Liquid-liquid spontaneous and / or induced separation system to produce solutions enriched with active thermostable proteins

Abstract

The present invention relates to a liquid-liquid spontaneous and / or induced separation system to produce solutions enriched with active proteins. This solution addresses in an efficient and controlled manner the challenge of separating active proteins, which are essential in different biotechnological and food processing applications.

Description

[0001] Spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active thermostable proteins

[0002] Description

[0003] Technology Sector

[0004] The technological sector to which the patent application refers falls within the fields of biotechnology and process engineering. The invention focuses on the spontaneous and / or induced separation of liquid-liquid phases to obtain solutions enriched with active thermostable proteins.

[0005] In short, the technological sector addressed in the patent application is a combination of biotechnology and process engineering technologies. The described system focuses on liquid-liquid separation to obtain solutions enriched with active proteins through containment media and integrated controlled heat transfer mechanisms. This approach has applications in various industries, including pharmaceuticals, food, and biotechnology, where protein purification is essential.

[0006] Previous State of the Art

[0007] The context of the state of the art prior to the invention of the spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched with active thermostable proteins takes on special relevance when considering the challenge of industrial scaling. In this scenario, the need to develop solutions that are not only effective at the laboratory level but also scalable for mass industrial applications becomes crucial. The state of the art is redefined below, taking this challenge into account:

[0008] Industrial Scale-Up of Liquid-Liquid Phase Separation: Traditional liquid-liquid separation methods have proven effective in laboratory settings, but the transition to industrial scale presents unique challenges. Optimizing separation conditions to ensure efficiency and cost-effectiveness on a large scale has been a focus of research.

[0009] Scalable Processing of Thermostable Proteins: Obtaining thermostable proteins in industrial quantities is essential to meet market demand. Methods that work efficiently on a small scale need to be adapted to ensure consistency and economic viability on a larger scale.

[0010] Large-Scale Controlled Heat Transfer: Industrial applications require heat transfer systems that are not only precise but also capable of handling substantial volumes efficiently. Adapting these systems to meet mass production requirements is critical.

[0011] Designing Containment and Continuous Systems at the Industrial Level: Implementing containment and continuous systems in industrial environments involves logistical and engineering challenges. The ability to design robust and efficient systems that can be effectively scaled is crucial.

[0012] Large-scale applications in the food and pharmaceutical industries: The industrial relevance of obtaining solutions enriched with thermostable proteins is amplified, and the need for scalable solutions becomes evident in sectors such as large-scale food and pharmaceutical production.

[0013] The proposed invention not only addresses laboratory-level efficacy but is also strategically positioned to overcome the obstacles associated with industrial scaling. The integration of containment or continuous media and controlled heat transfer mechanisms, as well as the optimization of process conditions, presents an innovative and practical solution for achieving successful application of this system in large-scale production environments.

[0014] Description of the Invention

[0015] The invention described in the patent application relates to a spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched with active proteins. This solution efficiently and controllably addresses the challenge of separating active proteins, which are essential in various biotechnological and biological product processing applications.

[0016] The invention consists of several key components and features:

[0017] Containment or Continuous Media: The invention includes containment media or continuous mechanisms specifically designed to facilitate liquid-liquid phase separation. These media can be made of materials that facilitate the specific interaction of proteins with the different liquid phases and ensure efficient separation. Furthermore, they can be structures or devices that allow the controlled flow of proteins in the phases in question.

[0018] Integrated Heat Transfer Mechanisms: The system of the invention incorporates mechanisms responsible for controlling heat transfer in the process. These mechanisms allow for the generation of controlled temperature gradients throughout the system, which is essential for the separation of active proteins.

[0019] Spontaneous and / or Induced Separation: The system of the invention comprises two separation processes: one spontaneous and one induced separation. Spontaneous separation occurs naturally due to differences in the physical and chemical properties of the liquid phases in simple containment media, while induced separation can be activated and controlled by the aforementioned thermal transfer mechanisms in containment media and / or successive continuous media. Obtaining Solutions Enriched in Active Proteins: The fundamental objective of this invention is to obtain solutions that are enriched in active proteins. This implies the effective purification of these proteins, maintaining their stability and biological activity even under particular conditions of temperature, presence of polymers, salt concentration, among others.

[0020] The proposed invention is highly versatile and can have applications in a variety of fields, including the production of pharmaceuticals, functional foods, and industrial biotechnology. By providing an effective solution for the purification of active proteins, this system significantly contributes to improving production processes and product quality in these industries.

[0021] In summary, the Spontaneous and / or Induced Liquid-Liquid Separation System for obtaining solutions enriched in active proteins is an invention that combines liquid-liquid separation, controlled thermal transfer and the obtaining of active thermostable proteins, offering significant benefits in terms of process efficiency and quality in multiple technological and biotechnological applications.

