Continuous gradient elution spectrum fractionation

Abstract

The present invention relates to a method for separating a product of interest from impurities and a device for carrying out the method. The method comprises the following steps in the indicated order: (1) loading a first volume of a feed solution comprising a product of interest and impurities onto a chromatography matrix; (2) contacting a chromatographic matrix with an eluent solution; (3) a) optionally collecting the elution fraction 1 (EF1) in the side fraction container (SFC), b) collecting the elution fraction 2 (EF2) in the product container, and c) optionally collecting the elution fraction 3 (EF3) in the SFC wherein at least one of EF1 and EF3 is collected; and (4) loading EF1 and / or EF3 and a second volume of feed solution onto the chromatography matrix simultaneously or successively. Steps (2) to (4) are repeated at least once, and the chromatographic matrices of steps (1) and (4) are 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 chromatography fractionation method which allows for efficient separation and purification of a product in a solution from impurities. Background Art

[0002] Chromatography is a widely used unit operation for analytical as well as preparative separations, especially for the production of high purity products like pharmaceuticals. In these cases, the desired target component often elutes closely with the impurities, e.g. Figure 1 In order to achieve high purity, narrow product fractionation is one of the main goals. However, narrow product fractionation leads to product losses because the target product often elutes with impurities in a bell-shaped manner. Therefore, one object of the present invention is to reduce this loss.

[0003] Several chromatographic methods are known in the art. In particular, continuous chromatography is described in the following literature: Subramanian (Continuous biomanufacturing. Innovative Technologies and Methods. 2018, Weinheim: WILEY-VCH), Zobel-Roos (Entwicklung, Modellierung und Validierung von integriertenkontinuierlichen Gegenstrom-Chromatographie-Prozessen [Development, Modeling and Validation of an Integrated Continuous Countercurrent Chromatography Process]. 1st edition, 2018. Herzogenrath: Shaker Verlag (Thermische Verfahrens- und Prozesstechnik [Thermal Processes and Process Technology])), Schmidt-Traub et al. (Preparative chromatography. 2nd edition, completely revised and updated 2012, Weinheim, Germany: WILEY-VCH [Wiley-VCH]).Continuous chromatography includes several subcategories, such as (i) simulated moving bed chromatography (SMB), which is described in the following literature: Imamoglu (Simulated moving bed chromatography (SMB) for application in bioseparation. Advances in biochemical engineering / biotechnology 2105, Vol. 76, pp. 211-231), Rodrigues (Simulated Moving Bed Technology. Principles, Design and Process Applications. 2015, Burlington: Elsevier Science); (ii) sequential chromatography, which is described in the following literature: Holzer (Sequential Multi-Column Chromatography. BPI. Düsseldorf, April 2013), Bisschops (BioSMB. TM Technology: Continuous Countercurrent Chromatography Enabling a FullyDisposable Process [BioSMB TMTechnology: Continuous countercurrent chromatography for a fully disposable process. Ganapathy Subramanian (ed.): Biopharmaceutical Production Technology. 1st edition, 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 A affinity chromatography. Journal of chromatography, vol. 1389, 2015, pp. 85-95), and Godawat et al. (Periodic counter-current chromatography-design and operational considerations for integrated and continuous purification). of proteins. [Periodic countercurrent chromatography - design and operational considerations for integrated and continuous purification of proteins. Biotechnology Journal [Biotechnology Journal] 2012, volume 7(12), pages 1496-1508); and (iii) multicolumn countercurrent solvent gradient purification (MCSGP), which is described in the following literature: Aumann and Morbidelli (A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process [Continuous multicolumn countercurrent solvent gradient purification (MCSGP) process]. Biotechnology and Bioengineering [Biotechnology and Bioengineering] 2007, volume 98(5), pages 1043-1055); and Morbidelli (Continuous Chromatography for the Purification of Monoclonal Antibodies. Uwe Gottschalk (ed.): Process scale purification of antibodies. Hoboken, NJ: John Wiley & Sons, 2009, pp. 223-238) and Steinebach et al. (Continuous counter-current chromatography for capture and polishing steps in biopharmaceutical production. Biotechnology Journal, 2016, Vol. 11(9), pp. 1126-1141).

[0004] Specifically, multi-column countercurrent solvent gradient purification (MCSGP) is a process originally conceived for continuous heart-cutting operations in gradient chromatography (Aumann and 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); et al. (A continuous, counter-current multi-column chromatographic process incorporating modifier gradients for ternary separations. Journal of chromatography, 2006, vol. 1126(1-2), pp. 338-346); (Purification of monoclonal antibodies by continuous chromatography. Also published as: Zürich, Diss., Technische Hochschule ETH Zürich [PhD thesis, Federal Institute of Technology (ETH), Zurich], No. 18066, 2009). Figure 2 As shown in, three columns are operated separately, and three columns are interconnected. These single columns elute pure product (red) and strong (green) and weak (blue) bound side components. The interconnected columns separate the overlapping areas of product and impurities. After a given time, each column is switched to the left by one position. In the connected pipeline, the column on the left elutes with the highest elution intensity, the column in the middle elutes with a lower elution intensity, and the column on the right is in equilibrium. These components flow from one column to another, where they will combine again. For this reason, the elution intensity of the liquid phase between the columns needs to be reduced. Therefore, these flows are diluted.

