WO2018062984A2 - New method for synthesising dendron-based chromatography resins - Google Patents

Abstract

The present invention relates to an improved method for the synthesis of dendron-based chromatography resins. The method comprises incorporating dendrons, molecules having a branched structure, into the chromatography resin. In a second step, the functional groups of the dendron are functionalised with ligands. The method can be applied to a wide variety of materials of different natures for the synthesis of chromatography resins for a wide variety of chromatography processes such as hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEXC), reversed-phase chromatography (RPC) and affinity chromatography (AC), inter alia.

Description

 NEW METHOD OF SYNTHESIS OF CHROMATOGRAPHIC RESINS A

 DENDRON BASE

FIELD OF THE INVENTION

The present invention discloses a new method for the synthesis of dendron-based chromatographic resins. The present scalded resin technology is primarily focused on the purification and recovery of high molecular weight molecules such as proteins; However, the present invention can also be used in the recovery of low molecular weight chemical compounds.

BACKGROUND OF THE INVENTION

 In the biotechnology industry, one of the greatest needs is the recovery and purification of proteins that often come from very complex sources. From the whole biotechnological process, from the recovery to the obtaining of the product, the part related to the purification of proteins can reach up to 80% of the total cost of the final product. Because of this, there is a great need to implement innovative and efficient strategies for protein purification that reduce production costs.

At present, chromatography is one of the most used methods for protein purification. This is due to the fact that these methods allow obtaining proteins with high degrees of recovery and purity as well as being scalable methods, which allows production to be taken to an industrial level. Among the main chromatographic methods used for protein separation are: interaction chromatography hydrophobic (HIC), ion exchange chromatography (IEXC), reverse phase chromatography (RPC) and affinity chromatography (AC).

 HIC is a technique widely used in the purification of monoclonal antibodies, it is based on the interaction of hydrophobic surfaces of proteins and cough ligands on the surface of the chromatographic resin. It is particularly used in the final phase of the purification process called polishing or "poHshing". In the market there are numerous resins based on hydrophobic interaction modified with ligands of different hydrophobic strength, for example, butyl, octyl and phenyl, among others.

 In the prior art (Glad G, Matoisel JL, Thevenin N. Activated solid support and method. 2012), the activation of chromatographic resins is carried out by incorporating epoxy groups in the matrix, which in turn are made react with the desired ligand. This method is commonly used for the synthesis of a wide variety of resins based on different chromatographic techniques such as IEXC, HIC, RPC and AC. However, the distribution of the ligands in the chromatographic resin is limited and their density on the surface of the resin is low.

 On the other hand, document DE 196 21 741 A1 discloses a stationary phase for chromatography that prevents loss of modifier by washing. Said resin is synthesized by the chemical binding of a dendron to a substrate; however, said method comprises the gradual construction of the dendron on the substrate.

Therefore, the prior art methods used for the synthesis of this type of chromatographic resins are long and tedious. Therefore, there is an unmet need to provide an improved method to synthesize dendron-based resins. BRIEF DESCRIPTION OF THE INVENTION

 The present invention discloses a new method for the synthesis of dendron-based chromatographic resins.

 Said method is characterized in that it comprises two steps: coupling of the dendron in the chromatographic resin and immobilization of the ligand in the dendron previously attached to the chromatographic resin.

 The present invention is applicable in different chromatographic techniques such as IEXC, HIC, RPC and AC. Both for the purification of high molecular weight molecules and proteins, as well as for the purification of low molecular weight molecules.

BRIEF DESCRIPTION OF THE FIGURES

The brief description of the figures is shown below:

 Figure 1 represents the chemical structures of the reagents used for the synthesis of chromatographic resins.

 Figure 2 is a schematic representation of the chemical synthesis of chromatographic resins with dendrons.

 Figure 3 shows a comparison between the UV / Vis spectra of various chromatographic resins. The numbers 1 and 2 correspond to benzyl and carbonite groups, respectively, as shown in the structure at the top of the figure.

Figure 4 shows a comparison of an FTIR analysis of chromatographic resins. The black line represents the unmodified resin; the dark gray line represents RG3; and the light gray line represents RG5. The numbers shown in the figure are 1: benzyl, 2: glycosidic bond in agarose, 3: ester bond, 4: carbonyl inner group, and 5: carbonite outer group.

