JP2018052822A - Method of preparing immunoglobulin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing an immunoglobulin which does not inhibit, prevent, avoid, or promote the appearance of aggregates in purification of immunoglobulin.SOLUTION: The method of preparing an immunoglobulin solution based on the first solution of an immunoglobulin having a purity of at least 96% in the presence of a polyether of glycol or a polymer, the method comprising the following steps of: a) adding caprylic acid or a salt thereof to the first solution; b) adjusting the pH of the solution obtained in step a); c) incubating the solution obtained in step b) for a time and a temperature necessary to deactivate a virus with an envelope; and d) carrying out ultra-filtration/diafiltration on the solution obtained in step c).SELECTED DRAWING: None

Description

  The present invention relates to a novel method for preparing immunoglobulins. The resulting immunoglobulin composition is suitable for parenteral administration, for example.

  Immunoglobulins are glycoproteins that are found in soluble form in vertebrate blood and other body fluids to recognize and nullify foreign substances such as bacteria, viruses, or parasites. Used by. Immunoglobulins have a variety of medical applications such as disease diagnosis, therapeutic treatment, and prenatal therapy. The most common medical applications of immunoglobulins are the three general groups of pathology: primary immune deficiency (humoral immune deficiency), secondary immune deficiency, or acquired immune deficiency (eg, prevention of viral infection, and In therapy), and autoimmune immunodeficiency (antibody development).

  Immunoglobulins can be administered by various routes such as, for example, intramuscular, intravenous, and subcutaneous routes. The intravenous route is of course preferred because it provides many advantages, particularly excellent therapeutic effects.

  Immunoglobulins are obtained from human plasma using the Cohn fractionation method (Cohn EJ. Et al., J Am Chem Soc, 1946, 62, 459-475), the Cohn-Oncley method (Oncley JL. Et al., J Am Chem Soc, 1949). , 71, 541-550), or other equivalent methods based on cryogenic ethanol fractionation, eg, the Kistler-Nitschmann method (Kistler P, Nitschmann H, 1962, 7, 414-424) By doing so, it is usually purified. Thus, an immunoglobulin-rich fraction (eg, fraction II + III, or fraction II, or precipitate A, or gamma globulin GG precipitate) obtained by any of the above methods is used. Modifications are introduced to thoroughly purify the immunoglobulins (IgG) and to make them acceptable for administration, preferably intravenous administration. Such modifications are introduced, for example, to remove aggregates and other impurities and to ensure product safety. However, the addition of multiple steps to the process for the preparation of immunoglobulins reduces the yield in the process and increases manufacturing costs. Increased demand for immunoglobulin products, primarily for intravenous administration, makes yield a critical aspect in their manufacturing process on an industrial scale.

  The methods described in the prior art for obtaining an acceptable immunoglobulin composition via the intravenous route include the following steps: precipitation with polyethylene glycol (PEG), ion exchange chromatography, virus inactivation These include physical / chemical methods involving chemicalization, or treatment with enzymes, and partial chemical modification of immunoglobulin molecules.

  Therefore, there is a need to ensure product safety by performing a stable step with the ability to remove pathogenic biological agents. A commonly used method involves the use of solvents / detergents to inactivate viruses with lipid envelopes, since they do not significantly reduce the biological activity of the protein. However, considering the toxicity of the solvent / detergent mixture, this reagent must be sufficiently removed before obtaining the final product, and this increases the time required for the manufacturing process and increases the yield. Reduce. The method described for the removal of the solvent / detergent is not simple and is due to direct hydrophobic interaction or indirect capture of the immunoglobulin in the ion exchange resin and the uncaptured solvent / The use of chromatographic adsorption techniques with the separation of detergents is usually required. In all cases, the method is costly and cumbersome with significant loss of protein.

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

  In US Pat. No. 4,446,134, sodium caprylate is used in combination with heat treatment as a virus inactivation procedure in a process for the preparation of amino acids and factor VIII. I think that the virucidal agent capable of degrading lipid membranes is undissociated caprylic acid, but the method of using the agent is generally known as inactivation by caprylate, and biochemistry The acid solution, and its ionized form, has the latter name, ie the name of caprylate.

  Caprylic acid has also been used as a precipitating agent to purify immunoglobulins (Steinbuch, M. et al., Arch. Biochem. Biophys., 1969, 134 (2), 279-284). The purity and yield of the immunoglobulin depends mainly on the concentration of caprylic acid added and the pH. Steinbuch, M .; Also state that it is advantageous to add an effective amount of caprylate in two different steps with removal of the precipitate between the two steps. This will give the method the ability to remove both viruses with and without the envelope as a result of the distribution of non-immunoglobulin proteins in the precipitate.

  The description describes the discovery of state-of-the-art technology followed by a combination of precipitation and caprylate followed by ion exchange chromatography for immunoglobulin purification (Steinbuch, M. et al., V. Supra ).

  In EP 0893450, in a double precipitation step, combining the effects of both two anion exchange columns in succession and the caprylate after the step of adding caprylate at a concentration of 15-25 mM: For purification of IgG using fraction II + III (obtained using the method based on the Cohn method described above), including the reduction of non-immunoglobulin proteins by precipitation and the ability to inactivate viruses using incubation. A method is disclosed. A later anion exchange step is used to remove caprylate in addition to removing other impurities (IgM, IgA, albumin, etc.), and thus double adsorption is relatively bulky. It is necessary to use an anionic resin.

  In WO 2005/082937, caprylate and / or heptanoate is also added to a solution or composition containing immunoglobulin and subsequently the solvent is applied to a column with an anion exchange resin. A method for preparing a composition comprising an immunoglobulin, comprising:

  However, as described in the prior art literature, we have the appropriate concentration and pH (eg, pH 5.0-5.2) to provide a treatment with virus inactivation capability. The use of caprylate causes the formation of high molecular weight protein aggregates, which are partially irreversible due to dilution and / or pH changes. Furthermore, these agglomerates can only be partially filtered by filtration, thus requiring certain subsequent separation steps, for example using chromatography or precipitation. Separation of these aggregates causes significant loss of protein and reduced yield in the industrial process of immunoglobulins.

  The inventors have also found that separation steps using ultrafiltration membranes under optimal process conditions, even if the presence of aggregates formed during treatment with caprylate is at a very low level. Direct application of hinders accurate removal of caprylate. These aggregates hinder or prevent the preparation of solutions of immunoglobulins at therapeutic concentrations (eg, 5% to 20%) due to the presence of colloids (turbidity) or instability in liquid form. Hinders or prevents subsequent steps of methods for the preparation of immunoglobulins, such as nanofiltration and sterile filtration.