[0005] In an improved variant of this method, the modification Figure 2 and adapted it to a two-column setup (Aumann and Morbidelli 2008; et al. (Online control of the twin-column countercurrent solvent gradient process for biochromatography. Journal of chromatography. Vol. 1293, 2013, pp. 51-59); et al. (Closed loop control of the multi-column solvent gradient purification process. Journal of chromatography, 2011, Vol. 1218(50), pp. 9028-9036); et al. 2013; Steinebach et al. 2016). This two-column setup requires eight steps, which are Figure 3As shown in. In the first step, the cycle begins with loading the overlapping portion of the weakly bound component (W) and the product (P) from column 1 to column 2. In order to take into account the elution strength of the fraction (e.g., the amount of salt or modifier such as polar solvent), the feed is diluted online with pure eluent (E). In the second step, when the purity standard is met, the product (P) is obtained from column 1, and column 2 is loaded with feed. In the third step, when the overlapping portion of the product and the strongly bound impurity (S) is about to elute, column 1 is connected to column 2 again, and the fraction is diluted online with eluent (E). In the fourth step, the gradient of column 2 is started, and the weakly bound component (W) is eluted and transferred to waste. The gradient of column 1 ends, and the strongly bound impurity (S) is discharged. The process is repeated once, but for columns 1 and 2, there is an exchanged role (steps 5 to 8). The selection of the cutting point and the online dilution are critical to the process. In addition, the columns must be synchronized. Feed loading and product elution (see step 2 or 6) must occur simultaneously and should take the same amount of time. Otherwise, impurities will shift. It is expected that weakly bound impurities from column 1 are loaded to column 2 before feed loading, while strongly bound components are loaded after feed loading.

[0006] The methods of the prior art and in particular the dual column MCSGP method have significant disadvantages. First, there is no continuous feed loading. Continuous purification methods are usually defined by at least one continuous flow. In, for example, dual column MCSGP, both flows are discontinuous, and feed loading and product elution ( Figure 4 ) occurs only in steps 2 and 6. Note that the eluted product is not loaded onto another column. Figure 4 As shown in , there is a lack of synchronization between the various steps. For an ideal dual-column MCSGP method, the feed loading on one column and the gradient on the other column should be synchronized, and the two steps should spend the same amount of time. However, in many cases, one step takes much longer time than the other step. In particular, the feed loading step usually takes much longer time than the elution step. Therefore, the method often shows that the time required for the loading step is 5 to 10 times longer than the elution step. Therefore, steps 2 and 6 will take as long as the time required for their loading columns. During this period, the other column can only elute the product at a very low flow rate, or it can elute the product at an ideal speed, but then has to stop. Both situations are disadvantageous due to, for example, diffusion processes. Similar problems occur after steps 4 and 8. After the gradient separation, each column is regenerated with high elution, followed by rebalancing (comparison Figure 4The blue and yellow lines in Figure 2 are shown in Figure 3. This re-equilibrium must be completed before loading the weakly bound overlap (steps 1 and 5). Therefore, the other column must wait again. Due to the very long column loading compared to the time required for the eluted product, the classical MCSGP method shows a relatively low productivity. In short, one column has to wait until the other column is loaded in the case of its gradient, and then, the column forces the other column to wait until it completes the post-elution step. This procedure is extremely inefficient and time-consuming.

[0007] Therefore, there is a need for novel chromatography methods that overcome the disadvantages of the prior art, such as slow and inefficient methods, and unwanted diffusion processes in unfed and uneluted (ie, at rest) columns. There is a further need for novel chromatography methods that reduce product losses. Summary of the invention

[0008] In a first aspect, the present invention provides a method for separating a product of interest from impurities. The method comprises the following steps in the indicated order:

[0009] (1) loading a first volume of a feed solution comprising a product of interest and impurities onto a chromatography matrix;

[0010] (2) contacting the chromatography matrix with an elution solution; (3)

[0012] a) optionally collecting eluted fraction 1 (EF1) in a side fraction container (SFC),

[0013] b) collecting elution fraction 2 (EF2) in a product container, and

[0014] c) optionally collecting elution fraction 3 (EF3) in SFC,

[0015] wherein at least one of EF1 and EF3 is collected;

[0016] (4) loading EF1 and / or EF3 and a second volume of feed solution onto the chromatography matrix simultaneously or sequentially;

[0017] wherein steps (2) to (4) are repeated at least once, at least twice, 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; and

[0018] The chromatographic matrices in steps (1) and (4) are the same or different chromatographic matrices.

[0019] According to a preferred embodiment, the chromatographic matrix of steps 1 and 4 is the same first chromatographic matrix; or the chromatographic matrix of step 1 is the first chromatographic matrix and the chromatographic matrix of step (4) is the second chromatographic matrix, and the chromatographic matrices are alternated between the second chromatographic matrix and the first chromatographic matrix each time steps (2) to (4) are repeated.

