阅读说明：本技术 来自样品的靶的尺寸排阻层析分离或脱盐中的并行分离和清洗 (Parallel separation and washing in size exclusion chromatographic separation or desalting of target from sample ) 是由 C.O.艾瑞克森 于 2018-02-23 设计创作，主要内容包括：提供了用于在模拟移动床层析系统中执行分离的同时在该系统中执行柱的清洗的系统和方法。(systems and methods are provided for performing a column wash in a simulated moving bed chromatography system while performing a separation in the system.)

1. A method for performing size exclusion chromatographic Separation (SEC) or desalting of a target from a sample in a system, comprising:

i) A step of providing the system with at least three columns (A, B, C, …), at least two of which can be connected in series simultaneously;

ii) a step of connecting a column accommodating the target in series with another of the columns,

ii) a step of washing at least one further column with a washing fluid while transferring the target from the column containing the target to the other one of the columns by a flow of buffer fluid.

2. The method according to claim 1, characterized in that the method further comprises the following subsequent steps: connecting an outlet of the other column to an inlet of the other column being cleaned, and subsequently collecting the target from the outlet of the column being cleaned.

3. the method according to claim 1, characterized in that the method further comprises the steps of: transporting the target from the outlet of the other column successively through the other column being cleaned and one or more additional columns in any order, and finally collecting the target from the last of the additional columns or the other column being cleaned.

4. the method of claim 1 or claim 3, for use in a Size Exclusion Chromatography (SEC) or desalination system wherein the at least three columns comprise four columns for separating a target from a sample, wherein the target passes through three or four columns before being collected, wherein each column has an inlet and an outlet fluidly connectable to a valve assembly for selectively simultaneously connecting one of the at least two chromatography columns and the other chromatography columns in series to a flow of wash fluid, wherein the valve assembly comprises a first inlet for inputting a buffer solution, a second inlet for inputting a wash fluid, an inlet for receiving fluid from the chromatography columns, and a first outlet to a waste valve assembly or target collection assembly for diverting fluid to a waste valve assembly or target collection assembly;

the system further comprises:

a first detector for detecting the presence of the target substance;

a second detector for detecting the presence of the target substance;

one or more pumps for pumping buffer and wash fluids;

a source of a first buffer solution;

A source of the cleaning fluid; and the number of the first and second groups,

Means for injecting the sample into the SEC column.

5. The method of claim 4, further comprising the steps of:

a) i) operating the valve assembly such that the inlet of a third column is connected to a source of a wash fluid, the outlet of a first column is connected to the inlet of a second column, the inlet of the first column and optionally the inlet of a fourth column are connected to the source of the first buffer, and the outlets of the second, third and fourth columns are directed to a waste assembly,

ii) arranging the detector between the outlet of the first column and the inlet of the second column,

iii) injecting a sample S comprising the target substance into the inlet of the first column;

iv) subsequently, pumping a wash fluid into the third column and pumping a first buffer into the inlets of at least the first and optionally the fourth column until the target molecule has passed the detector and has entered the second column;

b) i) operating the valve assembly such that the outlet of the second column is connected to the inlet of the third column, the inlet of the second column and optionally the inlet of the first column are connected to the source of the first buffer, the inlet of the fourth column is connected to the source of a wash fluid,

ii) arranging the detector between the outlet of the second column and the inlet of the third column,

iii) subsequently pumping a wash fluid into the fourth column while pumping a first buffer into the inlet of at least the second column until the target molecule has passed the detector and has entered the third column;

c) i) operating the valve assembly such that the outlet of the third column is connected to the inlet of the fourth column, the inlet of the third column and optionally the inlet of the second column are connected to the source of the first buffer, the inlet of the first column is connected to the source of a wash fluid,

ii) arranging the detector between the outlet of the third column and the inlet of the fourth column,

iii) subsequently pumping a wash fluid into the first column while pumping a first buffer into the inlet of at least the third column until the target molecule has passed the detector and has entered the fourth column,

d) i) operating the valve assembly such that the outlet of the fourth column is connected to a valve member for diverting fluid to a waste assembly or a target collection assembly, the inlet of the third column is connected to the source of cleaning fluid, the outlet of the first column is connected to the inlet of the second column, and the outlets of the second and third columns are connected to the waste assembly;

ii) arranging the first detector between the outlet of the first column and the inlet of the second column, and arranging the second detector between the outlet of the fourth column and the inlet of the collection assembly,

iii) pumping a wash fluid into the third column and pumping a first buffer into the inlet of at least the fourth column until the target molecule has passed the detector and has entered the collection assembly.