  As a result of the above, the inventors surprisingly included a caprylate treatment having the ability to inactivate viruses at a lower concentration of caprylate than that described in the prior art document, and the first solution For the preparation of immunoglobulins that are suitably purified and diluted and do not inhibit, prevent, avoid or promote the appearance of aggregates in the presence of at least one polyether or polymer of glycol Developed a method.

  In addition, the inventors have also found that the presence of at least one polyether or polymer of glycol in the method of the present invention indicates the activity and effectiveness of caprylate in terms of its ability for enveloped viruses. I found out that it would not interfere.

  In a further aspect, we have a caprylate and glycol polyether or polymer by using only ultrafiltration techniques, including treatments that also have the ability to inactivate under optimal conditions. For the first time, a method for obtaining an immunoglobulin is contemplated which contemplates the possibility of removal or reduction of reagents (already present during said treatment). This ultrafiltration step is acceptable for administration of the product in the final product, for example, via the intravenous, intramuscular, or subcutaneous route without producing immunoglobulin protein aggregates. It is possible to purify and concentrate to a certain level. This eliminates the need to introduce additional separation steps after treatment with caprylate, such as chromatography. In addition, the residual level of glycol polyether or polymer and caprylate after ultrafiltration achieves a concentration of immunoglobulin, eg, IgG up to 20 ± 2% and is liquid when properly formulated. It does not compromise stability during their storage in form.

  Given the simplification of the method of the present invention, this makes it possible to substantially improve the yield and significantly reduce the production costs compared to the previous methods described in the prior art literature, This does not impair the safety or purity level of the product.

  Thus, in a first aspect, the present invention relates to the addition of caprylic acid, or a salt thereof, to a purified solution of immunoglobulin in the presence of at least one polyether or polymer of glycol, and subsequent said It relates to a method for the preparation of an immunoglobulin solution comprising the removal or reduction of reagents using ultrafiltration / diafiltration.

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

  In a further aspect, the present invention is limited to the removal or reduction of the level of caprylic acid, or its salt and / or glycol polyethers, or polymers used for virus inactivation in protein production methods. It relates to the realization of a single step of external filtration / diafiltration.

Accordingly, the present invention is a method for preparing a solution of an immunoglobulin based on an initial solution of immunoglobulin having a purity of 96% or more in the presence of a polyether or polymer of glycol, said method comprising: Steps:
a) Caprylic acid, or a salt thereof, is added to the initial solution;
b) adjusting the pH of the solution obtained in step a);
c) incubating the solution obtained in step b) for the time and temperature required for inactivation of the enveloped virus; and d) for the solution obtained in step c) Disclosed is a method comprising performing a diafiltration step.

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

  In the method of the invention, the initial solution of immunoglobulin is derived from the fraction I + II + III, fraction II + III, or fraction II obtained according to the Cohn method, or the Cohn-Oncley method, or obtained according to the Kistler-Nitschmann method. Derived from precipitate A, or I + A, or GG, obtained according to a similar method variation, obtained or further purified to obtain an IgG purity of 96% or higher. Preferably, the initial solution of immunoglobulin is derived from Fraction II + III obtained according to the Cohn method, or a variation of similar methods, which is followed by PEG, as described in EP 1225180B1. And purified using anion chromatography. In accordance with this patent, any of the above fractions removes the precipitation method using PEG, followed by an additional purification step that removes the precipitate and uses an ion exchange column (eg, a column with DEAE Sepharose). Can be subjected to filtration. In all these cases, the initial solution of immunoglobulin is derived from human plasma.

  In a most preferred embodiment, the initial solution of immunoglobulin is derived from fraction II + III obtained by a method based on the Cohn method, which is further purified by any one of the methods described in the prior art literature. Non-precipitation conditions of the invention (ie 96% of IgG determined by electrophoresis on cellulose acetate (w / v) where the albumin content is preferably 1% or less (w / v) relative to the total protein) v) above purity)) to achieve a level of purification sufficient to be subjected to treatment with caprylate. Thus, the initial solution of immunoglobulin is treated with caprylate for the intended route of therapeutic administration, such that no additional purification is required after the step having the ability to inactivate the virus of the present invention. Fully purified before and after.

  The initial solution of immunoglobulins of the methods of the invention can also be obtained by genetic engineering techniques, such as expression in cell culture; chemical synthesis techniques; or transgenic protein production techniques.

  In the most preferred embodiment, the immunoglobulin referred to in the method of the invention is IgG. It is contemplated that IgG may be monoclonal or polyclonal. In the most preferred embodiment, the IgG is polyclonal.

  The glycol polyethers or polymers of the present invention are also known as alkane polyethers, or polyalkane oxides, also polyglycols, and may be, for example, ethyl or ethylene, and It is intended to mean propyl, or a derivative of propylene, well known as polyethylene glycol (PEG), or polypropylene glycol (PPG), or their equivalents. Also, the reagents do not impair their stability, or solubility, depending on their size, they are removed by ultrafiltration techniques, or because of their low toxicity, Must be compatible with immunoglobulins in the sense that they are suitable for therapeutic use.

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

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

  It is contemplated that the concentration of glycol polyether or polymer in the initial solution of immunoglobulin may need to be adjusted. Glycol polyether or polymer preparation can be achieved by diluting and / or adding the initial purified solution of the immunoglobulin.

  According to the composition of the initial solution of immunoglobulin, it is contemplated that a series of steps of purification or concentration adjustment is performed prior to step a) of the method of the invention, for example, the concentration of immunoglobulin, Adjustment to between 1-10 mg / ml, more preferably between 3-7 mg / ml, etc. This adjustment can be performed at any time by any method known in the art, for example, an established range (Biuret method, Bradford method, or particularly immunoturbidimetry, eg, optical density E ( 1%) = 13.8-14.0 (determined according to the total protein by UA). Thus, in a preferred embodiment, the initial solution of immunoglobulin is preferably an immunoglobulin concentration of 1-10 mg / ml, and more preferably 3-7 mg / ml; and / or adjustment of the purity of the immunoglobulin solution And it should preferably reach at least 96% IgG with respect to the total protein. This purification can be accomplished by techniques well known to those skilled in the art, such as precipitation with PEG, filtration, and subsequent anion exchange chromatography (DEAE Sepharose).

  In step a) of the method of the invention, caprylic acid, or a salt thereof, is preferably added, preferably using 9 mM, using the same concentrated solution, for example between 1.5M and 2.5M. A final concentration between -15 mM is achieved.