[0022] Description of the Figures

[0023] Figure 1. General embodiment of the system of the invention.

[0024] Figure 2. General process diagram of the invention.

[0025] Figure 3. Stability of the matrices obtained with the system of the invention.

[0026] Detailed description The present invention relates to a system of spontaneous and / or induced liquid-liquid separation for obtaining solutions enriched in active proteins.

[0027] In a general aspect, the system of the present invention comprises simple containment means in which spontaneous separation occurs, containment means and / or continuous media successive to the simple containment means and in which induced separation occurs, and integrated heat transfer mechanisms embedded in the containment means and / or the continuous media.

[0028] In this same aspect of the invention, the containment means correspond to a spontaneous separation means, comprising a conical or curved bottom, phase separation verification devices and phase change detectors.

[0029] In particular, these containment means also comprise a control unit, one or more input dosing mechanisms, a screening module with rotor, a cleaning module and a connection module.

[0030] In another aspect of the invention, the continuous means correspond to forced separation means, which comprise residence time regulation devices and phase separation verification devices.

[0031] In particular, these continuous means further comprise a control unit, one or more input dosing mechanisms, a screening module with rotor, a cleaning module and a connection module.

[0032] In another aspect of the invention, the integrated thermal transfer mechanisms are embedded, integrated and / or connected in the simple containment means and / or in the successive containment or continuous means and generate controlled temperature gradients.

[0033] In a particular aspect of the invention, the simple containment means correspond to a conical reservoir, and the continuous means are a liquid-liquid separator. In this same aspect of the invention, the simple containment means contain a protein solution to which salts and polymers are added sequentially at specific rates for a determined time at room temperature and until homogenization; they are then transferred to a containment means with temperature control in the range of 20 to 24 °C for spontaneous separation in up to 15 hours without homogenization.

[0034] In particular, the homogenization time ranges from 60 to 75 minutes.

[0035] In a particular aspect of the invention, during the spontaneous separation, an aqueous solution of phenol at approximately 3.0% v / v is prepared and slowly added to the simple containment media with strong stirring to reach a final phenol concentration of approximately 0.25% v / v and the mixture is subsequently homogenized for at least 1 h.

[0036] Specifically, all the required salts are added in a solid state in a sequential manner, always under vigorous stirring to ensure rapid dissolution. The final concentration of the salts to be achieved is: 12.5% ​​sodium chloride, 11.0% potassium phosphate, and 17.0% polymers (m / v). The addition of the salts must begin when the solution is already at a temperature of 20°C to ensure their proper dissolution. Each of the salts can be added immediately after the previous one, preferably waiting for the complete dissolution of the previous one. The salts can be added one after the other at a moderate speed to ensure their incorporation into the solution. Preferably, the proper dissolution of all the salts is ensured before adding the polymer.

[0037] More particularly, this stage also includes the addition of potassium phosphate, which is added as a mixture of salts: one monobasic (KH2PO4), and another dibasic (K2HPO4), in a proportion of 0.8%: 10.2%, such that a pH of 7.2-7.8 is provided at the end. Regarding the polymer, which is soluble in water and which preferably can be PEG, it is incorporated at a temperature above 40 ° C, very slowly in order to avoid denaturation of the proteins present in the containment media.

[0038] In this same preferred embodiment, the polymer addition temperature is between 56 and 60°C, while the system temperature does not exceed 30-34°C. The system is then homogenized for one hour to ensure the subsequent formation of two aqueous phases.

[0039] The spontaneous separation process occurs in a minimum of 6 hours, depending on the total volume, containment media, and system temperature. Temperature regulation between 20 and 24°C provides much more effective visualization of phase separation.

[0040] After homogenization, the mixture is transferred to a continuous forced separation system with temperature control in the range of 20 to 24 °C for up to 5 hours.

[0041] In a particular aspect of the invention, in forced separation the initial and final temperature is maintained between 20-24°C and the rotation times are determined batch by batch.

[0042] In one aspect of the invention, successive steps of spontaneous and forced separation provide a stable source for a two-phase system and the reduction of contaminating proteins, while obtaining matrices rich in active proteins.