6. the method of claim 1 or claim 2 for a Size Exclusion Chromatography (SEC) or desalination system comprising four columns for separating target from a sample, wherein the target passes through three columns before being collected, wherein each column has an inlet and an outlet and is fluidly connectable to a valve assembly, such as a multi-position valve, arranged for selectively simultaneously connecting only two of the chromatography columns in series and one of the other chromatography columns to a flow of wash fluid, wherein the valve comprises a first inlet port for inputting a buffer solution, a second inlet port for inputting wash fluid, an inlet port for receiving fluid from the chromatography columns, a first outlet port leading to a valve member for diverting fluid to a waste member or target collection member;

The system further comprises:

a first detector for detecting the presence of the target substance;

a second detector for detecting the presence of the target substance;

A plurality of pumps for pumping buffer and wash fluids;

A source of a first buffer solution;

A source of the cleaning fluid; and the number of the first and second groups,

Means for injecting the sample into the SEC column,

Wherein the method comprises the steps of:

a) i) positioning the valve assembly such that the inlet of a third column is connected to a source of a wash fluid, the outlet of a first column is connected to the inlet of a second column, the inlet of the first column and optionally the inlet of the fourth column are connected to the source of the first buffer, and the outlets of the second, third and fourth columns are directed to a waste assembly,

ii) arranging the detector between the outlet of the first column and the inlet of the second column,

iii) injecting a sample S comprising the target substance into the inlet of the first column;

iv) subsequently, pumping a wash fluid into the third column and pumping a first buffer into the inlets of at least the first and optionally the fourth column until the target molecule has passed the detector and has entered the second column;

b) i) positioning the valve such that the outlet of the second column is connected to the inlet of the third column, the inlet of the second column and optionally the inlet of the first column is connected to the source of the first buffer, the inlet of the fourth column is connected to the source of a wash fluid,

ii) arranging the detector between the outlet of the second column and the inlet of the third column,

iii) subsequently pumping a wash fluid into the fourth column while pumping a first buffer into the inlet of at least the second column until the target molecule has passed the detector and has entered the third column;

c) i) positioning the valve such that the outlet of the third column is connected to a valve member for diverting fluid to a waste assembly or a target collection assembly, the inlet of the third column is connected to the source of buffer, the outlet of the fourth column is connected to the inlet of the first column, and the outlets of the first and second columns are connected to the waste assembly;

ii) arranging the first detector between the outlet of the fourth column and the inlet of the first column, and the second detector between the outlet of the third column and the inlet of the collection assembly;

iii) pumping a wash fluid into the second column and pumping a first buffer into the inlet of at least the third column until the target molecule has passed the detector and has entered the collection assembly.

7. the method according to claim 5 or claim 6, characterized in that it comprises the steps of:

repeating the steps of the method, wherein in each repetition, the number of each column is reduced by one in base four such that the second column is reassigned to the first column, the first column is reassigned to the fourth column, the third column is reassigned to the second column, and the fourth column is reassigned to the third column.

8. A method according to any one of the preceding claims, wherein the flow rate into each column is controlled to be substantially the same.

9. A chromatography system comprising a plurality of columns which can be interconnected for performing chromatographic separations, said system comprising:

At least three substantially identical SEC or desalting columns (A, B, C …), each column having an inlet (Ai, Bi, Ci …) and an outlet (Ao, Bo, Co, …) and being fluidly connectable to a valve assembly for selectively connecting only two of the chromatography columns in series at any time, wherein the valve comprises a first inlet port for inputting a buffer solution, a second inlet port for inputting a buffer solution, a third inlet port for inputting a wash solution, a first outlet port to a waste assembly, a second outlet port to a target collection assembly, a third output, a fourth output and a fifth output simultaneously connectable to three of the four columns;

the system further comprises:

a first detector for detecting the presence of the target substance,

A second detector for detecting the presence of the target substance,

A first pump for injecting fluid into the first inlet port;

a second pump for injecting fluid into the second inlet port;

a third pump for injecting fluid into the third inlet port;

A source of a first buffer solution;

a source of the cleaning solution; and the number of the first and second groups,

Means for loading a sample into the column.

