Continuous gradient elution chromatography fractionation

Abstract

The present invention relates to a method for separating a desired product from impurities and an apparatus for carrying out the method. The method includes, in the order shown, (1) loading a first volume of a feed solution containing the desired product and impurities onto a chromatography matrix; (2) contacting the chromatography matrix with an elution solution; (3) a) optionally collecting elution fraction 1 (EF1) in a subfractionation container (SFC), b) elution fraction 2 (EF2) in a product container, and c) optionally collecting elution fraction 3 (EF3) in the SFC, wherein at least one of EF1 and EF3 is collected; and (4) simultaneously or subsequently loading EF1 and / or EF3 and a second volume of the feed solution onto the chromatography matrix. Steps (2) through (4) are repeated at least once, and the chromatography matrices in steps (1) and (4) can be the same or different.

Description

[Technical Field]

[0001] The present invention relates to the field of chromatography, and more particularly to a continuous gradient elution chromatographic fractionation method that allows for highly effective separation and purification of a product in solution from impurities. [Background technology]

[0002] Chromatography is a widely used unit operation for analytical and preparative separations, particularly for producing high-purity products such as pharmaceuticals. In these cases, the desired target component is often closely eluting with impurities, as shown in Figure 1. To achieve high purity, narrow product fractionation is one of the primary objectives. However, narrow product fractionation results in product loss because the target product often elutes with impurities in a bell-shaped fashion. Therefore, reducing this loss is one of the objectives of the present invention.

[0003] Several chromatographic methods are known in the art. Specific continuous chromatography methods are described by Subramanian (Continuous biomanufacturing. Innovative Technologies and Methods. 2018, Weinheim: WILEY-VCH), Zobel-Roos (Integrated continuous chromatography. 1. Augmented reality, 2018. Herzogenrath: Shaker (Thermische Verfahrens-und Prozesstechnik)), and Schmidt-Traub et al. (Preparative chromatography. 2., completely revised and updated ed. 2012, Weinheim, Germany: WILEY-VCH).Continuous chromatography includes (i) simulated moving bed chromatography (SMB) as described by Imamoglu (Simulated moving bed chromatography (SMB) for application in bioseparation. Advances in biochemical engineering / biotechnology 2105, Vol. 76, pp. 211-231) and Rodrigues (Simulated Moving Bed Technology. Principles, Design and Process Applications. 2015, Burlington: Elsevier Science); (ii) Holzer (Sequential Multi-Column Chromatography. BPI. Duesseldorf, April 2013) and Bischops (BioSMB™ Technology: Continuous Countercurrent Chromatography Enabling a Fully Disposable Process. Ganapathy Subramanian (Hg.): Biopharmaceutical Production Technology. 1). sted. 2012, Weinheim: WILEY-VCH, pp. 769-791), Whitford (Single-Use Systems As Principal Components in Bioproduction. BioProcess International 2010, Vol. 8(11), pp. 34-44), Angarita et al. (Twin-column CaptureSMB: a novel cyclic process for protein Affinity Chromatography. Journal of Chromatography, Vol. 1389, 2015, pp. 85-95) and Godawat et al. (Periodic countercurrent chromatography - design and operational considerations for integrated and continuous purification of proteins. Biotechnology Journal 2012, Vol. 7(12), pp. 1496-1508), and (iii) sequential chromatography as described by Aumann und Morbidelli (A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process. Biotechnology and Bioengineering 2007, Vol. 98(5), p. 1043-1055); Mueller-Spaeth und Morbidelli (Continuous Chromatography for the Purification of Monoclonal Antibodies. Uwe Gottschalk (Hg.): Process scale purification of antibodies. Hoboken, NJ: John Wiley&Sons, 2009, p. 223-238) and Steinebach et al.This includes several subcategories, such as multicolumn countercurrent solvent gradient purification (MCSGP), which is described in "Continuous countercurrent chromatography for capture and polishing steps in biopharmaceutical production. Biotechnology Journal 2016, Vol. 11(9), pp. 1126-1141."

[0004] Specifically, multicolumn countercurrent solvent gradient purification (MCSGP) is a process originally devised for continuous center-cut operations in gradient chromatography (Aumann & Morbidelli 2007, Aumann et al. (Parametric study of a 6-column countercurrent solvent gradient purification (MCSGP) unit. Biotechnology and Bioengineering, 2007, Vol. 98(5), pp. 1029-1042), Stroehlein et al. (A continuous, countercurrent multi-column chromatographic process incorporating modifier gradients for ternary separations. Journal of chromatography, 2006, Vol. 1126(1-2), pp. 338-346), Mueller-Spaeth (Purification of monoclonal antibodies by continuous chromatography. Zugl.: Zuerich, Diss., Eidgenoessische Technische Hochschule ETH Zuerich, Nr. 18066, 2009). As shown in Figure 2, three columns are operated separately and are interconnected. The individual columns elute the pure product (red) and strongly (green) and weakly (blue) bound minor components. The interconnected columns separate the overlapping region of the product and impurities. After a given time, each column is switched one position to the left. In the connected line, the left column is eluted with the highest elution strength, the middle column with a lower elution strength, and the right column is in equilibrium. Components flow from one column to the other, where they recombine. This requires a lower elution strength of the liquid phase between the columns. Thus, the flow is diluted.

