human plasma by using procedures based on: cohn fractionation methods (Cohn EJ. et al, J Am Chem Soc,1946,62,459-475), cohn-Oncley methods (Oncley JL. et al, J Am Chem Soc,1949,71,541-550), or other equivalent methods based on cold ethanol fractionation, such as the Kistler-Nitschmann method (Kistler P, nitschmann H,1962,7,414-424). Thus, an immunoglobulin-rich fraction obtained by any of the above methods (such as fraction II + III, or fraction II, or precipitate a, or gamma globulin GG precipitate) is used. Modifications have been introduced to more thoroughly purify immunoglobulins (IgG) and make them tolerable for administration, preferably intravenous administration. The modifications have been introduced, for example, to remove aggregates and other impurities, and to ensure the safety of the product. However, adding multiple steps to the procedure for preparing immunoglobulins reduces the yield of the procedure and increases manufacturing costs. The increasing demand for immunoglobulin products, mainly for intravenous administration, has made yield a critical aspect in processes for their production on an industrial scale.

Among the methods described in the prior art, the procedures for obtaining immunoglobulin compositions tolerable via the intravenous route include those using the following steps: precipitation with polyethylene glycol (PEG), ion exchange chromatography, physical/chemical methods with virus inactivation capability, or treatment with enzymes and partial chemical modification of immunoglobulin molecules.

Thus, it is necessary to ensure the safety of the product by implementing a robust step with the ability to eliminate pathogenic biological agents. Commonly used methods include the use of solvents/detergents to inactivate viruses with lipid envelopes, as this does not severely reduce the biological activity of the protein. However, in view of the toxicity of the solvent/detergent mixture, this agent must be completely eliminated before the final product is obtained, and this increases the time required for the process and reduces the yield. The procedures described for eliminating the solvent/detergent are not simple and generally require the use of chromatographic adsorption techniques, either directly through hydrophobic interactions or indirectly by capturing the immunoglobulin in an ion exchange resin and isolating the uncaptured solvent/detergent. In all cases, the process is expensive and laborious, involving significant loss of protein.

However, simpler and more efficient alternative treatments with the ability to inactivate viruses are known in the art. For example, caprylic fatty acid (also known as octanoic acid) or a salt thereof has been used.

In patent US4446134, sodium caprylate is used in combination with amino acids and heat treatment as a virus inactivation procedure in a process for the preparation of factor VIII. Although the virucidal agent capable of breaking down the lipid membrane is believed to be undissociated caprylic acid (undissociated caprylic acid), the procedure for using the agent is commonly referred to as caprylate inactivation, according to the biochemical nomenclature in which solutions of the acid and its ionic form are referred to by the name of the latter, i.e., caprylate (caprate).

Octanoic acid has also been used as a precipitant for purifying immunoglobulins (Steinbuch, M. Et al, arch. Biochem. Biophys.,1969,134 (2), 279-284). The purity and yield of the immunoglobulin mainly depends on the concentration and pH of the added octanoic acid. Steinbuch, m. et al also state that the addition of an effective amount of caprylate in two different steps, it is advantageous to remove the precipitate between the two steps. Proteins due to non-immunoglobulins partition in the pellet, which will provide the program with the ability to eliminate both enveloped and non-enveloped viruses.

A combination of precipitation with caprylate followed by ion exchange chromatography was also found in the prior art 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 the above mentioned procedure based on the Cohn method) comprising two consecutive anion exchange columns after the step of adding caprylate at a concentration of 15-25mM in a double precipitation step and combining the two effects of caprylate: reduction of non-immunoglobulin proteins by precipitation, and inactivation of the virus by incubation. The subsequent anion exchange step, besides removing other impurities (IgM, igA, albumin, etc.), also serves to eliminate caprylate and therefore requires double adsorption using relatively large amounts of anionic resin.

Patent application PCT WO2005/082937 also discloses a method for preparing a composition comprising immunoglobulins, comprising the steps of: to the solution or composition comprising the immunoglobulin is added a caprylate and/or a heptanoate (heptanoate) and subsequently the solution is applied in a column with an anion exchange resin.

Disclosure of Invention

However, the inventors of the present invention have realised that the use of an appropriate concentration of caprylate and pH (e.g. pH 5.0-5.2) to provide a treatment with virus inactivation capability (as already described in the prior art) results in the formation of protein aggregates with high molecular weight, which is partially irreversible by dilution and/or change of pH. Moreover, these aggregates can only be partially separated by filtration and therefore require special subsequent separation steps, for example by chromatography or precipitation. The separation of these aggregates results in significant loss of protein and a reduction in the yield of the industrial process for immunoglobulin production.

Furthermore, the inventors of the present invention have realized that the presence of aggregates formed during the caprylate treatment, even at very low levels, prevents the proper elimination of caprylate under optimal process conditions by directly applying a separation step using ultrafiltration membranes. These aggregates hinder or prevent the preparation of solutions of immunoglobulins at therapeutic concentrations (e.g., between 5% and 20%) due to the presence of colloids (turbidity) or instability in liquid form, thereby hindering or preventing subsequent steps of the process for preparing immunoglobulins, such as nanofiltration and sterile filtration.

As a result of the above, the inventors of the present invention developed a method for preparing an immunoglobulin solution which surprisingly comprises a treatment of caprylate with virus inactivation capacity at a lower concentration than that described in the prior art, and the initial solution is suitably purified and diluted and in the presence of at least one polyether or glycol polymer, the occurrence of aggregates is inhibited, prevented, avoided or not promoted.

Furthermore, the inventors of the present invention have found that the presence of at least one polyether or glycol polymer in the method according to the invention does not interfere with the activity and efficacy of the caprylate in its ability to inactivate enveloped viruses.

In a further aspect, the inventors of the present invention describe for the first time a method for obtaining immunoglobulins which also comprises a treatment with inactivation capacity under optimal conditions, covering the possibility of eliminating or reducing the caprylate and polyether or glycol polymer reagents (pre-existing during said treatment) by using only ultrafiltration techniques. This ultrafiltration step makes it possible to purify and concentrate the product to a level that is tolerable for its administration, for example via intravenous, intramuscular or subcutaneous routes, without producing immunoglobulin aggregates in the final product. This eliminates the need to introduce an additional separation step after the caprylate treatment, such as for example chromatography. Furthermore, the residual levels of polyether or glycol polymer and caprylate after ultrafiltration make it possible to achieve concentrations of immunoglobulins such as IgG of up to 20 ± 2%, which, if correctly formulated, are not unstable during their storage in liquid form.

This makes it possible, in view of the simplification of the process according to the invention, to significantly improve the yields and to very significantly reduce the production costs compared to the previous processes described in the prior art, without thus compromising the safety or purity level of the product.

Thus, in a first aspect, the present invention relates to a process for the preparation of an immunoglobulin solution, said process comprising the addition of caprylic acid or a salt thereof to a purified solution of immunoglobulins in the presence of at least one polyether or diol polymer, and the subsequent elimination or reduction of said agent by ultrafiltration/diafiltration.