10. A system according to claim 9, characterized in that the system comprises N columns and that control means are provided arranged to monitor the detectors and to control the pumps and means for loading samples into the columns.

11. The system according to claim 10, wherein the control means is adapted to perform a separation cycle, wherein the separation cycle is arranged to be performed by N-1 columns in series, and wherein the remaining columns are subjected to washing during the separation cycle.

12. The system according to claim 10, wherein the control means is adapted to perform a separation cycle, wherein the separation cycle is arranged to be performed by N columns in series, and wherein at least one column is subjected to washing during the separation cycle.

Technical Field

the present invention relates to chromatography, and more particularly to methods and systems for performing chromatography while simultaneously performing cleaning of one or more chromatography columns in a chromatography system comprising a plurality of columns. More particularly, the present invention relates to parallel washing of a column in a simulated moving bed chromatography system while it is performing Size Exclusion Chromatography (SEC) or desalting.

background

Size Exclusion Chromatography (SEC) separates molecules according to size differences as they pass through the SEC medium in a column. SEC media consist of a porous matrix of spherical particles with chemical and physical stability and inertness (i.e., lack of reactivity and adsorption properties). To perform the separation, media is packed into the column to form a packed bed. The packed bed is equilibrated with a buffer, which fills the pores of the matrix and the spaces between the particles. The stationary phase or liquid located inside the pores is in equilibrium with the mobile phase or liquid located outside the particles. Small molecules enter the pores of the medium and are delayed, while large molecules that are too large to enter the medium pass through the column without obstruction. An example of the results of a typical SEC run is shown in fig. 2b), where high molecular weight molecules leave the SEC column first and low molecular weight molecules leave the column last. Thus, unlike ion exchange or affinity chromatography, molecules are not bound to the chromatography medium and, therefore, the buffer composition does not directly affect the resolution (the degree of separation between the peaks of the molecules exiting the column). Thus, a significant advantage of SEC is that conditions can be varied to accommodate sample type or need for further purification, analysis or storage without changing the separation. The longer the column, the better the resolution because smaller molecules are delayed for an extended longer time as the length of the column increases, however, the column has a greater backpressure and there is a practical limit on how long the column can be. A process known as Simulated Moving Bed (SMB) is used to overcome this problem by dividing a column of the desired overall length into a plurality of columns connected in series. The pressure drop is thereby reduced to the fraction of the pressure drop that would be experienced on a single column with the same overall length as the plurality of columns. Fig. 1a) and 1b) show a theoretical comparison between the pressure drop dP over a system (shown in fig. 1a) comprising one long column 1 of length L and the pressure drop over a system (shown in fig. 1b) comprising three identical columns A, B, C of the same total length and volume as a single column. Fluid is supplied to the top of column 1 (column a, respectively) and fluid is extracted from the bottom of column 1 (column 3, respectively). The extracted fluid passes through detector D and then through a valve, which may direct the fluid to waste pathway W or storage S. The valve is controlled by detector D-when detector D detects the presence of a target molecule, the valve can be controlled to deliver fluid to a waste or storage section as appropriate. As can be clearly seen, the total length of columns a, B, C is the same as the length of column 1, and the theoretical pressure drop over each of the three columns a-C is only one third of the theoretical pressure drop over a single column 1. However, since only two columns need to be connected in series at any one time (e.g., column a and column B as shown in solid lines and column B and column C as shown in dashed lines), the actual pressure drop during a chromatography run is 2/3 dP.

the sample is usually eluted evenly, and therefore, no different buffer needs to be used during the separation. However, a washing step (also known as clean-in-place (CIP)) using a running buffer or alkaline solution is typically included at the end of the separation to remove molecules that may have remained on the column and prepare the column for a new run. Such a washing step is time consuming because the separation medium needs to be subjected to the washing fluid for a certain period of time (e.g. 10 minutes) and the column is not usable until the washing step is completed.

disclosure of Invention

In one aspect, the invention relates to a chromatography system comprising a plurality of substantially identically packed size exclusion chromatography columns for use in a method according to the invention.

in another aspect, the invention provides a method of operating a size exclusion chromatography system in which separation can be performed in parallel with a washing step.

drawings

fig. 1a) schematically shows a single column.

