Intermittent percolation cleaning

Abstract

The present invention relates to a method for replacing compounds and solvents contained in the liquid and solid phases of a heterogeneous liquid-solid phase reaction such as solid phase peptide synthesis (SPPS) or solid phase oligonucleotide synthesis (SPOS). The method is applied to a reactor containing a liquid and a solid phase, to which a replacement liquid is discontinuously supplied while the liquid phase is removed from the reaction chamber. The present invention also encompasses a method for synthesizing peptides or oligonucleotides by application of a solid phase peptide or oligonucleotide synthesis protocol further comprising a method for replacing compounds.

Description

[Technical field]

[0001] The present invention relates to a method for replacing compounds and solvents contained in the liquid and solid phases of a heterogeneous liquid-solid phase reaction such as solid phase peptide synthesis (SPPS) or solid phase oligonucleotide synthesis (SPOS). The method is applied to a reactor containing a liquid and a solid phase, to which a replacement liquid is discontinuously supplied while the liquid phase is removed from the reaction chamber. The present invention also encompasses a method for synthesizing peptides or oligonucleotides by application of a solid phase peptide or oligonucleotide synthesis protocol further comprising a method for replacing compounds. [Background technology]

[0002] Heterogeneous liquid-solid phase chemical reaction protocols are attractive for the synthesis of molecules containing repeated subunits. Liquid-solid phase protocols allow for the effective separation of the solid phase from the liquid phase and provide suitable conditions for the application of iterative cycles containing reaction steps for the successive stepwise introduction (addition) of subunits. Liquid-solid phase reaction protocols have been successfully implemented in the field of peptide synthesis and oligonucleotide synthesis. During the course of the synthesis, the nascent molecule (such as a growing peptide or oligonucleotide) is covalently attached to a solid support, providing conditions for the efficient removal of by-products by washing between the reaction steps of the iterative cycle. Heterogeneous liquid-solid phase chemical reaction protocols have had a major impact, for example, on the synthesis of peptides and oligonucleotides on a commercial scale, but this methodology consumes significant volumes of solvents required for the replacement of reagents, by-products and reaction solutions after each reaction step of the iterative cycle.

[0003] Solid-phase peptide synthesis is usually carried out in batch reactors such as stirred tank reactors (STRs) where there is no continuous flow from or to the reactor during the reaction. Tubular reactors such as packed-bed reactors offer several advantages over other reactors during the wash step. Packed-bed reactors allow the wash to proceed as a displacement operation rather than a dilution operation as in STRs, provided that the amount of "dead volume" between the reactor inlet and the resin beads is minimized.

[0004] Solid-phase peptide synthesis allows peptide chains to be built on a solid support by repeated cycles involving binding an amino acid to the support, deprotecting the amino acid, and coupling one or more subsequent amino acids to the amino acid or amino acid fragment covalently attached to the solid support. The solid supports used in solid-phase peptide synthesis are usually low-crosslinked gel resins. The most common support is polystyrene containing 1 or 2% divinylbenzene (DVB) as a crosslinker, but other solid supports include polyacrylates, polyacrylamides, and polyethylene glycols. These crosslinked supports are insoluble in organic solvents, but are solvated and swell in aprotic solvents such as toluene, dimethylformamide, and dichloromethane. In addition, peptide resins may also shrink / swell during assembly due to the extension of peptide chain length. Furthermore, these resins tend to be fairly soft in nature and therefore susceptible to physical wear.

[0005] During the peptide synthesis steps (coupling and deprotection), the preferred reactor is a stirred reactor, which makes it possible to have a homogeneous medium to control the reaction, whereas for washing the resin between the synthesis steps of the cycle, the preferred reactor is a column-type piston reactor, which makes it possible for the species to be removed to percolate in an optimal manner with a minimum of solvent.

[0006] Solid-phase oligonucleotide synthesis has many similarities with solid-phase peptide synthesis. Target nucleotides are formed by the successive reaction (coupling) of individual nucleosides on the solid phase by the application of repetitive reaction steps constituting a cycle. More specifically, oligonucleotides are typically formed by the implementation of derivatives of nucleosides that contain protecting groups, in particular phosphoramidites. The key feature of phosphoamidites is their reactivity towards nucleophiles (e.g., the deprotected hydroxyl group at the 5' carbon of the pentose sugar) catalyzed by weak acids such as tetrazole. Tetrazole-catalyzed phosphoramidite coupling increases the efficiency to over 99%, allowing the synthesis of long oligonucleotides, up to 100 nucleotides or more. Typically, oligonucleotides are synthesized from the 3' to the 5' end, starting from a suitable solid phase to which protected nucleosides are covalently linked by a linker / spacer at the 3' carbon. The reactive groups of the nucleosides, i.e., the hydroxyl group of the base, the phosphate group and the amine, are typically protected. Suitably, the 5' hydroxyl group is protected by dimethoxytrityl (DMT), isobutyryl or benzoyl is used to protect the amines of the base, and the phosphoramidite protects the hydroxyl group of the 3' carbon. Phosphoramidite solid-phase synthesis begins with the 3' nucleotide and undergoes a series of cycles consisting of four reaction steps that are repeated until the final 5' nucleotide of the target oligonucleotide is attached. The four reaction steps of phosphoramidite solid-phase synthesis typically include deprotection, coupling, capping and stabilization. The solid phase of choice for oligonucleotide synthesis is and remains porous silicate, such as controlled-pore-glass (CPG). However, more recently, polystyrene-based solid phases have also attracted interest (e.g. porous cross-linked aminoethyl polystyrene resin). Oligonucleotide synthesis is usually carried out in a reaction column containing a solid support. Reagents are sequentially run through the column.

[0007] Similar to solid-phase peptide synthesis, solid-phase oligonucleotide synthesis also consumes significant amounts of liquids that are used to displace excess reactants, by-products and reaction solvents after the reaction step of the cycle. A reduction in the amount of displacement liquids (washing liquids) is warranted, since the displacement liquids represent half of the organic material used, which is equal to about 25% of the process mass intensity (PMI = amount of raw material divided by amount of API [active pharmaceutical substance]).

[0008] Continuous flow reactors are usually deployed for solid phase oligonucleotide synthesis, in particular where the solid phase is a porous silicate. One type of continuous flow reactor is the fixed bed reactor. As alluded to above, the solid phase, which is usually a polymer with some cross-links, tends to change volume, specifically due to the solvent, but also due to the growing peptide or oligonucleotide. It is not uncommon for the resin in SPPS to swell more than 100% in volume, but also to shrink more than 100%. In some way, the swelling and shrinkage of the resin must be accommodated within the continuous flow reactor.

[0009] US9169287 discloses a continuous flow reactor for solid phase peptide synthesis by applying a packed bed reactor. To accommodate the resin volume change, only a part of the reactor is filled with resin, while a significant volume of liquid is present above the resin bed. The large amount of liquid above the resin bed is highly unfavorable for reaction steps such as coupling reactions and washing runs, due in part to backmixing effects and dilution, resulting in the use of large excesses of reagents and large consumption of washing liquids. The synthesis of US9169287 would not be favorable for large-scale production of peptides.

[0010] US200206714 presents a continuous flow reactor for solid phase peptide synthesis. The solution to accommodate resins with significant changes in volume over the reaction cycle while keeping the liquid phase volume low is to provide adjustment of the total reactor volume by a movable wall actuated by a piston. The proposal of US200206714 presents a highly complex reactor that can be difficult to operate advantageously for large-scale production.

[0011] Furthermore, the use of flow reactors generally does not provide uniform reaction conditions during the reaction step and can locally vary the concentration of reagents in the resin, resulting in variations in reaction progress across the bed, thereby affecting purity.

[0012] Furthermore, the continuous flow reactor has a height much greater than its diameter and has a significant amount of wall surface area that prevents the expansion and contraction of the resin bed. As the resin bed expands, it is forced against the walls of the vessel, reducing the void space between the resin particles. Thus, high pressure droplets are created to force all of the cleaning agent into the bed in the required time. This high pressure can compress the soft gel beads, potentially restricting flow, damaging the resin beads, creating fines that can plug filters, and potentially damaging or destroying filter frits. If high pressure is not used, the flow through the bed will be too slow, making the cleaning step take longer and reducing the productivity of the process.

[0013] DE 2017351 discloses that reactors based on a rotating bowl design have also been developed. Rotating bowl or centrifugal reactors can allow for increased liquid velocity relative to the resin particles. In this "washing machine" reactor, a porous basket is first filled with resin and then rotated to a moderate speed while immersed in liquid. Centrifugal forces cause the resin particles to form a bed on the inner wall of the basket, causing moderate fluid recirculation through the resin bed. The bowl is filled with water (i.e., filled with liquid) as well as the resin bed. The drag forces imposed on the basket by the liquid bath impose high torque on the drive motor and also cause heat generation. For these reasons, the rotation speed of the Birr reactor is relatively slow and the relative velocity of the fluid to the velocity of the solids is limited. The limited rotation speed almost certainly results in a non-uniform resin bed that is shallow at the top and deep near the bottom. This technology is also complicated to scale up for large-scale production.