[0020] According to another preferred embodiment, the volume of EF1 and / or EF3 collected 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.

[0021] According to a particularly preferred embodiment, during step (2), the concentration of the eluent contained in the elution solution increases over time, and wherein the eluent weakens the interaction between the product of interest and the chromatography matrix.

[0022] According to one embodiment, EF1 and / or EF3 contain the product of interest and impurities, wherein the concentration of the product of interest is increased by at least about 2 times, preferably at least about 5 times, more preferably at least about 10 times, compared to the solution loaded in step 1. In this embodiment, EF1 is collected at a first predetermined concentration X of the product of interest in the eluate. EF1-P and the product of interest is at a predetermined concentration Y in the eluate EF1-P The collection of EF3 is stopped when the product of interest is at the first predetermined concentration X in the eluate. EF3-P and the product of interest is at a predetermined concentration Y in the eluate EF3-P Additionally or alternatively, EF1 is collected when the impurity has a first predetermined concentration X in the eluate. EF1-I and the impurity concentration Y in the eluate is EF1-I When the impurities in the eluate are stopped, and / or the collection of EF3 is stopped, the impurities in the eluate are collected at a first predetermined concentration X EF3-I and the impurity concentration Y in the eluate is EF3-I Additionally or alternatively to this embodiment of the invention, EF2 comprises a substantially pure product of interest, and EF2 is collected at a predetermined concentration x of the product of interest in the eluate. EF2-P and the product of interest is at a predetermined concentration Y in the eluate EF2-P and / or EF2 is collected at a predetermined concentration of impurities in the eluate X EF2-I and the impurity concentration Y in the eluate is EF2-I Stop when.

[0023] According to a preferred embodiment, step (3) further comprises the step of diluting EF1 and / or EF3. The dilution preferably occurs in SFC. Also preferably, EF1 and / or EF3 are diluted with one or more of feed, chromatography buffer and water.

[0024] 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).

[0025] According to a particularly preferred embodiment, EF1 and / or EF3 collected from the first chromatographic matrix are collected in a first SFC, and EF1 and / or EF3 collected from the second chromatographic matrix are collected in a second SFC.

[0026] According to yet another embodiment, in steps (1) and (4), loading is stopped before any product of interest is eluted from the chromatography matrix with the flow-through.

[0027] According to one embodiment, steps (1) and (4) comprise binding the product of interest to a chromatography matrix.

[0028] According to a preferred embodiment, the chromatography matrices of step (1) and step (4) are of the same type.

[0029] According to another embodiment, the chromatography mode used in step (1) and step (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.

[0030] According to a preferred embodiment, the chromatography mode of step (1) and step (4) is reverse phase chromatography.

[0031] According to a preferred embodiment, the eluent contained in the elution solution is a polar eluent. Preferably, the polar eluent is selected from the group consisting of acetonitrile, benzyl alcohol, methanol, acetic acid, ethylene glycol, tetrahydrofuran, ethanol, 1-propanol and 2-propanol.

[0032] According to a preferred embodiment, the chromatographic matrix of step (1) and step (4) is a chromatographic column.

[0033] According to a particularly preferred embodiment, the product of interest is a polypeptide or a protein.

[0034] According to one embodiment of the present invention, a chromatography device comprises: one or more chromatography matrices having a first end and a second end; a feed container; one or more side fraction containers (SFC); a conduit device connecting the second end of the one or more chromatography matrices to the one or more side fraction containers; a conduit device connecting the one or more side fraction containers to the first end of the one or more chromatography matrices; a conduit device connecting the feed container to the one or more side fraction containers; a conduit device connecting the feed container to the first end of the one or more chromatography matrices. The volume of the one or more side fraction containers in this embodiment is about 0.05 to about 8 volumes of the chromatography matrices.

[0035] Further aspects and embodiments are disclosed in the dependent claims and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Chromatogram of a protein eluted from a prior art chromatography column. The target component (red) elutes closely to impurities (light blue and green). The dark blue line indicates the summed signal as seen by the detector.

[0037] Figure 2 : Schematic diagram of the 6-column MCSGP operation described in 2014.

[0038] Figure 3 :according to Schematic diagram of the dual-column MCSGP steps in one cycle (Journal of chromatography 2013, vol. 1293, pp. 51-59).

[0039] Figure 4 : The timetable of different process steps of conventional double column MCSGP. The chart is divided at the y-axis value 0. The upper part shows the feed loading (orange line) and elution gradient (blue line) of column one. The lower part shows the feed loading (light blue line) and elution gradient (yellow line) of the second column. The gray line is the cut point of the overlapping part of the product and impurities transferred from one column to another column as indicated by the red / gray arrows. The red arrow shows the product elution.

[0040] Figure 5 : Flow chart of the method of the present invention.

[0041] Figure 6 : Schematic diagram of a preferred embodiment of the device according to the invention, which has two matrices (column 1 and column 2) and two side fraction containers (P1, P2).

