Macromolecule purification process with combined selective precipitation, concentration and dialysis steps under constant stability conditions, with which stable and high-purity formulations are obtained

Abstract

The proposed invention relates to an integral macromolecule purification process based on macromolecule- and / or protein-rich matrices. The process comprises the following steps: selective precipitation, successive filtration, ultrafiltration to eliminate salts and continuous dialysis to adjust the pH and maintain constant stability conditions. Recovery by filter press and the option to pasteurise in order to purify some macromolecules add refining and safety layers. In the case of other macromolecules, an optional chromatography separation phase is included. Concentration via ultrafiltration, treatment with solvent / detergent, industrial scale chromatography and subsequent dialysis highlight the versatility of the method. The invention provides a robust solution to obtain stable and high-purity formulations to be used in potential applications in different fields, from pharmaceutical production to biomedical research.

Description

[0001] Macromolecule purification process with stages of selective precipitation, concentration and dialysis combined under constant stability conditions, which results in stable and high purity formulations.

[0002] Description

[0003] Technology Sector

[0004] The technological sector related to the macromolecule purification process of the present invention lies at the intersection of biotechnology, chemical engineering, and materials science. This sector is driven by the need to develop advanced technologies that enable the efficient purification of macromolecules, such as proteins, nucleic acids, or polysaccharides, with the goal of obtaining stable, high-purity formulations.

[0005] Previous State of the Art

[0006] The prior art of the invention focuses on existing methods for the purification of macromolecules, with a particular focus on processes that incorporate selective precipitation, concentration, and dialysis steps. These methods are essential for obtaining stable, high-purity formulations, crucial requirements in diverse applications, from drug production to biomedical research.

[0007] To date, conventional macromolecule purification methods have faced significant challenges in terms of efficiency and yield. Traditional techniques often involve multiple steps and require variable conditions, which can affect the stability and purity of the final product.

[0008] Selective precipitation has been a key component in many purification processes, allowing the separation of impurities and other unwanted molecules. However, existing methods often lack the ability to achieve optimal selectivity or maintain stable conditions throughout the entire process.

[0009] Regarding concentration, methods such as ultrafiltration and centrifugation have been widely used. However, these techniques present challenges associated with the loss of active ingredient, variability in efficiency, and the possibility of cross-contamination.

[0010] Dialysis, on the other hand, has proven effective for the removal of small molecules and salts. Despite this, the efficient integration of dialysis into an overall purification process has been an area of ​​constant improvement.

[0011] The proposed invention addresses these limitations by specifically combining the steps of selective precipitation, concentration, and dialysis into a single, integrated process. The advantage of this approach is the implementation of constant stability conditions throughout all phases of the process, ensuring integrity and purity of the final product.

[0012] This invention is expected to provide an efficient and robust solution for the purification of macromolecules, overcoming the limitations encountered in existing methods. Obtaining stable, high-purity formulations is essential for a wide range of applications, from pharmaceutical production to biotechnology research, and this invention is anticipated to have a significant impact on the improvement of macromolecule purification processes.

[0013] Description of the Invention The present invention represents a significant advance in the field of macromolecule purification, by offering a comprehensive process that guarantees constant stability conditions for such large molecules. This method encompasses several main steps, including selective precipitation, concentration, dialysis, and selective purification, all meticulously combined to achieve final formulations of high purity and exceptional stability.

[0014] In the first phase, selective precipitation is carried out by applying specific conditions, such as controlled temperature and pH variations, along with the precise addition of reagents. This step allows for the selective formation of aggregates, facilitating the efficient separation of impurities and contributing to product purity.

[0015] The concentration phase, a critical part of the invention's process, is carried out using advanced techniques such as ultrafiltration. This step increases the density of the macromolecules of interest and contributes to obtaining highly concentrated and, therefore, potentially more effective formulations.

[0016] The process then includes a dialysis stage, where the macromolecules undergo careful removal of impurities and adjustments to their chemical environment. This step is essential to ensure the integrity and stability of the macromolecules throughout the process.

[0017] Process control and automation play a central role in this invention, ensuring consistent and reproducible conditions at every stage. Precise parameter optimization, supported by advanced analytical technologies such as mass spectrometry and chromatography, is implemented to ensure the quality and purity of the final product.

[0018] This purification process finds significant applications in diverse areas, from biopharmaceutical production to biomedical research, where obtaining stable and pure macromolecules is of vital importance. Furthermore, the invention complies with regulatory standards and regulations, establishing itself as a significant contribution in the fields of biotechnology and chemical engineering. Overall, this innovation redefines efficiency and quality in the macromolecule purification process, promoting significant advances in industry and scientific research.