[0014] US Patent No. 5,186,824 discloses a centrifugal reactor based on a water-filled "hollow rotor". The liquid flow paths in the reactor are axial rather than radial, and the geometry is irregular relative to the liquid flow field. The point at which the liquid is introduced depends on the density of the liquid relative to the density of the last liquid added. Also, there is little room for the resin to expand. Expansion and contraction tend to result in uneven exposure of the resin to the liquid phase. Complete and uniform contact and removal of the liquid from the rotor can be very difficult to achieve.

[0015] WO 2021 / 158444 relates to a reactor design for solid-phase peptide synthesis. A feature of the reactor design is the application of several reactors in series. The series reactor design has the potential to reduce solvent, but at the expense of requiring multiple reactors with high complexity, which may be difficult to operate for large-scale production.

[0016] US Patent Application Publication No. 2021 / 0094982 discloses a reactor system for solid-phase peptide synthesis. The system can use percolation for solvent removal. Percolation is carried out by introducing a wash liquid into the reactor at the same flow rate as the outlet flow rate, thereby maintaining the liquid height at a constant level. It is stated that maintaining a constant liquid height during percolation by keeping the inflow and outflow at the same flow rates provides the most effective wash mode, allowing for a significant reduction in solvent consumption.

[0017] The continuous flow of wash liquid makes it difficult to monitor the level of the liquid phase above the resin bed. By continuously removing the liquid phase from the reactor and simultaneously applying a discontinuous supply of wash (displacement) liquid to the reactor, the liquid level (i.e., the height of the liquid phase above the solid phase (resin bed)) can be accurately monitored during periods when wash liquid is not being fed to the reactor, without at the same time significantly affecting the superficial velocity of the wash liquid above the solid phase (resin bed).

[0018] US2021 / 0094982 presents experimental data on a percolation washing protocol where the inflow of the wash liquid and the outflow of the liquid are set at the same flow rate. The table in paragraph 0144 shows the effect on the volume of wash liquid and the time required to reach the set end point (where the amount of piperidine in the liquid phase leaves the reactor) as well as different flow rates of the wash solvent (and by inference, also the flow rate of the outflow of the liquid from the reactor). In US2021 / 0094982, optimal washing is defined as the preferred end point. At a flow rate of 50 ml / min, optimal washing is reached within 20 minutes with a consumption of 930 ml of wash solvent. At a flow rate of 300 ml / min, the optimum is reached within 4 minutes with a consumption of 1200 ml of wash solvent. Clearly, both the washing time and the wash liquid consumed are important. At a set endpoint, a decrease in wash time is offset by an increase in solvent consumption, while a decrease in solvent consumption is offset by an increase in wash time and a decrease in overall throughput.

[0019] One conclusion from the empirical data underlying the present invention is the discovery that the metric provided by the superficial velocity is a better proxy for overall efficiency than the flow rate of the wash solvent. Unlike the flow rate, the superficial velocity is independent of the reactor size (universally applicable to reactors of different volumes), taking into account both the consumption of the wash solvent and the wash time. Moreover, the applicant has surprisingly also found that there is a specific preferred range that constitutes the optimum value of the superficial velocity. In a particular range of superficial velocity, the overall efficiency that takes into account both the spent volume and time of the wash liquid is specifically favored.

[0020] The percolation cleaning of US Patent Application Publication No. 2021 / 0094982 applies a constant liquid level as close as possible to the resin bed without generating turbulence. To provide a constant liquid level, the inflow and outflow of liquid must be continuous and have the same flow rate at steady state. Providing a constant liquid level close to the resin bed is difficult to achieve without a sophisticated regulation system incorporating sensors. The volume of the resin bed may change as a function of the solvent (solvent composition) and the growth of the peptide chain length. Furthermore, the properties of the solid phase change as the compound of interest covalently bound to the solid phase increases in size by the introduction of subunits. The introduction of a cleaning liquid (displacement liquid) after the reaction step of a repeated cycle may gradually change the composition of the liquid phase and affect the swelling properties of the solid phase. In conclusion, it may be necessary to monitor not only the liquid level but also the level of the solid phase (resin bed) for the reasons presented. Therefore, keeping the liquid level constant as close as possible to the solid phase depends on accurate monitoring of the level of the solid phase (i.e., the liquid-solid phase boundary) and the liquid level (i.e., the gas-liquid phase boundary). In US 2021 / 0094982, the liquid level is measured during a continuous inflow of cleaning liquid, which impairs the accuracy of the measurement.

[0021] The present invention implements a discontinuous inflow of displacement liquid, which allows monitoring of the solid phase level and the liquid surface without disturbing the continuous supply of washing liquid. Moreover, the flow rate and duration of the discontinuous supply of displacement liquid can be easily adjusted (without affecting the superficial velocity of the displacement liquid above the resin bed [solid phase]) if necessary, for example, to wash the solid phase (resin) of the walls of the reactor.

[0022] Furthermore, the displacement of reaction solvents, reagents and by-products by application of discontinuous supply of displacement / washing solution and continuous purging of the liquid phase from the reactor further improves the reduction of spent washing solution over the washing protocol of continuous inflow of washing solution and continuous outflow of liquid. Summary of the Invention

[0023] The present invention relates to a method for replacing compounds (and / or solvents) contained in a liquid phase and a solid phase present in particulate form dispersed in the liquid phase, of a reaction step of a heterogeneous chemical reaction for the formation of a target product synthesized by successive introduction of subunits (compounds) by repeated (repeated) cycles, each cycle comprising a reaction step, comprising at least providing means for preventing the solid phase from escaping the reaction chamber when the solid phase is removed from the reactor, and removing the liquid phase from the reactor, the replacement liquid being discontinuously fed into the reactor and a permanent layer of the liquid phase being maintained on the solid phase.

[0024] The present invention also encompasses methods for the synthesis of peptides and oligonucleotides by application of solid state synthesis protocols, including the methods for substituting compounds presented herein.

[0025] Peptides and oligonucleotides are biological polymers that contain repeating subunits (building blocks) that are similar in many dimensions, including chemical structure. These subunits share similar functional groups, such as carboxylic acid and alpha-amino functional groups for peptide subunits (amino acids) and bases, sugars and phosphate groups for oligonucleotide subunits (nucleosides).

[0026] The synthesis protocol of choice for the production of oligonucleotides and peptides is solid-phase synthesis, in which the oligonucleotide or peptide is covalently attached to the solid phase. Nucleosides or amino acids (or any residues thereof) are coupled stepwise to a successively growing oligonucleotide or peptide covalently attached to the solid phase. Solid-phase synthesis involves reaction steps of a heterogeneous chemical reaction involving liquid phase and solid phase, in which reactants (nucleosides or amino acids) are provided in liquid phase while the peptide or oligonucleotide is covalently attached to the solid phase.

[0027] The solid phase may be in particulate form, suitably dispersed in a liquid phase. The solid phase may be compressible, ranging from highly solvated polymeric resins to essentially incompressible particles or rigid structures with high surface area. Furthermore, solid phase synthesis may be operated in batch, continuous mode, or a combination of both.

[0028] Solid phase synthesis protocols, applicable to both oligonucleotide and peptide synthesis, typically involve repetitive cycles involving a number of different procedural steps, including deprotection, i.e., removal of protecting groups, coupling reactions in which additional subunits (nucleosides or amino acids) are covalently attached to the growing polymer of peptide or oligonucleotide, and steps involving the replacement of by-products, reactants and solvents.

[0029] A cycle for the synthesis of peptides usually includes a step of deprotection, which typically means that the protecting group linked to the N-terminal α-amino group of the peptide-resin residue is cleaved, leaving an unprotected α-amino function. In a subsequent step, the carboxylic acid moiety of the N-terminal α-amino protected amino acid is covalently attached to the unprotected α-amino function of the peptide-resin residue under formation of an amide coupling. By-products and excess reagents are removed after successful deprotection and coupling by a washing procedure.

[0030] Solid-phase oligonucleotide synthesis typically involves the use of phosphoramidite synthesis methods that include the use of phosphoramidite nucleoside building blocks (subunits) that can be derived from protected 2'-deoxynucleosides (dA, dC, dG, T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, such as LNA or BNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the product sequence. The use of nucleoside phosphoramidites instead of naturally occurring nucleotides dramatically improves selectivity and yield. Repeated cycles typically include the steps of deblocking or detritylation, coupling, capping (or sulfurization), and oxidation.

[0031] The solid phase used in oligonucleotide synthesis is usually a non-swelling or low-swelling solid phase (support).The solid phase suitable for oligonucleotide synthesis is porous silicate, for example, porous glass (CPG).Recently, non-silicate solid phases have been introduced in SPOS, such as polystyrene-based resins, including macroporous polystyrene (MPPS), which tend to change volume as process conditions (for example, solvent and oligonucleotide growth) change.

[0032] Heterogeneous chemical reactions can be operated in continuous or batch mode.

[0033] The present invention relates to the substitution of compounds when a reaction step in a heterogeneous liquid-solid phase chemical reaction protocol is deemed to have reached a predetermined reaction endpoint.

[0034] The reaction end point is considered to be the point at which the reaction has reached a useful yield. The compound that is displaced is any compound that is undesirable for the next reaction step. Such undesirable compounds can include excess reactants, by-products, and solvents.