 Figure 5 shows a chromatogram of the separation of lipase, B-lactoglobulin and a-chymotrypsin, using a dendronized resin synthesized with the method disclosed in the present invention.

 Figure 6 shows a chromatogram of the separation of a Sacchammyces cerevisiae proteome, using a dendronized resin synthesized with the method disclosed in the present invention. The stepped line represents the salt gradient and the concentrations of ammonium sulfate (M) are indicated in each step.

 Figure 7 shows an SDS-PAGE electrophoresis analysis of the fractions obtained. FT: waste, l-IV: fractions, MM: marker, C: crude extract.

 Figure 8 shows the adsorption capacities of RG3 and RG5 in static mode. The squares correspond to unmodified resin in 2M ammonium sulfate. The circuits and diamonds are 1.5M and 2M concentrations of ammonium sulfate, respectively. The solid line represents the Langmuir model obtained from the adjustment of the experimental data.

Figure 9 shows bovine serum albumin rupture (BSA) curves in dendron-based chromatographic resins. The two concentrations used of ammonium sulfate were 1.5 M and 2 M, which are represented by dotted lines and solid lines, respectively. The different lines represent the concentrations of BSA used: 1 mg / mL, 2 mg / mL and 3 mg / mL. Each column of graphs corresponds to each of the flow rates used (1 and 2 mL / min). DETAILED DESCRIPTION OF THE INVENTION

The methods used in the prior art for the synthesis of dendron-based chromatographic resins are long and tedious. Therefore, there is a need to provide an improved method to synthesize this type of chromatographic resins.

 In response to the shortcomings of the prior art, the present invention discloses a new method for the synthesis of dendron-based chromatographic resins.

 The branched nature of dendrons provides the advantage of incorporating a greater amount of ligands which generates groups or "dusters" of ligands in the periphery of free molecules to interact with proteins. This allows to have a greater number of interaction sites and a greater interaction between protein and resin.

In the context of the present invention, the term "dendron", also known as dendimer or dentritic molecule, refers to a polymer with branched chemical structure, consisting of radially associated monomers from a core (also called center or "core ¹ ) The final ends of the branches, that is, on the periphery, can be activated with any functional group.

 In this sense, in the context of the present invention, the term "dendronized resin" refers to the chromatographic resin modified with the dendron.

On the other hand, in the context of the present invention, the term chromatographic resin refers to the support, adsorbent, material or chromatographic matrix. In this invention, dendrons are incorporated by covalent lens attachment with a chromatographic resin. In this sense, any branched molecule that is classified as dendron, formed of any chemical (polyester, polyamide, polysilane, polyimine, etc.) and with any degree of branching or generation, can be coupled to any chromatographic resin as long as the groups functional resin and dendron can react.

 In one embodiment of the present invention, dendrons are selected from the group consisting of polyesters, polyamides, polysilanes, polyimines, pellets, polyphenylacetylenes, or any combination thereof.

 In the context of the present invention, the term polyamide refers to both polyamides and polyamide amines (PAMAM).

 In a preferred embodiment of the present invention, dendrons are polymers of 2,2-Bis (hydroxymethyl) propionic acid (bis-MPA).

 In one embodiment of the present invention, the coupling of the dendron in the chromatographic resin is carried out by the chemical reaction between an N-hydroxysuccinimide (NHS) functional group present in the resin, and an amino functional group as the dendron core, forming In this way an amido bond.

 In another embodiment, the coupling of the dendron in the chromatographic resin is carried out the chemical reaction between a hydroxyl group present in the resin, and a carboxylic group as the core of the dendron, thus forming an ester bond.

Once the chromatographic resin has been chemically modified with dendrons, the next step is the incorporation of a ligand to said resin dendron hoisted. The incorporation of this ligand is carried out by a reaction between it and the functional groups present in the dendrons. Thus, the present invention can be used to synthesize chromatographic resins for hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEXC), reverse phase chromatography (RPC) and / or affinity chromatography (AC), of according to the chemical characteristics of the ligand used to functionalize the dendronized resin. So the ligand can be a molecule or compound of any chemical nature.

 In this context, in the present invention, the chromatographic resin may comprise hydrophobic interaction resin, ion exchange resin, reverse phase resin, affinity resin, or any combination thereof.