In a further aspect, the invention relates to the use of caprylic acid or a salt thereof in the presence of at least one polyether or glycol polymer for virus inactivation in a protein production process and subsequent elimination or reduction of said agent by ultrafiltration/diafiltration.

In a further aspect, the invention relates to performing a single step of ultrafiltration/diafiltration for eliminating or reducing the level of caprylic acid or salt thereof and/or polyether or glycol polymer used for virus inactivation in the protein production process.

The present invention therefore discloses a process for preparing an immunoglobulin solution in the presence of a polyether or glycol polymer, based on an initial solution of immunoglobulins with a purity greater than or equal to 96%, said process being characterized in that it comprises the following steps:

a) Adding caprylic acid or a salt thereof to the initial solution;

b) Adjusting the pH of the solution obtained in step a);

c) Incubating the solution obtained in step b) at a temperature necessary for inactivation of the enveloped virus for a time necessary for inactivation of the enveloped virus; and

d) Subjecting the solution obtained in step c) to an ultrafiltration/diafiltration step.

The process according to the invention may also comprise a step of final formulation of the solution obtained in step d).

In the method according to the 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-oncoley method, or from precipitate a or I + a or GG obtained according to the Kistler-Nitschmann method, or a variant thereof, which fraction I + II + III, fraction II + III or fraction II, precipitate a or I + a or GG, or a variant thereof has been additionally purified to obtain an IgG purity greater than or equal to 96%. Preferably, the initial solution of immunoglobulins is derived from a fraction II + III or a variant thereof obtained according to the Cohn method, which fraction II + III or variant thereof has been subsequently purified by precipitation with PEG and anion chromatography, as described in document EP1225180B 1. According to this patent, any of the above fractions may be subjected to a precipitation procedure using PEG, followed by filtration to eliminate the precipitate and an additional purification step using an ion exchange column (e.g., a column with DEAE Sepharose). In all these cases, the initial solution of immunoglobulins is derived from human plasma.

In a most preferred embodiment, the initial solution of immunoglobulins is derived from a fraction II + III obtained by a procedure based on the Cohn method, which fraction II + III is additionally purified by any of the methods described in the prior art to achieve a sufficient level of purification to withstand 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, having an albumin content preferably less than or equal to 1% (w/v) relative to the total protein. As such, the initial solution of immunoglobulin is sufficiently purified for its intended therapeutic route of administration before and after the caprylate treatment, so that no additional purification is required after the step of viral inactivation of the invention.

The immunoglobulin of the initial solution of the method according to the invention may also be obtained by: genetic recombinant techniques, such as by expression in cell culture; chemical synthesis technology; or transgenic protein production techniques.

In a most preferred embodiment, the immunoglobulin mentioned in the method according to the invention is an IgG. It is envisaged that the IgG may be monoclonal or polyclonal. In a most preferred embodiment, the IgG is polyclonal.

It is contemplated that the polyether or glycol polymer of the present invention may be an alkane polyether or an oxide of a polyalkane, also known as a polyglycol, and refers to derivatives such as ethyl or ethylene and propyl or propylene, better known as polyethylene glycol (PEG) or polypropylene glycol (PPG), or equivalents thereof. Furthermore, the agents must be compatible with the immunoglobulins Bai Xiangrong in the sense that they do not compromise the stability or solubility of the immunoglobulins and, due to their size, they can be advantageously eliminated by ultrafiltration techniques, or, due to their low toxicity, they are compatible with the therapeutic use of the immunoglobulins.

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

The content of the above polyether or glycol polymer in the initial solution of immunoglobulin is preferably between 2% and 6% (w/v), and more preferably between 3% and 5% (w/v).

It is envisaged that it may be necessary to adjust the concentration of the polyether or glycol polymer in the initial solution of immunoglobulin. Said conditioning of said polyether or glycol polymer may be achieved by diluting the initial purified solution of immunoglobulin and/or by adding said polyether or glycol polymer.

Depending on the composition of the initial solution of immunoglobulins, it is envisaged that, before step a) of the method according to the invention, a series of purification or concentration adjustment steps are carried out, such as, for example:

-adjusting the concentration of immunoglobulins to between 1 and 10mg/ml, more preferably between 3 and 7 mg/ml. This adjustment can be achieved by any procedure known in the art, for example by diluting or concentrating the protein concentration to an established range as the case may be (e.g. determined from total protein by optical density =13.8-14.0UA at 280nm E (1%), by Biuret method, by Bradford method, or in particular by immunoturbidimetry). Thus, in a preferred embodiment, the initial solution of immunoglobulins has a concentration of immunoglobulins preferably between 1 and 10mg/ml, and more preferably between 3 and 7 mg/ml; and/or

Adjusting the purity of the immunoglobulin solution, which should preferably reach at least 96% IgG relative to total protein. This purification can be achieved by techniques well known to the person skilled in the art, such as, for example, by precipitation with PEG, and filtration and subsequent anion exchange chromatography (DEAE Sepharose).

In step a) of the method according to the invention octanoic acid or a salt thereof is added, preferably with a concentrated solution thereof, for example between 1.5M and 2.5M, to achieve a final concentration preferably between 9mM 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 5.1.

In a preferred embodiment, in step c), the obtained solution is incubated for at least 10 minutes, more preferably 1 to 2 hours, still more preferably 2 hours. Furthermore, the incubation is carried out at a temperature between 2 ℃ and 37 ℃, more preferably between 20 ℃ and 30 ℃.

In a preferred embodiment, before step d) of the process according to the invention, the content of polymers or aggregates having a high molecular weight in the solution obtained in said step c) is less than or equal to 0.2%, and more preferably less than 0.1%. This percentage of molecular aggregates of polymers or immunoglobulins relative to total protein was determined by size exclusion HPLC gel column based on optical density values at 280 nm. Said percentage of molecular aggregates of polymers or immunoglobulins can be evaluated, for example, using the analytical methods described in the monograph of the european pharmacopoeia on intravenous gamma globulin.

Preferably, the solution of immunoglobulins is clarified using a depth filter before performing step d) of ultrafiltration/diafiltration.

With respect to step d), it is envisaged that preferably the ultrafiltration/diafiltration in the method according to the invention has an initial step by reduced volume diafiltration and concentration, followed by diafiltration applied at constant volume.

The following facts are taken into account: the concentration of protein is optimal and preferably less than or equal to 30mg/ml, ultrafiltration/diafiltration can be performed on an industrial scale preferably by a process of simultaneous dialysis and concentration, reduction of the product volume and diafiltration instead, so that the consumption of reagents is slightly lower and the process is more efficient. In any case, the person skilled in the art is readily able to determine the most suitable and practical way to carry out this step of ultrafiltration/diafiltration, chosen from among the various operating procedures known in 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 process according to the invention is preferably composed of polysulfone, regenerated cellulose or equivalents thereof, such as, for example, under the trademark PSK(Millipore,USA)、(Pall,USA)、Kvik-(General Electric, USA). However, the molecular weight cut-off selected for the membrane may vary depending on a number of factors, such as the manufacturer of choice. The person skilled in the art can easily determine the membrane chosen, which will be adjusted by the needs of each case, depending on, for example, the concentration of octoate and polyether or glycol polymer in the solution to be processed.