[0035] The displacement liquid reduces and, appropriately, eliminates compounds that are deemed undesirable in the next reaction step. The term displacement should therefore be interpreted to include any physical phenomenon that causes the reduction of undesirable compounds. Displacement procedures that include the addition of a displacement liquid to a solid phase thus encompass phenomena such as reduction, removal, washing, percolation, etc.

[0036] As detailed above, the method is specifically configured to involve repeated cycle reaction steps of a heterogeneous liquid-solid phase chemical reaction protocol to form a target product synthesized by successive introduction of similar subunits. The method is suitably carried out in the same reaction chamber / vessel, in which the cycle reaction steps are also carried out. Thus, the reaction chamber typically includes the features necessary to carry out any one of the cycle reaction steps for the synthesis of the target molecule, and may also include the elements specifically for the method. Thus, one advantage of the method is that it is carried out in the same reaction chamber in which the reaction steps are carried out, reducing the complexity of the method.

[0037] According to one embodiment useful for the most general definition of the invention, the method comprises continuously removing a liquid phase from the reactor via an outlet and discontinuously feeding a displacement liquid to the chamber via an inlet, the height (h) of the liquid above the solids varying over time with the proviso that the height (h) is never zero.

[0038] According to a further aspect, the reactor of the present method comprises an inlet, an outlet, a liquid phase, a solid phase, and a means for preventing the solid phase from escaping the reaction chamber when the liquid phase is removed from the reactor; and continuously removing the liquid phase from the reactor via an outlet, and a displacement liquid is discontinuously fed to the chamber through the inlet such that a continuous layer of the liquid phase is maintained above the solid phase.

[0039] According to one embodiment, the target compounds are selected from peptides and oligonucleotides, and the subunits are selected from amino acids and nucleosides and any derivatives thereof.

[0040] In the context of the present invention, an amino acid can be any organic compound that contains an amine and a carboxylic acid. One group of amino acids contains an α-amine and a carboxylic acid. Amino acids and amino acid derivatives have the same meaning and can be used interchangeably herein. Important amino acids are proteinogenic amino acids (often called natural amino acids) that are biosynthetically incorporated into proteins during translation. Proteinogenic amino acids may be chemically modified.

[0041] A nucleoside in the context of the present invention may be any organic molecule useful in the synthesis of a polynucleotide chain. Nucleoside derivatives include organic molecules that contain either a derivative of the nucleic acid base (nitrogenous base) and / or a derivative of the sugar or a moiety that replaces the sugar.

[0042] A peptide (also called a polypeptide) is a molecule that contains at least two amino acids or amino acid derivatives. At some point, determined by the number of amino acids, a molecule is designated as a protein rather than a peptide. Usually, molecules with less than 100 amino acids (or amino acid derivatives) are designated as peptides.

[0043] Amino acids and derivatives thereof also encompass amino acid fragments. The amino acid fragments can be any number of fragments of the target peptide, including from 2 amino acids to a fragment having a number of amino acids equal to the number of amino acids of the target peptide minus 1, all of which are the target peptide.

[0044] Amino acids and their derivatives also include non-proteinogenic amino acids, as well as any non-amino acid compounds that can enhance the utility of a target peptide for biological applications.

[0045] According to yet a further embodiment, the target compound is selected from peptides and the subunits are selected from amino acids and their derivatives. Amino acids and their derivatives may also include subunits not included in the conventional definition of amino acids.

[0046] According to one aspect, the present invention relates to a method for replacing compounds (and / or solvents) contained in the liquid and solid phases of a reaction step of solid phase peptide synthesis, comprising providing a reactor in which the reaction step takes place, the reactor comprising the liquid and solid phases, means for preventing solid particles from escaping the reactor when the liquid phase is removed from the reactor, and continuously removing the liquid phase from the reactor while discontinuously (intermittently) feeding a replacement liquid to the reactor, wherein a continuous layer of the liquid phase is maintained above the solid phase. According to one aspect, the present invention relates to a method for replacing compounds (and / or solvents) contained in the liquid and solid phases of a reaction step of solid phase peptide synthesis, comprising providing a reactor in which the reaction step takes place, the reactor comprising a liquid phase and a solid phase, means for preventing solid particles from escaping the reactor when the liquid phase is removed from the reactor, and means for promoting mass transfer between the liquid and solid phases, continuously removing the liquid phase from the reactor, and (meanwhile) discontinuously (intermittently) feeding a replacement liquid to the reactor, wherein a continuous layer of the liquid phase is maintained above the solid phase.

[0047] A further aspect of the method includes settling of the solid phase, thereby forming a phase boundary between the liquid and solid phases that is measurable both visually and by a suitable sensor. Settling of the solid phase occurs when the stirring of the solid and liquid phases by a means for promoting mass transfer between the liquid and solid phases, for example by a stirrer, vortex mixing, nitrogen bubbling or a recirculation pump, is interrupted. Thus, a further aspect of the method is that the continuous removal of the liquid phase from the reactor and the discontinuous supply of replacement liquid to the reactor are carried out when the means for promoting mass transfer between the liquid and solid phases, for example by a stirrer, vortex mixing, nitrogen bubbling or a recirculation pump, are interrupted. Settling of the solid phase implies that homogenization of the liquid and solid phases occurs in the reaction step preceding the replacement operation, by application of a means for promoting mass transfer between the liquid and solid phases, for example by a stirrer, vortex mixing, nitrogen bubbling or a recirculation pump.

[0048] According to yet a further embodiment, the reactor deployed is a tank reactor (e.g. a stirred tank reactor) or a column reactor.

[0049] According to a further embodiment, all the reaction steps of the cycle are carried out in the reactor that is also used to replace the compound of the invention. If the process of the reaction steps includes providing a solid phase in the form of particles evenly distributed in the liquid phase, homogenization by means to promote mass transfer between the liquid phase and the solid phase, the application of the replacement process of the invention mimics a fixed bed reactor while still using one and the same reactor.

[0050] A further embodiment relates to a method comprising washing a solid phase by percolation, wherein the solid phase is contained in a reactor, and the method comprises continuously removing a liquid phase from the reactor and discontinuously feeding a displacement / wash liquid to the reactor while maintaining a continuous layer of the liquid phase on the solid phase.

[0051] In a further embodiment, the displacement liquid is introduced into the reactor at any time after the means for promoting mass transfer between has terminated (is no longer engaged). Preferably, the displacement liquid is introduced when the height of the liquid phase above the solid phase reaches a predetermined minimum threshold (h(min)).

[0052] According to one embodiment, the liquid phase is removed from the reactor using a protocol that results in a superficial velocity of the displacement liquid over the solid phase that does not vary by more than about 30%, more than about 20%, or more than about 10%.

[0053] According to a further embodiment, the liquid phase is continuously removed from the reactor to provide a superficial velocity of the displacement liquid over the solid phase that does not vary by more than about 30%, suitably more than about 20%, or more than about 10%.

[0054] According to one embodiment, the solid phase is selected from silica-containing materials and polymeric materials comprising reactive sites that allow the polymeric material (solid phase) to be covalently linked to the (proximal) subunit of the target compound (nascent compound), optionally via a linker.

[0055] According to yet a further embodiment, the solid phase is selected from cross-linked polystyrenic polymers comprising reactive sites by which the polystyrenic polymer (solid phase) can be covalently linked to a (proximal) subunit of the target compound (nascent compound), optionally via a linker. [Brief description of the drawings]

[0056] [Figure 1] FIG. 1 shows a schematic reactor for carrying out the process of the present invention. [Diagram 2] FIG. 2 shows the height of the liquid phase as a function of time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] An important feature of the present invention is the separation of the supply of displacement liquid to the reactor and the withdrawal of the liquid phase from the reactor, i.e. the inlet and outlet flows of the reactor. Thus, the continuous output flow rate is adjusted to achieve the desired superficial velocity of the liquid phase over the solid phase while the inflow is adjusted in a discontinuous manner. The inflow of displacement liquid is adjusted so that there is always a layer of liquid phase over the solid phase.

[0058] According to one embodiment, the liquid phase is continuously removed from the reaction chamber through the outlet while displacement liquid is discontinuously fed to the reaction chamber through the inlet, whereby a continuous layer of the liquid phase is always maintained above the solid phase, preferably until a predefined displacement end point is reached.

[0059] The substitution end point is defined as a given amount of any compound or compounds that are detrimental to the next reaction step. In solid-phase peptide synthesis, a deprotecting agent is used to deprotect the (most) distal protected amino acid to the resin (solid phase). It is important to remove the deprotecting agent before the subsequent coupling step to an amount that eliminates or minimizes the formation of non-target peptides. If the α-amine is Fmoc protected, the deprotecting agent may be piperidine. Thus, the substitution end point may be defined as the amount of piperidine.

[0060] According to another embodiment, the gas-liquid phase boundary and the liquid-solid phase boundary are determined, thereby establishing the height (h) of the liquid phase above the solid phase, suitably provided that the height (h) is never zero.

[0061] The height (h) suitably varies over time, preferably by up to about 1000%, preferably by up to about 500%, more preferably by up to about 300%, more preferably by up to about 200%.

[0062] When the replacement liquid is discontinuously supplied to the reaction chamber, the flow rate of the replacement liquid is typically higher than the flow rate of the purged liquid phase to ensure that the solid phase is always covered by a layer of liquid phase.