 The reaction of the incorporation of the ligand to the resin can be accelerated by means of a cabodiimide catalyst, for example: 1-eti-3- (3- dimethylaminopropyl) carbodiirnide (EDC), 4- (dimetJlamino) pyridinium-4-toluene- suffonate (DPTS), 4-dimethylamino-pyridine (DAMP), Ν, Ν'- didohexylcarbodiimide (DCC). Ν, Ν'-Dilsopropylcarbodiimlde (DIC) or 1.1-carbonyldiimidazole (CDI)

 In this regard, the present invention is directed to the purification and recovery of high molecular weight molecules such as proteins; However, it can also be used in the recovery of low molecular weight chemical compounds.

In one embodiment of the present invention, the ligand is any hydrophobic chemical molecule or compound that comprises a carboxylic group. The incorporation of this ligand is carried out through a esterification reaction between the carboxylic acid of the ligand and the hydroxyl groups (-OH) present in the dendrons.

 In the present invention, it is possible to use as a ligand any carboxylic acid with a hydrophobic chain containing a number of carbons n + 1, where n is a number between 1 and 1Θ. Said carboxylic acid is both an alHatic (open chain) and cyclic molecule.

 In a preferred embodiment of the present invention, the ligand is valeric acid.

 On the other hand, the following invention can be carried out both on a laboratory level in test tubes, and directly on the chromatographic column containing the resin to be modified. This can be achieved by recirculating a solution containing the dendron or ligand through the column containing the chromatographic resin.

 In a preferred embodiment of the invention, a resin with formula RA, where R is the commercial NHS-activated Sepharose 4 Fast FlowQ resin (NHS-activated Sepharose 4FF), and A is the NHS group capable of reacting with amino groups (-NH2 ), is chemically modified with bis-MPA dendrons of third (G3) and fifth (G5) generation. These dendrons contain an NH2 group (part that binds to the matrix) at its center and OH groups (parts where the ligands chemically bind) at their endpoints. The representation of the chemical structures of the reagents used for the synthesis of the dendron-based chromatographic resin can be seen in Figure 1.

In a first step, the dendrons (G3 or G5) covalently bind to the matrix through the formation of an amide bond between the NH2 group of the dendron and the carboxyl group that remains active after the leaving NHS group is released from the matrix in alkaline conditions. In a second step, the ligand is chemically bound by an esterification reaction between the carboxyl group of the ligand and the free hydroxyl groups present in the dendrons. In this embodiment, valoneo acid is used as a ligand, which contains five carbon atoms, four of which remain free after the reaction, acting as a butyl radical, hydrophobic ligands.

 To facilitate a better understanding of the present invention, the following examples are provided. In no way do the examples limit or define the scope of the invention.

Example 1

 Hydrophobic character commands.

 In the present invention, it is possible to use any carboxylic acid as a ligand.

 For example, phenylacetic acid can be used as a Henyl-type ligand, since the carboxylic group can be attached to the hydroxyl groups in the dendron, leaving the benconic ring free to act as a ligand.

Other compounds of hydrophobic nature) that can be used as ligands are polypropylene glycol (PPG) and poly-ethylene glycol (PEG) of any chemical. As long as they are activated with a carboxyl group, which allows the ligand to be attached to the dendron hydroxyl groups by means of an ester bond. Some examples of PEG hydrophobic ligand (of any molecular weight) include: methoxy-PEG-acetic acid, methoxy-PEG-propionic acid, methoxy-PEG-butanoic acylo and methoxy-PEG-hexanoic acid. Other types of molecules such as Y-shaped PEG activated with a carboxylic group can also be used as hydrophobic ligands. Other examples of carboxylic acids that can be used as hkJrophobic ligands are shown in Table 1.

 Table 1. hkJrophobic ligands.

Item 2

 Synthesis of a dendron-based chromatographic resin.

A resin with formula RA, where R is the commercial NHS-activated Sepharose 4 Fast Fiow® resin (NHS-activated Sepharose 4FF), and A is the NHS group capable of reacting with amino groups (-NKb), was chemically modified with bis-MPA dendrons of third (G3) and fifth (G5) generation. A diagram of the route followed for the chemical synthesis of the dendron-based chromatographic resin is presented in Figure 2.

 The dendron-based chromatographic resins, hereinafter, are referred to as RG3 (resins with third generation bis-MPA dendrons) and RG5 (resins with fifth generation bis-MPA dendrons). The method to synthesize them is described below:

 Stage 1. Resin washing. This step is carried out by washing the resin (13-26 pmoL NHS / mL drained resin) with cold 1 mM HC1.