Preferably, the ultrafiltration/diafiltration of step d) is effected through a membrane having a molecular weight cut-off of less than or equal to 100kDa, more preferably 100 kDa.

In a most preferred embodiment, the ultrafiltration/diafiltration of step d) is performed in two stages:

a first stage wherein the pH is adjusted to between 5.0 and 6.0 to reduce or eliminate most of the octanoate, and a second stage wherein the pH is adjusted to a pH of less than 5.0, preferably to between 4.0 and 5.0 to reduce or eliminate most of the polyether or glycol polymer.

In a preferred embodiment, in the first stage of the step of ultrafiltration/diafiltration, diafiltration is effected using a diafiltration medium which comprises an alkaline salt of a carboxylic acid, for example acetic acid, at a concentration of greater than or equal to about 5 mM. In a most preferred embodiment, the diafiltration described above is performed using a sodium acetate solution at a concentration greater than or equal to 5mM adjusted to the pH described above, i.e. between 5.0 and 6.0.

The multiple of the diafiltration volume to be performed in the first stage of ultrafiltration/diafiltration of step d) can be easily determined by the skilled person depending on the amount of caprylate initially used and the acceptable final amount. Preferably, at least three volumes of diafiltration medium are used, which is preferably a 5mM sodium acetate solution at pH 5.0-6.0, as mentioned above. Preferably, about 90% or more of the initial octanoate is eliminated in the first stage of ultrafiltration/diafiltration, so that the concentration of octanoate is reduced to about 1mM or less in the first stage.

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

Preferably, the diafiltration in the second stage of ultrafiltration/diafiltration is performed using a buffer solution at the pH indicated above, which contains a basic metal salt formed from acetate (acetate), phosphate (phosphate) or equivalent, or an amino acid and/or a polyol, such as glycine and/or sorbitol.

As in the case of the first stage of diafiltration, the fold in the second stage for suitably reducing the dialysis volume of the polyether or glycol polymer used in the process according to the invention can be easily determined by the skilled person taking into account the reduction or elimination of the need for the polyether or glycol polymer. In a preferred embodiment, the amount of buffer to be exchanged in the diafiltration of the second stage of ultrafiltration/diafiltration of step d) is equal to or more than six volumes. In a most preferred embodiment, in said second stage, the exchange is carried out according to a multiple of the buffer volume necessary for a reduction equal to or greater than 100 times of the initial content of polyether or glycol polymer obtained before starting the ultrafiltration/diafiltration of step d).

After the caprylate and polyether or glycol polymer have been reduced in the ultrafiltration/diafiltration of step d), the solution can be adjusted to the desired final composition by adding the necessary excipients and/or stabilizers in the above mentioned final formulation step, thereby concentrating the product to achieve the final formulation. The addition of excipients and/or stabilizers to be carried out after the final formulation can be achieved as follows: directly by adding said excipients and/or stabilizers in solid form or in concentrated solution, or, still more preferably, by diafiltration using the necessary multiple of the exchange volume of the formulated solution to ensure the proper composition of the final product.

In another embodiment, the addition of excipients and/or stabilizers is carried out by replacing the sodium acetate dialysis buffer solution used in the second phase of step d) completely or partially with a solution containing excipients and/or stabilizers adjusted to the same pH value, preferably between 4.0 and 5.0, so that after the final concentration, the immunoglobulins have been formulated.

The person skilled in the art knows which types of excipients and/or stabilizers have to be added in order to achieve the desired stability. It is envisaged, for example, that the excipient and/or stabilizer may be one or more amino acids, such as glycine, preferably at a concentration of between 0.2 and 0.3M; one or more carbohydrates or polyols, such as sorbitol; or a combination thereof.

Finally, the final concentration of the immunoglobulin, preferably IgG, is adjusted to a concentration suitable for its intravenous, intramuscular or subcutaneous use, which concentration will be known to the person skilled in the art and may be, for example, between 5% and 22% (w/v). The concentration is achieved by any procedure known in the art, for example concentration by ultrafiltration. It is envisaged that if the concentration of immunoglobulins is achieved by ultrafiltration, the concentration may be carried out using the same membrane as used in the preceding diafiltration. Obviously, the three diafiltrations mentioned as well as the concentration can be carried out using different membranes.

The process according to the invention also envisages the possibility of introducing nanofiltration in order to increase the safety margin of the product. The presence of the product in the procedure can be achieved using commercially available filters (e.g., manufactured by Asahi-Kasei)Andmanufactured by PallAndmanufactured by SartoriusManufactured by MilliporeOr its equivalent) multiple stages of nanofiltration with pore sizes of 20nm or less and up to 50nm, preferably with pore sizes of 20nm or less, or even 15nm nanofilters may be used. An intermediate step in which the nanofiltration step may be performed is,for example, in an initial solution of immunoglobulin; or in materials treated with octoate after the ultrafiltration/diafiltration step (after the octoate and polyether or glycol polymer have been reduced); or in the material (final product) after concentration and formulation of the immunoglobulin, preferably IgG, solution. The skilled person will select the best option depending on the pore size of the membrane, the required filtration area depending on the procedure time, the product volume to be nanofiltered, and the protein recovery, etc.

The final product obtained according to the process of the invention perfectly meets the criteria of the european pharmacopoeia regarding the content of the same hemagglutinin. However, the method according to the invention also envisages the option of comprising a step of selectively and specifically capturing the anti-a and/or anti-B blood antibodies in order to maximize their reduction. This step is preferably performed using biospecific affinity resins, as already described in the prior art. For example, by using biospecific affinity resins with ligands formed from trisaccharides, a significant reduction in the level of allohemagglutinin can be achieved (Spalter et al, blood,1999,93,4418-4424). This additional capture may be optionally incorporated in any step of the methods of the invention, or may be performed before or after performing the methods of the invention, as determined by one of skill in the art.

Thus, with respect to the method for preparing the immunoglobulin solution according to the invention, in a most preferred embodiment, an initial solution of immunoglobulins having a purity of greater than or equal to 96% IgG is used. The solution is adjusted to a concentration of IgG preferably between 1mg/ml and 10mg/ml, and preferably between 3mg/ml and 7mg/ml, comprising (by addition in the previous step) PEG at a concentration of 4 + -1% (w/v) or to which PEG is added to a concentration of 4 + -1% (w/v). The pH of the solution is then adjusted to between 5.0 and 5.2 by acetic acid and sodium caprylate is added (e.g., using a concentrated solution of the sodium caprylate). In a preferred embodiment, a concentrated solution of caprylate is added slowly with stirring to the purified solution of IgG. After addition of caprylate calculated to bring the product to a final concentration of caprylate between 9 and 15mM, the final pH is then adjusted, if necessary, to between 5.0 and 5.2, and the solution is preferably incubated at a temperature of 2-37 ℃, more preferably at a temperature of 25. + -. 5 ℃ for at least 10 minutes, and preferably for 1 to 2 hours.

Clarification is then performed using a depth filter (e.g., cuno 90LA, 50LA, seitz EK, EK-1, EKs, or equivalent).