[0063] According to one embodiment, the supply of the substitution liquid is started when the height of the liquid phase above the solid phase reaches a predetermined minimum value, h(min), and is terminated when the height of the liquid phase above the solid phase reaches a predetermined maximum value, h(max).

[0064] The height of the liquid phase may be monitored visually by an operator, but preferably the height is provided by at least one sensor monitoring the gas-liquid and liquid-solid phase boundaries. By means of a suitable algorithm, the height h of the liquid layer can be derived from the two-phase boundaries.

[0065] Preferably, the minimum height h(min) is selected such that distribution of the displacement liquid over the liquid phase does not disturb the solid phase.

[0066] According to one embodiment, the inner cross-sectional area of ​​the reactor is about 10 cm 2 ~Maximum approx. 80000cm 2 It is.

[0067] According to a further embodiment, the minimum height h(min) of the liquid phase above the solid phase (resin bed) is about 0.5 cm, preferably about 1 cm, preferably about 2 cm, about 3 cm, about 4 cm, about 5 cm. The minimum height h(min) may range from about 0.5 cm to a maximum of about 20 cm, suitably from about 1 cm to about 10 cm. The maximum height h(max) is preferably about 2 cm, preferably about 4 cm, preferably about 5 cm, or about 6 cm, or about 7 cm, or about 10 cm, or about 20 cm. The maximum height h(max) of the liquid phase is suitably in the range from about 2 cm to a maximum of about 20 cm, suitably from about 4 cm to a maximum of about 10 cm. Another measure of the height of the liquid level is the arithmetic mean of the height. If h(min) is 1 cm and h(max) is 2 cm, then the average height, h(mean), is (1+2) / 2=1.5. According to one embodiment, the average height of the liquid phase, h(mean), ranges from 1.0 cm up to about 10 cm.

[0068] According to one embodiment, the ratio of h(min) to h(max) [ratio: h(max) / h(min)] is about 1.1 to about 6.

[0069] According to one embodiment, the superficial velocity of the displacement liquid (over the solid phase) in the reactor is maintained in the range of about 100 cm / h to a maximum of about 400 cm / h, preferably about 150 cm / h to a maximum of 300 cm / h, preferably about 175 cm / h to a maximum of about 250 cm / h. By superficial velocity is meant herein the hypothetical linear average velocity given by dividing the volumetric flow rate [volume / time] by the cross-sectional area [area] of the solid phase (fixed bed). The superficial velocity may also be called the percolation velocity. The percolation velocity is suitably adjusted taking into account the mass transfer kinetics of the compound to be displaced from the solid phase (resin) and the mass transfer kinetics of the pressure droplets of the solid phase (bed of solid phase in the form of particles dispersed in the liquid phase: resin bed).

[0070] The exit velocity of the liquid phase from the reaction chamber is continuous and preferably does not vary by more than 30%, preferably by more than 20%, suitably by more than 10%. The exit velocity is typically adjusted to provide a superficial velocity in the ranges specified herein.

[0071] According to one embodiment, the inflow / outflow ratio (ratio of flow rate of the displacement liquid to the flow rate of the removed liquid phase) is greater than 1, suitably from about 5 up to about 10.

[0072] When the level reaches a low threshold, solvent is added in a fraction of the defined volume. Once this volume has been added, the inflow is stopped and the liquid level is measured, which can be done under optimal conditions since the sensor beam or visual inspection of the level is not disturbed by the flow of inflowing solvent. Percolation reduces this level and when a low level is reached again, the feed pump is restarted.

[0073] One embodiment of the present invention relates to solid phase synthesis involving a solid phase in the form of particles dispersed in a liquid phase, where the reaction steps of the cycle are carried out when the solid phase is uniformly dispersed in the liquid phase. The solid phase can be said to be homogenized by means for promoting mass transfer between the liquid and solid phases, such as a stirrer, vortex mixing, nitrogen bubbling or a recirculation pump. Thus, the reactor must be equipped with a means for promoting mass transfer between the liquid and solid phases in order to homogenize the solid phase in the liquid phase during the reaction step. Usually, solid phase synthesis involving a reaction step involving homogenization of the solid phase involves a reactor operated in batch mode.

[0074] When the reaction of a reaction step, such as a deprotection or coupling step, reaches a predetermined reaction end point, it may be preferable to allow the reaction composition comprising a liquid phase and a solid phase (homogenized reaction composition) to settle until a clear phase boundary between the liquid particles and the solid particles is detectable or observable.

[0075] Preferably, before the displacement (wash) liquid is fed to the reaction chamber, the means for promoting mass transfer between the liquid and solid phases and for homogenizing the solid phase (if present) are discontinued and the reaction composition is allowed to settle until the liquid and solid particles have separated to an extent that they can be monitored visually or by at least one detector, and the gas-liquid phase boundary and the liquid-solid particle boundary can be monitored.

[0076] At the moment the replacement liquid is introduced, it is preferred that there is always a liquid layer on the solid phase, preventing the latter from drying out.

[0077] However, the displacement liquid may also be introduced at any time after the means for promoting mass transfer are interrupted. The displacement liquid may also be introduced before the means for promoting mass transfer are interrupted. Displacement may also be introduced into the reactor after the means for promoting mass transfer are interrupted but before a visual or measurable (detectable) phase boundary between the liquid and solid phases is established.

[0078] In principle, there is no boundary between the liquid phase and the solid phase in the strict sense of the term phase boundary. Rather, the solid phase, or rather the solid particles, are always immersed in the liquid phase or dispersed in the liquid phase. The term solid phase should therefore be understood as a solid phase in particle form dispersed in the liquid phase. If the reaction composition is not subjected to forces tending to distribute the solid particles uniformly in the liquid phase, the particles will gradually settle by gravity, thereby forming a liquid layer above the dispersion of solid particles. After a while, an equilibrium is created between the gravitational and repulsive forces between the solid particles, resulting in a certain level of dispersion containing the solid particles, i.e., the level of the solid phase at the liquid-solid boundary. At this equilibrium, a visually observable and detectable transition between the liquid phase and the dispersion can be shown as a pseudo-phase boundary. Here, this pseudo-boundary is shown as a liquid-solid boundary for the sake of simplicity.

[0079] The solid particles dispersed in the liquid phase of the heterogeneous liquid-solid phase chemical reaction protocol of the present invention may also be referred to as a bed of solid phase in particulate form or simply as a resin bed.

[0080] One of the distinctive features of the present invention is the provision of a discontinuous or intermittent supply of displacement / washing liquid and a continuous outflow of the liquid phase. By separating the inflow of the washing liquid and the outflow of the liquid phase, the flow rate of the washing liquid can be varied while maintaining an essentially constant superficial flow rate (or simply superficial velocity) of the liquid phase above the bed of solid particles, where superficial velocity is defined as:

number

[0081] The method may also be considered as a percolation method, ie an extraction procedure in which soluble components are removed from the solid phase by extraction by means of the supply of a displacement liquid.

[0082] A further embodiment, wherein the method further comprises settling of the solid phase, thereby forming a gas-liquid phase boundary, a liquid-solid phase boundary, and a liquid phase above the solid phase.

[0083] According to a further aspect, the invention comprises continuously removing a liquid phase from the reactor through an outlet, wherein a displacement liquid is intermittently (discontinuously) fed to the reactor through an inlet, and the height (h) of the liquid layer above the solid layer varies over time, but the height (h) is never zero.

[0084] According to a further aspect, the settling is forced by the continuous outflow of the liquid phase. If the reaction composition is not subjected to a force that tends to distribute the solid particles uniformly in the liquid phase, the particles will gradually settle by gravity, thereby forming a liquid layer on top of the dispersion of solid particles. The outflow of the liquid phase forces the settling, and after a while, an equilibrium is reached between the gravitational forces and the repulsive forces between the solid particles, resulting in a certain level of dispersion containing the solid particles, i.e., the level of the solid phase at the liquid-solid boundary. At this equilibrium, a visually observable and detectable transition between the liquid phase and the dispersion can be shown as a pseudo-phase boundary. Here, this pseudo-boundary is shown as a liquid-solid boundary for the sake of simplicity.

[0085] According to yet a further aspect, discontinuous percolation cleaning is carried out in a reactor in which all reaction steps of a cycle of a heterogeneous chemical reaction are carried out.

[0086] Reactor The reactor or reaction chamber is preferably a reactor for carrying out a heterogeneous liquid-solid phase chemical reaction protocol for forming a target molecule synthesized by successive introduction of subunits. More specifically, the reactor can be any reactor suitable for solid-phase peptide synthesis and solid-phase oligonucleotide synthesis. According to one embodiment, the reactor can be any reactor applicable for solid-phase peptide synthesis. The reactor can be a reactor for batch mode or continuous mode, or a reactor that can be operated in both batch mode and continuous mode.

[0087] Preferably, the reactor is selected from a column reactor and a tank reactor. Reactions with unfavourable reaction kinetics can be carried out in a tank reactor, suitably in batch operation. Reactions with favourable reaction kinetics can be carried out in a column reactor, suitably in continuous operation.