[0019] Description of the Figures

[0020] Figure 1. General embodiment of the invention.

[0021] Figure 2. Preferred embodiment of the invention.

[0022] Figure 3. Preferred embodiment of the invention.

[0023] Detailed description

[0024] In a general aspect, the process of the invention comprises the steps of selective precipitation, concentration, and dialysis combined, which yield stable, highly pure formulations. Optionally, a selective purification step is also incorporated.

[0025] In this same aspect, the present invention incorporates at least two sequential concentration stages, two diafiltration stages, two separation stages, and at least one pasteurization stage. Additionally, at least two viral inactivation stages or mechanisms are included.

[0026] Particularly, in the general embodiment of the invention two diafiltrations precede the final concentration of the formulation.

[0027] The process of the invention can start from matrices rich in different macromolecules or proteins, and after the precipitation stage, a concentration stage is performed in all embodiments of the invention, followed by an initial diafiltration. In the same aspect of the invention, a final concentration stage is performed before the formulation stage, which may correspond to the second or fourth concentration stage.

[0028] In this same regard, the selective precipitation stage begins with the reception of protein- or macromolecule-rich matrices as raw material and the application of specific conditions, such as controlled temperature and pH variations, along with the precise addition of reagents. This step allows for the selective formation of aggregates and facilitates the efficient separation of impurities.

[0029] Specifically, in this precipitation stage, temperatures are controlled and maintained between room temperature and / or up to 60°C, then filtration and ultrafiltration are performed, followed by a formulation stage and another final filtration.

[0030] Optionally, for certain macromolecules, ultrafiltration is followed by chromatography, followed by successive steps of: concentration by ultrafiltration, solvent-detergent treatment, another chromatography, and another concentration with dialysis and ultrafiltration.

[0031] In this same regard, the final formulation is optionally pasteurized after filling, which occurs after final filtration. In one particular embodiment, up to two pasteurization stages are performed: after formulation and bulk filtration, and after filling.

[0032] Furthermore, the process of the invention comprises operations or mechanisms of viral inactivation and microbial control by temperature.

[0033] In the detailed aspect of each of the stages of the process of the invention, the conditions are described below:

[0034] Reception of macromolecule- and / or protein-rich matrices: In this stage, macromolecule-rich matrices are received from another initial treatment process. Each matrix may contain different components, and the separation of these components constitutes the essence of the macromolecule purification process of the invention. This initial step establishes the basis for the differentiated treatment of each matrix during the purification process.

[0035] Specifically, at the beginning of this stage, and regardless of whether or not different batches of the matrices to be purified are received, a homogenization process is carried out for 30 ± 5 minutes. Optionally, the temperature is raised and constant stirring is applied.

[0036] Impurity Removal - Precipitation at Room Temperature and up to 60°C The matrix undergoes a precipitation step at a specific temperature from room temperature to 60°C. During this process, a saturated fatty acid is used as a reagent to induce the precipitation of impurities present in the solution. Selective precipitation at temperatures within the established range facilitates the separation of the macromolecule or protein of interest from the impurities, thus improving product purity.

[0037] Specifically, this precipitation aims to remove a large portion of the contaminating proteins from the matrix, and this stage, in turn, constitutes a viral inactivation process.

[0038] In a first instance, injectable quality water is added to the matrix or matrix mixture (various batches) until the protein concentration is adjusted to around 4%. The precipitation of the diluted matrix is ​​carried out by adding the saturated fatty acid directly to the protein solution gradually with continuous and strong stirring, in such a way as to achieve a final concentration of approximately 6% v / v of acid, the solution is adjusted to an acidic pH of around 5.2 with 1 M HCl and the mixture is incubated with continuous stirring for up to 1 hour.

[0039] Alternatively, this precipitation step is carried out by gradually adding emulsifying and chelating agents to the protein solution with continuous, vigorous stirring, until a final concentration of approximately 0.3 to 0.6 mmol of these additives per gram of protein is achieved. Optionally, once the components are fully integrated, ethanol is slowly added with vigorous stirring until a final concentration of approximately 10% v / v is reached. The temperature of the mixture is increased from room temperature to 60°C and incubated for 60 minutes with continuous stirring.

[0040] In this same particular embodiment, finally, the mixture is cooled to around 25°C and the pH is adjusted to approximately 5.1 by the slow addition of 1 M HCl with strong stirring.