The solution thus obtained is then processed through an ultrafiltration/diafiltration unit formed by a membrane comprising polysulfone, for example manufactured by MilliporeOr from PallThe ultrafiltration/diafiltration apparatus is preferably in the form of a stackable cassette. The solution is recirculated through each ultrafiltration/diafiltration unit, preferably in a volume of about 100-500L/h and at a temperature of 5 ± 3 ℃. The pressure drop between the inlet pressure and the outlet pressure (atmospheric pressure) is preferably between 1 and 3 bar. Then, the first ultrafiltration stage of the ultrafiltration/diafiltration step is started to eliminate the caprylate, preferably applying an exchange of at least three volumes of buffer, preferably formed by a solution of sodium acetate at a concentration equal to or greater than 5mM and at a pH between 5.0 and 6.0. Preferably, for each volume of buffer added or consumed, the volume of product solution is reduced to half the initial volume, except for the last addition.

After the first stage of diafiltration (by dilution and concentration or equivalent treatment), the pH of the obtained solution is adjusted to between 4.0 and 5.0 using, for example, acetic acid. Diafiltration at constant volume is then started, preferably with six or more volumes of a buffer solution formed from sodium acetate at a concentration equal to or greater than 5mM and a pH between 4.0 and 5.0.

The above dialysis buffer solution formed by sodium acetate may optionally be replaced completely or partially by an amino acid, e.g. glycine solution, at a concentration of 0.2-0.3M, optionally in combination with carbohydrates and polyols, e.g. sorbitol, preferably adjusted to the same pH value between 4.0 and 5.0, so that after the final concentration the immunoglobulin has been formulated.

After applying the above dialysis solution, preferably at least six volumes (more preferably between six and ten volumes) of pH between 4.0 and 5.0, the product can be formulated as follows if it has not already been formulated: by adding the excipient(s) and/or stabilizer(s), such as, for example, glycine or other amino acids, and carbohydrates such as sorbitol, or combinations thereof, directly to the obtained solution in the form of a solid state or concentrated solution of the excipient(s) and/or stabilizer(s). The IgG solution obtained by volume reduction is then concentrated to achieve the appropriate IgG concentration for intravenous, intramuscular, or subcutaneous use.

The concentrated solution, suitably adjusted with respect to the concentration and pH of the one or more excipients and/or the one or more stabilizers, is applied by absolute filtration using a filter having a pore size of 0.2 μm, and optionally nanofiltered. Finally, the IgG solution is aseptically dosed (contaminated) in injectable preparations, ampoules, vials, bottles or other glass containers, which are subsequently sealed (hermetic sealing). Another option is dosing (dosification) in a compatible rigid or flexible plastic container such as a bag or bottle.

The dosed product was passed for quarantine and appearance inspection and subsequently stored at a temperature between 2 ℃ and 30 ℃ for up to at least 2 years.

Furthermore, as mentioned above, the present invention also discloses for the first time the use of caprylic acid or a salt thereof in the presence of at least one polyether or glycol polymer for viral inactivation during protein production, wherein the polyether or glycol polymer and caprylic acid or a salt thereof are subsequently eliminated by ultrafiltration.

Preferably, the protein is selected from the group of proteins comprising: an immunoglobulin; albumin; coagulation factors such as factor VII, factor VIII and factor IX; and von willebrand factor. Still more preferably, the protein is an immunoglobulin. In a most preferred embodiment, the protein is an IgG.

Detailed Description

The 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 merely to exemplify the description thereof.

Examples

Example 1. Method according to the invention for obtaining a virologically safe (viral safe), aggregate free and with sufficient yield of immunoglobulin solution for industrial applications from plasma

The starting material was a 16 liter immunoglobulin solution containing IgG as the major protein component, obtained by the method described in european patent EP1225180B 1. Briefly, the solution is obtained by extracting gamma globulin from fraction II + III using the Cohn method. To perform this extraction of gamma globulin from fractions II + III, said fractions were previously isolated by fractionation of human plasma using ethanol. It was then suspended in the presence of carbohydrate and the accompanying content of most of the protein was reduced by precipitation with PEG-4000. Finally, the final purification of the fractions is carried out by adsorption on an ion exchange resin column (DEAE Sepharose). The column effluent thus obtained (resin i.e.the fraction not adsorbed in DEAE) had an electrophoretic purity in cellulose Acetate (ACE) of immunoglobulin 98. + -.2%, a pH of 6.0, a turbidity of 2.6 Nephelometric Turbidity Units (NTU) and an IgG concentration of about 5mg/ml.

The solution obtained is adjusted to a pH of 5.1 and to a temperature of 2 ℃ to 8 ℃ by adding acetic acid. This solution of immunoglobulin is then brought to a final concentration of 13mM by adding a concentrated solution of sodium caprylate.

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

Cooling the caprylate-treated solution to an approximate temperature of 8 ℃ for subsequent use with a depth filter: (Ultrafilter, denmark). About 20 liters of filtered liquid was obtained from the clarification (including rinsing), with an IgG concentration of about 4mg/ml and less than3NTU turbidity.

The clarified solution was obtained by using a membrane with a nominal molecular weight cut-off of 100 kDa: (Millipore, USA) by ultrafiltration. Ultrafiltration is carried out in two distinct stages: in the first phase, the material with a pH of 5.1 is dialyzed by using a 5mM acetate solution adjusted to pH5.1 and subjected to a sequence of three steps of dialysis and concentration by concentration to approximately 30 UA. In the second phase, the solution with sufficient concentrations of protein, caprylate and PEG was adjusted to pH 4.5 ± 0.1 and then dialysis was started using 8 volumes of 5mM acetate solution at pH 4.5. The product was then formulated as follows: by dialysis with approximately 20 l of 200mM glycine solution at pH 4.2 and concentration in the same ultrafiltration unit to a value of 140.5UA, the aim was to obtain an IgG solution with a concentration of 10% (w/v).

Finally, the solution is used with a depth filter (a)Ultrafilter, denmark) and absolute filters or membranes with a pore size of 0.22 μm ((R)Millipore, USA; orPALL, USA).

Table 1 shows the characterization of the starting materials, various intermediates and final products according to the process described above. With respect to the results included in the table, it should be noted that turbidity was measured by turbidimetry; the percentage of molecular aggregates of detected polymers or immunoglobulins relative to the total protein, determined by size exclusion HPLC gel column based on optical density values at 280 nm; the concentration of caprylate was determined using an enzymatic method by colorimetric substrate quantification; the concentration of PEG was determined by HPLC filtration of the gel column using a refractive index detector; and the percent recovery of the method was calculated from the concentration of IgG quantified by turbidimetry.

The results of this example show that treatment of the above purified solution with caprylate does not cause any formation of immunoglobulin aggregates or other precipitates, keeping the molecular distribution of the product unchanged. Thus, no purification step is required to eliminate aggregates and/or precipitates after treatment with caprylate. This fact greatly facilitates the production process and allows the direct application of materials to ultrafiltration membranes.

Table 1 results obtained for the starting material, various intermediates and final products in the process of example 1.