[0088] The kinetics of the reaction steps in oligonucleotide synthesis, particularly the rate-limiting reactions, are such that column reactors are preferred. Column reactors for solid phase oligonucleotide synthesis are usually operated in continuous mode and may have configurations that allow for recirculation of reaction solutions.

[0089] The kinetics of the reaction steps in SPPS, specifically the rate-limiting coupling step (where the amide bond is formed), are such that a tank reactor is preferred. Tank reactors for SPPS are generally operated in batch mode.

[0090] The reactor may be equipped with a feedback system that allows the reaction solution to be recirculated.

[0091] According to one embodiment, the reactor is a batch type reactor. The reactor is suitably a tank reactor, preferably designated for batch operation (such as a stirred tank reactor).

[0092] Suitably, the reaction chamber comprises at least one inlet, one outlet, a liquid phase, and a solid phase. The reaction chamber may also comprise means for facilitating mass transfer between the liquid and solid phases, and means for allowing removal of the liquid phase without depleting the solid phase. The reaction chamber is suitably equipped with a replacement liquid distribution system.

[0093] The means for enhancing mass transfer between the liquid and solid phases may be mechanical devices acting on the liquid, including the solid phase; the introduction of irregular disturbances or turbulent motion of the liquid and solid phases. Examples of mechanical devices include impellers such as anchors, propellers, flat blade disk turbines, paddles, gate anchors and helical screws. Increased mass transfer may also be achieved by gas phase such as nitrogen bubbling, vortexing or recirculation.

[0094] The displacement liquid is appropriately distributed to the liquid phase above the solid phase by an appropriate liquid distribution system. The displacement liquid is fed to the liquid phase so that the solid phase can essentially provide a plug flow of the displacement liquid over the solid phase. The displacement liquid is preferably distributed to the liquid phase so that disturbance of the solid phase is minimized. The flow rate of the displacement liquid into the reactor is governed to some extent by the type of distribution system in the reactor. The distribution system may be configured with a nozzle capable of atomizing the liquid into a spray. The nozzle of the distribution system may be configured to be capable of adjusting the particle size of the liquid. If solid particles are present on the walls of the reactor above the liquid phase after the reaction step, it may be advisable to distribute the displacement liquid in a physical form that facilitates the removal of the solid particles from the walls. After washing the reactor walls, it is preferable to reduce the particle size of the liquid by adjusting the nozzle, thereby forming a fine spray that does not disturb the solid phase. Suitably, the inlet flow rate of the displacement liquid must be sufficient to allow the distribution system to function properly, ensure uniform distribution of the fluid, and allow adequate rinsing of the reactor walls to eliminate trace compounds and solid phase particles. Nozzle operation often requires a limited flow range to ensure optimal operation. It is therefore important that the inlet flow rate can be set within an optimal range, appropriately depending on the size of the distribution system, thus making the inflow independent of superficial velocity (percolation velocity) constraints. The superficial velocity is highly correlated with the liquid phase outlet flow rate.

[0095] The reactor is suitably a glass, stainless steel, Hastelloy or jacketed reactor with a preferred volume of about 0.5 up to about 5000 liters. A filtration device is typically placed at the bottom of the reactor to retain the solid phase (resin) while draining the liquid phase. This filtration device may be formed from a sintered stainless steel material, but can be made of porous polymer or filter sheets, or any other filtration system known to those skilled in the art.

[0096] According to one embodiment, the reactor may have at least one inlet for the introduction of resins, reagents and solvents used in the different reaction steps. The liquids are preferably introduced by one or more self-priming pumps. The introduction flow rates and the introduced volumes are suitably measured and quantified by mass flow sensors. Any liquids fed to the reactor may be heated or cooled as required before entering the reactor via a heat exchanger.

[0097] According to one embodiment, the reactor may be configured to allow real-time measurement of the evolution of chemical species in the liquid discharged from the reactor via measurement of a cell, which may be selected from a conductivity cell, a near-infrared cell (e.g., 1 mm to 30 mm optical path), a UV cell (e.g., 0.5 mm to 10 mm optical path), and a refractive index cell.

[0098] As previously suggested, the gas-liquid and liquid-solid phase boundaries can be monitored visually or by at least one sensor. Monitoring of these two phase boundaries can provide the height of the liquid phase above the solid phase. Suitable sensors are sensors that emit ultrasonic or electromagnetic radiation (radar sensors) and lasers (including lidar sensors) at UHF and microwave wavelengths. The height (h) of the liquid phase above the solid phase can be monitored by a suitable sensor or sensors. According to one embodiment, two sensors (detectors) are implemented, one dedicated to monitoring the liquid-solid phase boundary and the other dedicated to monitoring the gas-liquid phase boundary. Preferably, of the two sensors, both are implemented to simultaneously monitor the two phase boundaries. According to a further embodiment, one sensor is implemented that can monitor both phase boundaries. Detection of the liquid-solid and gas-liquid phase boundaries facilitates the regulation of the discontinuous replacement liquid supplied to the reaction chamber.

[0099] In addition to the application of sensors to monitor the height of the liquid phase, the reactor can be equipped with a variety of sensors, including sensors for measuring pressure, conductivity and pH.

[0100] A further advantage of the method of providing a discontinuous supply of replacement liquid to the reaction is that monitoring of the phase boundary and the height of the liquid phase above the solid phase can be performed when no replacement liquid is supplied to the reactor. Measurement of the height of the liquid phase during periods when no replacement liquid is supplied to the reactor provides a more accurate measurement of the phase boundary and therefore the height of the liquid phase. The liquid level is not affected by the replacement liquid. Also, the sensor is not disturbed by liquid particles in the gas phase (sprays of replacement liquid from the distribution system (system of nozzles)). Also, the discontinuous operation of the replacement liquid supply provides more freedom to adjust the flow rate of the replacement liquid. The flow rate of the replacement liquid can therefore be adjusted. For example, the flow rate of the replacement liquid can be reduced periodically. Also, the duration of the supply of replacement liquid can be adjusted. Thus, both the flow rate and the duration of the supply of replacement liquid to the reactor can be changed. The flow rate can be temporarily increased within or between delivery stages / periods. If the flow rate of the replacement liquid needs to be reduced, the duration may be increased so that a constant volume of replacement liquid is supplied to the reactor during each delivery stage.

[0101] According to one embodiment, the method is carried out in a solid phase peptide synthesis protocol. In particular, the method may be carried out after any of the reaction steps of the cycle (or between two reaction steps), more particularly after the deprotection step and after the coupling step.

[0102] Description of the embodiments implemented in the examples The dipeptide is synthesized using the solid-phase peptide synthesis protocol (SPPS) and the Fmoc protecting group of the α-amide. 4-Methylbenzhydrylamine resin hydrochloride (MBHA-resin) with a loading of 1.12 meq / g is used as the solid phase. The MBHA-resin is loaded into a stirred tank reactor (diameter 30 cm) with a filter bottom in the form of a sintered stainless steel material and washed with dimethylformamide (DMF). The reactor is equipped with a stirring blade. At the top of the reactor there are several inlets, of which one inlet is dedicated to the introduction of the resin and one inlet is dedicated to the introduction of the solvent (DMF, piperidine, etc.), introduced by a self-priming pump. The introduction flow rate and the introduced volume are measured and quantified by mass flow sensors. The flow rate can range from 35 l / h to 600 l / h (50 to 850 cm / h). To properly wash the reactor between each step of the synthesis, a device for dispersing a solvent, such as a displacement (washing) liquid, is placed at the end of the line, at the level of the reactor. This device operates properly between 20 and 1000 l / h. An inlet pipe dedicated to the introduction of the agent for deprotecting the amino acids can also be placed at the top of the reactor. This is done by a self-priming pump with a flow rate of 20 to 1000 l / h (28 to 1415 cm / h). The introduction flow rate and the introduced volume are measured and quantified by mass flow sensors. An inlet pipe for an additional solvent, whose volume and flow rate are controlled by mass flow sensors, can also be placed at the top of the assembly reactor.

[0103] The resin is neutralized by the addition of a 10% solution of N,N-diisopropylethylamine (DIEA) in DMF. The Fmoc-protected Rink amide linker is then added to the reactor and the linker is coupled to the resin using DIC / OXYMA. After successful coupling, the resin is washed in DMF.

[0104] The first repeated cycle involves deprotection of the Fmoc group by addition of a 25% piperidine / DMF solution. After deprotection, the resin is washed with DMF and the method of the present invention (discontinuous percolation wash). After successful deprotection, stirring is stopped and the resin (solid phase) is allowed to settle to form a resin bed and a liquid phase above the bed, thereby forming a resin bed that is uniformly and horizontally distributed in the reactor. The wash solvent is introduced uniformly on the surface of the liquid and on the walls of the reactor to remove any traces of piperidine, by-products (as dibenzofulvene) and solid phase. The solvent is introduced when a low level liquid threshold h (min) of 1 cm is reached. The reactor is continuously drained during the percolation wash. Percolation wash is performed until the desired end point of 100 ppm piperidine is reached. Figure 2 shows the discontinuous flow protocol of the wash solution with the height of the liquid phase as a function of time.