[0042] Figure 7 : Purity and yield of the method of the present invention over the number of cycles. Each cycle comprises one repetition of steps (2) to (4), wherein the first round is denoted as X.1 and the repetition of steps (2) to (4) is denoted as X.2. The first cycle 1.1 comprises step (1), and the subsequent cycles 2 to 5 do not comprise step (1). The blue line indicates purity. The grey line indicates the cycle yield associated with the amount of feed loaded. The yellow line indicates the total yield. The orange line indicates the yield associated with the total amount of protein loaded (feed plus side fractions). DETAILED DESCRIPTION

[0043] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0044] definition

[0045] Preferably, the terms used herein are defined as described in the following document: "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel and Editor, Helvetica Chimica Acta [Swiss Chemical Acta], CH-4010 Basel, Switzerland, (1995).

[0046] To practice the present invention, conventional methods of chemistry, biochemistry, cell biology and recombinant DNA techniques are employed, unless otherwise indicated, as explained in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0047] Throughout this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and variations such as "comprises" and "comprising" should be understood to imply 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 referents unless the context clearly dictates otherwise.

[0048] When used in conjunction with a numerical value, the term "about" is meant to encompass the numerical value within a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0049] As used herein, the term "and / or" means that it refers to both / all of the options cited in the context of this term.

[0050] The terms "matrix" and "chromatographic matrix" are used interchangeably herein and refer to the stationary phase in the chromatography. The stationary phase can exist in the form of a solid, liquid or gel material, preferably in the form of a resin or a combination of resins. The matrix can have the form of a column, a capillary, a plate or a sheet. Particularly preferred is a chromatographic matrix in the form of a column. Preferred examples of the chromatographic mode or method to be used in the context of the present invention include but are not limited to 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. It is within the capabilities of the technician to select the corresponding solid phase for the chromatographic mode to be applied.

[0051] The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bonded amino acid chain, regardless of length or post-translational modification. Proteins useful 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 other chemical groups in addition to 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 polypeptides can provide advantageous properties, such as one or more of enhanced stability, increased biological half-life, or increased water solubility compared to the parent polypeptide. Chemical modifications suitable for use in variants useful in the present invention include, but are not limited to, pegylation, glycosylation of a non-glycosylated parent polypeptide, covalent coupling with a therapeutic small molecule (such as glucagon-like peptide 1 agonists including exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins and liraglutide), or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications may occur during or after translation.

[0052] The term "amino acid" encompasses naturally occurring amino acids as well as amino acid derivatives. In the context of the present invention, a hydrophobic non-aromatic amino acid is preferably any amino acid having a Kyte-Doolittle hydropathic index higher than 0.5, more preferably higher than 1.0, even more preferably higher than 1.5 and which is not aromatic. 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 hydropathic index 1.8), methionine (Kyte Doolittle hydropathic index 1.9), isoleucine (Kyte Doolittle hydropathic index 4.5), leucine (Kyte Doolittle hydropathic index 3.8) and valine (Kyte Doolittle hydropathic index 4.2) or a derivative thereof having a Kyte Doolittle hydropathic index as defined above.

[0053] Unless stated otherwise, these descriptions and definitions are valid for the entire application.

[0054] Example

[0055] Hereinafter, the elements of the present invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any quantity to create other embodiments. Specifically, the embodiments described for the method of the present invention are equally applicable to the equipment of the present invention, because the latter is designed to carry out the method. This also applies to the embodiments of the equipment that can also be used in combination with the method of the present invention. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass the embodiments that clearly describe the embodiments and any number of disclosed and / or preferred element combinations. In addition, unless otherwise indicated by the context, it should be considered that any arrangement and combination of all described elements in the present application are disclosed by the description of the present application.

[0056] The present invention provides an improved chromatography method and an apparatus for carrying out the method. In the method of the present invention, in order to reduce product loss, overlapping parts of product and side components are captured and reloaded to increase yield.

[0057] According to a first aspect, the present invention provides a method for separating a product of interest from impurities, the method comprising the following steps in the indicated order: (1) loading a first volume of a feed solution comprising a 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 side fraction container (SFC), b) collecting elution fraction 2 (EF2) in a product container, and c) optionally collecting elution fraction 3 (EF3) in an SFC, wherein at least one of EF1 and EF3 is collected; and (4) loading a second or additional volume of feed solution onto the chromatography matrix simultaneously or sequentially with EF1 and / or EF3. According to the method of the present invention, steps (2) to (4) are repeated at least once. According to a preferred embodiment of the present invention, steps (2) to (4) are repeated at least twice, at least 3 times, at least 4 times or at least 5 times. Thus, steps 2 to 4 are repeated between 2 and 50 times or more, such as 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, yet even more preferably at least 24 times, and most preferably at least 29 times.

[0058] Therefore, the present invention basically provides two settings for the method and the device: the first setting includes a chromatographic matrix, and EF1 and / or EF3 are collected in one SFC before they are fed to the matrix again. The second setting includes two chromatographic matrices, and EF1 and / or EF3 from the first matrix are collected in the first SFC before they are fed to the second matrix, and EF1 and / or EF3 from the second matrix are collected in the second SFC before they are fed to the first matrix. Therefore, according to one embodiment, a single chromatographic matrix and a single side fraction container are used. According to an alternative embodiment, two chromatographic matrices and two side fraction containers are used. Two chromatographic matrices and a single side fraction container can also be used. Therefore, according to one embodiment, the present invention provides a method and a device, wherein two chromatographic matrices are used and EF1 and / or EF3 are collected in a single side fraction container.