[0041] Filtration: After precipitation, filtration is performed to separate precipitated impurities and other unwanted solids. This step helps refine the composition of the solution by removing unwanted particles and improving product clarity.

[0042] Specifically, the precipitate formed is removed by adding a filtration aid (cellulose filter or similar) and subsequent clarifying filtration (filter plates or similar).

[0043] The amount of filter aid must be previously calculated, taking into account that this amount is approximately 40% of the solids present after precipitation and incubation. Specifically, the standard concentration of total solids is set at 15%, and this has defined a factor of approximately 0.06 for the production process.

[0044] In this same aspect, the number of filter plates will depend especially on the Filter Press to be used (dimensions, frame volumes), taking into account the recommended surface area of ​​approximately 1 m 2 every 34-36 L of initial raw material.

[0045] Before starting the filtration process, the filter must be conditioned with a buffer, and the solution to be filtered must be passed through slowly to keep the filter pressure low (ideally less than 1 bar, but not more than 2 bar) and to ensure that the precipitated proteins are retained in the filter press. The cake must then be dried by increasing the working pressure, and the precipitate must be washed with a buffer or water to recover any macromolecules or proteins of interest along with the precipitate. The volume of buffer used in this wash is approximately 1 volume of the cake formed in the filter press. A larger amount of buffer or washing at an excessive flow rate can cause the transfer of contaminating proteins or a greater amount of acid, which is generally evident as an increase in the turbidity of the resulting solution.

[0046] Optionally, certain macromolecules undergo an additional recovery process after filtration using a filter press. This mechanical device allows for the effective separation of residual solids and liquids, ensuring the production of a more purified macromolecule or protein of interest.

[0047] Ultrafiltration - Salt Removal. The purified solution undergoes an ultrafiltration step to remove salts and lower molecular weight molecules. This step helps concentrate the macromolecule or protein of interest and eliminate unwanted compounds, further improving product quality.

[0048] The objective of this step is to remove the residual acid and the largest amount of PEG and other molecules, such as polymers and other compounds, in addition to other salts that remain in the clarified after precipitation and filtration.

[0049] The clarified matrix is ​​first concentrated to approximately 3.5 to 10% total protein to optimize the subsequent diafiltration process. Membranes with nominal pore sizes of 10 kDa to a maximum of 30 kDa are used in this step.

[0050] Regarding the membrane surface required for this operation, 1 m has been established. 2 membranes of a maximum of 30 kDa per 35-40 L of raw material initially used. The final surface area will depend on the speed at which the process is required; it is recommended that it be carried out quickly to reduce the risks associated with contamination and the temperature at which the process is being carried out.

[0051] Formulation: The purified macromolecule or protein is formulated according to the specifications required for its final application, ensuring the preparation of a final product with the desired characteristics. The macromolecule or protein solution is adjusted to 10 to 20%, and glycine, a fatty acid, and / or an amino acid such as n-acetyl tryptophan are added to approximately 300 mmol / L.

[0052] Pasteurization: Optionally, the stabilized solution is filtered through a sterile filter and then pasteurized at approximately 60°C for up to 10 hours. Once the solution has cooled, it is sterile filtered for subsequent aseptic filling.

[0053] In one particular modality, up to two stages of pasteurization are carried out, after formulation and bulk filtration and then filling.

[0054] Continuous dialysis: The concentrated macromolecule or protein undergoes a continuous dialysis process to adjust the pH of the solution. This step is crucial to maintain optimal conditions that promote the stability and integrity of the macromolecule or protein of interest.

[0055] Successive diafiltration with sodium chloride and then water: An additional diafiltration is performed using sodium chloride and water to remove any remaining impurities or reagents. This step ensures that the purified macromolecule or protein meets the required purity standards.

[0056] Alternatively, diafiltration is performed first with sodium chloride and then with water.

[0057] Pasteurization: At this stage, pasteurization is considered optional for some macromolecules or proteins. This additional heat treatment can help ensure the elimination of unwanted microorganisms, ensuring the sterility and safety of the product.

[0058] Additional Filtration: After pasteurization, additional filtration is performed to remove any particulate matter or impurities that may have been generated during the thermal process. These detailed steps demonstrate a comprehensive approach to the purification of macromolecules or proteins of interest and other macromolecules, highlighting the importance of precision at each stage to achieve stable, high-purity formulations.