 Stage 2. Reaction of dendron coupling to the resin. At this stage, a dendron solution (1.2-2.6 equivalents with respect to the amount of NHS groups in the resin / 0.5 g resin) in 0.5 mL of reaction buffer (100 mM carbonate buffer at pH 8.3 / NaCI) was prepared 0.5M). In the case of the third generation dendron G3, dimethylformamide (DMF) (1% of the total volume of the reaction buffer) was added to solubilize it. Then, the dendron solution (0.5-1.0 mL) was mixed with the resin (0.5 g) in 2 mL polypropylene tubes. The reaction was kept under constant stirring for 12 hours. After the reaction, the tubes were centrifuged at 10,000 rpm for 10 minutes and the supernatant was removed. In the case of the resin modified with the G3 dendron, the resulting resin contained ~ 10 pmoL dendron / 0.5 g wet resin and ~ 80 equivalents of groups -OH pmoL dendron G3. The resin modified with the G5 dendron contained 5 pmoL dendron / 0.5 g wet resin and -180 equivalent of -OH groups.

Stage 3. Immobilization of the ligand. At this stage the ligand in the resin was immobilized, specifically the ligand binds to the hydroxyl groups free of dendrons. For this, the dendronized resin was subjected to a dehydration process with solutions of acetone: H2O (1: 2, 1: 1 and 3: 1) and pure acetone. Each of these solutions, starting with the one with the lowest concentration of acetone, was added to the resin and stirred for a few seconds. Subsequently, the resin was filtered and the following solution was added until it reached pure acetone. After this, the resin was washed with DMF and the excess solvent was removed by vacuum filtration. Subsequently, 2-5 equivalents of the ligand (valoric acid), with respect to the amount of -OH groups in the dendron, were dissolved in 1 ml_ of a DMF / bufrer solution of 5% sodium carbonate and pH 8.3. Next, 10% and 40% excess of the 1-etik3- (3 <lirr ^ laininopropyl) carbodiimide (EDC) and 4 ^ dimethylamino) pin \ iinium-4-toluene-eurronate (DPTS) catalysts were added, respectively , with respect to the amount of valeric acid. The reaction mixture was kept under constant stirring at room temperature for 12 hours.

 Stage 4. Resin wash. Finally, the resin was washed with pure DMF and then with pure acetone. Subsequently, the resin was rehydrated by washing with acetone solutions: H20 (2: 1, 1: 1 and 1: 2) and finally a water wash was performed.

 Each of the following examples refers to the use of the modified resin with the third generation dendron (G3) in the separation of proteins from both model samples, that is, pure proteins analyzed separately or as an artificial mixture thereof, as well as the separation of a complex non-artificial sample.

Example 3

Chemical characterization of dendron-based chromatographic resins The dendron-based chromatographic resins were characterized by UV-Vis spectrophotometry and FTIR analysis. The absorption spectra were recorded with a Genesys 10S UV-Vis spectrophotometer. For this, 100 mg (wet base) of resin were suspended in 1 mL of distilled water and transferred to a quartz cell 1 cm long. The samples were read in a wavelength range of 190-700 nm. The infrared analysis was carried out on an FT-IR / FT-NIR spectrophotometer.

 The UV-Vis spectrum of dendronized resins and unmodified resins are shown in Figure 3.

 The two modified resins showed absorption bands around 208 nm, which is consistent with the absorption spectrum of the carbonyl groups of the valeric acid; as well as with the band of the pure G5 in the same wavelength. This indicates the presence of the esterified valeric acid on the resin as well as the presence of abundant carbonyl ester groups in the dendrons.

 On the other hand, infrared absorption spectra are shown in Figure 4.

The presence of dendrons coupled in the resin is revealed by the appearance of a peak around 800-840 cnv ¹ , which is lower for RG3, and corresponds to the phenyl ring; This signal is not present in the unmodified resins confirming in this way that by means of the present method, the dendron was coupled to the resin. Serials at 1218 and 1280 cm ^{* 1} displayed by RG3 and RG5, respectively, not present in the unmodified resin, correspond to a typical stretching of ester groups present in dendronized resins. Peaks at around 1635 cm ^{* 1} indicate the presence of a corresponding internal carbonyl group with ester groups in both structures of dendrons, while the serials at 1704 and 1730 cnr ¹ correspond to the peripheral carbonyl groups of the valeric acid immobilized to RG3 and RG5, respectively. A summary of the analysis is shown in Table 2.