[0059] According to a particularly preferred embodiment, EF1 (the overlapping portion of product and impurities before the main product eluted in EF2) is collected in a side fraction container (SFC), and EF3 (the overlapping portion of product and impurities after the main product eluted in EF2) is discarded. This discarding of EF3 can be performed in each repetition cycle of the method of the present invention, or alternatively in every other repetition cycle, in every three, every four or every five repetition cycles. According to another embodiment of the present invention, EF1 is discarded and EF3 is collected in a side fraction container. This discarding of EF1 can be performed in each repetition cycle of the method of the present invention, or alternatively in every other repetition cycle, in every three, every four or every five repetition cycles. Therefore, the method of the present invention may further include the step of discarding EF1 and / or EF3. 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 times when performing the method of the present invention, this discarding can be repeated according to corresponding needs. Discarding in the context of the present invention means that the fraction is not collected in the side fraction container according to the present invention and the discarded fraction is not fed to any further chromatographic matrix of the method or apparatus of the present invention. In a particularly preferred embodiment, in the method of the present invention, mainly EF1 is collected in the side fraction container and most of EF3 is discarded. More preferably, only EF1 is collected in the side fraction container. This preferred embodiment is particularly useful in the case where a protein is the product of interest.

[0060] According to the present invention, the chromatographic matrices of steps (1) and (4) may be the same or different chromatographic matrices. In the method of the present invention, the product is preferably bound to the chromatographic matrix in steps (1) and (4).

[0061] The feed solution comprising the product of interest and impurities is preferably provided from a storage vessel.

[0062] In embodiments where two chromatographic matrices are used, it is preferred that these chromatographic matrices are changed or switched after the first cycle of steps (1) to (4) of the method of the invention. According to a preferred embodiment, at each repetition of steps (2) to (4), the chromatographic matrix alternates between the second chromatographic matrix and the first chromatographic matrix. In other words, in step (1), the first matrix is ​​loaded with a first volume of feed solution comprising the product of interest and impurities, and in step (4), the second matrix is ​​loaded with elution fraction (EF) 1 and / or EF3 and a second volume of feed solution. When steps (2) to (4) are repeated, the second chromatographic matrix loaded with EF1 and / or EF3 and a second volume of feed solution is eluted, and (as described in steps (2) and (3)) EF2 is collected in a product container, while EF1 and / or EF3 are transferred to a side fraction container, from which EF1 and / or EF3 are again loaded to the first matrix together with a certain volume of feed solution.

[0063] According to one embodiment of the present invention, EF1 and / or EF3 collected from a first chromatography matrix are collected in a first SFC (P1), and EF1 and / or EF3 collected from a second chromatography matrix are collected in a separate second SFC (P2).

[0064] According to one embodiment of the present invention, EF1 and / or EF3 can be diluted. The dilution is preferably performed in step (3), and therefore performed before EF1 and / or EF3 are loaded onto the chromatographic matrix. Therefore, according to a preferred embodiment, EF1 and / or EF3 are diluted in SFC. EF1 and / or EF3 can be diluted with any suitable substance or composition. Preferably, EF1 and / or EF3 are diluted with feed, chromatographic buffer, water or any combination thereof. If water is used for dilution, the water is preferably deionized. Diluting EF1 and / or EF3 can prevent precipitation of products or other substances in EF1 and / or EF3.

[0065] According to a preferred embodiment, the second or additional 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 additional volume of feed solution loaded onto the chromatography matrix in step (4) is smaller than the first volume in step (1).

[0066] The product container, storage container and the one or more side fraction containers (SFC) can be any suitable container made of any suitable material known in the art. It is entirely within the capabilities of the technician to determine which type of container is particularly suitable for which separation process, and many suitable storage containers are commercially available. Since the present invention is not limited to separating any specific product or compound from impurities, the product container and the one or more side fraction containers are not further limited. However, it should be understood that the container as used in the present invention should be distinguished from a simple conduit. Therefore, the container of the present invention allows the volume collected on its axial length to be greater than the volume in the conduit of the same axial length. Preferably, the volume of the container of the present invention is at least twice the volume of the conduit with the same length as the container. More preferably, the volume of the container is at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times the volume of the conduit with the same length. The container as used in the context of the present invention is preferably connected to the chromatographic matrix by one or more conduits. The terms "container" and "tank" are used interchangeably herein.

[0067] The chromatographic matrix to be used in the present invention can be of the same type or different types (if two matrices are used). The chromatographic matrix is ​​selected according to individual needs and the product to be purified. The matrix can have the form of a column, a capillary, a plate or a sheet. Particularly preferred is a chromatographic matrix in the form of a column. The chromatographic matrix is ​​preferably selected so that the product to be purified can be attached to the matrix in steps (2) and (4) of the method of the present invention. The chromatographic mode is preferably 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. According to a preferred embodiment, the chromatographic mode is reverse phase chromatography. The chromatographic matrix is ​​preferably in the form of a chromatographic column.