  In a preferred embodiment, in step b), the resulting solution is adjusted to a pH of 5.0 to 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. The temperature at which the incubation is performed is 2 ° C to 37 ° C, more preferably 20 ° C to 30 ° C.

  In a preferred embodiment, prior to step d) of the method of the invention, the amount of polymer, or aggregate, having a high molecular weight in the solution obtained in step c) is less than 0.2%, more preferably Less than 0.1. This ratio of polymer or immunoglobulin molecular aggregates to total protein is determined by an exclusion HPLC gel column according to the optical density value at 280 nm. The proportion of polymer or immunoglobulin molecular aggregates can be assessed using, for example, the analytical methods described in the European Pharmacopeia intravenous gamma globulin monograph.

  Preferably, the immunoglobulin solution is clarified using a depth filter prior to performing the ultrafiltration / diafiltration of step d).

  With regard to step d), it is preferably contemplated that the ultrafiltration / diafiltration in the method of the invention comprises the first step of dialysis and concentration by volume reduction followed by application of diafiltration at a constant volume. .

  Ultrafiltration / diafiltration is produced in such a way that the consumption of reagents is somewhat lower and the process is more efficient in view of the fact that the protein concentration is optimal and preferably 30 mg / ml It can be carried out on an industrial scale by reducing the volume of the material and then diafiltering, preferably by means of simultaneous dialysis and concentration. In any case, the person skilled in the art is selected from various operating methods known in the art (eg dilution / concentration, or diafiltration / concentration, constant volume diafiltration, or improvements and combinations of the above). The optimal and practical method of performing this step of ultrafiltration / diafiltration can be easily determined.

  The ultrafiltration / diafiltration membrane used in step d) of the method of the present invention preferably consists of polysulfone, regenerated cellulose or the like, for example Biomax® (Millipore, USA), Omega® (Trademark) (Pall, USA), Kvik-flow (registered trademark) (General Electric, USA). However, the molecular weight cutoff selected for the membrane may vary depending on various factors, such as the manufacturer selected. The person skilled in the art can easily determine the membrane to be selected and it adapts to the needs in each case depending on, for example, the caprylate in the solution to be treated and the polyether of glycol or the concentration of the polymer. Will be done.

  Preferably, the ultrafiltration / diafiltration of step d) is accomplished using a membrane having a molecular weight cutoff of 100 kDa or less, more preferably 100 kDa.

  In the most preferred embodiment, the ultrafiltration / diafiltration of step d) is carried out in two stages: between pH 5.0 and 6.0 in order to reduce or remove most of the caprylate In order to reduce or remove most of the glycol polyethers or polymers, and the first stage adjusted to a pH of 5.0 or less, preferably between 4.0 and 5.0. Done in the second stage, which is adjusted to.

  In a preferred embodiment, in the first stage of the ultrafiltration / diafiltration step, diafiltration is performed using a diafiltration medium comprising an alkali salt of a carboxylic acid, eg, acetic acid, at a concentration of about 5 mM or more. Is called. In a most preferred embodiment, the diafiltration is carried out using a solution of sodium acetate at a concentration of 5 mM or higher, adjusted in pH as described above (ie, between 5.0 and 6.0). Is called.

  The amount of diafiltration volume performed in the first stage of ultrafiltration / diafiltration in step d) is easily determined by the person skilled in the art according to the amount of caprylate used initially and the final amount allowed. Can. Preferably, at least 3 volumes of diafiltration medium are used, and the diafiltration medium is preferably a 5 mM sodium acetate solution, pH 5.0-6.0, as described above. Preferably, in the first stage of ultrafiltration / diafiltration, in this first stage, about 90% or more of the initial caprylate is removed so that the concentration of caprylate is reduced to about 1 mM or less. .

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

  Preferably, the diafiltration in the second stage of ultrafiltration / diafiltration comprises an alkali metal salt formed by acetate, phosphate, or the like, or an amino acid and / or polyol, such as glycine and / or sorbitol, It is performed using a buffer solution having the pH value described above.

  As in the first stage of diafiltration, in the second stage, the amount of glycol polyether or the amount of dialysis volume used to adequately reduce the polymer used in the process of the present invention is It can be readily determined by one skilled in the art in view of the required reduction or removal of the polyether, or polymer. In a preferred embodiment, the amount of buffer exchanged in the second stage dialysis of ultrafiltration / diafiltration in step d) is 6 volumes or more. In the most preferred embodiment, in the second stage, the exchange is a polyether of glycol, or a polyether of glycol more than 100 times the initial amount of polymer, before starting the ultrafiltration / diafiltration of step d). Or according to the volume of buffer required to obtain a reduction in polymer.

  Once the caprylate and glycol polyether or polymer are reduced in the ultrafiltration / diafiltration of step d), the product is concentrated in the final formulation step described above to achieve final formulation. In order to achieve the desired final composition by adding the necessary excipients and / or stabilizers. The addition of excipients and / or stabilizers that occurs after final formulation is by adding excipients and / or stabilizers in solid or concentrated solutions, or more preferably, the final product. Can be achieved directly using diafiltration with the required amount of the exchange volume of the formulation solution.

  In another embodiment, the addition of excipients and / or stabilizers is the sodium acetate dialysis buffer used in the second stage of step d) so that the immunoglobulin is already formulated after final concentration. By replacing the whole or part with a solution containing excipients and / or stabilizers, preferably adjusted to the same pH value between 4.0 and 5.0, Done.

  The person skilled in the art knows what kind of excipients and / or stabilizers should be added in order to achieve the desired stability. For example, the excipient and / or stabilizer may be one or more amino acids, such as glycine, preferably at a concentration between 0.2 and 0.3M; one or more carbohydrates or polyols, such as It is contemplated that it may be 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 is known to those skilled in the art and is for example 5% -22 % (W / v). The concentration is achieved by any method known in the art, such as concentration by ultrafiltration. Where immunoglobulin concentration is achieved by ultrafiltration, it is contemplated that the concentration may be performed using a membrane similar to that used in previous diafiltration. Of course, the three diafiltration methods and concentration may also be performed using different membranes.

  The method of the present invention also contemplates the possibility of introducing a nanofiltration step to increase the product safety margin. There are multiple stages in the process, where the product is a commercially available filter (eg, Planova® and Bioex®) having a pore size of 20 nm or less to 50 nm, preferably 20 nm or less. (Trademark) (Asahi Kasei), DV (registered trademark) and SV4 (registered trademark) (Pall), Virosart (registered trademark) (Sartorius), Vpro (registered trademark) (Millipore), or the like) Or even 15 nm nanofilters can be used. The intermediate steps in which the nanofiltration step can be performed are, for example, in the initial solution of immunoglobulin; or in the material treated with caprylate after the ultrafiltration / diafiltration step (capillate and glycol polyethers, Or (when the polymer is reduced); or in the material after concentration and formulation of the immunoglobulin, preferably IgG, (final product). Those skilled in the art will select the optimal option depending on, among other things, the pore size of the membrane, the filtration area required according to the time of the process, the volume of the nanofiltered product, and the protein yield.