Fig. 1b) schematically shows a system comprising a plurality of columns arranged in series with the same total length as the columns in fig. 1 a).

Fig. 2a) shows a typical graph of the absorbance of UV light with a wavelength of 280 nm as a function of time for a SEC run in a SEC column, correspondingly showing a typical graph of the conductivity as a function of time for a desalting run in a desalting column.

Figure 2b) shows a typical graph of absorbance as a function of column volume through a SEC column.

fig. 3a) schematically shows an example of a SEC or desalination system according to the invention.

Fig. 3b) shows a schematic enlarged view of the multi-position valve of fig. 3 a).

Fig. 4-7 show steps in a method according to the invention comprising a plurality of cycles using the system of fig. 3.

Fig. 8-11 show steps in another method according to the present invention comprising a plurality of cycles using the system of fig. 3.

Detailed Description

The present invention relates to a method for separating at least one target molecule or group of target molecules (hereinafter, for the sake of brevity, simply referred to as "target") from a sample in a simulated moving bed chromatography system (SMB system) comprising a plurality of columns containing SEC medium using size exclusion chromatography, in which method at least one column containing medium in the system is washed during the separation process.

one embodiment of such a system is schematically shown in fig. 3. In this embodiment, the system 30 includes four chromatography columns A, B, C, D. Each column contains a separation medium (33). Preferably, the columns have substantially the same performance, and preferably, the same separation media and packing protocol are used in each column. Having substantially identical columns ensures that the results of the chromatography cycle are independent of the order in which the columns are used. Each column follows the convention and has inlets Ai, Bi, Ci, Di at one end and outlets Ao, Bo, Co, Do at the opposite end. The system includes a multi-position valve assembly 35, the multi-position valve assembly 35 including a plurality of ports P1, P2 …, Pm, each of which may be connected by fluid lines to one of the other components in the system. Each of the column inlets and outlets can be connected by fluid lines to a single one of the ports of the multi-position valve 35. Valve 35 can be arranged to connect the outlet of any column to the inlet of any other column (e.g., to connect the outlet Ao of column a to the inlet Bi of column B). In this way, fluid in any column can be continuously transferred to any other column. The valves are further arranged so that buffer can be simultaneously supplied to the inlets of two or more of the other columns and wash fluid is supplied to the inlets of the remaining columns. This can be achieved by, for example, a valve 35, the valve 35 having a rotor with internal channels that can rotate within a stator to provide the desired connections between the ports of the valve.

the system is provided with a sample or other fluid 39 (hereinafter, for brevity, it will be referred to as "sample") containing a target 41 and a syringe or container and a pump or gravity feed line 42 or other means for supplying the sample to the fluid line or other inlet to the sample introduction port 'pgample' of the valve.