[0105] After successful deprotection and after discontinuous washing, the Fmoc-protected amino acid (Fmoc-AA-OH) is introduced into the reactor. The coupling of the Fmoc-protected amino acid is carried out with DIC / OXYMA in DMF (0.5 M). After coupling, the stirring is stopped and the resin (solid phase) is allowed to settle to form a resin bed and a liquid phase above the bed, thereby forming a resin bed that is uniformly and horizontally distributed in the reactor. The washing solvent is introduced uniformly on the surface of the liquid and on the walls of the reactor to remove all traces of reactants (Fmoc-AA-OH), coupling agents (DIC / OXYMA) and by-products (as diisopropylurea). The washing solvent is introduced discontinuously at 600 l / h when a low-level liquid threshold of 3.0 cm h (min) is reached. The reactor is continuously drained during percolation washing with a superficial velocity of 212 cm / h (150 l / h).

[0106] Figure 1 shows a schematic reactor for carrying out the method of the invention. The reactor comprises an inlet (1) and a solvent distribution system (2). Through the inlet and the solvent distribution system, the displacement liquid is fed to the reactor. At the bottom of the reactor there is an outlet (7) for discharging the liquid phase. In the reactor there is a gas phase (3), a layer of liquid phase (4) on a solid (resin) phase (5). A filtration device (6) is arranged at the bottom of the reactor, allowing the discharge of the liquid phase through the outlet (7) without removing the solid phase.

[0107] FIG. 2 shows the height of the liquid phase above the resin as a function of time. The solid (resin) phase layer (16) and the filtration device (17) as well as the liquid phase, e.g. (15), are also included diagrammatically. (8) indicates the level axis, (18) is the time axis. (9) is the high level liquid threshold [h(max)] and (10) is the low level liquid threshold, h(min). The triangles (12) define the duration of the displacement liquid fed to the reactor and the increase in the level of the liquid phase over time. The start of the displacement liquid inflow is triggered by the liquid phase reaching the low level liquid threshold [h(min)] (10). When the level of the liquid phase reaches the high level threshold h(max) (9), the displacement liquid inflow is stopped. The displacement liquid inflow remains inactive until the liquid phase reaches the low level liquid threshold h(min) (10). The triangles (13) define the duration for which the displacement liquid is shut off and the decrease in the liquid phase over time. (14) indicates the variable layer of the liquid phase during discontinuous percolation. (11) indicates the resin bed level, and therefore the liquid-solid boundary.

[0108] Profile of discontinuous percolation cleaning carried out in a 30cm diameter stainless steel reactor. First, the resin bed level (after draining) is measured and recorded using a level sensor. This step is performed after draining the reactor. Before the first unblocking, or Before the final deblocking (after draining the penultimate deblocking solution) Before coupling To do so.

[0109] After the deblocking / coupling step, the stirrer stops at a defined position to avoid interference with the radar probe and to allow level measurement during the percolation step. For this purpose, sensors are installed on the stirrer to ensure a defined position.

[0110] Percolation is started immediately after the deblocking / coupling step without draining the piperidine / coupling solution.

[0111] The bottom valve of the reactor is opened and the drain pump is started at a flow rate set by the user. Drainage is continuous with a constant outflow during the entire percolation sequence.

[0112] When the level sensor reaches the low-level liquid threshold h(min), the inlet valve opens and the inlet pump starts introducing a user-defined volume. The inflow must be higher than the outflow to ensure efficient fluid distribution by the nozzle and flushing of the piperidine / coupling solution.

[0113] The level set point above the resin bed is very important so that under no circumstances the solvent level can fall below the resin bed level, otherwise the resin can dry out and the percolation quality can be affected.

[0114] This cycle can be monitored online as follows: FT-NIR Threshold UV Threshold Or for offline monitoring: Chloranil test pH Spectroscopic analysis (e.g. DNFB) until a stopping criterion selected by the user is reached.

[0115] When the end of the percolation set point is reached, draining stops and the DMF dosing pump is stopped (if dosing is in progress).

[0116] Homogenization is performed immediately after percolation, without draining the wash from the reactor, after which the residual concentration (piperidine or coupling solution) is analyzed by online or offline methods (DNFB, chloranil), and this quantification defines whether additional batch washings need to be performed to reduce the residual concentration or whether the next step needs to be started.

[0117] Discontinuous percolation cleaning carried out in a 6cm diameter glass reactor. First, without draining the reactor, the resin bed level is measured manually.

[0118] Percolation is started immediately after the deblocking / coupling step without draining the piperidine / coupling solution.

[0119] The bottom valve of the reactor is opened and the drain pump is started at a flow rate set by the user. Drainage is continuous with a constant outflow during the entire percolation sequence.

[0120] When the liquid level reaches the low level liquid threshold h(min), the inlet pump starts to inject the solvent until it reaches the maximum height h(max). The inflow must be higher than the outflow to ensure that the solvent level does not fall below the level of the resin bed. Otherwise, the resin may dry out and the percolation quality may be affected.

[0121] This cycle is repeated and the washes are collected in different fractions and analyzed by off-line methods (DNFB, chloranil) to determine the residual concentrations at the reactor outlet.

[0122] When the end of the percolation set point is reached, draining is stopped.

[0123] Homogenization is performed immediately after percolation, without draining the wash from the reactor, after which the residual concentration (piperidine or coupling solution) is analyzed by offline methods (DNFB, chloranil), and this quantification defines whether additional batch washings need to be performed to reduce the residual concentration or whether the next step needs to be started.

[0124] example overview Example 1 relates to a comparison of discontinuous percolation washing according to the invention with continuous percolation washing and batch washing, using a 6 cm diameter glass reactor filled with 4-methylbenzhydrylamine hydrochloride resin (MBHA-polystyrene).

[0125] In examples 2-6, a dipeptide resin, Fmoc-Ala-Gly-Rink amide-MBHA-resin, is used as a model resin to mimic the conditions of solid phase peptide synthesis. Examples 2-5 show the conditions after the deprotection step of the cycle.

[0126] Example 6 provides the conditions after the coupling step of the cycle.

[0127] Examples 2 and 6 relate to a comparison of batch cleaning with discontinuous percolation cleaning according to the present invention.

[0128] Examples 3-5 show the effect of several parameters of discontinuous percolation washing (superficial velocity, varying height of the liquid phase above the solid phase, change in maximum height of the liquid phase above the solid phase) on the washing volume and time at a given endpoint.

[0129] System Description In the following examples, two different reactors are used: a glass reactor with a diameter of 6 cm and a stainless steel reactor with a diameter of 30 cm.

[0130] Both reactors are equipped with a stirred tank reactor (STR) that includes an impeller. In addition, the reactor has a filtration device at the bottom to retain the solid phase while draining the liquid. At the top of the reactor there are several inlets, one of which is dedicated to the introduction of the resin and another is used for the introduction of various solvents such as synthesis solvents and displacement liquids. At the bottom of the reactor there is an outlet that is used to drain the liquid. The volume and flow rate of the solvent / liquid are measured by mass flow sensors. The reactors further comprise a distribution system that distributes the incoming liquid in such a way that the walls of the reactor are washed. The stainless steel reactor is also equipped with a sensor for measuring the phase boundary and thereby the height of the liquid phase above the solid phase.

[0131] The same reactor is used in each example comparing discontinuous percolation cleaning with batch cleaning.

[0132] In example 1 a glass reactor with a diameter of 6 cm is used. In all other examples a stainless steel reactor with a diameter of 30 cm is used.

[0133] All examples use the same base resin (solid phase): 4-methylbenzhydrylamine hydrochloride resin (MBHA-polystyrene).

[0134] Batch cleaning explained The batch wash step is performed by continuous batches consisting of introducing the wash solvent, stirring for 5 minutes, and then draining. The end point of the batch wash step after deprotection is determined by measuring the residual concentration of piperidine in the wash solution by near-IR quantification. After coupling, the end point is established by measuring the absorbance of the residual coupling solution (e.g., coupling agent, amino acid, and by-products) in the wash solution by UV quantification.

[0135] Explanation of discontinuous percolation cleaning. Discontinuous percolation washing is carried out on a resin arranged as a fixed bed in a reactor with a filtering bottom and distributed evenly and horizontally on its surface to avoid any preferential passage of the washing solvent through the resin bed.

[0136] Example 1 In this example, the discontinuous percolation wash of the present invention is compared to a continuous percolation wash and a batch wash in terms of wash volume and duration to achieve a predetermined end point, defined as residual piperidine in the discharged liquid phase.

[0137] In this example, different washing procedures are tested during the post-deprotection step conditions of a cycle using DMF / piperidine for deprotection of base-labile α-amine protecting groups (eg, Fmoc).

[0138] A 6 cm diameter glass stirred tank reactor (STR) is filled with 4-methylbenzhydrylamine hydrochloride resin to a height of 6 cm. The deprotection agent DMF / piperidine (25%) solution is then added to the reactor and the resin is homogenized in the solution by stirring for less than 10 minutes. The solution is drained from the reactor and the deprotection agent DMF / piperidine (25%) solution is added to the reactor and the resin is homogenized in the solution by stirring for less than 5 minutes. The deprotection solution is drained from the reactor in the case of batch washing. The homogenized resin with the deprotection solution is the starting point for the evaluation of two different percolation washing operations.