[0005] In an improved version of this method, the six-column setup shown in Figure 2 was modified and adapted to a twin-column setup (Aumann and Mortidelli 2008; Kraettli et al. (Online control of the twin-column countercurrent solvent gradient process for biochromatography. Journal of Chromatography. Vol. 1293, 2013, pp. 51-59); Kraettli et al. (Closed-loop control of the multi-column solvent gradient purification process. Journal of Chromatography, 2011, Vol. 1218(50), pp. 9028-9036); Mueller-Spaeth et al. 2013; Steinebach et al. 2016). This twin-column setup requires eight steps, as shown in Figure 3. In the first step, the cycle begins by loading the overlap of weakly binding components (W) and product (P) from column 1 to column 2. To account for the elution strength of the fractions (e.g., the amount of modifiers, such as salts or polar solvents), the feed is diluted in-line with pure eluent (E). In the second step, once the purity criteria are met, the product (P) is obtained from column 1, and column 2 is loaded with the feed. In the third step, when the overlap between the product and strongly binding impurities (S) is about to elute, column 1 is reconnected to column 2, and the fractions are diluted in-line with eluent (E). In the fourth step, the gradient of column 2 is started, and weakly binding components (W) are eluted and transported to waste. The gradient of column 1 is then terminated, and strongly binding impurities (S) are discharged. This process is repeated once, but the roles of columns 1 and 2 are switched (steps 5-8). The selection of the cutpoint and the in-line dilution are crucial for this process. Additionally, the columns must be synchronized. Feed loading and product elution (see steps 2 or 6) must occur simultaneously and take the same time. Otherwise, impurities will shift.It is intended that weakly binding impurities from column 1 are loaded onto column 2 before feed loading, and strongly binding components are loaded after feed loading.

[0006] Prior art methods, specifically the two-column MCSGP process, have obvious drawbacks. First, there is no continuous feed loading. A continuous purification process is typically defined by at least one continuous flow. For example, in a twin-column MCSGP, both flows are discontinuous, and feed loading and product elution (red arrows in Figure 4) occur only in steps 2 and 6. Note that the eluted product is not loaded onto the other column. Second, as shown in Figure 4, there is a lack of synchronization between the individual steps. For an ideal twin-column MCSGP process, the feed loading of one column and the gradient of the other column must be synchronized, so that both steps take the same amount of time. However, in many cases, one step takes much longer than the other. In particular, the feed loading step usually takes much longer than the elution step. Therefore, the method often exhibits a loading step that requires 5 to 10 times the length of the elution step. Therefore, steps 2 and 6 take as long as the columns need to be loaded. Meanwhile, the other column can only perform product elution at a very low flow rate, or it can perform product elution at an ideal rate but then must be stopped. Both are disadvantageous, for example, due to diffusion processes. Similar problems occur after steps 4 and 8. After gradient separation, each column undergoes high-elution regeneration and re-equilibration (compare the blue and yellow lines in Figure 4). This re-equilibration must be completed before loading the weakly bound stack (steps 1 and 5). Therefore, the other column must wait again. Because column loading is very long compared to the time required to elute the product, the classical MCSGP process exhibits relatively low productivity. In summary, one column must wait in its gradient until the other column is fully loaded, and immediately thereafter, the same column forces the other column to wait until the post-elution step is complete. This procedure is very inefficient and time-consuming. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need for new chromatographic methods that overcome the drawbacks of the prior art, such as slow and inefficient methods and undesirable diffusion processes in columns that are not fed and not eluted (i.e., retained). There is also a need for new chromatographic methods that reduce product loss. [Means for solving the problem]

[0008] In a first aspect, the present invention provides a method for separating a desired product from impurities, the method comprising, in the given order: (1) loading a first volume of a feed solution containing the desired product and impurities onto a chromatography matrix; (2) contacting the chromatography matrix with an elution solution; (3)a) optionally, elution fraction 1 (EF1) in a subfraction container (SFC); b) Elution Fraction 2 (EF2) in the product container, and c) optionally, elution fraction 3 (EF3) during SFC wherein at least one of EF1 and EF3 is recovered; (4) simultaneously or sequentially loading EF1 and / or EF3 and a second volume of feed solution onto a chromatographic matrix; Including, Steps (2) to (4) are repeated at least once, at least twice, at least four times, preferably at least nine times, at least fourteen times, more preferably at least nineteen times, at least twenty-four times, and most preferably at least twenty-nine times; The chromatographic matrices in steps (1) and (4) may be the same or different chromatographic matrices.

[0009] According to a preferred embodiment, the chromatographic matrix in steps 1 and 4 is the same first chromatographic matrix, or the chromatographic matrix in step 1 is the first chromatographic matrix and the chromatographic matrix in step (4) is a second chromatographic matrix, and each time steps (2) to (4) are repeated, the chromatographic matrix alternates between the second chromatographic matrix and the first chromatographic matrix.

[0010] According to further preferred embodiments, the volume of EF1 and / or EF3 recovered in SFC in step (3) is at least about 0.05, at least about 0.25, at least about 0.5, at least about 1, at least about 1.5 or at least about 2.0 volumes of the chromatography matrix.

[0011] According to a particularly preferred embodiment, the concentration of the eluting agent contained in the elution solution is increased over time during step (2), the eluting agent weakening the interaction between the product of interest and the chromatographic matrix.