[0068] The present invention does not require any particular product to be separated, and essentially any product can be separated and / or purified from impurities using the methods and apparatus of the present invention. However, preferred products to be separated using the methods and apparatus of the present invention are proteins or polypeptides, preferably recombinant proteins or polypeptides expressed in a cell expression system. Another preferred product to be separated is a nucleic acid molecule, preferably mRNA.

[0069] According to one embodiment of the present invention, the volume of EF1 and / or EF3 collected 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 chromatography matrix.

[0070] According to another preferred embodiment of the present invention, during step (2), the concentration of the eluent contained in the elution solution increases over time. This allows the interaction between the product of interest and the impurities and the chromatographic matrix to be gradually weakened. Starting with a lower concentration of eluent, weakly bound impurities will be released from the chromatographic matrix first. As the concentration of the eluent increases, the product of interest 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 will be determined by the technician according to the specific circumstances, and will especially depend on the type of product to be separated / purified and the type of impurities. Therefore, in some embodiments, the concentration of the eluent contained in the elution solution increases in one, two, three or more elution steps (2), and in other embodiments, the concentration of the eluent contained in the elution solution increases in substantially all elution steps (2) or does not increase at all. It is within the capabilities of the technician to determine when and to what extent the concentration of the eluent contained in the elution solution increases.

[0071] 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 collected in the eluate increases by at least 2 times when compared to the feed solution loaded onto the chromatography matrix in step (1). According to a preferred embodiment, the concentration of the product of interest collected in the eluate increases by at least 5 times and more preferably by at least 10 times when 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 at different stages of the method can be determined for the start of the collection of EF1, EF2 and / or EF3. For example, the collection of EF1 is preferably performed at a first predetermined concentration / absorption x of the product of interest in the eluate. EF1-P and at a predetermined concentration / absorption Y of the product of interest in the eluate EF1-P Additionally or alternatively, EF3 is collected at the first predetermined concentration / absorbance X of the product of interest in the eluate. EF3-P and at a predetermined concentration / absorption Y of the product of interest in the eluate EF3-P In addition to determining the concentration / absorption of the product of interest, the concentration / absorption of the impurity may also be determined. Therefore, the collection of EF1 is preferably performed when the impurity has a first predetermined concentration / absorption X in the eluate. EF1-I and at a predetermined concentration / absorption Y of the impurity in the eluate EF1-I Additionally or alternatively, EF3 is collected at a first predetermined concentration / absorption X of the impurity in the eluate. EF3-I and at a predetermined concentration / absorption Y of the impurity in the eluate EF3-IAccording to the present invention, EF2 preferably contains the product of interest. Therefore, the collection of EF2 can be stopped at a predetermined concentration / absorption X of the product of interest in the eluate. EF2-P and at a predetermined concentration / absorption Y of the product of interest in the eluate EF2-P As described above, the absorption and / or concentration of the product and / or impurity of interest at different stages of the process can be determined for the start of collection of a separate fraction containing EF2. Therefore, according to another embodiment of the present invention, the collection of EF2 is performed at a predetermined concentration / absorption of the impurity in the eluate. EF2-I and at a predetermined concentration / absorption Y of the impurity in the eluate EF2-I Stop when.

[0072] According to a preferred embodiment of the present invention, the loading in steps (1) and / or (4) is stopped before any product of interest is eluted from the chromatography matrix with the flow-through. This enables a higher concentration of the product of interest to be obtained with a higher purity and prevents the loss of the product in the whole process, thereby contributing to an overall increase in efficiency.

[0073] The eluent contained in the elution solution is preferably a polar eluent. 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.

[0074] According to an embodiment, the method includes the step of preparing a feed solution before step (1). Preferably, the preparation includes initial separation or rough purification of the product of interest, and the concentration of the product of interest in the feed solution can be increased. Therefore, the feed solution is preferably optimized by performing integrated countercurrent chromatography with a solution containing the product of interest. Integrated countercurrent chromatography is known to technicians and is a combination of ion exchange chromatography and hydrophobic interaction chromatography. This step increases the concentration of the product in the feed solution.

[0075] According to another embodiment of the present invention, the concentration of the product of interest and / or impurity in the product container and the one or more side fraction containers, or other time points during the method or in the equipment of the present invention is determined, for example, by online process analysis technology. Determining the concentration of a substance (such as a product of interest and an impurity) is completely within the capabilities of the technician, and can be completed, for example, by determining the absorption at a specific wavelength. Based on the absorption at a specific wavelength, the concentration of the corresponding substance can be calculated. Therefore, according to one embodiment, the present invention includes determining the absorption of the product of interest and / or impurity in the product container and the one or more side fraction containers, or other time points during the method or in the equipment of the present invention.

[0076] 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 person based on his experience and / or based on the instructions of the manufacturer of the chromatography matrix and / or solid phase used, for example.