In this way, the subsequent ultrafiltration process satisfactorily achieves the aim of effectively 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 immunoglobulins to obtain a suitable composition for its therapeutic use.

As can be seen in table 1, the protein recovery from the starting effluent to the 10% concentrated product obtained in this example was 89.4%, indicating the feasibility of this process on an industrial scale. This recovery is greater than the value obtained by the conventional process according to the prior art and as described in patent application PCT WO2005/073252 (70% recovery, based on a yield of 4.8g/l compared to the initial 6.8 g/l).

Example 2. Effect of the purity of the initial solution of immunoglobulin in the treatment with caprylate.

In this example, the effect 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 invention was evaluated.

Two independent experimental test groups were created:

in group a, the starting material is 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, referred to as 4% peg filtrate, was obtained by the same procedure as described in example 1 up to the step preceding DEAE sepharose chromatography. Thus, material B was obtained after precipitation of the extraction suspension of fractions II + III with PEG, and had an approximate electrophoretic purity (ACE) of 90% igg.

The two starting materials (group a and group B), with an equivalent PEG content of about 4%, were subjected to a treatment with caprylate at a concentration of 13mM and a pH between 5.0 and 5.2 and purified as shown in example 1.

Table 2 details the main characteristics of the starting materials used in the two test groups (a and B respectively), as well as of the materials produced in the step following the treatment with octanoate.

The results obtained and collected in table 2 show that the addition of caprylate to a material of lower purity (approximately 90% igg, see group B) at an effective concentration for inactivation (13 mM) resulted in precipitation of the components of the solution, resulting in a drastic increase in turbidity (above 500 NTU). Thus, the molecular distribution results of the solution showed that the fraction with high molecular weight accompanied the precipitation of the protein.

Addition of caprylate to the low purity material in the amounts and under the conditions described hereinbefore (13 mM caprylate, pH between 5.0 and 5.2) resulted in a precipitated suspension which necessitated additional separation and purification steps to separate the proteins with high molecular weight and the precipitated aggregates. Thus, the molecular composition of the group B products treated with caprylate, i.e. having an aggregate content of more than 1%, shows the impracticability of processing this product into a purified end product unless additional purification or separation steps are included, such as steps with PEG precipitation, chromatography or equivalent methods. Finally, this fact shows the feasibility of using caprylate as an agent with virus inactivation capacity under non-precipitating conditions only when it is added to a material with sufficient purity.

Example 3 Effect of the composition of the starting Material on the formation of aggregates

The purpose of this experiment was to evaluate the effect of the composition of the initial solution of immunoglobulin to which the caprylate treatment was applied.

Two independent experimental test groups a and B were created starting from materials of equal purity (97.9 ± 1.5%) but with different compositions.

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 is referred to as the concentrate and dialyzed effluent, which is the same column effluent as that mentioned for group A, but after concentration and dialysis. Thus, the DEAE column effluent (mentioned above in example 1 and corresponding to group a of the present example) is subjected to dialysis and further steps of concentration by ultrafiltration, whereby the PEG content is reduced by a factor of about 6 and the protein is concentrated to an approximation of 4%, i.e. 40mg/ml.

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

Table 3 details the main characteristics of the materials processed in the experimental groups a and B mentioned above, as well as the characteristics of the material produced in the step after treatment with caprylate and of the diafiltered and concentrated final product for each experimental group.

As can be seen in table 3, the results demonstrate: under the conditions specified, the purified solution of immunoglobulin (group a, column effluent) in the presence of a concentration of 5 ± 2mg/ml and a PEG concentration of 40 ± 10mg/ml (4 ± 1%) was treated with caprylate without causing any change or aggregation of the immunoglobulin solution, keeping the molecular distribution of the product unchanged during and after the addition of caprylate, with an undetectable proportion of aggregates of less than 0.1%.

However, when these same conditions of treatment with caprylate were applied to materials with low PEG content (< 1%) (group B), a substantial increase of immunoglobulin aggregates was observed after addition of caprylate. Moreover, elimination of this aggregate content by ultrafiltration under the conditions of use is not possible and comparable polymer levels are measured in the final product.

Considering that the main difference features between the starting materials used in experimental groups a and B are protein concentration and PEG concentration, additional tests were performed with the aim of determining the effect of each of these parameters on the subsequent treatment with caprylate.

In this experiment, the starting point was a single batch of concentrated and dialyzed effluent (initial material of group B above), which was divided into four different 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 approximately 4% of the same protein concentration, 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% protein. As to the PEG content, it was adjusted to a concentration of about 0.6% (w/w) in group B3, while the PEG content was readjusted to 4. + -. 1% (w/w) in group B4.

The resulting material obtained in the four experimental groups was adjusted to a pH of 5.10. + -. 0.05 and a caprylate concentration of 15mM and then incubated at 25 ℃ for 2 hours. The results obtained are shown in table 4.

Table 4 results obtained for the starting material and after incubation with caprylate for groups B1, B2, B3 and B4 of example 3.

The results shown in table 4 indicate that during treatment with caprylate under established conditions PEG protection was observed along with sufficient protein dilution. Notably, when the starting material was at an approximate protein concentration of 5 ± 2mg/ml and a PEG concentration of 4%, undetectable levels of aggregates were obtained (< 0.1%) after treatment with caprylate.

Example 4. Effect of ph on solubility of immunoglobulin solutions treated with caprylate.

It is known that elimination of PEG from immunoglobulin solutions, and concentration of the immunoglobulin to a suitable concentration for intravenous use thereof, must preferably occur at a pH of about 4.5.

Furthermore, considering the insolubility of caprylic acid at a pH value below its pKa (4.89), the effect of pH on the solubility of the immunoglobulin solution treated with caprylate was evaluated in this experiment, with the aim of establishing a suitable pH value for starting its ultrafiltration.

For this purpose, a batch of column effluent obtained according to the initial process detailed in example 1 was processed to obtain an immunoglobulin solution treated with 13mM caprylate and clarified.

This intermediate, which constitutes the material before the ultrafiltration step, is acidified by adding acetic acid from the pH treated with caprylate (5.1) to a pH of about 4.5. Subsequently, the appearance and solubility of the solution were evaluated for each evaluated pH value, and the generation of colloidal particles was quantified by turbidimetric measurement of turbidity.

Table 5 shows the appearance and turbidity results obtained for each pH value evaluated.

Table 5 turbidity and visual appearance results obtained for different pH values analyzed in example 4.

pH	Turbidity (NTU)	Visual appearance
			5.1	5.6	Is 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

As seen in table 5, the results obtained show that when the immunoglobulin solution treated with 13mM caprylate was acidified to a pH below pH 5.0, the appearance of a precipitate of blush was observed, with a significant increase in turbidity. This effect is most likely due to the formation of insoluble octanoic acid in the form of a colloid, which makes it infeasible to start the ultrafiltration process at a pH below 5.0.

The results obtained demonstrate that: when the purified solution is subjected to the caprylate treatment in an effective concentration range for virus inactivation (9-15 mM caprylate) and under the conditions described above, the subsequent ultrafiltration step is preferably started at a pH greater than or equal to the pH of the virus inactivation treatment, i.e. 5.1, in order to increase the concentration of the caprylate in ionic and soluble form and thus to promote its permeability through the ultrafiltration membrane.