[0012] According to one embodiment, EF1 and / or EF3 contain the product of interest and impurities, and the concentration of the product of interest is increased by at least about 2-fold, preferably at least about 5-fold, and more preferably at least about 10-fold compared to the solution loaded in step 1. In this embodiment, the recovery of EF1 is determined by the first predetermined concentration X of the product of interest in the eluate. EF1-P and a predetermined concentration Y of the desired product in the eluate EF1-P and / or the recovery of EF3 is stopped at a first predetermined concentration X of the product of interest in the eluate. EF3-P and a predetermined concentration Y of the desired product in the eluate EF3-P Additionally or alternatively, the recovery of EF1 is stopped at a first predetermined concentration X of impurities in the eluate. EF1-I and a predetermined concentration Y of impurities in the eluate EF1-I and / or the recovery of EF3 is stopped at a first predetermined concentration X of impurities in the eluate. EF3-Iand a predetermined concentration Y of impurities in the eluate EF3-I Additionally or alternatively to this embodiment of the invention, EF2 comprises a substantially pure product of interest, and recovery of EF2 is stopped at a predetermined concentration X of the product of interest in the eluate. EF2-P and a predetermined concentration Y of the desired product in the eluate EF2-P and / or the recovery of EF2 is stopped at a predetermined concentration of impurities in the eluate, X EF2-I and a predetermined concentration Y of impurities in the eluate EF2-I will be stopped.

[0013] According to a preferred embodiment, step (3) further comprises diluting EF1 and / or EF3. The dilution is preferably carried out by SFC. Also preferably, EF1 and / or EF3 are diluted with one or more of the feed, chromatography buffer and water.

[0014] According to one embodiment, the second volume of step (4) is the same as the first volume of step (1). According to an alternative embodiment, the second volume of step (4) is smaller than the first volume of step (1).

[0015] According to a particularly preferred embodiment, EF1 and / or EF3 recovered from the first chromatographic matrix are recovered in a first SFC, and EF1 and / or EF3 recovered from the second chromatographic matrix are recovered in a second SFC.

[0016] According to yet another embodiment, the load is stopped in steps (1) and (4) before any product of interest is eluted from the chromatographic matrix in the flow-through.

[0017] According to one embodiment, steps (1) and (4) involve binding the product of interest to a chromatographic matrix.

[0018] According to a preferred embodiment, the chromatographic matrices of steps (1) and (4) are of the same type.

[0019] According to another embodiment, the chromatographic method of steps (1) and (4) is selected from the group consisting of reverse phase chromatography, hydrophobic interaction chromatography, affinity chromatography, ion exchange chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, chiral chromatography, hydrophilic interaction liquid chromatography, size exclusion chromatography, and dielectric chromatography.

[0020] According to a preferred embodiment, the chromatographic method in steps (1) and (4) is reverse phase chromatography.

[0021] According to a preferred embodiment, the eluent contained in the elution solution is a polar eluent, preferably selected from the group consisting of acetonitrile, benzyl alcohol, methanol, acetic acid, ethylene glycol, tetrahydrofuran, ethanol, 1-propanol and 2-propanol.

[0022] According to a preferred embodiment, the chromatographic matrix in steps (1) and (4) is a chromatographic column.

[0023] According to certain preferred embodiments, the product of interest is a polypeptide or protein.

[0024] According to one embodiment of the present invention, a chromatography device includes one or more chromatography matrices having first and second ends, a feed container, one or more subfraction containers (SFCs), conduit means connecting the second ends of the one or more chromatography matrices to the one or more subfraction containers, conduit means connecting the one or more subfraction containers to the first ends of the one or more chromatography matrices, conduit means connecting the feed container to the one or more subfraction containers, and conduit means connecting the feed container to the first ends of the one or more chromatography matrices. The one or more subfraction containers in this embodiment have a volume that is about 0.05 to about 8 volumes of the chromatography matrix.

[0025] Further aspects and embodiments are disclosed in the accompanying claims and the following detailed description of the invention. [Brief explanation of the drawings]

[0026] [Figure 1] Chromatogram of a protein eluting from a state-of-the-art chromatography column. The target component (red) elutes closely with impurities (light blue and green). The dark blue line shows the total signal seen by the detector. [Figure 2] Schematic of the six-column MCSGP operation as described in Mueller-Spaeth, 2014. [Figure 3] Schematic diagram of the twin-column MCSGP process over one complete cycle by Kraettli et al. (Journal of Chromatography 2013, Vol. 1293, pp. 51-59). [Figure 4]Schedule of different process steps for a conventional twin-column MCSGP. The scheme is divided at the y-axis value 0. The top shows the feed loading (orange line) and elution gradient (blue line) of column 1. The bottom shows the feed loading (light blue line) and elution gradient (yellow line) of the second column. The gray line is the cut point for the overlap of product and impurities migrating from one column to the other, as indicated by the red / gray arrows. The red arrow indicates the elution of the product. [Figure 5] 1 is a flow chart of the process of the present invention. [Figure 6] Schematic diagram of a preferred embodiment of the device of the present invention having two matrices (columns 1 and 2) and two subfraction containers (P1, P2). [Figure 7] Purity and yield of the method of the present invention over cycle number. Each cycle includes one repetition of steps (2)-(4), with the first round denoted as X.1 and the repetition of steps (2)-(4) denoted as X.2. The first cycle 1.1 included step (1), and subsequent cycles 2-5 did not include step (1). The blue line indicates purity. The gray line indicates cycle yield relative to the amount of feed loaded. The yellow line indicates overall yield. The orange line indicates yield relative to the total amount of protein loaded (feed + subfractions). DETAILED DESCRIPTION OF THE INVENTION

[0027] Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] definition Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0029] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology and recombinant DNA technology will be used, as described in the art (e.g., Molecular Cloning: A Laboratory Manual, 2002). nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0030] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0031] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0032] As used herein, the term "and / or" is meant to refer to either or both / all of the alternatives recited in connection with the term.