[0077] According to a particularly preferred embodiment, the present invention provides a chromatographic device, comprising one or more chromatographic matrices having a first end and a second end, a feed container, one or more side fraction containers (SFC), wherein the second end of the one or more chromatographic matrices is in fluid connection with the one or more side fraction containers, wherein the one or more side fraction containers are in fluid connection with the first end of the one or more chromatographic matrices, wherein the feed container is in fluid connection with the one or more side fraction containers, and wherein the feed container is in fluid connection with the first end of the one or more chromatographic matrices. The fluid connection is preferably carried out via a conduit device. More preferably, the fluid connection can be regulated via one or more valve devices. By using a valve, a fluid flow can be guided between the various components of the device via a conduit device. According to a preferred embodiment, the volume of the one or more side fraction containers is about 0.05 to about 8 volumes of the chromatographic 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 the chromatographic matrix.

[0078] Figure 6 A preferred method and simultaneously preferred apparatus of the present invention are schematically depicted in . In step (1), a feed solution containing the product of interest and impurities is loaded from a feed tank to a first chromatographic matrix (column 1). Then in step (2), the loaded first chromatographic matrix is ​​contacted with an elution solution to obtain elution fractions 1 to 3 in step (3). EF2 containing the product is introduced to a product container (product tank), and EF1 and / or EF3 are introduced to a first side fraction container (P1). In step (4), EF1 and / or EF3 from the first side fraction container (P1) are loaded to a second chromatographic matrix (column 2) simultaneously with or subsequently to an additional volume of feed solution containing the product of interest and impurities from the feed tank. This is Figure 6, where the conduit under loading is shown in a thicker manner than the conduit without any loading. The method then continues by repeating steps (2) to (4), however with the roles of the first chromatography matrix and the second chromatography matrix switched, where the loaded second chromatography matrix is ​​contacted with the elution solution in step (2) and the elution fractions in step (3) are collected from the second chromatography matrix. Similarly, EF1 and / or EF3 are directed to a second side fraction container (P2), from which they are directed again to the first chromatography matrix, where they are loaded onto the first chromatography matrix simultaneously or subsequently with a further volume of feed solution containing the product of interest and impurities from the feed tank to the first chromatography matrix, which starts another cycle of steps (2) to (4).

[0079] The present invention is characterized in that a container is used as a time buffer of the elution fraction in the method and apparatus, which allows the chromatographic matrix to perform its steps and tasks individually. In this way, the overlapping parts of the product and impurities are not directly loaded onto the matrix, but are stored in corresponding containers until the matrix can be loaded. Although the whole process generally remains synchronized in each cycle, the previous synchronization step can be performed separately. Therefore, the present invention preferably synchronizes each step by using a side fraction container, a storage container for the feed solution, and a product container for the purified / separated product. The method provides excellent performance in terms of purity and yield and simultaneously obtains further flexibility. Since the columns no longer need to wait for each other during the process step, the method is faster and more productive. In addition, this desynchronization especially allows continuous feed loading, which is a major benefit in chromatographic purification.

[0080] Examples

[0081] The examples are designed to further illustrate the invention and for better understanding. They should not be interpreted as limiting the scope of the invention in any way.

[0082] Example 1:

[0083] Chromatography in self-filling 600-16 column (Getech Laboratory Technology Co., Ltd., Bickenbach, Germany) GmbH) with reverse phase resin (RP-resin). The bed height was set to 30 to 40 cm. Asymmetry of AS = 1.44 was achieved. For the hydrodynamic experiments, The 150-10 column was packed to a bed height of 10 cm.

[0084] Feed was prepared from a frozen pool of recombinant protein, which was mixed with deionized water at a 1:2 volume ratio after thawing. Deionized water was obtained from Pro(Göttingen, Germany( The product was obtained from Sartorius Lab Instruments GmbH & Co. KG in Germany.

[0085] For preparative operation The system was performed using a VWR International, Radnor, PA, USA, which consisted of two P110 pumps, a P314 UV detector, and a Knauer Wissenschaftliche Physik, Berlin, Germany. The peak fractionation was performed with a Foxy Jr. sample collector (Teledyne Isco, Lincoln, NE, USA).

[0086] VWR-Hitachi LaChrom for analytical chromatography The analysis was performed using a VWR 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).

[0087] like Figure 6 The dual matrix column setup schematically depicted in has been used to purify recombinant proteins. Figure 7 The purity and yield of the process according to the invention are shown in Table 1 as a function of the number of cycles (the first cycle comprises steps (1) to (4) = cycle 1.1, and the repetition of steps (2) to (4) = cycle 1.2; the subsequent cycles do not comprise step (1)). The purity remains at a high level, while the overall yield increases with each cycle. The results of the arrangement according to the invention ("continuous") are compared with the results of a conventional process ("batch") in which the side fractions are not fed to the chromatography matrix.

[0088] Table 1: Comparison of process parameters and results between the continuous gradient elution chromatography fractionation method ("continuous") and the conventional method ("batch")

[0089] Batch continuous deviation Flow rate (feed) l / min 0.0095 0.0083 -12.5％ Loading amount (per column) l 1.4 1.4 0％ Feed / day (maximum) l 6.27 11.97 +91％ Feed / hour (average value) l 0.26 0.5 +91％ purity [％] 99.5 99.9 Yield [％] 73.1 95.8 +31％ Output / day g 2.3 5.7 +150.2％ productivity g / l / d 33.51 41.9 +25.1％ Eluent consumption l / g 1.78 1.36 -23.6％

[0090] As can be seen from Table 1, the process and apparatus of the present invention results in a significant increase in yield and therefore productivity, while reducing eluent consumption, compared to conventional processes.