external in dendrons

Element 4

 Analytical estimates of the concentration of dendrons and ligand in dendron-based resins

The G3 and G5 dendrons were quantified by UV spectrophotometer. For this, the UV-visible spectrum of both dendrons and the -NHS group, which leaves the resin during the reaction, were first recorded in order to determine the wavelengths at which maximum absorption occurred. The dendron concentration was calculated by solving a simple linear equation taking into account the molar extinction coefficients of the dendrons and NHS as well as the absorbency measures recorded. The Absorbance measurements of the 100 pL samples were recorded in a microplate reader (Epoch Microplate Reader, Biotek, VY, USA).

 On the other hand, the estimation of the amount of valeric acid immobilized in the resin was carried out by HPLC (They vary), equipped with a Luna 5 \ C18 column of 100 A and 150x4.6 mm (Phenomenex, CA, USA) . The mobile phase used was 60% methanol: 40% H2O at a pH of 2.3.

 Table 3 presents the density of dendrons and ligand in the resin that was achieved with the present invention.

Table 3. Dendron and ligand density on the surface of synthesized resins.

 "Repettoflidad cough data from tote to tote.

The natural structure of these dendrons allowed to increase the amount of ligand on the surface of the resin. And in this sense, ligand density depends directly on the number of branches. In this way, it was possible to incorporate a larger amount of the G3 dendron (10 pmoL / mL resin) into the resin compared to G5, due to the spherical effects of the latter (G5 is approximately 5 times larger than G3). It is important to mention despite the amounts of dendron coupled to the resin, the present invention allowed the incorporation of a greater amount of ligands compared to commercial HIC resins produced by the methods disclosed in the prior art, whose ligand densities vary between 9 and 53 MmoL ligand / mL resin, depending on the type of ligand (butyl or phenyl). Accordingly, the present invention can resolve prior art deficiencies related to low ligand density at the time of developing new chromatographic resins.

 In this sense, since the density of ligand is related to the adsorption capacity of the resin, the higher the density of the ligand, the greater the adsorption capacity. Therefore, with the present invention it is possible to use smaller amounts of resin to adsorb larger amounts of protein or any other molecule of interest. This would also allow the use of smaller chromatographic columns, accelerating the entire chromatographic process.

Hemplo S

Separation of model proteins with the dendron-based chromatographic resin

 The following solutions were used to carry out this analysis: Buffer A: This solution was used for the conditioning of the column and for the preparation of the samples. It has a pH of 7 and is composed of 20 mM tris-HCI and 1.75 M ammonium sulfate.

 Buffer B: [This solution was used for elution of resin samples. Specifically, it was used as a diluent to reach different concentrations of ammonium sulfate during the sample elution process. The solution has a pH of 7 and is composed of 20 mM tris-HCI.

 Sample: It can be any reconstituted protein sample in buffer A.

In this study three model proteins were used: lipase (Sigma Aldrich, CaL No. L3126), β-lactoglobulin (Sígma Aldrích, Cat. No. L3908) and a-chymotrypsin (Sigma Aldrích, Cat. No. C4129)

 The separation of the lipase, β-lactoglobulin and a-chymotrypsin model proteins with the hydrophobic resin modified with the third generation dendron, RG3 was carried out as described below:

 Stage 1. Resin packaging and conditioning. For this example, a 1 mL capacity column (TricornTM 5/50, GE Healthcare) was used. The column was placed vertically on a universal support and with the help of an automatic 1 mL pipette the resin (G3 in this case) was slowly poured until the column was filled to the 1 mL mark. The column was connected to the equipment, an Akta puré® chromatography equipment (GE Healthcare, Uppsala, Sweden) was used, and a low flow rate of 0.5 mL / min was applied, which was increased to a maximum of 4 mL / min in order to compress the resin inside the column. Finally, the column was conditioned with buffer A (20 mM Tris-HCI at pH 7 and 1.75 M ammonium sulfate); This operation was performed at a flow of 1 mL / min.

 Stage 2. Preparation of the sample. Samples (Mizados) of pure protein were reconstituted: lipase (4 mg), β-lactoglobulin (3 mg) and a-chymotrypsin (3.5 mg) in 1 mL of buffer A: 20 mM Tris-HCI at pH7 / ammonium sulfate 1.75 M.