[0033] The terms "matrix" and "chromatography matrix" are used interchangeably herein and refer to a stationary phase in chromatography. The stationary phase may be in the form of a solid, liquid, or gel material, preferably in the form of a resin or a combination of resins. The matrix may have the form of a column, a capillary tube, a plate, or a sheet. A chromatography matrix in the form of a column is particularly preferred. Preferred examples of chromatographic modes or methods used in connection with the present invention include, but are not limited to, reversed-phase chromatography, hydrophobic interaction chromatography, affinity chromatography, ion-exchange chromatography, cation-exchange chromatography, anion-exchange chromatography, mixed-mode chromatography, chiral chromatography, hydrophilic interaction liquid chromatography, size-exclusion chromatography, and dielectric chromatography. It is within the capabilities of a person skilled in the art to select the respective solid phase for applying a chromatographic mode.

[0034] The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-linked amino acid chain, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) can be further modified by chemical modification. This means that such chemically modified polypeptides contain chemical groups other than the 20 naturally occurring amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of a polypeptide may provide advantageous properties compared to the parent polypeptide, such as one or more of improved stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to variants usable in the present invention include, but are not limited to, PEGylation, glycosylation of a non-glycosylated parent polypeptide, covalent attachment to therapeutic small molecules such as exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins, and glucagon-like peptide 1 agonists, including liraglutide, or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications can occur co- or post-translationally.

[0035] The term "amino acid" encompasses naturally occurring amino acids and amino acid derivatives. In the context of the present invention, a hydrophobic non-aromatic amino acid is any amino acid that is not aromatic and preferably has a Kyte-Doolittle hydrophobicity index of greater than 0.5, more preferably greater than 1.0, even more preferably greater than 1.5. Preferably, in the context of the present invention, the hydrophobic non-aromatic amino acid is selected from the group consisting of the amino acids alanine (Kyte-Doolittle hydrophobicity index 1.8), methionine (Kyte-Doolittle hydrophobicity index 1.9), isoleucine (Kyte-Doolittle hydrophobicity index 4.5), leucine (Kyte-Doolittle hydrophobicity index 3.8) and valine (Kyte-Doolittle hydrophobicity index 4.2) or their derivatives with a Kyte-Doolittle hydrophobicity index as defined above.

[0036] These explanations and definitions are valid for the entire application unless otherwise stated.

[0037] Embodiment The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. Specifically, embodiments described for methods of the present invention equally apply to devices of the present invention, since the devices are designed to perform the methods. The same applies to device embodiments that can be used in conjunction with methods of the present invention. The various described examples and preferred embodiments should not be construed as limiting the present invention to only those embodiments explicitly described. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0038] The present invention provides an improved chromatography method and apparatus for carrying out the method, which captures and reloads product and minor component stacks to reduce product loss and increase yield.

[0039] According to a first aspect, the present invention provides a method for separating a product of interest from impurities, comprising, in the order shown, (1) loading a first volume of a feed solution containing the product of interest and impurities onto a chromatography matrix; (2) contacting the chromatography matrix with an elution solution; (3) a) optionally collecting elution fraction 1 (EF1) in a subfractionation container (SFC), b) elution fraction 2 (EF2) in a product container, and c) optionally collecting elution fraction 3 (EF3) in the SFC, wherein at least one of EF1 and EF3 is collected; and (4) simultaneously or subsequently loading a second and / or additional volume of the feed solution onto EF1 and / or EF3 on the chromatography matrix. According to the method of the present invention, steps (2) through (4) are repeated at least once. According to preferred embodiments of the present invention, steps (2) through (4) are repeated at least two, at least three, at least four, or at least five times. Thus, steps 2 to 4 are repeated 2 to 50 or more times, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more times, preferably at least 9 times, more preferably at least 14 times, even more preferably at least 19 times, even more preferably at least 24 times, and most preferably at least 29 times.

[0040] Thus, the present invention basically provides two configurations for the method and device. The first configuration involves one chromatographic matrix, and EF1 and / or EF3 are collected in one SFC before being fed back to the matrix. The second configuration involves two chromatographic matrices, and EF1 and / or EF3 from the first matrix are collected in the first SFC before being fed to the second matrix, from which EF1 and / or EF3 are collected in the second SFC before being fed to the first matrix. Thus, according to one embodiment, a single chromatographic matrix and a single sub-fraction container are used. According to an alternative embodiment, two chromatographic matrices and two sub-fraction containers are used. It is also possible to use two chromatographic matrices and a single sub-fraction container. Thus, according to one embodiment, the present invention provides a method and device in which two chromatographic matrices are used, and EF1 and / or EF3 are collected in the sub-fraction containers.