  The final product obtained by the method of the present invention is in full compliance with European Pharmacopoeia standards relating to the content of allogeneic hemagglutinin. However, the methods of the present invention also contemplate options that include selective and specific capture steps of anti-A and / or anti-B antibodies to maximize their reduction. This step is preferably performed using a biospecific affinity resin, as described in the prior art literature. For example, by using a biospecific affinity resin having a ligand formed by a trisaccharide, a significant reduction in the level of allogeneic hemagglutinin can be achieved (Sparter et al., Blood, 1999). 93, 4418-4424). This additional capture may be incorporated at any step of the method of the present invention, optionally at the discretion of one of ordinary skill in the art, or may be performed before or after performing the method of the present invention.

  Thus, with regard to the method for the preparation of immunoglobulin solutions of the present invention, in the most preferred embodiment, the first solution of immunoglobulin having an IgG purity of 96% or higher is used. This solution is preferably adjusted to a concentration of IgG between 1 mg / ml and 10 mg / ml, and preferably between 3 mg / ml and 7 mg / ml, and it contains PEG 4 ± 1% (w / V) at the concentration (by addition in the previous step) or added. The pH of the solution is then adjusted between 5.0 and 5.2 with acetic acid and sodium caprylate is added (eg, using a concentrated solution of sodium caprylate). In a preferred embodiment, the concentrated solution of caprylate is slowly added to the purified solution of IgG and stirred. After adding all the caprylate calculated to bring the final concentration of caprylate between 9-15 mM to the product, the final pH is then between 5.0 and 5.2, if necessary. And the solution is preferably incubated at a temperature between 2-37 ° C. and more preferably at a temperature of 25 ± 5 ° C. for at least 10 minutes, and preferably 1-2 hours. The

  Clarification is then performed using a depth filter (eg, Cuno 90LA, 50 LA, Seitz EK, EK-1, EKS, or equivalent).

  Thus, the resulting solution is then subjected to a membrane containing polysulfone, such as an ultrafiltration / diafiltration device formed by Biomax® (Millipore) or Omega® (Pall), preferably In the form of a stackable cassette. The solution is circulated through each ultrafiltration / diafiltration unit, preferably at a volume between about 100-500 L / h and at a temperature of 5 ± 3 ° C. The pressure drop (atmospheric pressure) of the pressure between the inlet and the outlet is preferably between 1 and 3 bar. The first diafiltration step of the ultrafiltration / diafiltration step is then preferably at a concentration of 5 mM or more and at a pH between 5.0 and 6.0 to remove caprylate. Preferably, the application begins with the application of an exchange of at least 3 volumes of buffer formed by a solution of sodium acetate. Preferably, with each volume of buffer added or consumed, the volume of the product solution is reduced to half of the initial volume, except for the last addition.

  After the first stage of diafiltration (by dilution and concentration, or equivalent), the pH of the resulting solution is adjusted to between 4.0 and 5.0 using, for example, acetic acid. Diafiltration at a constant volume is then carried out at a concentration of 5 mM or more, at a pH between 4.0 and 5.0, preferably using a volume of 6 or more of the buffer solution formed by sodium acetate. To be started.

  The dialysis buffer solution formed by the sodium acetate is an amino acid, eg glycine, optionally a carbohydrate at a concentration of 0.2-0.3M, so that after final concentration, the immunoglobulin is already formulated. And optionally substituted, in whole or in part, by a solution combined with a polyol, such as sorbitol, preferably adjusted to the same pH value between 4.0 and 5.0. Good.

  Preferably, after application of at least 6 volumes (more preferably between 6 and 10 volumes) of the dialysis solution at a pH between 4.0 and 5.0, if the product is not formulated, Excipients and / or stabilizers, such as glycine, or other amino acids, and carbohydrates, such as sorbitol, or combinations thereof, in the form of solids or concentrated solutions of the excipients and / or stabilizers, The product is formulated by adding it directly to the resulting solution. 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, adjusted appropriately for excipient and / or stabilizer concentration and pH, is applied by complete filtration using a 0.2 μm pore size filter, and optionally by nanofiltration. is there. Finally, the IgG solution is aseptically placed in an injection, ampoule, vial, bottle, or other glass container, which is then sealed. Another option is injection into a compatible hard or flexible plastic container, such as a bag or bottle.

  Products prepared as described above undergo quarantine and visual inspection before being transferred to storage for storage for at least 2 years at temperatures between 2-30 ° C.

  Furthermore, as mentioned above, the present invention also provides the use of caprylic acid, or a salt thereof, in the presence of at least one polyether or polymer of glycol for virus inactivation in protein production processes. For the first time, where the polyethers or polymers of glycol and caprylic acid, or salts thereof, are subsequently removed using ultrafiltration.

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

  The invention will be described in greater detail with reference to various exemplary embodiments. However, these examples are not intended to limit the scope of the invention, but to illustrate the description.

Example 1
Method of the invention for obtaining a solution of immunoglobulin from plasma that is virally safe, free of aggregates and having a yield suitable for industrial applications. The starting material is a 16 L immunoglobulin solution. Yes, and it contains IgG as the main protein component and was obtained by the method described in EP 1225180B1. In summary, the solution was obtained by extracting gamma globulin from fraction II + III using the Cohn method. To perform this extraction of gamma globulin from fraction II + III, the fraction was previously isolated by fractionation of human plasma using ethanol. Subsequently, it was suspended in the presence of carbohydrates and the accompanying content of numerous proteins was reduced by precipitation with PEG-4000. Finally, the final purification of the fractions was performed by adsorption on an ion exchange resin column (DEAE Sepharose). Thus, the column eluate obtained (resin, ie the fraction not adsorbed to DEAE) was 98 ± 2% immunoglobulin electrophoretic purity in cellulose acetate, pH 6.0, turbidimetric 2.6 It has a turbidity of total turbidity unit (NTU) and an IgG concentration of about 5 mg / ml.

  The resulting solution was adjusted to a pH of 5.1 by adding acetic acid and adjusted to a temperature between 2-8 °. This solution of immunoglobulin was then brought to a final concentration of 13 mM by adding a concentrated solution of sodium caprylate.