the system further comprises: a source of buffer fluid 43 connectable to the buffer inlet port of the valve; a source of a cleaning fluid 45 (e.g., sodium hydroxide solution) connectable to the cleaning fluid inlet port of the valve; a first sensor line comprising a first sensor 47A, the first sensor 47A being connectable to the inlet port of the valve and the detector outlet port; a second sensor line comprising a second sensor 47B, the second sensor 47B connectable to the inlet port of the valve and the detector outlet port. The sensors 47A, 47B may be any type of suitable sensor, for example for detecting proteins, the sensors 47A, 47B may be UV absorption detectors sensitive to one UV light wavelength or preferably two or more UV light wavelengths. Such UV detectors are well known in the art and detect the absorption of UV light by proteins through a fluid. Where the system is to be used for other purposes, such as desalination, the sensor may be a conductivity sensor of the type well known in the art. Pumps 49A, 49B or other fluid-driven means (e.g., gravity) are provided to enable the buffer fluid and the wash fluid to flow to the respective ports of the multi-position valve 35. The multi-position valve 35 is further provided with one outlet port to the waste path W and another outlet to the target collection vessel S or path. A control unit 51, such as a computer, microprocessor, control circuit with appropriate software, or a manually operated control unit 51, is provided to control the multi-position valves and pumps in order to achieve the desired sample flow through the system. It will be appreciated that the system shown in figure 3b) schematically illustrates a variety of components which are individually known and therefore a detailed description of the construction of these components is not included herein. Furthermore, alternative components that will provide acceptable performance will be apparent to the skilled person. For example, the use of a multi-position valve 35 is preferred because the valve assembly provides a compact valve, but the multi-position valve can be replaced by an array of individually on/off, on/off type valves, for example to provide an overall lower cost valve assembly if the system is to be manufactured as disposable hardware. In this case, a simple fluid pressure actuated diaphragm valve may be used, or a solenoid operated valve may be employed, all with the same effect as the multi-position valve described herein. Such an alternative valve array would be controlled by the control unit 51 such that the valves open and close in the correct sequence to provide the desired flow path(s) as described below.

In order to achieve a good resolution of the target and to maximize the utilization of the chromatography system, a method is provided comprising at least one separation cycle according to the steps described hereinafter, which method may be performed manually or by controlling the pump(s) and valve(s) using control components and suitable software and hardware. The method is intended to ensure that when the columns are used in multiple cycles, each column is subjected to approximately the same wear (i.e. each column is utilized equally), and the method is intended to ensure that during the washing step the media in each column is exposed to the washing fluid for a period of time sufficient to ensure adequate washing. This is achieved by providing a wash fluid to at least one of the columns while the target is flowing in and/or between two other columns. Switching of flow between columns is initiated by studying the signal generated by a sensor positioned in the fluid line connecting the outlet from the first column to the inlet of the second column. When the signal indicates that the target has left the first column and a time sufficient for the target to enter the second column has elapsed, the multi-position valve is actuated to connect the inlet of the second column to the source of the buffer and the outlet of the second column to the inlet of the third column in the series. This causes the target to pass through the second column and into the third column. At the same time, the wash buffer may be input into the first column via the inlet of the first column, while the outlet of the first column is connected to the waste. This provides the following advantages: the undesired smaller molecules that have been delayed in the columns are sent directly to the waste without passing through any subsequent columns, thereby reducing the load on these columns. In one example of the method, the target can be separated in a cycle that includes passing the sample through all but one of the plurality of columns in succession (e.g., three of four columns).

the method may be adapted for substantially continuous use such that as soon as a target from one sample initially loaded onto a first column is separated and collected, the system is ready to load a new sample onto a different column than the previous column that initially received the previous sample. Preferably, the loading of the sample is arranged such that each column initially receives the sample in turn before the first column resumes loading with the sample. This means that the columns are subject to approximately the same wear.

an example of a method for size exclusion chromatography to separate a target from a sample is shown in figures 4 to 7, wherein the sample may be loaded in a series of cycles, wherein each series comprises four cycles, and wherein in each cycle the sample is loaded onto a different column. Each cycle includes three steps.

Fig. 4a) shows the initial and final state of the four columns in the first step of the first cycle. In the initial state of the first step of the first cycle, as shown on the left in fig. 4a), columns a and D are filled with buffer, and columns B and C are filled with washing fluid (e.g. NaOH). The multi-position valve is arranged such that the inlets of column a, column C and column D are connected to the buffer stream, the outlet of column a is connected to the inlet of column B via a UV detector, and the outlets of column B, column C and column D are connected to the waste. This first step is initiated by injecting the sample into the buffer stream entering the inlet of column a. This causes the sample to travel through column a. The detector monitors the fluid exiting column a and, when a signal is detected indicating that the target has passed the detector and entered column B, the first step of the first cycle is complete. This is shown in the terminating state on the right in fig. 4 a). The multi-position valve is then adjusted so that the second step of the first cycle can begin.