Example 5. The acetate content in the dialysis solution has an effect on the reduction of caprylate by ultrafiltration/diafiltration.

A series of independent ultrafiltration/diafiltration processes were performed in the presence of different concentrations of acetate in the buffer solution used for the dialysis product.

The starting material used, referred to as the concentrate and dialyzed effluent, was the same as in example 3, group B. The starting material has an IgG purity of 98 ± 2%, an approximate protein concentration of 40mg/ml and an approximate PEG content of 0.6%, was subjected to caprylate treatment and subsequently to ultrafiltration/diafiltration using a membrane with a nominal molecular weight cut-off of about 100 kDa.

The applied ultrafiltration/diafiltration step includes a first stage of concentration to approximately 4% (w/v) IgG, a second stage of dialysis using eight volumes of dialysis solution, and finally concentration to an approximate value of 9-10% (w/v) IgG.

The first of the ultrafiltration/diafiltration tests was performed using water for injection, while the subsequent tests were performed using buffer solutions with increasing concentrations of acetate, more specifically 2, 5, 20 or 50mM acetate, respectively, and in all cases with an adjusted pH between 5.0 and 5.5.

Table 6 results obtained from the ultrafiltration/diafiltration step using different concentrations of acetate in the dialysis buffer.

(1) Value determined after dialysis with 8 dialysis volumes

(2) Permeability calculated by the following formula:

multiple of dialysis volume = ln (Cf/Co)/(R-1); where Cf is the concentration after dialysis with the multiple of the dialysis volume in question, co is the concentration before dialysis, and R is the retention factor.

The results of table 6 show that the aim of effectively reducing caprylate to the appropriate level in the final concentrated product is satisfactorily achieved using a membrane with a molecular weight cut-off of about 100kDa, applying 8 dialysis volumes of buffer solution with acetate, a pH between 5.0 and 5.5 and an ultrafiltration/diafiltration procedure with a minimum concentration of about 5mM and acetate of at most 50 mM.

In contrast, when the solution used for dialysis was water for injection or a buffer solution having an acetate level of 2mM, caprylate in the filtrate was not effectively eliminated.

This proves that: considering the correct level of caprylate detected in the final concentrated product, the method of ultrafiltration/diafiltration using a membrane with a molecular weight cut-off of about 100kDa under the conditions described above effectively reduced caprylate originating from previous treatments.

Example 6 simultaneous elimination of chemicals (PEG and caprylate) by a single step of ultrafiltration.

A batch of IgG was processed according to the method described in example 1 to obtain a solution inactivated with caprylate and clarified. The solution with approximate protein concentration of 0.5% and pH of 5.1 used a molecular weight cut-off of 100kDaType polysulfone membranes (Millipore, USA) are processed in ultrafiltration/diafiltration units. Ultrafiltration/diafiltration is performed in two distinct stages, as described in example 5:

-in a first phase, at pH5.1, 5.6 or 5.8, the material is subjected to sequential steps of dialysis and concentration by diafiltration with not less than three volumes of 5mM acetate buffer solution adjusted to pH5.1, 5.6 or 5.8, and the protein is concentrated to an approximation of 2%.

In the second stage, after the caprylate content has been reduced to about one tenth, the solution is adjusted to a pH of 4.5. + -. 0.1 or 5.1. The product was then adjusted to a sufficient concentration of protein and PEG to begin dialysis, and dialysis was started with 8 volumes of 5mM acetate buffer solution at a pH of 4.5 or 5.1.

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

Table 7 shows the percentage of passage (passage) of PEG and caprylate obtained at the beginning of each stage of ultrafiltration/diafiltration and at different pH values:

table 7. Passage of PEG and caprylate in the two stages of the ultrafiltration/diafiltration step at different pH values analyzed.

The results in table 7 show that in stage I, at the start of the ultrafiltration/diafiltration step, the octanoate shows a very high breakthrough value between pH5.1 and pH 5.8. These values lead to a very high reduction of caprylate during said phase I of the ultrafiltration/diafiltration step (more than 10 times reduction of caprylate is obtained with respect to the initial content). In contrast, the passage of PEG in said phase I is very low (< 20%), and its overall elimination in the presence of caprylate at pH >5 is practically not feasible.

On the other hand, as can be seen in table 7, in phase II the PEG passage at pH 4.5 is very high, with a value of 82%. It was furthermore found that during this phase II also the octanoate was reduced, which allows a breakthrough of practically 100%, considering that it was present at the start of this phase at a residual level of ≦ 1 mM.

Table 8 details the evolution of the concentration of protein, PEG and caprylate in each stage of the ultrafiltration/diafiltration step and in the final formulation step.

TABLE 8 Virus inactivation step, stages I and II of the ultrafiltration/diafiltration step, solution after formulation at pH 4.2, and the amount of PEG and caprylate in the final solution (measured by concentration and optical density)

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

In the case of caprylate, the reduction factor in phase I (pH 5.1) of the ultrafiltration/diafiltration step was 55 and in phase II (pH 4.5) of the ultrafiltration/diafiltration step was 13, giving a total reduction factor (phase I and phase II) of about 700 (initial absorbance of 2.2 compared to 0.003 of the absorbance obtained at the end of the ultrafiltration/diafiltration step).

The results show that the agent with virus inactivation capacity (caprylate or caprylate) as well as the precipitation agent (PEG) can be effectively reduced by a single ultrafiltration step using a membrane with a molecular weight cut-off of about 100kDa, selecting the physical and chemical conditions (pH, protein concentration, fold of dialysis volume, dialysis buffer, etc.) to be applied in each stage of the ultrafiltration/diafiltration step, and yielding a final product with some residual concentration of the above two agents suitable for intravenous use, concentrated to 10% IgG.

Example 7 evaluation of the Virus inactivating Capacity of caprylate in the Presence of PEG.

Column effluent or dialysis and concentrated effluent (obtained according to examples 1 and 3, respectively) were taken as starting material for each independent experiment to evaluate the ability of caprylic acid or caprylate salts to eliminate or inactivate viruses with lipid envelopes in the presence of PEG.

The two materials had an immunoglobulin purity of 98. + -. 2% and a protein concentration of between 5 and 10mg/ml, whereas the PEG contents differed by 40mg/ml and 1.5mg/ml respectively.

The virus inactivation test is performed using 40-60nm of Bovine Viral Diarrhea Virus (BVDV) of the Flaviviridae family, which has a lipid envelope and general tolerance to physical and chemical agents.

In each test, the corresponding starting material was inoculated with virus to a value of less than or equal to 0.5% and subjected to a virus inactivation treatment at 15 ℃ or 25 ℃ with an caprylate concentration of 9mM or 13mM for 2 hours.

Quantification of viral load of BVDV in the different samples produced was performed by TCID50 test (50% tissue culture infectious dose) using MBDK cell line. The fold reduction of virus (RF) for the virus inactivation step was determined as follows: the factor of the viral load detected in the inoculated starting material divided by the amount of virus detected in the sample obtained at the end of the treatment is given as log ₁₀ And (4) showing.