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

  The caprylate treated solution was cooled to a temperature of about 8 ° C. for subsequent clarification using a depth filter (CUNO®, Ultrafilter, Denmark). About 20 L of filtered liquid with an IgG concentration of about 4 mg / ml and a turbidity of 3 NTU or less was obtained from the clarification (including rinsing).

  The clarified solution was dialyzed by ultrafiltration using a membrane (Biomax® Millipore, USA) with a nominal molecular weight cutoff of 100 kDa. Ultrafiltration was performed in two different stages. In the first stage, a material having a pH of 5.1 is used in a three step series of dialysis by using a 5 mM acetic acid solution adjusted to pH 5.1 and using a concentration to about 30 UA. And subjected to concentration. In the second stage, the solution with the appropriate concentration of protein, caprylate, and PEG is brought to pH 4.5 ± 0.1 and dialysis followed by 8 volumes of 5 mM acetic acid solution at pH 4.5. Use and started. The product is then formulated using dialysis using about 20 L of 200 mM glycine solution at pH 4.2 to obtain a solution of IgG having a concentration of 10% (w / v). For the purpose, it was concentrated in the same ultrafiltration unit to a value of 140.5 UA.

  Finally, the solution is applied to a depth filter (CUNO®, Ultrafilter, Denmark) and an absolute filter, or a membrane having a pore size of 0.22 (CVGL®, Millipore, USA; or DFL®) (Trademark), PALL, USA).

  Table 1 shows the characterization of the starting material, multiple intermediate products, and the final product according to the above method. For the results shown in the table, the turbidity was measured by turbidimetry; the proportion of polymer or immunoglobulin molecular aggregates was measured according to the optical density value at 280 nm for the total protein detected. That the concentration of caprylate was determined using an enzymatic method by quantification of a colorimetric substrate; the concentration of PEG was determined using a differential refractive index detector It should be noted that the HPLC filtration gel column was determined: and the method yield (%) was calculated according to the quantified IgG concentration by turbidimetry.

  The results of this example show that treatment of the purified solution with caprylate does not induce any formation of immunoglobulins or other precipitates and maintains the product molecular distribution unchanged. Show. Thus, no purification step was required to remove aggregates and / or precipitates after treatment with caprylate. This fact greatly facilitates the product manufacturing process and allows direct application of the material to the ultrafiltration membrane.

  Thus, the subsequent ultrafiltration process achieves the goal of effectively reducing manufacturing chemical reagents (ie, PEG and caprylate) and allows the formation and concentration of purified immunoglobulin solutions. And obtain a suitable composition for its therapeutic use.

  As can be seen from Table 1, the yield of the protein obtained in this case from the starting eluate to a 10% concentrated product is 89.4%, indicating the feasibility of the process on an industrial scale. . This recovery rate is described in prior art documents as described in WO 2005/073252 (70% yield, based on a yield of 4.8 g / L compared to the initial 6.8 g / L). Greater than the value obtained by the conventional method according to

Example 2
In this example, the effect of the purity of the initial solution of immunoglobulin on the treatment with caprylate, and the accompanying protein in the starting material subjected to the method of the present invention are evaluated. Went in existence.

Two independent test groups were created:
In group A, the starting material was DEAE Sepharose column eluate, electrophoretic purity (ACE) of 98 ± 2%, ie the starting material described in Example 1.
In Group B, the starting material, designated 4% PEG filtrate was obtained by the method described in Example 1 up to the step before DEAE Sepharose chromatography. Thus, material B is obtained after precipitation with PEG of the extract suspension of fraction II + III and has an electrophoretic purity (ACE) of about 90% IgG.

  Both starting materials (Group A and Group B) having a PEG content equivalent to about 4% were treated with a concentration of 13 mM and caprylate between 5.0 and 5.2 and shown in Example 1 Purified.

  Table 2 shows the starting materials used in both test groups (A and B, respectively) and the properties of those materials produced in the steps after treatment with caprylate.

  The results obtained and collected in Table 2 show that the addition of caprylate at a concentration effective for inactivation (13 mM) to material of low purity (approximately 90% IgG, see group B) Causes precipitation of the components of the solution, resulting in a dramatic increase in turbidity (> 500 NYU). Thus, the molecular distribution results for the solution showed precipitation of a portion of the accompanying protein with high molecular weight.

  Addition of caprylate to low purity material under the previously described amounts and conditions (13 mM caprylate, pH between 5.0 and 5.2) results in a precipitated suspension. In order to separate high molecular weight proteins, and precipitated aggregates, it is necessary to include additional steps of separation and purification. Thus, the molecular composition of group A products treated with caprylate, ie having an aggregate content greater than 1%, is an additional step of purification, or separation, such as precipitation with PEG, chromatography, or Unless an equivalent method is included, it indicates that it is not feasible to process this product into a purified final product. Finally, this fact shows the feasibility of using caprylate as a reagent with virus inactivation ability in non-precipitating conditions only when caprylate is added to sufficiently pure material.

Example 3
Effect of starting material composition on aggregate formation The purpose of this example was to evaluate the effect of the composition of the initial solution of immunoglobulin undergoing treatment with caprylate.

  Starting with two independent test groups, materials of comparable purity (97.9 ± 1.5%), groups A and B with different compositions were made.

  In group A, the starting material is the column eluate (obtained according to the first method described in Example 1) and has a protein concentration of 5 ± 2 mg / ml and a PEG-4000 concentration of 4 ± 1%. .

  In Group B, the starting material, designated, concentrated and dialyzed eluate, is the same column eluate as described in Group A, but after concentrated and dialyzed. is there. Thus, the DEAE column eluate (described in Example 1 above and corresponds to Group A of this example) is reduced to a PEG content of about 6 times and the protein is about 4%, ie 40 mg. The solution was subjected to dialysis by ultrafiltration and an additional step of concentration to concentrate to / ml.

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

  Table 3 shows the main properties of the materials processed in the above test groups A and B, and the subsequent caprylate processing steps and the dialyzed and concentrated end product for each experimental group. , Show the properties of the material produced.

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

  However, when these same conditions for treatment with caprylate were applied to materials with low PEG content (<1%) (Group B), the substantial increase in immunoglobulin aggregates was Observed after addition. Furthermore, under the conditions used, ultrafiltration could not remove this agglomerate content and comparable polymers were measured in the final product.

  Given that the main different characteristics between the starting materials used in experimental groups A and B are the protein concentration and the PEG concentration, additional tests were conducted for each of these parameters, with subsequent capri We went to find out the impact on rate processing.