In the initial state of the second step of the first cycle, as shown on the left in fig. 4b), all columns are filled with buffer. The multi-position valve is arranged such that the inlets of column a and column B are connected to the buffer stream, the inlet of column D is connected to the washing fluid stream, the outlet of column B is connected to the inlet of column C via a UV detector, and the outlets of column a, column C and column D are connected to the waste. This second step starts and the flow of buffer into the inlet of column B causes the target to travel through column B and into column C. The detector monitors the fluid exiting column B and, when a signal is detected indicating that the target has passed the detector and entered column C, the second step of the first cycle is completed. This is shown in the terminating state on the right in fig. 4 b). The multi-position valve is then adjusted so that the third step of the first cycle can begin.

In the initial state of the third and final step of the first cycle, as shown on the left in fig. 4C), column a, column B and column C are filled with buffer, while column D is filled with washing fluid. The multi-position valve is arranged such that the inlets of column C and column D are connected to the buffer stream, the inlet of column B is connected to the washing fluid stream, the outlet of column D is connected via a UV detector to the inlet of column a, the outlets of column a and column B are connected to the waste, and the outlet of column C is connected via a second detector to an outlet valve (not shown) that can direct the fluid leaving column C to the waste or to a container for collecting the target. Initially, the outlet valve directs fluid exiting column C to a waste. This third step starts and the flow of buffer into the inlet of column C causes the target to travel through column C. Once the second detector detects the presence of target molecules in the fluid exiting the column C, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected (step 3), the third step of the first cycle is complete. This is shown in the right terminating state of fig. 4 c).

if further separation is to be performed on the system, a second cycle may begin. The steps of this cycle are shown in fig. 5a) to 5 c).

Fig. 5a) shows the initial and final state of the four columns in the first step of the second cycle. In the initial state of the first step of the second cycle, as shown on the left in fig. 5a), columns C and D are filled with buffer and columns a and B are filled with washing fluid. The multi-position valve is arranged such that the inlets of column B, column C and column D are connected to the buffer stream, the outlet of column D is connected to the inlet of column a via a UV detector, and the outlets of column a, column B and column C are connected to the waste. This first step is initiated by injecting the sample into the buffer stream entering the inlet of column D. This causes the sample to travel through column D. The detector monitors the fluid exiting the column D and, when a signal is detected indicating that the target has passed the detector and entered the column a, the first step of the first cycle is complete. This is shown in the right terminating state of fig. 5 a). The multi-position valve is then adjusted so that the second step of the second cycle can begin.

In the initial state of the second step of the second cycle, as shown on the left in fig. 5b), all columns are filled with buffer. The multi-position valve is arranged such that the inlets of column a and column D are connected to the buffer stream, the inlet of column C is connected to the washing fluid stream, the outlet of column a is connected to the inlet of column B via a UV detector, and the outlets of column B, column C and column D are connected to the waste. This second step starts and the flow of buffer into the inlet of column a causes the target to travel through column a and into column B. The detector monitors the fluid exiting column a and, when a signal is detected indicating that the target has passed the detector and entered column B, the second step of the second cycle is completed. This is shown in the terminating state on the right in fig. 5 b). The multi-position valve is then adjusted so that the third step of the second cycle can begin.

In the initial state of the third and final step of the second cycle, as shown on the left in fig. 5C), column a, column B and column D are filled with buffer, while column C is filled with wash fluid. The multi-position valve is arranged such that the inlets of column B and column C are connected to the buffer stream, the inlet of column a is connected to the washing fluid stream, the outlet of column C is connected via a UV detector to the inlet of column D, the outlets of columns a and D are connected to the waste, and the outlet of column B is connected via a second detector to an outlet valve (not shown) that can direct the fluid leaving column B to the waste or to a container for collecting the target. Initially, the outlet valve directs fluid exiting column B to a waste. This third step starts and the flow of buffer into the inlet of column B causes the target to travel through column B. Once the second detector detects the presence of target molecules in the fluid exiting the column B, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected (step 3), the third step of the second cycle is complete. This is shown in the right terminating state of fig. 5 c).

if further separation is to be performed on the system, a third cycle may begin. The steps of this cycle are shown in fig. 6a) to 6 c).