[0043] In another aspect of the invention, the resulting matrices remain stable for a period of 72 hours, with a maximum of 120 hours, provided they are stored at room temperature (18-25°C) and with continuous stirring. During this period, both the appearance and the main physicochemical parameters such as protein concentration, pH, conductivity, and specific protein levels remain stable. Examples

[0044] Example 1

[0045] At room temperature and under controlled conditions, the system of the invention was evaluated and the following data were obtained:

[0046] Stage Process Parameter

[0047] Addition of 3% phenol solution required to adjust the phenol concentration in the batch to 0.25% phenol

[0048] Phenol addition flow rate 0.25 ± 0.15 L / min

[0049] Contact time 60-75 min

[0050] Formation two phases Batch temperature 19 ± 1 °C

[0051] Amount of NaCl to be added to achieve a concentration of 12.5% ​​in the batch

[0052] Homogenization time 20 - 25 minutes

[0053] Amount of K2HPO4 to be added to achieve a concentration of 10.2% in the batch

[0054] Homogenization time 50-60 minutes

[0055] Amount of KH2PO4 to be added to achieve a concentration of 0.8% in the batch

[0056] Mixing time 30 - 40 minutes

[0057] Amount of polymer to be added to achieve a concentration of 17% in the batch

[0058] Batch temperature for PEG addition 25 ± 2 °C Polymer temperature 58 ± 2 °C

[0059] Batch temperature for homogenization 32 ± 2 °C

[0060] Homogenization time 60 - 75 minutes

[0061] Resting time for phase separation > 6 hours

[0062] Batch temperature for phase separation 22 ± 2 °C

[0063] Fractionation tank pressure 0.2 bar

[0064] Induced separation test

[0065] At room temperature and under controlled conditions, induced separation tests were carried out and the following data were obtained:

[0066] Test temperature: 22°C Interface No. 2.5 2 1.3 1

[0067] Sep

[0068] Solids 0.5 0.5 0.5 0.5 0.5

[0069] Spontaneous separation repetition tests

[0070] At room temperature and under controlled conditions, spontaneous separation tests were carried out and the following data were obtained:

[0071] Two-phase system Vr 0.60 1.00 pH 7.20 7.80

[0072] Matrix 1 Total protein % 5.50 8.00

[0073] Albumin % >4.0 igG <0.4

[0074] Protein composition > 60%

[0075] Microbial load Informative (CFU / mL) LAL (EU / mL) <10

[0076] Conductivity 90-130 mS / cm

[0077] Matrix 2 Total Protein % 4.00 6.00

[0078] Albumin %

[0079] Protein composition > 40%

[0080] Microbial Load Informative (CFU / mL)

[0081] LAL (EU / mL) <10 igG

[0082] Conductivity 50-90 mS / cm

[0083] Factor XI Informative

[0084] Example 2

[0085] Determination of salts, temperature and homogenization time

[0086] At room temperature and under controlled conditions, a constant for the aggregation of salts and polymers in the containment media was determined.

[0087] In all cases (NaCl, K2HPO4, KH2PO4) and polymer, a constant associated with the volume of one of the matrices was determined. The value of this constant ranges from 0.008 to 0.17.

[0088] The following process parameters were determined:

[0089] Process Parameters to be Recorded Weight of NaCl Calculation

[0090] Weight of K2HPO4 calculation

[0091] Weight of KH2PO4 | calculation

[0092] PEG 1000 Weight | Calculation

[0093] Homogenization temperature i 30-34°C

[0094] Homogenization time i 60-75 min

[0095] Rehearsal

[0096] Example 3

[0097] Stability of the obtained matrices

[0098] In several tests at room temperature and under controlled conditions, it has been proven that the matrices obtained can remain stable for a period of 72 hours, with a maximum of 120 hours, provided they are kept under continuous agitation.

[0099] During this period, both the appearance and the main physicochemical parameters such as protein concentration, pH, conductivity, and specific protein levels remain stable, and no changes in electrophoretic patterns that could indicate protein denaturation or degradation are observed. Continuous homogenization has been observed to be extremely important to prevent salt precipitation, changes in pH, and possible destabilization of proteins in each of the matrices (Figure 3).

Claims

Claims 1. Spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins, characterized in that it comprises simple containment means, containment means and / or successive continuous means and integrated heat transfer mechanisms.

2. The spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins according to claim 1 , wherein the simple containment means correspond to a spontaneous separation means, comprising a conical or curved bottom, phase separation verification devices and phase change detectors. The spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins according to claim 1 , wherein the containment means are successive continuous.

3. The spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins according to claim 1, wherein the successive continuous forced separation means comprise a temperature control in the range of 20 to 24 °C in up to 5 hours.

4. The spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins according to the claim, wherein in the forced separation the initial and final temperature is maintained between 20-24°C and the rotation times are determined batch by batch.

5. The spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins according to claim 1, wherein the integrated thermal transfer mechanisms are embedded in the simple containment media and / or in the media containment or successive continuous and generate controlled temperature gradients.

6. The spontaneous and / or induced liquid-liquid separation system for obtaining solutions enriched in active proteins according to claim 1, wherein the matrices obtained can remain stable for a maximum period of 120 hours.

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