  In this experiment, the starting point is a single batch of concentrated and dialyzed eluate (first material of group B above) and it is divided into four distinct experimental groups: B1, B2, B3, And B4.

  Group B1 material was treated with a protein concentration of about 4% and a PEG concentration of about 0.6%.

  Group B2 material was treated with the same protein concentration of approximately 4%, but the PEG concentration 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. Regarding the PEG concentration, in Group B3, the concentration was about 0.6% (w / w), but in Group B4, the PEG concentration was readjusted to 4 ± 1% (w / w).

  The resulting material obtained in the four experimental groups was brought to a pH of 5.10 ± 0.05 and a caprylate concentration of 15 mM and then incubated at 25 ° C. for 2 hours.

  The results shown in Table 4 indicate that the PEG protection effect during treatment with caprylate under defined conditions was observed in combination with sufficient protein dilution. When the starting material was approximately a protein concentration of 5 ± 2 mg / ml and a PEG concentration of 4%, an undetectable value of aggregate was obtained after treatment with caprylate (<0.1%) It is worth noting.

Example 4
Effect of pH on the solubility of solutions of immunoglobulin treated with caprylate Removal of PEG in the solution of immunoglobulin and concentration of the immunoglobulin to a concentration suitable for their intravenous use is preferably At a pH value of about 4.5.

  Furthermore, in view of the insolubility of caprylic acid at pH values below pKa (4.89) in this experiment, the effect of pH on the solubility of caprylate-treated immunoglobulin solutions was measured by ultrafiltration. In order to determine an appropriate pH value to initiate

  To achieve this objective, a batch of column eluate obtained according to the first method described in detail in Example 1 is processed, treated with 13 mM caprylate and clarified immunoglobulin. Solution was obtained.

  This intermediate, and it constitutes the material prior to the ultrafiltration step and is acidified by the addition of acetic acid to a pH value of about 4.5 from the pH of treatment with caprylate (5.1). . Subsequently, the appearance and solubility of the solution was evaluated for each evaluated pH, and the production of colloidal particles was quantified by turbidimetric nephelometry.

  Table 5 shows the appearance and turbidity obtained for each of the evaluated pH values.

  The results obtained are shown in Table 5, when the solution of immunoglobulin treated with 13 mM caprylate is acidified to a pH of less than 5.0, the appearance of white precipitates Observed with a clear increase in degree. This effect was attributed to the formation of insoluble caprylic acid in colloidal form, which made it impossible to start the ultrafiltration process at a pH below 5.0.

  The results obtained are shown when the purified solution is subjected to treatment with caprylate in the effective concentration range for virus inactivation (9-15 mM caprylate) and under the conditions described above. Increase the concentration of ionized and soluble forms at a pH above the pH of the virus inactivation treatment (ie 5.1) and thus facilitate its permeability through the ultrafiltration membrane That is the evidence that it is preferable to start.

Example 5
Effect of acetic acid content in dialysis solution on the reduction of caprylate by ultrafiltration / diafiltration A series of independent ultrafiltration / diafiltration processes are used to vary the acetic acid in buffer used for product dialysis. Performed in the presence of concentration.

  The starting materials used, designated, concentrated and dialyzed eluates were the same as those of Example 3, Group B. The starting material, having an IgG purity of 98 ± 2%, a protein concentration of about 40 mg / ml, and a PEG content of about 0.6% is subjected to treatment with caprylate and subsequently a nominal molecular weight of about 100 kDa It was subjected to ultrafiltration / diafiltration using a membrane with a cut-off.

  The applied ultrafiltration / diafiltration steps consisted of a first stage of concentration to about 4% (w / v) IgG, a second stage of dialysis using 8 volumes of dialysis solution, and a final of about 9 Concentration to IgG values of -10% (w / v).

  Initial ultrafiltration / diafiltration tests were performed using distilled water for injection, but subsequent tests were performed with increasing concentrations of acetic acid, more specifically 2, 5, 20, or 50 mM, respectively. In all cases and using a buffer adjusted to a pH between 5.0 and 5.5.

(1) Value measured at 8 dialysis volume after dialysis (2) The following formula:
Number of dialysis volumes = ln (Cf / Co) / (R-1)
(Where Cf is the concentration after dialysis at the number of dialysis volumes in question, Co is the concentration before dialysis, and R is the retention factor)
Transparency calculated using

  The results in Table 6 use a membrane with a molecular weight cut-off of about 100 kDa, with acetic acid at a pH between 5.0 and 5.5 and with a minimum concentration of acetic acid of about 5 mM and at least 50 mM. We show that the ultrafiltration / diafiltration method applying 8 dialysis volumes of buffer sufficiently achieves an efficient reduction of caprylate to the appropriate level in the final concentrated product.

  In contrast, if the solution used for dialysis is distilled water for injection or a buffer with 2 mM acetic acid level, caprylate is not efficiently removed in the filtrate.

  This is because the ultrafiltration / diafiltration method using a membrane with a molecular weight cut-off of about 100 kDa, under the conditions previously described, detected the correct level of the reagent in the final concentrated product. Is evidence that it is effective in reducing caprylate from previous treatments.

Example 6 Simultaneous removal of chemical reagents (PEG and caprylate) using single-step ultrafiltration A batch of IgG was processed according to the method described in Example 1, inactivated with caprylate and clarified. Solution was obtained. The solution, having a protein concentration of about 0.5%, and a pH of 5.1, is a polysulfone membrane Biomax® type (Millipore, USA) with a 100 kDa molecular weight cutoff. Processed using a filtration / diafiltration device. Ultrafiltration / diafiltration was performed in two different stages as described in Example 5.

  In the first stage, performed at pH 5.1, 5.6, or 5.8, the material is at least 3 volumes of 5 mM acetate buffer adjusted to pH 5.1, 5.6, or 5.8. Was subjected to a series of dialysis and concentration steps, using a diafiltration of and a concentration of protein to a value of about 2%.

  In the second stage, after the amount of caprylate was reduced to about 1/10, the solution was brought to a pH of 4.5 ± 0.1, or 5.1. The product was then brought to the appropriate concentration of protein and PEG, dialysis was started, and dialysis was started with 8 volumes of 5 mM acetate buffer at a pH of 4.5 or 5.1.

  Finally, the product was formulated using dialysis with 6 volumes of glycine solution at a pH of 4.2 and a concentration of 200 mM and concentrated to obtain a 10% solution of IgG.

  Table 7 shows the resulting PEG and caprylate passage rates at the beginning of each stage of ultrafiltration / diafiltration and at various pH values.