fig. 6a) shows the initial and final state of the four columns in the first step of the third cycle. In the initial state of the first step of the third cycle, as shown on the left in fig. 6a), columns B and D are filled with buffer, and columns a and D are filled with wash fluid. The multi-position valve is arranged such that the inlets of column a, column B and column C are connected to the buffer stream, the outlet of column C is connected to the inlet of column D via a UV detector, and the outlets of column a, column B and column D are connected to the waste. The first step is initiated by injecting the sample into the buffer stream entering the inlet of column C. This causes the sample to travel through column C. The detector monitors the fluid exiting the column C and, when a signal is detected indicating that the target has passed the detector and entered the column D, the first step of the first cycle is complete. This is shown in the terminating state on the right in fig. 6 a). The multi-position valve is then adjusted so that the second step of the third cycle can begin.

in the initial state of the second step of the third cycle, as shown on the left in fig. 6b), all columns are filled with buffer. The multi-position valve is arranged such that the inlets of column C and column D are connected to the buffer stream, the inlet of column B is connected to the washing fluid stream, the outlet of column D is connected to the inlet of column a via a UV detector, and the outlets of column a, column B and column C are connected to the waste. This second step starts and the flow of buffer into the inlet of column D causes the target to travel through column D and into column a. The detector monitors the fluid exiting the column D and, when a signal is detected indicating that the target has passed the detector and entered the column a, the second step of the third cycle is completed. This is shown in the terminating state on the right in fig. 6 b). The multi-position valve is then adjusted so that the third step of the third cycle can begin.

in the initial state of the third and final step of the third cycle, as shown on the left in fig. 6C), column a, column C and column D are filled with buffer, while column B is filled with wash fluid. The multi-position valve is arranged such that the inlets of column a and column B are connected to the buffer stream, the inlet of column D is connected to the washing fluid stream, the outlet of column B is connected via a UV detector to the inlet of column C, the outlets of column C and column D are connected to the waste, and the outlet of column a is connected via a second detector to an outlet valve (not shown) that can direct the fluid leaving column a to the waste or to a container for collecting the target. Initially, the outlet valve directs fluid exiting column a to a waste. This third step starts and the flow of buffer into the inlet of column a causes the target to travel through column a. Once the second detector detects the presence of target molecules in the fluid exiting column a, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected (step 3), the third step of the third cycle is complete. This is shown in the right terminating state of fig. 6 c).

If further separation is to be performed on the system, a fourth cycle may begin. The steps of this cycle are shown in fig. 7a) to 7 c).

Fig. 7a) shows the initial and final state of the four columns in the first step of the fourth cycle. In the initial state of the first step of the fourth cycle, as shown on the left in fig. 7a), columns a and B are filled with buffer, and columns C and D are filled with washing fluid. The multi-position valve is arranged such that the inlets of column a, column B and column D are connected to the buffer stream, the outlet of column B is connected to the inlet of column C via a UV detector, and the outlets of column a, column C and column D are connected to the waste. The first step is initiated by injecting the sample into the buffer stream entering the inlet of column B. This causes the sample to travel through column B. The detector monitors the fluid exiting column B and, when a signal is detected indicating that the target has passed the detector and entered column C, the first step of the first cycle is complete. This is shown in the terminating state on the right in fig. 7 a). The multi-position valve is then adjusted so that the second step of the fourth cycle can begin.

In the initial state of the second step of the fourth cycle, as shown on the left in fig. 7b), all columns are filled with buffer. The multi-position valve is arranged such that the inlets of column B and column C are connected to the buffer stream, the inlet of column a is connected to the washing fluid stream, the outlet of column C is connected to the inlet of column D via a UV detector, and the outlets of column a, column B and column D are connected to the waste. This second step starts and the flow of buffer into the inlet of column C causes the target to travel through column C and into column D. The detector monitors the fluid exiting the column C and, when a signal is detected indicating that the target has passed the detector and entered the column D, the second step of the fourth cycle is completed. This is shown in the terminating state on the right in fig. 7 b). The multi-position valve is then adjusted so that the third step of the fourth cycle can begin.