[0059] Filling: The final product, which is the purified and, in some cases, pasteurized macromolecule or protein, undergoes the filling process in containers suitable for storage and distribution.

[0060] Optionally, as indicated in the Pasteurization stage, another pasteurization can be carried out after this filling stage.

[0061] Optionally, for some macromolecules or proteins of interest, the following steps are included before the filling step:

[0062] Ion Exchange Chromatography. The objective of this step is to remove contaminating proteins.

[0063] The diafiltered macromolecule or protein solution is diluted to a protein concentration of approximately 2% with a buffer and then passed through an ion exchange resin, previously conditioned with a buffer. Any macromolecules or proteins that may have been trapped on the resin are then recovered. Finally, the retained proteins are eluted by passing through a more conductive buffer.

[0064] Ultrafiltration II: The post-chromatography solution is adjusted to a pH of around 5.3 and the proteins are concentrated to approximately 4% using an ultrafiltration system with membranes of up to 30 kDa with a similar capacity to that previously used.

[0065] Detergent Solvent Treatment: The macromolecule or protein solution is treated with a detergent solvent mixture for approximately 6 hours at a temperature of 20-25°C under continuous stirring.

[0066] The reaction is stopped by subsequent absorption of the reagents onto a hydrophobic resin, previously equilibrated with a buffer. The precipitate is then washed with buffer III to recover the retained macromolecule or protein, and this wash is recovered along with the previously obtained filtrate.

[0067] Ultrafiltration III The post-chromatography solution is concentrated up to 4% of macromolecules or proteins, with a 30 kDa ultrafiltration system but with a lower capacity than the one previously used (approximately 1 m is recommended). 2 of 30 kDa membranes per 90-100 L of raw material initially used). Once the sample is concentrated, it is dialyzed with 5 volumes of 0.3 M Glycine solution.

[0068] Examples

[0069] Example 1

[0070] Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described steps and the following data were obtained:

[0071] Macromolecule 1

[0072] STAGE Process parameter

[0073] MIXING OF Homogenization time 30 ± 5 minutes

[0074] MATRICES

[0075] DILUTION OF Amount of water to reach the protein concentration

[0076] PROTEINS AT 4.2 ± 0.2 desired g / dL Homogenization time after dilution 20 ± 5 min ACID ADJUSTMENT Amount of acid to add to adjust the concentration to 6%

[0077] (VIRAL INACTIVATION) in the batch

[0078] Acid addition flow rate 0.30 ± 0.05 L / min

[0079] Homogenization after addition of acid 20 ± 5 min

[0080] HCI addition flow rate 0.30 ± 0.05 L / min

[0081] Batch temperature: 21 ± 1° C

[0082] Contact time 1 hour

[0083] FILTRATION Required amount of diatomaceous earth (40%) with respect to solids

[0084] CLARIFIER (15%) Pressure inside the filter <2 Bar

[0085] Washing volume: 1 cake volume

[0086] CONCENTRATION OF Quantity of permeate to be extracted to concentrate the batch

[0087] PROTEINS A 3.5 ± 0.5 Inlet pressure < 2.5_bar g / dL

[0088] DIALYSIS WITH BUFFER 8 diavolumes

[0089] Inlet pressure < 2.5 bar

[0090] DILUTION OF Buffer I required amount to adjust batch concentration

[0091] PROTEINS A 2 ± 0.2 Homogenization time 20 minutes g / dL

[0092] FILTRATION N°1 Maximum pressure 2 bar

[0093] Homogenization time 20 minutes

[0094] CONDITIONING Working pressure <1.5 bar

[0095] Flow Column 120 cm / h

[0096] ION EXCHANGE Column balance 5 VC

[0097] Example 2

[0098] Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described steps and the following data were obtained:

[0099] Macromolecule 1 - Essay 2

[0100] Stage Process Parameter

[0101] CHROMATOGRAPHY Flow 9

[0102] ION EXCHANGE

[0103] Column working pressure < 1.5 bar

[0104] Homogenization time 20 minutes Protein elution 2-3 VC

[0105] pH ADJUSTMENT TO 5.3 ± HCI addition flow rate 0.30 ± 0.05 L / min 0.1

[0106] PROTEIN CONCENTRATION Amount of permeate to be extracted to concentrate the batch A 4.0 ±

[0107] Inlet pressure < 2.5 bar 0.5 g / dL

[0108] TREATMENT Necessary amount of Tween 80 and TnBP for the treatment SOLVENT /