  The results in Table 7 show that at the start of ultrafiltration / diafiltration, in stage I, caprylate showed very high pass values between pH 5.1 and pH 5.8. These values resulted in a very high reduction of caprylate during stage I of the ultrafiltration / diafiltration step (observed a 10-fold reduction in caprylate relative to the initial content). In contrast, the passage of PEG was very low (<20%) in Stage I and its total removal was virtually infeasible at pH> 5 in the presence of caprylate.

  On the other hand, in stage II, as can be seen in Table 7, the passage of PEG was very high at pH 4.5, a value of 82%. Also, during this phase II, caprylate was also present at the beginning of this phase, considering that caprylate was present at a residual level of <1 mM and allowed substantially 100% passage. I understood that it was reduced.

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

  According to the PEG and caprylate values recorded at each step and stage, and taking into account the protein concentration at each step, the total reduction factor of about 350 times (stage I and stage II) (ultrafiltration / Given the initial absorbance of 6.9 compared to the absorbance of 0.02 obtained at the end of the diafiltration step), the PEG reduction factor is calculated in Phase I of the ultrafiltration / diafiltration step (at pH 5.1). ) And 4 in stage II (at pH 4.5) of the ultrafiltration / diafiltration step.

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

  The results show that the physical and chemical conditions in which the reagent (caprylic acid or caprylate) having the ability to inactivate the virus and the precipitation reagent (PEG) are applied at each stage of the ultrafiltration / diafiltration step. (Especially pH, protein concentration, number of dialysis volumes, dialysis buffer) can be selected and efficiently reduced by a single step of ultrafiltration using a membrane with a molecular weight cut-off of about 100 kDa and venous It was shown to yield a final product of 10% IgG with some residual concentration of both reagents suitable for internal use.

Example 7 Evaluation of virus inactivation ability with caprylate in the presence of PEG As starting material, column eluate or dialyzed and concentrated eluate (obtained according to Examples 1 and 3, respectively) A variety of independent experiments were used to evaluate the ability of caprylic acid or caprylate in the presence of PEG to remove or inactivate viruses with lipid envelopes.

  Both materials had an immunoglobulin purity of 98 + 2% and a protein concentration of 5-10 mg / ml, while their PEG content differed from 40 mg / ml and 1.5 mg / ml, respectively.

  The virus inactivation test uses a 40-60 nm flaviviridae bovine viral diarrhea virus (BVDV) with a lipid envelope and average resistance to physical and chemical agents. And went.

  In each test, the corresponding starting material is inoculated with the virus to 0.5% or less and a 9 mM or 13 mM caprylate concentration is applied for 2 hours at a temperature of 15 ° C to 25 ° C. It used for the inactivation process.

Quantification of viral load of BVDV in the different samples generated was performed using the TCID50 test (50% tissue culture infectious dose) using the MBDK cell line. The virus reduction factor (RF) of the virus inactivation step divided by the amount of virus detected in the sample obtained at the end of the treatment, expressed as log ₁₀ , divided by the viral load detected in the inoculated starting material. Decided as a quotient.

  Table 9 details the properties of the starting material for each test and the resulting RF.

  The virus reduction results obtained in all tests (see Table 9) show that in both starting materials, even for the minimum 9 mM concentration of caprylate after testing at different temperatures (15 and 25 ° C.) Shows high ability for inactivation of BVDV. Furthermore, given that these tests yielded comparable results for each PEG concentration analyzed with the materials evaluated in both cases, the observed interference by PEG on the virus inactivation ability of caprylate was Showed no.

Example 8 FIG. Characterization of Intravenous Immunoglobulin Solution Obtained According to the Production Method of the Present Invention This example illustrates the biological and function of an immunoglobulin solution with 10% (w / v) protein obtained by the method of the present invention. The purpose is to define the characteristics.

  Two batches of DEAE column eluate were processed according to the method detailed in Example 1 in order to obtain a caprylate inactivated virus solution at about 200 L plasma scale.

  Once clarified, the solution with caprylate is dialyzed in a differentiated stage as described in Example 6 with the aim of achieving removal of the main process residues (PEG and caprylate). And concentrated by ultrafiltration. Subsequently, the purified solution was adjusted to a pH of 4.5 ± 0.1 at a protein concentration of about 2.5% and a constant volume of about 6 volumes of a buffer consisting of 1% sorbitol and 240 mM glycine. Then, it was formulated by dialysis. Finally, the solution is concentrated by ultrafiltration and adjusted to an optical density of 140 ± 5 UA (280 nm), corresponding to 10% (w / v) protein, and 5.25 ± 0.25. Adjusted to final pH.

  The resulting product (IGIV 10% (w / v)) stabilized with sorbitol and glycine and once clarified and filtered using a sterile grade membrane (0.22 μm) was administered intravenously. In order to determine the most relevant analytical parameters of immunoglobulin solution quality, invariance, and stability, it was placed in a glass container with a chlorobutyl stopper. The average analytical values obtained for the two batches and the European Pharmacopoeia value designations are shown in Table 10.

  Eur. Ph. : European Pharmacopoeia; n. e. : Not specified; TGT FXI: Thrombin generation test (using factor IX deficient plasma); NAPTT PKA: Prekallikrein activator; ACA: Anti-complement activity

  The above results indicate that the resulting product has some uninjured functional properties, such as the absence of polymer, PKA, or ACA activity, particularly plasma, with no damaging functional properties, such as percentage of IgG subclass, and With respect to parameters such as conservation of Fc fragment integrity, the purification process of the present invention is substantially invariant and at the same time exhibits an excellent purity profile (low anti-A / anti-B allogeneic hemagglutinin). Titration, IgM concentration, coagulation promoting activity, etc.).

  The overall method of the present invention to obtain 10% (w / v) IGIV, including virus inactivation with caprylate in the presence of PEG, and subsequent separation and final formulation steps, Is feasible as a whole, formulated as a 10% (w / v) IGIV protein solution, and expandable to a concentrated end product, fully compatible with the values defined in the European Pharmacopoeia The conclusion is reached that gives the final product to

  A stability test was performed and it is essential for commercialization of the product, stabilizing 10% (w / v) intravenous immunoglobulin solution for 2 years at room temperature (25 ° C-30 ° C) In order to achieve a formulation that has sorbitol (5%), glycine (equivalent), or a combination thereof, the pH range of 4.2-6.0 has been demonstrated.

Example 9 Applicability of treatment with caprylate on IgG rich fractions obtained by alternative methods was evaluated using other process intermediates obtained using alternative purification methods. With the effectiveness of application with caprylate under the conditions described in the document.