[0041] According to a particularly preferred embodiment, EF1 (the product and impurity overlap before the main product elutes in EF2) is collected in a subfraction container (SFC), and EF3 (the product and impurity overlap after the main product elutes in EF2) is discarded. This discarding of EF3 can be performed in each repeat cycle of the method of the present invention, or alternatively, every other repeat cycle, every third, every fourth, or every fifth repeat cycle. According to a further embodiment of the present invention, EF1 is discarded and EF3 is collected in a subfraction container. This discarding of EF1 can be performed in each repeat cycle of the method of the present invention, or alternatively, every other repeat cycle, every third, every fourth, or every fifth repeat cycle. Thus, the method of the present invention may additionally comprise a step of discarding EF1 and / or EF3. This discarding can be repeated as needed when carrying out the method of the present invention, as long as EF1 and / or EF3 are collected at least twice, at least three times, at least four times, at least five times, or more. In the context of the present invention, discarding means not collecting the fractions in the subfraction container according to the present invention and not feeding the discarded fractions to any further chromatographic matrix of the method or the device of the present invention. In a particularly preferred embodiment, the method of the present invention mainly collects EF1 in the subfraction container and discards most of EF3. More preferably, only EF1 is collected in the subfraction container. This preferred embodiment is particularly useful when a protein is the product of interest.

[0042] According to the present invention, the chromatographic matrices in steps (1) and (4) can be the same or different chromatographic matrices. In the process of the present invention, the product is preferably bound to a chromatographic matrix in steps (1) and (4).

[0043] A feed solution containing the desired product and impurities is preferably provided from a storage vessel.

[0044] In embodiments using two chromatographic matrices, these are preferably changed or switched after the first cycle of steps (1) to (4) of the method of the present invention. According to a preferred embodiment, each time steps (2) to (4) are repeated, the chromatographic matrix alternates between the second chromatographic matrix and the first chromatographic matrix. In other words, the first matrix is ​​loaded in step (1) with a first volume of feed solution containing the desired product and impurities, and the second matrix is ​​loaded in step (4) with elution fractions (EF) 1 and / or EF3 and a second volume of feed solution. By repeating steps (2) to (4), the second chromatographic matrix loaded with EF1 and / or EF3 and a second volume of feed solution is eluted, and EF2 is collected in the product container and EF1 and / or EF3 are transferred to a subfraction container, from which the first matrix is ​​again loaded with EF1 and / or EF3 together with a volume of feed solution, as described in steps (2) and (3).

[0045] According to one embodiment of the present invention, EF1 and / or EF3 recovered from a first chromatographic matrix are recovered in a first SFC (P1), and EF1 and / or EF3 recovered from a second chromatographic matrix are recovered in a separate second SFC (P2).

[0046] According to one embodiment of the present invention, EF1 and / or EF3 may be diluted. This dilution is preferably carried out in step (3), thus before EF1 and / or EF3 are loaded onto the chromatography matrix. Therefore, according to a preferred embodiment, EF1 and / or EF3 are diluted with SFC. EF1 and / or EF3 may be diluted with any suitable substance or composition. Preferably, EF1 and / or EF3 are diluted with feed, chromatography buffer, water, or any combination thereof. When water is used for dilution, it is preferred that the water is deionized. Diluting EF1 and / or EF3 can prevent precipitation of products or other substances in EF1 and / or EF3.

[0047] According to a preferred embodiment, the second or further volume of feed solution loaded onto the chromatography matrix in step (4) has the same volume as the first volume in step (1). Alternatively, the second or further volume of feed solution loaded onto the chromatography matrix in step (4) is smaller than the first volume in step (1).

[0048] The product container, storage container, and subfraction container (SFC) can be any suitable container made from any suitable material known in the art. It is well within the ability of one skilled in the art to determine which type of container is particularly suited to which separation process, and many suitable storage containers are commercially available. Because the present invention is not limited to separating any particular product or compound from impurities, the product container and subfraction container will not be further defined. However, it should be understood that the containers used in the present invention are to be distinguished from simple conduits. Thus, the containers of the present invention allow for the collection of a volume over their axial length that is greater than the volume within a conduit of the same axial length. Preferably, the containers of the present invention have a volume at least twice the volume of a conduit of the same length as the container. More preferably, the containers have a volume at least three, four, five, six, seven, eight, nine, or ten times the volume of a conduit of the same length. The containers used in connection with the present invention are preferably connected to a chromatography matrix by one or more conduits. The terms "vessel" and "tank" are used interchangeably herein.

[0049] The chromatographic matrices used in the present invention can be of the same type or different types (if two matrices are used). The chromatographic matrix is ​​selected depending on the individual needs and the product to be purified. The matrix can be in the form of a column, a capillary tube, a plate, or a sheet. A chromatographic matrix in the form of a column is particularly preferred. The chromatographic matrix is ​​preferably selected so that the product to be purified can bind to the matrix in steps (2) and (4) of the method of the present invention. The chromatographic method is preferably selected from the group consisting of reversed-phase chromatography, hydrophobic interaction chromatography, affinity chromatography, ion exchange chromatography, cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, chiral chromatography, hydrophilic interaction liquid chromatography, size exclusion chromatography, and dielectric chromatography. According to a preferred embodiment, the chromatographic method is reversed-phase chromatography. The chromatographic matrix is ​​preferably in the form of a chromatographic column.

[0050] The present invention does not require the isolation of any particular product; essentially any product can be separated and / or purified from impurities using the methods and apparatus of the present invention. However, preferred products separated with the present methods and apparatus are proteins or polypeptides, preferably recombinant proteins or polypeptides expressed in a cellular expression system. A further preferred product separated is a nucleic acid molecule, preferably mRNA.

[0051] According to one embodiment of the present invention, the volume of EF1 and / or EF3 recovered in SFC in step (3) is at least 0.05, at least 0.25, at least 0.5, at least 1, at least 1.5 or at least 2.0 volumes of the chromatographic matrix.