Table 9 details the characteristics of the starting materials, as well as the obtained RF, for each test.

Table 9. Virus inactivation results observed in caprylate treatment tests on virus inocula in the presence or absence of PEG.

The virus reduction results obtained in all tests (see table 9) show the high capacity to inactivate BVDV9 in both starting materials after treatment at different temperatures (15 and 25 ℃) even for the minimum 9mM concentration of caprylate. Moreover, these tests show that at each PEG concentration analyzed, there is no observed interference of PEG in the virus inactivation capacity of the caprylate, given that equivalent results were obtained for both materials evaluated.

Example 8 characterization of intravenous immunoglobulin solutions obtained according to the production method of the invention.

It is intended to establish the biochemical and functional characteristics of the immunoglobulin solution with 10% (w/v) protein obtained by the method of the invention.

On an approximate scale of 200 liters of plasma, two batches of DEAE column effluent were processed according to the method detailed in example 1 to obtain a virus solution inactivated with caprylate.

The solution with caprylate, after being clarified, is dialyzed and concentrated by ultrafiltration in distinct stages, as described in example 6, with the aim of achieving elimination of the main process residues (PEG and caprylate). Subsequently, the purified solution at an approximate protein concentration of 2.5% was prepared by dialysis at a constant volume against about 6-fold volume of a buffer solution consisting of sorbitol 1% and glycine 240mM, adjusted to pH 4.5 ± 0.1. Finally, the solution was concentrated by ultrafiltration and adjusted to an optical density of 140 ± 5UA (280 nm), equivalent to 10% (w/v) protein, and to a final pH of 5.25 ± 0.25.

The product obtained (IGIV 10% (w/v)) was stabilized with sorbitol and glycine and, after clarification and filtration using a sterile-grade membrane (0.22 μm), quantified in glass vials with chlorobutyl stoppers by determining the most relevant analytical parameters of quality, i.e. invariance and stability of the immunoglobulin solution 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 characterization of intravenous immunoglobulin solutions at 10% (w/v)

Ph.: european pharmacopoeia; n.e.; not established; TGT FXI: thrombin generation assay (using plasma deficient in factor IX); NAPTT PKA: a prekallikrein activator; ACA: anti-complementary activity.

The above results enhance that the obtained product is substantially unchanged by the purification process of the invention in terms of parameters such as absence of polymer, undesired biological activity such as PKA or ACA activity, etc., retaining some functional characteristics with respect to plasma such as IgG subclass and Fc fragment integrity intact (intact), and at the same time showing an excellent purity profile (low titer of anti-a/anti-B homohemagglutinin, concentration of IgM, procoagulant activity, etc.).

In summary, the steps for obtaining IGIV 10% (w/v), incorporating viral inactivation with caprylate in the presence of PEG and its subsequent isolation, and finally formulating the overall process of the invention, are fully feasible and scalable to the final product formulated and concentrated to an IGIV 10% (w/v) protein solution, providing a final product that fully complies with the values established in the european pharmacopoeia.

Stability studies carried out, which are essential for the commercial viability of the product, show the suitability of a 10% (w/v) solution of stabilized intravenous immunoglobulin, formulated with sorbitol (to 5%), glycine (to isotonicity) or a combination of both, at a pH range between 4.2 and 6.0, for two years at ambient temperature (25 ℃ -30 ℃).

Example 9 suitability of treatment with caprylate for IgG-enriched fractions obtained by alternative methods.

The effectiveness of using the octanoate 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 IgG-rich plasma intermediate called fraction II suspension from Cohn-oncoley ethanol fractionation as starting material.

This intermediate was obtained by the same plasma fractionation method described in the present invention up to fractions II + III. The procedure is then continued with ethanol reprecipitation of the extract suspension of fractions II + III, followed by separation of III, finally obtaining fraction II with a purity greater than 96%. The suspension of fraction II was used as starting material for these experiments after purification with bentonite and dialysis with water to remove the ethanol component.

In the two experiments performed, the material derived from the two plasma batches was divided into two different groups a and B according to its PEG content. In group B, the starting material was adjusted to a nominal PEG concentration of 40mg/ml by adding a concentrated solution of PEG-4000.

Subsequently, the two materials derived from the two groups (a and B) were diluted to an approximate protein concentration of 5mg/ml, adjusted to a pH value of 5.1 and subjected to treatment with caprylate until a nominal concentration of 13mM and a pH between 5.0 and 5.2 was reached, as described in the method of the invention.

Table 11 details the main characteristics of the starting materials used in the two test groups (a and B, respectively), as well as the characteristics of the materials produced after treatment with the octoate.

(1) PEG and caprylate values correspond to those obtained by analytical determination.

The results show the feasibility of using immunoglobulin solutions sufficiently purified by different methods, inactivation treatment with caprylate under the specified conditions, without causing the formation of immunoglobulin aggregates or other irreversible precipitates, which greatly facilitates the subsequent purification process.

The results show that, in combination with sufficient dilution and sufficient degree of purity of the protein, the protective effect of PEG on the production of immunoglobulin polymers is evident.

This experimental example demonstrates the feasibility of using caprylate under non-precipitating conditions, and/or aggregation-promoting conditions, only as an agent with virus-inactivation capability, when added to a material of sufficient purity and following the specified conditions with respect to protein and PEG concentration.

While the present invention has been shown and described with reference to embodiments thereof, it is to be understood that these embodiments are not limitations of the present invention, as many variations in manufacturing or other details may exist which will be apparent to those skilled in the art upon interpreting the subject matter disclosed in this specification and claims. Accordingly, all modifications and equivalents may be resorted to, falling within the broadest scope of the claims that follow, and are intended to be included within the scope of the invention.

Claims

1. Method for preparing an immunoglobulin solution, said method being carried out on the basis of an initial solution of immunoglobulins having a purity greater than or equal to 96% in the presence of a polyether or glycol polymer, characterized in that it comprises the following steps:

a) Adding caprylic acid or a salt thereof to the initial solution to a concentration of between 9mM and 15 mM;

b) Adjusting the pH of the solution obtained in step a) to a pH between 5.0 and 5.2;

d) A step of ultrafiltration/diafiltration of the solution obtained in step c);

wherein the initial solution of immunoglobulins has an immunoglobulin concentration of between 3 and 7 mg/ml; and is

Wherein the polyether or glycol polymer is polyethylene glycol (PEG) and the concentration of the PEG in the initial solution is between 3% and 5% (w/v).

2. The method according to claim 1, characterized in that it further comprises a step of final formulation of the immunoglobulin solution obtained in step d).

3. The method according to claim 1 or 2, characterized in that said initial solution of immunoglobulins is derived from fraction I + II + III, fraction II + III or fraction II obtained according to the Cohn or Cohn-oncoley method, or from precipitate a or I + a or GG obtained according to the Kistler-Nitschmann method, or a variant thereof, which fraction I + II + III, fraction II + III or fraction II, precipitate a or I + a or GG, or a variant thereof, has been additionally purified in order to obtain an IgG purity greater than or equal to 96%.