  Two independent experiments were performed using as a starting material a predetermined suspension of fraction II from the IgG rich plasma intermediate, Cohn-Oncley ethanol fraction.

  This intermediate was obtained up to fraction II + III by the same method as the plasma fractionation method described herein. The procedure was then continued with alcohol reprecipitation of the fraction II + III extract suspension, followed by separation of fraction III, and finally, fraction II having a purity greater than 96%. The fraction II suspension was purified once with bentonite and dialyzed with water to remove the alcohol and used as starting material for these experiments.

  In the two experiments performed, materials from two plasma batches were separated into two different groups, A and B, according to their PEG content. In group B, the starting material was brought to a nominal PEG concentration of 40 mg / ml by adding a concentrated solution of PEG-4000.

  Thereafter, both materials from both groups (A and B) were diluted to a protein concentration of about 5 mg / ml, adjusted to a pH value of 5.1, and 13 mM nominal as described herein. It was subjected to treatment with caprylate until a concentration and pH between 5.0 and 5.2 was reached.

  Table 11 details the main properties of the starting materials used in both test groups (A and B, respectively), and the properties of the materials that resulted after treatment with caprylate.

  (1) PEG and caprylate values correspond to values obtained using analytical quantification.

  The results show the feasibility of inactivation treatment with caprylate under certain conditions using well purified immunoglobulin solutions by different methods, immunoglobulin aggregates, or other irreversible precipitates It does not induce product formation and it shows that the subsequent purification process is greatly facilitated.

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

  This experimental example is a reagent that has the ability to inactivate viruses under non-precipitation and / or aggregation-promoting conditions if a material with sufficient purity is added and the specific conditions regarding protein and PEG concentration are met. As an example, the feasibility of using caprylate is shown.

  The present invention has been presented and described with reference to the same embodiments, but will be apparent to those skilled in the art after making the interpretation of the subject matter disclosed herein and in the claims, or otherwise. With respect to the details, it will be understood that these embodiments are not intended to limit the invention, since there are several variations. Accordingly, all modifications or equivalents are intended to be included within the scope of the invention if they are considered to be within the broadest scope of the invention as set forth in the following claims.

Claims (24)

A method for preparing a solution of an immunoglobulin based on an initial solution of an immunoglobulin having a purity of 96% or more in the presence of a polyether or polymer of glycol, said method comprising the following steps:
a) Caprylic acid, or a salt thereof, is added to the initial solution;
b) adjusting the pH of the solution obtained in step a);
c) incubating the solution obtained in step b) for the time and temperature required for inactivation of the enveloped virus; and d) for the solution obtained in step c) A preparation method comprising performing diafiltration.   The method according to claim 1, characterized in that it further comprises the step of final formulation of the solution of immunoglobulin obtained in step d).   The initial solution of the immunoglobulin is derived from fraction I + II + III, fraction II + III, or fraction II, obtained according to the Cohn method or Cohn-Oncley method, or obtained according to the Kistler-Nitschmann method, or Method according to claim 1 or 2, characterized in that it is derived from a precipitate A, or I + A, or GG obtained according to a variation of the method, further purified to obtain an IgG purity of 96% or more. .   The initial solution of the immunoglobulin is derived from the fraction II + III, obtained according to the Cohn method, or a variation of the method, followed by purification by precipitation with PEG and anion chromatography. The method of claim 3, wherein:   The method according to any one of claims 1 to 4, characterized in that the initial solution of immunoglobulin has an immunoglobulin concentration of between 1 and 10 mg / ml.   5. A method according to claim 4, characterized in that the initial solution of immunoglobulin has an immunoglobulin concentration of between 3 and 7 mg / ml.   The method according to any one of claims 1 to 6, characterized in that the polyether or polymer of glycol is selected from polyethylene glycol (PEG), polypropylene glycol (PPG), or combinations thereof. .   The method according to claim 7, characterized in that the concentration of PEG in the initial solution is between 2% (w / v) and 6% (w / v).   The method according to claim 7, characterized in that the concentration of PEG in the initial solution is between 3% (w / v) and 5% (w / v).   The method according to any one of claims 7 to 9, characterized in that the PEG is a PEG having a nominal molecular weight of 4000 Da.   11. The method according to any one of claims 1 to 10, characterized in that in step a) caprylic acid or a salt thereof is added to achieve a concentration between 9 mM and 15 mM.   12. A method according to any one of claims 1 to 11, characterized in that in step b) the obtained solution is adjusted to a pH between 5.0 and 5.2.   13. The method according to claim 12, characterized in that in step b) the obtained solution is adjusted to a pH of 5.1.   14. Method according to any one of claims 1 to 13, characterized in that in step c) the solution is incubated at a temperature between 2 <0> C and 37 <0> C for at least 10 minutes.   14. The method according to claim 13, characterized in that in step c) the solution is incubated at a temperature between 20 <0> C and 30 <0> C for at least 2 hours.   16. A method according to any one of the preceding claims, characterized in that the initial solution of immunoglobulin has an albumin content of 1% (w / v) or less relative to the total protein.   17. A method according to any one of the preceding claims, characterized in that the initial solution of immunoglobulin is derived from human plasma.   18. The initial solution of the immunoglobulin is obtained by genetic engineering techniques, chemical synthesis techniques, or transgenic protein production techniques, or obtained in cell culture. The method described in 1.   19. A method according to any one of the preceding claims, characterized in that the ultrafiltration / diafiltration of step d) is performed using a 100 kDa membrane. The ultrafiltration / diafiltration of step d) comprises the following two steps:
In order to reduce or remove most of the caprylic acid, the pH is adjusted between 5.0 and 6.0, the first stage; and most of the glycol polyether or polymer to reduce The process according to any one of claims 1 to 19, characterized in that it is carried out in a second stage, wherein the pH is adjusted to a value of 5.0 or less for removal.   21. The method according to claim 20, characterized in that in the second stage ultrafiltration / diafiltration of step d), the pH is adjusted between 4.0 and 5.0.   In the final formulation step, excipients and / or stabilizers selected from one or more amino acids, one or more carbohydrates, or polyols, or combinations thereof are added, The method of claim 2.   23. A method according to any one of the preceding claims, characterized in that the final concentration of immunoglobulin is adjusted to a concentration suitable for intravenous, intramuscular or subcutaneous use of said immunoglobulin.   Use of caprylic acid or a salt thereof in the presence of at least one glycol polyether or polymer in the presence of at least one polyether for virus inactivation in an immunoglobulin production process, wherein said polyether of glycol Or the polymer and caprylic acid, or salt thereof, are subsequently removed by ultrafiltration.