Stage 3. Sample injection. The sample, previously reconstituted in buffer A, was filtered through a syringe filter with a pore size of 0.2 μιη. Subsequently, 0.2 mL of the filtered sample was injected into the chromatography system equipped with a 0.2 mL injector and the separation process started. Stage 3. Elution and sample separation. Once the sample was injected, the sample that was adsorbed on the resin was eluted. Elution of the proteins that were adsorbed on the resin was carried out by decreasing the salt concentration. For this, a 5-step gradient was applied at a flow rate of 1.5 mL / min. The salt concentration (ammonium sulfate), at the beginning, that is, in the first step, was 1.75 M and decreased to 1.4, 1.05, 0.7 and 0.35 M, for the last 4 steps, respectively, until reaching a 0 % ammonium sulfate, a condition at which the hydrophobic interaction between proteins and resin was very unlikely. In each step, 10 mL of the corresponding salt concentration was applied (Figure 5).

Element 6

 Separation of a Saccharomvces cervisiae proteome with the dendron-based chromatographic resin.

 To evaluate the separation ability of the dendron-based chromatographic resin, a complex sample was also used: a complete proteome of Saccharomyces cervisiae yeast.

 Stage 1. Resin packaging and conditioning. Refer to step 1 of example 3.

 Stage 2. Preparation of the sample. Commercial yeast was washed with distilled water for 12 hours. It was then centrifuged at 10,600 x g and the supernatant discarded. The biomass (15 g) was reconstituted in 15 mL of buffer B and the yeast cells were lysed with a 20 kHz sonicator

(QSonica, model Q125, Newtown, CT, USA) equipped with a CL-18 probe. The equipment was operated at a 60% pulse width of 20 seconds with intervals of 10 seconds for 15 minutes. Next, the suspension is centrifuged at 10,600 xg and the supernatant (10 mL) was concentrated using 5 mL Amicon tubes for concentration (Merck Millipore). Two batches of concentrated supernatant (2.5 mL) were passed through a desalination column PD10 (GE Healthcare). After repeating this process for several times, the protein solution was concentrated to a volume of 10 mL and sufficient ammonium sulfate was added to reach a concentration of 1.75M.

 Stage 3. Sample injection. The sample, previously dissolved in buffer A, was filtered through a syringe filter with a pore size of 0.2 pm. Subsequently, 5 mL of the filtered sample was injected into the chromatography system equipped with a 5 mL injector and the separation process was initiated.

 Stage 4. Elution and sample separation. Elution of the sample was carried out in the same manner as in Example 3, with the only difference that this time this process was carried out at a flow rate of 1 mL / min. In this example, the chromatography equipment was programmed to collect 2 mL fractions in order to analyze them by electrophoresis (Figure 6).

Stage ^p to 4. Electrophoresis analysis. The 2 mL fractions obtained were concentrated and analyzed by SDS-PAGE electrophoresis analysis, an acronym in English for "sodium dodecyl sulfate potyacrylamide gel electrophoresis" (polyacrylamide gel electrophoresis with sodium dodecyl sulfate). For this, protein samples were routinely prepared by mixing the sample with the LaemmU sample buffer (6X), which contained 27 mM dithiothreitol, in a 5: 1 ratio. These mixtures were heated at 95 ° C for 5 minutes. Subsequently, the samples (10-15 pL) were loaded on a 4-20% polyacrylamide gel (BioRad, Cat. No. 4561094). The separation was carried out at 150 Volts (25 mA) for 35 minutes in a MinkPROTEAN® Tetra Cell (BioRad) electrotbresis equipment. Protein bands were stained using colloidal Coomassie staining (Figure 7).

Example 7

 Adsorption capacity of the dendron-based chromatographic resin in aesthetic and dynamic mode.

 Adsorption experiments of the dendron-based chromatographic resins were carried out both in static mode and in dynamic mode using bovine serum albumin (BSA) as protein models.

 For static mode experiments (SAC), adsorption was carried out in batches. For this, 100 mg of resin, wet base, were transferred to microtubes of 2 ml_ and were equilibrated with a solution of ammonium sulfate (AS) 1.5 or 2 M. Solutions of 2 mL of BSA (0-3 mg / mL) they were prepared in 1.5 or 2 M ammonium sulfate (AS), and then mixed with the resin. The resin was separated from the solution by centrifugation. The non-adsorbed protein (equilibrium concentration) was measured in the supernatant with a plaque assay based on the Bradford method. The adsorption capacities of RG3 and RG5 in static mode are shown in Figure 8.