Claims

1. A method for separating a product of interest from impurities, the method comprising the following steps in the order indicated: (1) loading a first volume of a feed solution comprising the product of interest and impurities onto a chromatography matrix; (2) contacting the chromatography matrix with an elution solution; (3) a) optionally collecting eluted fraction 1 (EF1) in a side fraction container (SFC), b) collecting elution fraction 2 (EF2) in a product container, and c) optionally collecting elution fraction 3 (EF3) in SFC, wherein at least one of EF1 and EF3 is collected; (4) loading EF1 and / or EF3 and a second volume of the feed solution onto a chromatography matrix simultaneously or sequentially; wherein steps 2 to 4 are repeated at least once, at least twice, at least 4 times, preferably at least 9 times, at least 14 times, more preferably at least 19 times, at least 24 times, most preferably at least 29 times; and The chromatographic matrices of steps 1 and 4 are the same or different chromatographic matrices.

2. The method of claim 1, wherein: (i) the chromatographic matrix of steps 1 and 4 is the same first chromatographic matrix; or (ii) the chromatographic matrix of step 1 is a first chromatographic matrix and the chromatographic matrix of step 4 is a second chromatographic matrix, and at each repetition of steps 2 to 4, the chromatographic matrices alternate between the second chromatographic matrix and the first chromatographic matrix.

3. The method according to claim 1 or 2, wherein: The volume of EF1 and / or EF3 collected 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. A method as claimed in any one of the preceding claims, wherein: During step 2, the concentration of the eluent contained in the elution solution increases over time, and wherein the eluent weakens the interaction between the product of interest and the chromatography matrix.

5. A method as claimed in any one of the preceding claims, wherein: (i) EF1 and / or EF3 contain the product of interest and impurities, wherein the concentration of the product of interest is increased by at least 2 times, preferably at least 5 times, more preferably at least 10 times compared to the solution loaded in step 1, wherein a) The first predetermined concentration X of the product of interest in the eluate collected by EF1 EF1-P and the product of interest in the eluate at a predetermined concentration Y EF1-P and / or the collection of EF3 at a first predetermined concentration X of the product of interest in the eluate EF3-P and the product of interest in the eluate at a predetermined concentration Y EF3-P and / or b) the first predetermined concentration X of the impurities collected in the eluate of EF1 EF1-I and the predetermined concentration Y of these impurities in the eluate EF1-I and / or EF3 is collected at a first predetermined concentration X of these impurities in the eluate. EF3-I and the predetermined concentration Y of these impurities in the eluate EF3-I and / or (ii) EF2 comprises a substantially pure product of interest, wherein: a) The collection of EF2 at a predetermined concentration X of the product of interest in the eluate EF2-P and the product of interest in the eluate at a predetermined concentration Y EF2-P and / or b) The predetermined concentration X of the impurity collected in the eluate of EF2 EF2-I and the impurity concentration Y in the eluate is EF2-I Stop when.

6. A method as claimed in any one of the preceding claims, wherein: Step 3 further comprises the step of diluting EF1 and / or EF3, preferably wherein the dilution occurs in the SFC, and / or Wherein EF1 and / or EF3 are diluted with one or more of the feed, chromatography buffer and water.

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

8. The method according to any one of claims 2 to 7, wherein: EF1 and / or EF3 collected from the first chromatographic matrix are collected in a first SFC, and EF1 and / or EF3 collected from the second chromatographic matrix are collected in a second SFC.

9. A method as claimed in any one of the preceding claims, wherein in steps 1 and 4, loading is stopped before any product of interest is eluted from the chromatography matrix with the flow-through; wherein steps 1 and 4 comprise binding the product of interest to the chromatographic matrix; and / or Wherein these chromatographic matrices of step 1 and step 4 are of the same type.

10. A method as claimed in any one of the preceding claims, wherein: The chromatography mode of step 1 and step 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.

11. A method as claimed in any one of the preceding claims, wherein: These chromatography matrices of step 1 and step 4 are reverse phase chromatography matrices.

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

13. A method as claimed in any one of the preceding claims, wherein: These chromatographic matrices of step 1 and step 4 are chromatographic columns.

14. A method as claimed in any one of the preceding claims, wherein: The product of interest is a polypeptide or a protein.

15. A chromatography device comprising one or more chromatography matrices having a first end and a second end; Feed container; one or more side fraction containers (SFC); conduit means connecting the second end of the one or more chromatography matrices to the one or more side fraction containers; conduit means connecting the one or more side fraction containers to the first end of the one or more chromatographic matrices; conduit means connecting the feed vessel to the one or more side fraction vessels; conduit means connecting the feed container to the first end of the one or more chromatographic matrices; wherein the volume of the one or more side fraction containers is about 0.05 to about 8 volumes of the chromatography matrices.

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