[0052] According to a further preferred embodiment of the present invention, the concentration of the eluent contained in the elution solution is increased over time during step (2). This allows for a stepwise weakening of the interaction of the target product and impurities with the chromatographic matrix. Starting with a lower concentration of eluent, weakly bound impurities are released from the chromatographic matrix first. As the concentration of the eluent increases, the target product is preferably released from the matrix before the strongly bound impurities are also eluted. Whether and when to increase the concentration of the eluent contained in the elution solution can be determined on a case-by-case basis by those skilled in the art and depends, inter alia, on the type of product and the type of impurities to be separated / purified. Thus, in some embodiments, the concentration of the eluent contained in the elution solution is increased in one, two, three, or more elution steps (2), while in other embodiments, the concentration of the eluent contained in the elution solution is increased in essentially all elution steps (2) or not at all. Determining the time and extent to increase the concentration of the eluent contained in the elution solution is within the capabilities of those skilled in the art.

[0053] When eluted from the chromatography matrix, EF1 and / or EF3 contain the product of interest and impurities. According to one embodiment, the absorption and / or concentration of the product of interest recovered in the eluate is increased by at least two-fold compared to the feed solution loaded onto the chromatography matrix in step (1). According to a preferred embodiment, the absorption concentration of the product of interest recovered in the eluate is increased by at least five-fold, more preferably at least ten-fold, compared to the feed solution loaded onto the chromatography matrix in step (1). The absorption and / or concentration of the product of interest and / or impurities can be determined at different stages of the method to initiate the collection of EF1, EF2 and / or EF3. For example, the collection of EF1 is preferably initiated at a first predetermined concentration / absorption X of the product of interest in the eluate. EF1-P and a predetermined concentration / absorption Y of the desired product in the eluate. EF1-P Additionally or alternatively, the collection of EF3 is stopped at a first predetermined concentration / absorption of the product of interest in the eluate, X EF3-Pand a predetermined concentration / absorption Y of the desired product in the eluate. EF3-P In addition to determining the concentration / absorption of the desired product, the concentration / absorption of impurities can also be determined. Thus, the recovery of EF1 is preferably stopped at a first predetermined concentration / absorption X of the impurity in the eluate. EF1-I and a given concentration of impurities in the eluate / absorption Y EF1-I Additionally or alternatively, the collection of EF3 is stopped at a first predetermined concentration / absorption of the impurity in the eluate, X EF3-I and a given concentration of impurities in the eluate / absorption Y EF3-I According to the present invention, EF2 preferably contains the desired product. Therefore, the recovery of EF2 is stopped at a predetermined concentration X of the desired product in the eluate. EF2-P and a predetermined concentration Y of the desired product in the eluate EF2-P As mentioned above, the absorption and / or concentration of the product of interest and / or impurities can be determined at different stages of the method in order to initiate the collection of individual fractions containing EF2. Thus, according to a further embodiment of the invention, the collection of EF2 can be stopped at a predetermined concentration / absorption X of the impurities in the eluate. EF2-I and a given concentration of impurities in the eluate / absorption Y EF2-I will be stopped.

[0054] According to a preferred embodiment of the present invention, the loading in steps (1) and / or (4) is stopped before any desired product is eluted from the chromatographic matrix in the flow-through, which allows for obtaining a higher concentration of the desired product with a higher purity, preventing product loss throughout the process and contributing to an improvement in overall efficiency.

[0055] The eluent contained in the elution solution is preferably a polar eluent, and according to a particularly preferred embodiment of the present invention, the eluent contained in the elution solution is selected from the group consisting of acetonitrile, benzyl alcohol, methanol, acetic acid, ethylene glycol, tetrahydrofuran, ethanol, 1-propanol and 2-propanol.

[0056] According to one embodiment, the method includes a step preceding step (1) of preparing a feed solution. Preferably, this preparation includes an initial separation or crude purification of the desired product, which can increase the concentration of the desired product in the feed solution. Therefore, the feed solution is preferably selected by performing integrated countercurrent chromatography with a solution containing the desired product. Integrated countercurrent chromatography is known to those skilled in the art as a combination of ion exchange chromatography and hydrophobic interaction chromatography. This step increases the concentration of the product in the feed solution.

[0057] According to a further embodiment of the present invention, the concentration of the desired product and / or the concentration of impurities in each of the product and subfraction containers or at other points or devices during the process of the present invention is determined, for example, by in-line process analytical techniques. Determining the concentrations of substances such as the desired product and impurities is well within the capabilities of those skilled in the art and can be done, for example, by determining the absorption at specific wavelengths. Based on the absorption at specific wavelengths, the concentration of each substance can be calculated. Thus, according to one embodiment, the present invention includes determining the absorption of the desired product and / or impurities in each of the product and subfraction containers or at other points or devices during the process of the present invention.

[0058] The parameters for running the chromatography matrix depend on the type of product to be separated (product of interest) and can be set by the skilled artisan according to his experience and / or according to, for example, the manufacturer's instructions for the chromatography matrix and / or solid phase used.