[0109] Incubation time 6 hours DETERGENT (INACTIVATION Temperature 22.5 ± 2.5°C

[0110] VIRAL)

[0111] Pre-chromatography temperature 20 ± 1 °C

[0112] FILTRATION N°2 Maximum pressure 2 bar

[0113] CONDITIONING Column volume SDR COLUMN REMOVAL

[0114] Working pressure <1.5 bar S / D

[0115] Flow 10 cm / h

[0116] Column balance 5 VC

[0117] CHROMATOGRAPHY Maximum flow 10 cm / h FOR REMOVAL OF

[0118] Working pressure < 2 bar S / D

[0119] Homogenization time 20 ± 5 minutes

[0120] PROTEIN CONCENTRATION Amount of permeate to be extracted to concentrate the batch A 4.0 ± 0.5

[0121] Inlet pressure < 2.5 bar g / dL

[0122] DIALYSIS WITH GLYCINE 5 Diavolumes 0.3 mM AND

[0123] Amount of permeate to be extracted to concentrate the batch

[0124] CONCENTRATION AT 11.5 ± 0.5 g / dL Inlet pressure < 2.5 bar FINAL FORMULATION Amount of 0.3 M Glycine to adjust protein concentration to 10%

[0125] Homogenization time 30 minutes

[0126] FINAL FILTRATION Batch temperature 25 ± 1 °C

[0127] Maximum pressure 2 bar

[0128] Example 3

[0129] Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described stages and the following data were obtained:

[0130] Macromolecule 1

[0131] LIE LSE TEST STAGE

[0132] Post-IEC protein Total protein % 0.50 1.00 pH 6.80 7.20 IgG (mg / dL) Informative IgA (mg / dL) <33

[0133] Protein composition (%) Size distribution >90% monomers and dimers and <3% molecular polymers and aggregates

[0134] Microbial load Informative (CFU / mL)

[0135] X Factor <0.3

[0136] Eluted Total Protein % 0.09 1 .45 Chromatography IgG (mg / dL) Informative IgA (mg / dL) Informative

[0137] Protein pH 5.3 pH 5.20 5.40

[0138] Total Protein Concentrate % 3.50 4.50 proteins

[0139] Microbial Load Informative

[0140] (CFU / mL)

[0141] Density >1.0

[0142] Protein permeates % < 0.06 concentration Treatment S / D pH 5.20 5.40

[0143] pH balance 5.00 6.00 SDR column Post protein Total protein % 1 .50 2.50

[0144] SDR Trace Polysorbate 80 <50

[0145] (PPm)

[0146] Trace TnBP (ppm) pH 4.50 6.50

[0147] Example 4

[0148] Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described steps and the following data were obtained:

[0149] Macromolecule 1

[0150] LIE LSE TEST STAGE Microbial load - <100

[0151] (CFU / mL)

[0152] Size distribution >90% monomers and dimers and <3% molecular polymers and aggregates

[0153] Trace TnBP (ppm) - < 10

[0154] Traces of Polysorbate 80 - <50

[0155] (PPm)

[0156] Protein Permeates % - < 0.06 concentration Dialyzed with glycine Total Protein % 3.50 4.50 pH 4.50 5.00

[0157] Glycine (mmol / L) 270.00 330.00

[0158] Dialysis permeates Protein % - < 0.06 with glycine Total protein concentrate % 11.00 12.00 proteins pH 4.50 5.00

[0159] LAL - <1

[0160] Microbial load - <100

[0161] (CFU / mL)

[0162] PEG 1000 Traces - <50

[0163] (PPm)

[0164] Traces of Polysorbate 80 - <50

[0165] (PPm)

[0166] Trace TnBP (ppm) - <10

[0167] Protein Permeates % - < 0.06 Macromolecule or Total Protein % 9.00 11.00 protein of interest stabilized Glycine (mMolar) 270.00 330.00 LAL (EU / mL) - < 1 Total Protein Concentrate % 3.50 4.50 proteins Example 5

[0168] Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described stages and the following data were obtained:

[0169] Macromolecule 2

[0170] STAGE PROCESS PARAMETERS

[0171] THERMOCOAGULATION SETTINGS | 1200 MM i 100 MM EDTA DOSAGE AMOUNT í AMOUNT OF WATER TO ADJUST j PROTEIN CONCENTRATION TO 4