4. The method according to claim 3, characterized in that the initial solution of immunoglobulins is derived from fraction II + III or a variant thereof obtained according to the Cohn method, which fraction II + III or variant thereof has been subsequently purified by precipitation with PEG and anion chromatography.

5. The method according to any one of claims 1, 2 and 4, characterized in that said PEG is a PEG with a nominal molecular weight of 4000 Da.

6. The method of claim 3, characterized in that said PEG is a PEG having a nominal molecular weight of 4000 Da.

7. The method according to any one of claims 1, 2,4 and 6, characterized in that in step b) the obtained solution is adjusted to a pH of 5.1.

8. The method according to claim 3, characterized in that in step b), the obtained solution is adjusted to a pH of 5.1.

9. The method according to claim 5, characterized in that in step b) the obtained solution is adjusted to a pH of 5.1.

10. The method according to any one of claims 1, 2,4, 6 and 8-9, characterized in that in step c) the solution is incubated at a temperature between 2 ℃ and 37 ℃ for at least 10 minutes.

11. A method according to claim 3, characterized in that, in step c), the solution is incubated at a temperature between 2 ℃ and 37 ℃ for at least 10 minutes.

12. The method according to claim 5, characterized in that in step c) the solution is incubated at a temperature between 2 ℃ and 37 ℃ for at least 10 minutes.

13. The method according to claim 7, characterized in that in step c) the solution is incubated at a temperature between 2 ℃ and 37 ℃ for at least 10 minutes.

14. The method according to claim 10, characterized in that in step c) the solution is incubated at a temperature between 20 ℃ and 30 ℃ for 2 hours.

15. Method according to any one of claims 11-13, characterized in that in step c) the solution is incubated at a temperature between 20 ℃ and 30 ℃ for 2 hours.

16. The method according to any one of claims 1, 2,4, 6, 8-9 and 11-14, characterized in that said initial solution of immunoglobulins has an albumin content less than or equal to 1% (w/v) with respect to the total protein.

17. The method according to claim 3, characterized in that said initial solution of immunoglobulins has an albumin content less than or equal to 1% (w/v) with respect to the total protein.

18. The method according to claim 5, characterized in that said initial solution of immunoglobulins has an albumin content less than or equal to 1% (w/v) with respect to the total protein.

19. The method according to claim 7, characterized in that said initial solution of immunoglobulins has an albumin content less than or equal to 1% (w/v) with respect to the total protein.

20. The method according to claim 10, characterized in that said initial solution of immunoglobulins has an albumin content less than or equal to 1% (w/v) with respect to the total protein.

21. The method according to claim 15, characterized in that said initial solution of immunoglobulins has an albumin content less than or equal to 1% (w/v) with respect to the total protein.

22. The method according to any one of claims 1, 2,4, 6, 8-9, 11-14 and 17-21, characterized in that the initial solution of immunoglobulins is derived from human plasma.

23. The method according to claim 3, characterized in that said initial solution of immunoglobulins is derived from human plasma.

24. The method according to claim 5, characterized in that said initial solution of immunoglobulins is derived from human plasma.

25. The method according to claim 7, characterized in that said initial solution of immunoglobulins is derived from human plasma.

26. The method according to claim 10, characterized in that said initial solution of immunoglobulins is derived from human plasma.

27. The method according to claim 15, characterized in that said initial solution of immunoglobulins is derived from human plasma.

28. The method according to claim 16, characterized in that said initial solution of immunoglobulins is derived from human plasma.

29. The method according to any one of claims 1, 2,4, 6, 8-9, 11-14, 17-21 and 23-28, characterized in that the immunoglobulins of said initial solution of immunoglobulins are obtained by genetic recombination techniques, chemical synthesis techniques or transgenic protein production techniques, or in cell culture.

30. The method according to claim 3, characterized in that 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 culture.

31. The method according to claim 5, characterized in that 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 culture.

32. The method according to claim 7, characterized in that 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 culture.

33. The method according to claim 10, characterized in that 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 culture.

34. The method according to claim 15, characterized in that the immunoglobulins of said initial solution of immunoglobulins are obtained by genetic recombinant techniques, chemical synthesis techniques or transgenic protein production techniques, or in cell culture.

35. The method according to claim 16, characterized in that the immunoglobulins of said initial solution of immunoglobulins are obtained by genetic recombinant techniques, chemical synthesis techniques or transgenic protein production techniques, or in cell culture.

36. The method according to claim 22, characterized in that the immunoglobulins of said initial solution of immunoglobulins are obtained by genetic recombinant techniques, chemical synthesis techniques or transgenic protein production techniques, or in cell culture.

37. The method according to any one of claims 1, 2,4, 6, 8-9, 11-14, 17-21, 23-28 and 30-36, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

38. The process according to claim 3, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

39. The process according to claim 5, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

40. The process according to claim 7, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

41. The process according to claim 10, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

42. The process according to claim 15, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

43. The process according to claim 16, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

44. The process according to claim 22, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

45. The process according to claim 29, characterized in that the ultrafiltration/diafiltration of step d) is performed using a 100kDa membrane.

46. The process according to any one of claims 1, 2,4, 6, 8-9, 11-14, 17-21, 23-28, 30-36 and 38-45, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

-a first stage wherein the pH is adjusted to between 5.0 and 6.0 to reduce or eliminate most of the caprylate;

-and a second stage wherein the pH is adjusted to a value of less than or equal to 5.0 to reduce or eliminate a substantial portion of the polyether or glycol polymer.

47. The process according to claim 3, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

48. The process according to claim 5, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

49. The process according to claim 7, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

50. The process according to claim 10, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

51. The process according to claim 15, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

52. The process according to claim 16, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

53. The process according to claim 22, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

54. The process according to claim 29, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

55. The process according to claim 37, characterized in that the ultrafiltration/diafiltration of step d) is carried out in two stages:

56. The process according to claim 46, characterized in that in the second stage of ultrafiltration/diafiltration of step d) the pH is adjusted to between 4.0 and 5.0.

57. The process according to any one of claims 47-55, characterized in that in the second stage of ultrafiltration/diafiltration of step d), the pH is adjusted to between 4.0 and 5.0.

58. The method according to claim 2, characterized in that in the final formulation step, excipients and/or stabilizers are added, selected from one or more amino acids, one or more carbohydrates or polyols, or a combination thereof.

59. The method according to any one of claims 1, 2,4, 6, 8-9, 11-14, 17-21, 23-28, 30-36, 38-45, 47-56 and 58, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use thereof.

60. The method according to claim 3, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use.

61. The method according to claim 5, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use.

62. The method according to claim 7, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use.

63. The method according to claim 10, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use.

64. The method according to claim 15, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use.

65. The method according to claim 16, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for their intravenous, intramuscular or subcutaneous use.

66. The method according to claim 22, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use thereof.

67. The method according to claim 29, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use thereof.

68. The method according to claim 37, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use thereof.

69. The method according to claim 46, characterized in that the final concentration of immunoglobulins is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use thereof.

70. The method according to claim 57, wherein the final concentration of immunoglobulins is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use thereof.