[0059] According to a particularly preferred embodiment, the present invention provides a chromatography device comprising one or more chromatography matrices having first and second ends, a feed container, and one or more subfraction containers (SFCs), wherein the second ends of the one or more chromatography matrices are in fluid communication with the one or more subfraction containers, the one or more subfraction containers are in fluid communication with the first ends of the one or more chromatography matrices, the feed container is in fluid communication with the one or more subfraction containers, and the feed container is in fluid communication with the first ends of the one or more chromatography matrices. The fluid connection is preferably via conduit means. More preferably, the fluid connection can be regulated via one or more valve means. Valves can be used to direct fluid flow between individual components of the device via the conduit means. According to preferred embodiments, the one or more subfraction containers have a volume of about 0.05 to about 8 volumes of chromatography matrix, such as about 0.1 to about 6, about 1 to about 4, about 2 to about 6, about 2 to about 4, and most preferably about 2 to about 3 volumes of chromatography matrix.

[0060] One preferred method and simultaneously preferred apparatus of the present invention is shown schematically in Figure 6. In step (1), a feed solution containing the desired product and impurities is loaded from a feed tank onto a first chromatographic matrix (column 1). The loaded first chromatographic matrix is ​​then contacted with an elution solution in step (2) to obtain elution fractions 1 to 3 in step (3). EF2, which contains the product, is directed to a product container (product tank), and EF1 and / or EF3 are directed to a first subfraction container (P1). In step (4), EF1 and / or EF3 from the first subfraction container (P1) are simultaneously or subsequently loaded with an additional volume of feed solution containing the desired product and impurities from the feed tank onto a second chromatographic matrix (column 2). This is the situation shown in Figure 6, where the conduit under the load is shown thicker than the conduit without the load. The process then continues by repeating steps (2)-(4), but with the roles of the first and second chromatographic matrices swapped, in that the loaded second chromatographic matrix is ​​contacted with the elution solution of step (2) and the elution fraction of step (3) is collected from the second chromatographic matrix. Similarly, EF1 and / or EF3 are directed to a second subfraction container (P2) and from there redirected to the first chromatographic matrix, where they are simultaneously or subsequently loaded with an additional volume of feed solution containing the desired product and impurities from the feed tank into the first chromatographic matrix, initiating another cycle of steps (2)-(4).

[0061] The present invention features a method and apparatus that uses containers as time buffers for elution fractions, allowing chromatography matrices to proceed through their steps and tasks independently. In this way, product and impurity stacks are stored in their respective containers until the matrix is ​​loaded, rather than being loaded directly onto the matrix. While the former synchronized steps can be performed separately, the overall process generally remains synchronized throughout each cycle. Therefore, the present invention preferably desynchronizes the individual steps by using subfraction containers, storage containers for the feed solution, and product containers for the purified / separated product. This process provides superior performance in terms of purity and yield while also providing additional flexibility. Because columns no longer need to wait for each other during process steps, the process is faster and more productive. Additionally, desynchronization allows for continuous feed loading, a key advantage in chromatography purification. [Example]

[0062] The examples are designed to further illustrate and facilitate a better understanding of the present invention, and should not be construed as limiting the scope of the invention in any way.

[0063] Example 1 Chromatography was performed using reversed-phase resin (RP-resin) on a self-packed Superformance® 600-16 column (Goetec-Labortechnik GmbH, Bickenbach, Germany). The bed height was set at 30-40 cm. An asymmetry of AS = 1.44 was achieved. For hydrodynamic experiments, a Superformance® 150-10 column was packed to a bed height of 10 cm.

[0064] The feed was prepared from a frozen pool of recombinant protein that was thawed and mixed with deionized water in a volume ratio of 1:2. The deionized water was obtained from Arium® Pro (Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany).

[0065] Preparative runs were performed using a LaPrep® system (VWR International, Radnor, PA, USA) consisting of two P110 pumps, one P314 UV detector, and a Knauer Smartline 3900 autosampler (Knauer Wissenschaftliche Geraete GmbH, Berlin, Germany). Peak fractionation was performed using a Foxy Jr. sample collector (Teledyne Isco, Lincoln, NE, USA).

[0066] Analytical chromatographic separations were performed using a VWR-Hitachi LaChrom Elite® system (VWR International, Radnor, PA, USA) equipped with two high-pressure gradient pumps L-2130, an L-2200 autosampler, an L-2350 column oven, and an L-2450 diode array detector (DAD).

[0067] Two matrix column configurations, as shown schematically in Figure 6, have been used to purify recombinant proteins. The purity and yield for the method of the present invention over the number of cycles (the first cycle includes steps (1)-(4) = cycle 1.1 and a repetition of steps (2)-(4) = cycle 1.2, while subsequent cycles do not include step (1)) are shown in Figure 7. Purity is maintained at a high level while the overall yield increases with each cycle. The results of the inventive setup ("continuous") compared to those of a conventional method ("batch") that does not feed a subfraction to the chromatography matrix are shown in Table 1 below.

[0068] [Table 1]

[0069] As can be seen from Table 1, the method and apparatus of the present invention results in significantly increased yield and therefore productivity compared to conventional methods, while at the same time reducing consumption of eluent.

Claims

1. 1. A method for separating a desired product from impurities, comprising, in the order shown: (1) loading a first volume of a feed solution containing the desired product and impurities onto a chromatography matrix; (2) contacting the chromatography matrix with an elution solution; (3) a) optionally, elution fraction 1 (EF1) in a subfraction container (SFC); b) Elution Fraction 2 (EF2) in the product container, and c) optionally, elution fraction 3 (EF3) during SFC wherein at least one of EF1 and EF3 is recovered; (4) simultaneously or sequentially loading EF1 and / or EF3 and a second volume of said feed solution onto a chromatographic matrix; Including, Steps 2-4 are repeated at least 1 time, at least 2 times, at least 4 times, preferably at least 9 times, at least 14 times, more preferably at least 19 times, at least 24 times, and most preferably at least 29 times; The method wherein the chromatographic matrices of steps 1 and 4 are the same or different chromatographic matrices.