[0172] IG / DL

[0173] | ADDITION FLOW RATE 0.25 ± 0.05 L / MIN

[0174] HOMOGENIZATION TIME 25 ± 5

[0175] MINUTES

[0176] THERMOCOGULATION AMOUNT OF PURE ETHANOL FOR

[0177] ACHIEVE 10% CONCENTRATION

[0178] ETHANOL ADDITION FLOW RATE 0.20 ± 0.05

[0179] L / MIN

[0180] THERMOCOAGULATION CONDITIONS 60 ± 1°CX 60 MIN BATCH TEMPERATURE 25 ± 1°C FLOW RATE ADDITION OF 1 M HCL 0.20 ± 0.05

[0181] PH ADJUSTMENT 5.1 ± 0.1 L / MIN

[0182] PRECIPITATION TIME 60 ± 10 MIN

[0183] CLARIFYING FILTRATION REQUIRED AMOUNT OF FILTRATION AID HOMOGENIZATION TIME 30 ± 5

[0184] AMOUNT OF N-ACETYL TO BE DOSED

[0185] FINAL ADJUSTMENTS TRYPTOPHAN 1000 MM

[0186] ADDITION FLOW RATE 0.25 ± 0.05 L / MIN

[0187] HOMOGENIZATION TIME 20 TO 25

[0188] MINUTES

[0189] AMOUNT OF SODIUM REQUIRED FOR

[0190] THE ADJUSTMENT

[0191] HOMOGENIZATION TIME 25 ± 5

[0192] MINUTES

[0193] AMOUNT OF WATER REQUIRED FOR

[0194] PRE FILTRATION ADJUSTMENT

[0195] BULK PASTEURIZATION j PASTEURIZATION CONDITIONS

[0196] FINAL FILTRATION | BATCH TEMPERATURE 25 ± 1°C MAXIMUM PRESSURE 2 BAR

[0197] PASTEURIZATION PACKAGING | PASTEURIZATION CONDITIONS 60 ± 0.5°C X 10 HOURS

[0198] Example 6

[0199] Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described stages and the following data were obtained:

[0200] Macromolecule 2

[0201] LIE LSE TEST STAGE

[0202] PHASE MIX Total protein % 5.50 8.00

[0203] LOWER LAL (EU / mL) - <10

[0204] Bacterial load - <100 (CFU / mL)

[0205] DILUTED LOWER PHASE AND Total Protein % 3.50 4.50

[0206] TIGHT

[0207] THERMOCAGULATED Total protein % 3.00 4.50

[0208] THERMOCAGULATED PH pH 5.00 5.20

[0209] TIGHT

[0210] FJ. AFTER FILTRATION Total protein % 1.00 2.00

[0211] Albumin % 0.70 2.00

[0212] ALBUMIN CONCENTRATE Total protein % 22.00 24.00

[0213] LAL (EU / mL) - <1.7

[0214] Bacterial load - <10

[0215] (CFU / mL) pH 6.70 7.30

[0216] COMPOSITION >95%

[0217] PROTEIN

[0218] Example 7 Under controlled conditions, the process of the invention was tested in one of its preferred modalities with each of the described steps and the following data were obtained:

[0219] Macromolecule 2

[0220] Additive(s) (mmol / g protein) 0.070 0.095

[0221] N-Acetyltryptophan (mmol / g 0.070 0.095 protein)

[0222] Sodium (mmol / L) < 160 SODIUM ADJUSTMENT pH 6.70 7.30

[0223] Sodium (mmol / L) 120.00 160.00

[0224] PROTEIN Total protein % 19.00 21 .00 STABILIZED

Claims

Claims 1. A process for purifying macromolecules with combined selective precipitation, concentration and dialysis stages under constant stability conditions, comprising at least two sequential concentration stages, two diafiltration stages, two separation stages and at least one pasteurization stage.

2. Process for purifying macromolecules according to Claim 1, which additionally comprises including at least two stages or mechanisms of viral inactivation.

3. Process for purifying macromolecules according to Claim 1, wherein the two diafiltrations precede the final concentration of the formulation.

4. Process for purifying macromolecules according to Claim 1, wherein after the precipitation stage a concentration stage occurs in all embodiments of the invention, followed by an initial diafiltration.

5. Process for purifying macromolecules according to Claim 1, before the formulation stage a final concentration stage is carried out, which may correspond to the second or fourth concentration.

6. Process for purifying macromolecules according to Claim 1, in the selective precipitation stage it begins by receiving matrices rich in proteins or macromolecules as raw material and the temperatures are controlled and maintained between ambient temperature and / or up to 60°C, then filtration and ultrafiltration are carried out, followed by a formulation stage and another final filtration.

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