[0139] Discontinuous percolation: The agitator is stopped and the resin bed is distributed evenly and horizontally in the reactor. The washing solvent (displacement liquid), DMF, is introduced evenly on the liquid surface and on the walls of the reactor to remove all traces of deprotection agent (piperidine) that need to be removed. When a low level liquid threshold h(min) of 1 cm is reached, the solvent is introduced into the reactor until a high level liquid threshold h(max) of 3 cm is reached. The reactor is continuously drained during percolation washing at a flow rate of 1.3 liters / hour to obtain a superficial velocity of 45 cm / hour on the resin bed. The percolation washing is carried out until the desired end point of 100 ppm piperidine is reached.

[0140] Batch wash: The wash solution (DMF) is introduced uniformly and stirred for 5 minutes. The reactor is then drained. The batch wash is repeated until the desired end point of 100 ppm piperidine is reached.

[0141] Continuous percolation: The agitator is stopped and the resin bed is distributed evenly and horizontally in the reactor. The washing solvent is introduced evenly on the surface of the liquid and on the walls of the reactor to remove all traces of the agent (piperidine) that are to be removed. The solvent is continuously introduced and drained during the percolation wash with a superficial velocity of 45 cm / h, and the liquid level is stable at 3 cm above the resin bed. The flow rate of the washing solvent is therefore equal to the flow rate of the liquid phase exiting the reactor. Continuous percolation washes are performed until the desired end point of 100 ppm piperidine is reached.

[0142] The volumes and durations of the washing fluid (displacement fluid) at the end points set for the three washing procedures are shown in Table 1. [Table 1]

[0143] Examples 2-6: Examples 2-5 all use the same MBHA-resin as in Example 1 with the additional dipeptide covalently attached to the resin by a Rink amide linker. The dipeptide resin, Fmoc-Ala-Gly-Rink amide-MBHA-resin, is prepared according to the protocol shown in Table 2. In Example 6, NH2-Gly-Rink amide-MBHA-resin is prepared and Fmoc-Ala-OH is coupled.

[0144] Table 2 describes the solid phase synthesis of the dipeptide linker resins used in Examples 6 to 10. [Table 2] Table 2: Overview of the SPPS procedure to provide Fmoc-Ala-Gly-Rink amide-MBHA-resin.

[0145] Example 2 Piperidine cleaning efficiency as a function of cleaning method.

[0146] In this example, the discontinuous percolation wash of the present invention is compared to a batch wash in terms of wash volume and duration to achieve a predetermined displacement (percolation) end point, defined as residual piperidine in the discharged liquid phase.

[0147] Batch and discontinuous percolation washes are tested during post-deprotection step conditions of the cycle using DMF / piperidine for deprotection of base-labile α-amine protecting groups (eg, Fmoc).

[0148] A stainless steel stirred tank reactor (STR) with a diameter of 30 cm and containing a bottom filter is filled with Fmoc-Ala-Gly-Rink Amide-MBHA-resin to a height of 10 cm. The deprotection agent DMF / piperidine (25%) solution is then added to the reactor and the resin is homogenized in the solution by stirring for less than 10 minutes. The solution is drained from the reactor and the deprotection agent DMF / piperidine (25%) solution is added to the reactor and the resin is homogenized in the solution by stirring for less than 5 minutes. The deprotection solution is drained from the reactor in case of batch washing. The homogenized resin with the deprotection solution is the starting point for the evaluation of the percolation washing operation.

[0149] Discontinuous percolation: The agitator is stopped and the resin bed is distributed evenly and horizontally in the reactor. The washing solvent, DMF, is introduced evenly on the surface of the liquid and on the walls of the reactor to remove all traces of deprotection agent (DMF / piperidine) that need to be removed. When the low level liquid threshold h(min) of 3.0 cm is reached and ends at the high level liquid threshold h(max) of 4.0 cm, the washing solvent (1 liter of DMF) is introduced into the reactor at a flow rate of 600 l / h. The supply of the washing liquid is interrupted for about 20-25 seconds. The reactor is continuously drained during the percolation wash at a flow rate of 150 l / h, which represents a superficial velocity of 212 cm / h on the resin bed. The percolation wash is carried out until the desired end point of 100 ppm piperidine is reached.

[0150] Batch wash: uniformly introduce wash solution (6 liters of DMF) and stir for 5 minutes. Then drain the reactor. Repeat batch wash until desired end point of 100 ppm piperidine is reached. [Table 3]

[0151] Example 3 Effect of superficial velocity on piperidine washing.

[0152] The flow rate of the washing solvent is optimized according to the diffusion rate of piperidine (species) to allow the piperidine (species) to be removed to move from the solid phase (resin) to the liquid phase (washing solvent). Thus, a too high flow rate means excessive consumption of washing solvent and increased washing duration. Moreover, if the outflow is too high, piperidine may not diffuse into the liquid phase fast enough, which may increase the piperidine content after homogenization. In this case, additional batch washing is required to reach the final end point.

[0153] The same stainless steel reactor as in example 2 is used. In addition, it is filled with Fmoc-Ala-Gly-Rink Amide-MBHA-resin (height 10 cm) and homogenized with DMF / piperidine solvent according to the protocol of example 2.

[0154] Stop the agitator and distribute the resin bed evenly and horizontally in the reactor. Introduce the washing solvent (DMF) evenly on the liquid surface (resin) and on the walls of the reactor to remove all traces of the drug to be removed. Introduce the solvent (1 liter of DMF) at 600 l / h when the low level liquid threshold of 3.0 cm h (min) is reached and interrupt at the high level liquid threshold as shown in Table 4. Interrupt the supply of washing liquid for about 20-25 seconds. Drain the reactor continuously during percolation washing at a flow rate that produces a superficial velocity in Table 4. Percolation washing is carried out until the desired end point of 100 ppm piperidine is reached. [Table 4]

[0155] For percolation at 424 cm / h, the percolation was stopped after 5 min when the piperidine content was below 100 ppm (corresponding to a consumption of 20.8 liters of DMF). However, after homogenization, the piperidine content increased due to delayed diffusion from the solid phase to the liquid phase. As a result, an additional batch wash (6 liters of DMF, stirred for 5 min) was performed to reduce the concentration below the desired end point. The superficial velocity of the wash should be adapted to the diffusion rate between the liquid and solid phases. If the superficial velocity is too high for diffusion, the end point will be reached before the diffusion equilibrium is obtained, requiring additional percolation or batch wash.

[0156] Example 4 Effect of minimum liquid level on resin on piperidine wash efficiency.

[0157] The same stainless steel reactor as in Example 2 is used. In addition, the Fmoc-Ala-Gly-Rink Amide-MBHA-resin (height 10 cm) is charged into the reactor and homogenized with DMF / piperidine solvent according to the protocol of Example 2. The stirrer is stopped and the washing solvent, DMF, is introduced uniformly so as not to disturb the resin bed and so that the surface of the resin bed remains horizontal. In order to limit remixing phenomena and thus optimize the washing step, a discontinuous solvent introduction is performed when a low level liquid threshold h (min) is reached. In order to verify the influence of the liquid level on the resin, the flow rate of the washing solvent introduced into the reactor is made the same as the flow rate of the liquid phase discharged from the reactor.

[0158] The resin bed is distributed evenly and horizontally in the reactor. The washing solvent (DMF) is introduced evenly on the surface of the liquid and on the walls of the reactor to remove any traces of the drug that need to be removed. The washing solvent is introduced at a flow rate of 600 l / h when the low level liquid threshold h(min)min is reached and is interrupted at the high level liquid threshold, as shown in Table 5. The reactor is continuously drained with a superficial velocity of 212 cm / h, which corresponds to a flow rate of 150 l / h of the discharged liquid phase. Percolation washing is performed until the desired end point of 100 ppm piperidine is reached. [Table 5]

[0159] Example 5 Effect of maximum liquid level on resin on piperidine wash efficiency.

[0160] The same stainless steel reactor as in Example 2 is used. Furthermore, Fmoc-Ala-Gly-Rink Amide-MBHA-resin (height 10 cm) is charged into the reactor and homogenized with DMF / piperidine solvent according to the protocol of Example 2. The stirrer is stopped and the washing solvent, DMF, is introduced uniformly so as not to disturb the resin bed and so that the surface of the resin bed remains horizontal. In order to limit backmixing phenomena and thus optimize the washing step, a discontinuous washing solvent introduction is performed when a low level liquid threshold h(min) is reached. After the solvent introduction, the liquid level reaches a maximum level h(max) depending on the inflow and partial volume.

[0161] The resin bed is distributed evenly and horizontally in the reactor. The washing solvent (DMF) is introduced evenly on the surface of the liquid and on the walls of the reactor to remove any traces of the drug that need to be removed. When the low level liquid threshold h(min) of 3.0 cm is reached and interrupted at the high level liquid threshold h(max) as shown in Table 6, the solvent (DMF) is introduced at a flow rate of 600 l / h. The reactor is continuously drained with a superficial velocity of 212 cm / h. The liquid phase is discharged at a flow rate of 150 l / h. Percolation washing is performed until the desired end point of 100 ppm piperidine is reached. [Table 6]

[0162] Example 6 Coupling solution washing efficiency as a function of washing method.