2. (i) the chromatographic matrix of steps 1 and 4 is the same first chromatographic matrix; or 2. The method of claim 1, wherein (ii) the chromatographic matrix in step 1 is a first chromatographic matrix, and the chromatographic matrix in step 4 is a second chromatographic matrix, and wherein with each iteration of steps 2 through 4, the chromatographic matrix alternates between the second chromatographic matrix and the first chromatographic matrix.

3. 3. The method of claim 1 or 2, wherein the volume of EF1 and / or EF3 recovered in the SFC in step 3 is at least 0.25, at least 0.5, at least 1, at least 1.5, or at least 2.0 volumes of the chromatography matrix.

4. 4. The method of claim 1, wherein the concentration of an eluent contained in the elution solution is increased over time during step 2, and the eluent weakens the interaction between the product of interest and the chromatographic matrix.

5. (i) EF1 and / or EF3 contain the product of interest and impurities, and the concentration of the product of interest is increased by at least 2-fold, preferably by at least 5-fold, more preferably by at least 10-fold compared to the solution loaded in step 1; a) the recovery of EF1 is determined by a first predetermined concentration X of the desired product in the eluate; ＥＦ１－Ｐ and a predetermined concentration Y of the product of interest in the eluate ＥＦ１－Ｐ and / or the recovery of the EF3 is stopped at a first predetermined concentration X of the product of interest in the eluate. ＥＦ３－Ｐ and a predetermined concentration Y of the product of interest in the eluate ＥＦ３－Ｐ and / or b) the recovery of the EF1 is performed by increasing the concentration of the impurity in the eluate by a first predetermined concentration X ＥＦ１－Ｉ and at a predetermined concentration Y ＥＦ１－Ｉ and / or the recovery of the EF3 is stopped at a first predetermined concentration X of the impurity in the eluate. ＥＦ３－Ｉ and at a predetermined concentration Y ＥＦ３－Ｉ and / or (ii) EF2 contains a substantially pure product of interest; a) The recovery of the EF2 is determined by the predetermined concentration X of the desired product in the eluate. ＥＦ２－Ｐ and a predetermined concentration Y of the product of interest in the eluate ＥＦ２－Ｐ and / or b) The recovery of the EF2 is carried out at a predetermined concentration X of impurities in the eluate. ＥＦ２－Ｉ and at a predetermined concentration Y of impurities in the eluate ＥＦ２－Ｉ The method according to any one of claims 1 to 4, wherein the method is stopped by

6. Step 3 further comprises diluting EF1 and / or EF3, preferably said dilution being carried out with said SFC; and / or The method of any one of claims 1 to 5, wherein EF1 and / or EF3 are diluted with one or more of the feed, chromatography buffer and water.

7. (i) the second volume in step 4 is the same as the first volume in step 1; or The method of any one of claims 1 to 6, wherein (ii) the second volume in step 4 is smaller than the first volume in step 1.

8. 8. The method according to claim 2, wherein EF1 and / or EF3 recovered from the first chromatographic matrix are recovered in a first SFC, and EF1 and / or EF3 recovered from the second chromatographic matrix are recovered in a second SFC.

9. In steps 1 and 4, the load is stopped before any product of interest is eluted from the chromatographic matrix in the flow-through; Steps 1 and 4 comprise binding the product of interest to the chromatographic matrix; and / or The method according to any one of claims 1 to 8, wherein the chromatographic matrices of steps 1 and 4 are of the same type.

10. 10. The method according to claim 1, wherein the chromatographic method in steps 1 and 4 is selected from the group consisting of reversed-phase chromatography, hydrophobic interaction chromatography, affinity chromatography, ion-exchange chromatography, cation-exchange chromatography, anion-exchange chromatography, mixed-mode chromatography, chiral chromatography, hydrophilic interaction liquid chromatography, size-exclusion chromatography, and dielectric chromatography.

11. The method according to any one of claims 1 to 10, wherein the chromatographic matrix in steps 1 and 4 is a reversed-phase chromatographic matrix.

12. 12. The method according to claim 11, wherein the eluent contained in the elution solution is a polar eluent, in particular selected from the group consisting of acetonitrile, benzyl alcohol, methanol, acetic acid, ethylene glycol, tetrahydrofuran, ethanol, 1-propanol and 2-propanol.

13. The method according to any one of claims 1 to 12, wherein the chromatographic matrix in steps 1 and 4 is a chromatographic column.

14. The method according to any one of claims 1 to 13, wherein the product of interest is a polypeptide or a protein.

15. 1. A chromatography apparatus comprising: one or more chromatographic matrices having first and second ends; supply container, one or more Sub-Fraction Containers (SFCs); conduit means connecting said second ends of said one or more chromatography matrices with said one or more subfraction containers; conduit means connecting said one or more subfraction containers with said first ends of said one or more chromatography matrices; conduit means connecting said feed vessel with said one or more subfraction vessels; conduit means connecting said supply vessel with said first end of said one or more chromatography matrices; wherein said one or more sub-fraction containers have a volume of from about 0.05 to about 8 volumes of said chromatographic matrix.

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