Subsequently, the experimental data (not shown) was adjusted by a mathematical model of Langmuir. The results of the analysis are shown in Table 4. Table 4. Adsorption capacity of the dendron-based chromatographic resin in static mode.

 "Repeatability of data from batch to batch.

 Where q is the amount of resin bound protein in mg / mL, and Ka represents the affinity constant in mg / mL.

On the other hand, the saturation curves for dynamic adsorption capacity (DBC) were obtained with 1 ml_ of a packed Tricom ™ 5/50 column with both RG3 and RG5 resins. The column was connected to a chromatography system) Fin (GE Healthcare, Uppsala, Sweden) and equilibrated with a buffer A composed of 20 mM Tris-HCI at a pH of 7 containing AS 1.6 or 2 M. Protein solutions of 40 were injected mL (1-3 mg BSA / mL) to the column using a 50 mL superloop (GE Healthcare, Uppsala, Sweden). The analysis was carried out at two different flow rates (1 and 2 mL / min) and two concentrations of AS (1.5 and 2M). The chromatographic runs comprised six stages that included balancing (5 mL), injecting (40 mL), washing (10 mL), eluting (5 mL), a second wash (10 mL) and another balancing step (5 mL). After saturating the column, the adsorbed protein was eluted by pumping a buffer B (20 mM Tris-HCI at pH 7) without salt in The column, in one stage. To calculate the protein concentration, each point of the curve was integrated by obtaining the delta volumes (mL) of two consecutive points on the x-axis and multiplying by the height (mUA) of each point. [These areas were compared with the total area under the curve (mUA'mL), considered as the total starting protein material loaded in the column in order to estimate the mass (mg). Finally, the mass was divided by the corresponding delta volume to calculate protein in mg / mL The 10% DBC break (10% DBC) was calculated using the following expression:

 Where co is the concentration of protein fed in mg / mL, Vio% is the volume in mL applied to 10% break, Vo is the empty volume in mL of the column, F is the flow rate in mL / min, and VB is the volume (mL) of the bed.

 Dynamic adsorption curves of dendron-based chromatographic resins are shown in Figure 9. DBC values are shown in Table 6.

 Table 5. Adsorption capacity of the dendron-based chromatographic resin in dynamic mode.

^■ Values obtained at 10% saturation. The data presented are the average of three runs with their standard deviation.

 "Repeatability of the data from run to run.

 It follows from the previous table that high DBCs were obtained for the assessed flows. In this regard, it is noteworthy to mention that the present invention provides a method for synthesizing chromatographic resins based on dendrons that have a high DBC at high flow rates. Therefore, this technology has the potential to accelerate chromatographic processes, saving time and reagents; as well as increase the capacity of the entire operation since more sample can be processed in each chromatographic run.