[0163] The same stainless steel reactor as in examples 2-5 is used. The reactor is filled with NH2-Gly-Rink Amide-MBHA-resin (height 10 cm) and a coupling solution containing Fmoc-Ala-OH / DIC / Oxyma (3 / 3 / 3 equiv.) in DMF (0.5 M) is added. The resin is homogenized by stirring until the reaction reaches completion (reaction end point) as monitored by offline analysis (i.e., Kaiser test). The Kaiser test is based on the reaction of ninhydrin with the primary amine from the N-terminal amine group of the deprotected peptide resin. The coupling solution is then drained from the reactor in case of batch washing.

[0164] The (displacement) endpoint was determined by UV after 5 batch washes (5.2 L each) and the percolation washes were then run until the same endpoint was reached (0.254 uA for a 0.5 mm path at 301 nm wavelength).

[0165] Discontinuous percolation: the agitator is stopped and the resin bed is distributed evenly and horizontally in the reactor. The washing solvent is introduced evenly on the surface of the liquid and on the walls of the reactor to remove all traces of the agent (piperidine) that need to be removed. The solvent is introduced at 600 l / h when a low level liquid threshold h(min) of 3.0 cm is reached and the introduction is stopped when a high level liquid threshold h(max) of 4 cm is reached. The reactor is continuously drained during the percolation wash with a superficial velocity of 212 cm / h (and a flow rate of the discharged liquid of 150 l / h).

[0166] Batch wash: The wash solution (5.2 liters of DMF) is uniformly introduced and stirred for 5 minutes. The reactor is then drained. [Table 7]

Claims

1. 1. A method for substituting compounds contained in a liquid phase and a solid phase present in particulate form dispersed in a liquid phase in a reaction step of a heterogeneous chemical reaction protocol, for the formation of a target compound synthesized by successive introduction of subunits (compounds) by repeated (iterative) cycles, each cycle comprising a reaction step, the method comprising: providing a reactor in which said reacting step takes place, said reactor comprising a gas phase, a liquid phase, a solid phase, and means for draining said liquid phase from said reactor without essentially removing said solid phase from said reactor; removing the liquid phase from the reactor, wherein a displacement liquid is discontinuously fed to the reactor so that a layer of the liquid phase is always maintained above the solid phase; A method comprising:

2. 10. The method of claim 1, wherein the liquid phase from the reactor is removed continuously.

3. 2. The method of claim 1, comprising determining a gas-liquid phase boundary and a liquid-solid phase boundary, thereby establishing a height (h) of the liquid phase above the solid phase, wherein the height (h) is never zero.

4. The method of claim 3 wherein the height (h) varies over time.

5. 4. The method of claim 3, wherein the height (h) of the liquid phase varies by up to about 1000%, preferably by up to about 500%, more preferably by up to about 300%, more preferably by up to about 200%.

6. 4. The method of claim 3, wherein the minimum height h(min) of the liquid phase above the solid phase is about 0.5 cm, preferably about 1 cm, preferably about 2 cm, about 3 cm, about 4 cm, about 5 cm.

7. 4. The method according to claim 3, wherein the maximum height h(max) of the liquid phase above the solid phase is about 20 cm, preferably about 10 cm, preferably about 10 cm, preferably about 7 cm, preferably about 6 cm, preferably about 5 cm, preferably about 4 cm, preferably about 2 cm.

8. 4. The method of claim 3, wherein the difference between h(max) and h(min) is from about 0.1 cm to a maximum of about 20 cm, preferably from about 0.1 cm to a maximum of about 10 cm, preferably from about 0.2 cm to a maximum of about 4 cm.

9. 4. The method of claim 3, wherein the ratio of h(min) to h(max) [ratio: h(max) / h(min)] is from about 1.1 to about 6.

10. 2. The method of claim 1, wherein the superficial velocity of the displacement liquid (over the solid phase) in the reactor ranges from about 100 cm / hr to a maximum of about 400 cm / hr, preferably from about 150 cm / hr to a maximum of about 300 cm / hr, and preferably from about 175 cm / hr to a maximum of about 250 cm / hr.

11. 2. The method of claim 1, wherein the flow rate of the displacement liquid into the reactor is higher than the flow rate of the liquid phase discharged from the reactor.

12. 12. The method of claim 11, wherein the flow rate of the displacement liquid into the reactor is from about 110% to a maximum of about 500% of the flow rate of the liquid phase exiting the reactor.

13. The method of claim 1 , wherein the gas-liquid phase boundary and the liquid-solid phase boundary are monitored by at least one detector.

14. The method of claim 13 , wherein the detector is selected from an ultrasonic, radar, or laser sensor.

15. 14. The method of claim 13, wherein acquisition of data from the at least one detector occurs when the displacement liquid is not supplied to the reactor.

16. 2. The method of claim 1, wherein the target compound is selected from peptides and oligonucleotides, and the subunits are selected from amino acids, nucleosides, and derivatives thereof.

17. The method of claim 1 , wherein the target compound is a peptide and the subunits are amino acids and derivatives thereof.

18. 18. The method of claim 17, wherein the heterogeneous chemical reaction is solid phase peptide synthesis (SPPS).

19. 17. The method of claim 16, wherein the reactor comprises means for facilitating mass transfer between the liquid and solid phases and at least between the inlet and outlet.

20. 20. The method of claim 19, wherein the means for facilitating mass transfer between the liquid and solid phases is not activated when the displacement liquid is drained from the reactor.

21. 20. The method of claim 19, wherein the means for facilitating mass transfer between the liquid and solid phases is turned off when the reaction step of the cycle reaches a predetermined reaction endpoint and remains off (inoperative) for a period (time) defined by the initial supply of replacement liquid until the replacement endpoint is reached.

22. 20. The method of claim 19, wherein the means for promoting mass transfer between the liquid phase and the solid phase is turned on after a displacement endpoint is achieved, thereby resulting in a homogenized dispersion of the solid phase in the liquid phase, and whether the endpoint is maintained is established.

23. 17. The method of claim 16, wherein the solid phase is selected from a silica-containing material and a polymeric material comprising reactive sites capable of covalently binding subunits of the target compound, optionally via a linker.

24. 10. The method of claim 1, wherein the liquid phase removed from the reactor is monitored by at least one detector for monitoring at least one species in the removed liquid phase to establish an endpoint, such as a reaction endpoint or a displacement endpoint.

25. 25. A method for synthesizing peptides or oligonucleotides by application of a solid phase peptide synthesis protocol, comprising a method as defined by any one of claims 1 to 24.

26. 1. A method for displacing compounds contained in a liquid phase and a solid phase in reaction steps of a solid phase peptide synthesis reaction protocol to form a target peptide synthesized by successive introduction of amino acids and their derivatives by repeated (iterative) cycles, each cycle comprising a reaction step in a liquid phase and a solid phase present in particulate form dispersed in the liquid phase, the method comprising: providing a tank reactor in which said reacting step takes place, said tank reactor comprising an inlet, an outlet, means for facilitating mass transfer between said liquid and solid phases, a gas phase, a liquid phase, a solid phase, and means for discharging said liquid phase from said reactor without essentially removing said solid phase from said reactor; removing the liquid phase from the reactor through the outlet, wherein the reactor is discontinuously supplied with a displacement liquid through the inlet, so that the liquid phase is always maintained above the solid phase until a predetermined endpoint is reached; A method comprising:

27. 27. The method of claim 26, wherein the liquid phase is continuously removed from the reactor.

28. 27. The method of claim 26, further comprising settling the solid phase, thereby forming a liquid-solid phase boundary, a liquid phase above the solid phase, and a gas-liquid phase boundary.

29. 29. The method of claim 28, wherein the reactor comprises at least one sensor capable of detecting the solid-liquid phase boundary and the gas-liquid phase boundary, thereby providing a height h of the liquid phase above the solid phase.

30. 30. The method of claim 29, wherein the minimum height h(min) of the liquid phase above the solid phase is about 0.5 cm, preferably about 1 cm, preferably about 2 cm, about 3 cm, about 4 cm, about 5 cm.

31. 30. The method of claim 29, wherein the maximum height h(max) of the liquid phase above the solid phase is about 20 cm, preferably about 10 cm, preferably about 7 cm, preferably about 6 cm, preferably about 5 cm, preferably about 4 cm, preferably about 2 cm.

32. 30. The method of claim 29, wherein the difference between h(max) and h(min) is from about 0.1 cm to a maximum of about 20 cm, preferably from about 0.1 cm to a maximum of about 10 cm, preferably from about 0.2 cm to a maximum of about 4 cm.

33. 33. The method of any one of claims 26 to 32, wherein the superficial velocity of the displacement liquid (over the solid phase) in the reactor ranges from about 100 cm / hr to a maximum of about 400 cm / hr, preferably from about 150 cm / hr to a maximum of about 300 cm / hr, preferably from about 175 cm / hr to a maximum of about 250 cm / hr.

34. 1. A method for displacing compounds (and / or solvents) contained in the liquid and solid phases of a reaction step in solid-phase peptide synthesis, comprising the steps of: providing a reactor in which said reaction step occurs, said reactor comprising a liquid phase and a solid phase, and means for draining said liquid phase from said reactor without essentially removing said solid phase from said reactor; and discontinuously (intermittently) supplying a displacement liquid to said reactor while said liquid phase is removed from said reactor, wherein a continuous layer of the liquid phase is maintained over said solid phase.

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