Claims

NOVELTY OF THE INVENTION Having described the present invention as above, it is considered as novelty and by k) therefore, what is contained in the following is claimed as property: CLAIMS  1. A method for the synthesis of a dendron-based chromatographic resin, characterized in that it comprises the steps of:  (a) Attach at least one dendron to a chromatographic resin by reacting the dendron core with the chromatographic resin, wherein the dendron is a branched molecule of any chemical and with any degree of branching;  (b) Immobilize at least one ligand in the dendronized resin obtained in step (a) by reacting the ligand with the Ibres functional groups of the dendrons, wherein the ligand is any molecule that can chemically react with the branches of the dendron.  2. The method according to claim 1, characterized in that the dendron is selected from the group consisting of polyesters, polyamides, polysilanes, polyimines, polyethers, polyphenylacetylenes, or any combination thereof.  3. The method according to claim 2, characterized in that the dendron is a polyester.  4. The method according to claim 3, characterized in that the dendron is a polyester of generation 1 to 8. 5. The method according to claim 4, characterized in that the dendron is a generation 3 polyester. 6. The method according to claim 4, characterized in that the dendron is a generation 5 polyester.  7. The method according to claim 1, characterized in that the dendron comprises 2,2-Bis (hydroxymethyl) propionic acid (bis-MPA).  8. The method according to claim 1, characterized in that the dendron core consists of an amino group.  9. The method according to claim 1, characterized in that the dendron core consists of a carboxyl group.  10. The method according to claim 1, characterized in that said chromatographic resin is selected from the group consisting of hydrophobic interaction resin, ion exchange resin, reverse phase resin, affinity resin, or any combination thereof.  11. The method according to claim 1, characterized in that said chromatographic resin is used in the purification and recovery of high molecular weight molecules such as proteins.  12. The method according to claim 1, characterized in that said chromatographic resin is used in the purification and recovery of low molecular weight molecules.  13. The method according to claim 1, characterized in that the ligand is an aliphatic molecule.  14. The method according to claim 1, characterized in that the ligand is a cyclic molecule. 15. The method according to claim 1, characterized in that the ligand has a carbon number n + 1, wherein n is a number between 1 and 19. 16. The method according to claim 1, characterized in that the ligand is hydrophobic in nature comprising at least one carboxylic acid.  17. The method according to claim 16, characterized in that the ligand is selected from at least one of the following acetic acid, propanoic acid, butyric acid, valeric acid, caproic acid, enantic acid, benzoic acid, caprific acid, acid valproic acid, phenylacetic acid, pelargonic acid, capric acid, undecyl acid, lauric acid, tridecylic acid, myristic acid, pentadecrylic acid, palmftic acid, margaric acid, stearic acid, arachidic acid, or any combination thereof.  18. The method according to claim 17, characterized in that the ligand is valeric acid.  19. The method according to claim 16, characterized in that the ligand is polypropylene glycol (PPG).  20. The method according to claim 16, characterized in that the ligand is poly-ethylene glycol (PEG).  21. The method according to claim 20, characterized in that the ligand is selected from at least one of the following methoxy-PEG-acetic acid, methoxy-PEG-propionic acid, methoxy-PEG-butanoic acid, methoxy-PEG-acid hexanoic, or any combination thereof.  22. The method according to claim 1, characterized in that in step a) the coupling is carried out for at least 1 hour at a temperature between 20 ± 5 ° C. 23. The method according to claim 1, characterized in that in step b) the immobilization is carried out for at least 1 hour at a temperature of between 20 ± 5 ° C. 24. The method according to claim 1, characterized in that the ratio between resin and dendron is 1: 1.  25. The method according to claim 1, characterized in that the chromatographic resin is activated with an N-hydroxysuccinimide (NHS) group.  26. The method according to claim 1, characterized in that the ratio between dendrons and resin is preferably 1.2-2.6 equivalents / 0.5 g of resin.  27. The method according to claim 1, characterized in that the ratio between dendrons and ligand is 2 equivalents of the ligand with respect to the amount of -OH groups in the dendron.  28. The method according to claim 1, characterized in that in step (b) the catalyst is such a cabodiimkJa such 1-etü-3- (3- dimethylaminopropyl) carbodiimide (EDC), 4- (dimethylamine) pyridinium-4- toluene sulfonate (DPTS), 4-dimethylamino-pyridine (DAMP), Ν, Ν'-dicyclohexylcarbodiimide (DCC), N.N'-Diisopropylcarbodiimide (DIC) or 1,1-carbonyldiimidazole (CDI), or any combination of the same.  29. The method according to claim 1, characterized in that in step (b), the catalyst 1-ethyl ^ 3 ^ irneti laminen ropil) ∞rfocdiim ^ (EDC) is in a 10% excess with respect to the amount of ligand. 30. The method according to claim 1, characterized in that in step (b), the catalyst 4- (dimethylamino) pyridinium-4-toluene-sutophonate (DPTS) is in a 40% excess with respect to the amount of ligand. 31. The method according to claim 1, characterized in that in step a) the coupling is carried out at a pH of 8.1 ± 0.2.  32. The method according to claim 1, characterized in that in step a) the coupling is carried out in the presence of DMF.  33. The method according to claim 1, characterized in that in step (a), the chemical bond between the chromatographic resin and the dendron forms an amido bond.  34. The method according to claim 1, characterized in that in step (a), the chemical bond between the chromatographic resin and the dendron forms an ester bond.  35. The method according to claim 1, characterized in that said method is carried out on a chromatographic column.  36. A dendron-based chromatographic resin obtained by the method described in claims 1 to 35, characterized in that it has a dendron density of 4.9 to 11.5 pmoL dendron / mL resin, and a ligand density of 71.2 to 186.5 pmoL ligand / mL resin.