In the initial state of the third and final step of the fourth cycle, as shown on the left in fig. 7C), column B, column C and column D are filled with buffer, while column a is filled with wash fluid. The multi-position valve is arranged such that the inlets of column a and column D are connected to the buffer stream, the inlet of column C is connected to the washing fluid stream, the outlet of column a is connected to the inlet of column B via a UV detector, the outlets of column B and column C are connected to the waste, and the outlet of column D is connected via a second detector to an outlet valve (not shown) that can direct the fluid leaving column D to the waste or to a container for collecting the target. Initially, the outlet valve directs the fluid exiting the column D to a waste. This third step starts and the flow of buffer into the inlet of column D causes the target to travel through column D. Once the second detector detects the presence of target molecules in the fluid exiting the column D, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected (step 3), the third step of the fourth cycle is complete. This is shown in the right terminating state of fig. 7 c).

during four cycles, each column has been used once for the initial injection of the sample and once for the final fractionation of the target. Thus, the load on each column is already substantially equal.

If further separation is to be performed on the system, a new series of cycles may begin. Since the contents of the column in the final state of the fourth cycle (i.e., the buffer in columns a and D and the wash fluid in columns B and C) are the same as the contents of the column in the initial state of the first cycle, the new series of cycles preferably follows the same sequence as the previous series of cycles.

In a general method according to the invention using four chromatography columns, three of which will be exposed to the target in a separation cycle, in the first step in the cycle, a sample containing the target (e.g. protein) to be separated from other molecules in the sample is loaded in a first of the four columns and subsequently transported to a clean second column, while the third and fourth columns are flushed with buffer.

Once the target has been detected in the fluid line between the first column and the second column and has entered the second column, the second step of the cycle begins: the multi-position valve is operated such that the second column is connected in series to the third column and the target is transported to the third column, while the first column is flushed with a buffer and the fourth column is cleaned with a washing solution.

Once the target has been detected in the fluid line between the second and third columns and has entered the third column, the third step in the cycle begins: the valve is operated such that the fourth column is connected in series to the first column and the wash fluid from column 4 is fed into the first column while the target molecules are transported through the third column. The cleaning fluid is also supplied into the second column. Initially, the outlet of the third column is connected to the waste via a sensor. Once the sensor detects the target, the output from the sensor is switched to a target collection vessel and the target is collected there. At the same time, the first column is cleaned with a wash solution from the fourth column, while the third and fourth columns are washed with a buffer.

when further separations are to be performed, each separation is then started by injecting the sample into the last column that has been filled with buffer after the washing step.

In a second example of a method according to the present invention, the target may be separated in a cycle comprising passing the sample through all of the plurality of columns (e.g., four of the four columns) in succession.

An example of a method for size exclusion chromatography to separate a target from a sample in which the sample may be loaded in a series of cycles, wherein each series comprises four cycles and in each cycle the sample is loaded onto a different column, is shown in figures 8 to 11. Each cycle uses all four of the columns to achieve high resolution, and each cycle includes four steps.

Fig. 8a) shows the initial and final state of the four columns in the first step of the first cycle. In the initial state of the first step of the first cycle, as shown on the left in fig. 8a), column a and column D are filled with buffer, and column C is filled with a washing fluid (e.g. NaOH), and column B is washed with buffer from column a to remove the washing fluid. The multi-position valve is arranged such that the inlets of column a and column D are connected to the buffer stream, the inlet of column C is connected to the washing fluid stream, the outlet of column a is connected to the inlet of column B via a UV detector, and the outlets of column B, column C and column D are connected to the waste. This first step is initiated by injecting the sample into the buffer stream entering the inlet of column a. This causes the sample to travel through column a. The detector monitors the fluid exiting column a and, when a signal is detected indicating that the target has passed the detector and entered column B, the first step of the first cycle is complete. This is shown in the right-hand end state of fig. 8a), in which column C is now substantially filled with cleaning fluid. The multi-position valve is then adjusted so that the second step of the first cycle can begin.

shortly after the initial state of the second step of the first cycle, as shown on the left in fig. 8B), column a and column B were filled with buffer, column D had started to be filled with wash fluid, and column C had started to empty wash fluid. Thus, the multi-position valve is arranged such that the inlets of column a and column B are connected to the buffer stream, the inlet of column D is connected to the washing fluid stream, the outlet of column B is connected to the inlet of column C via a UV detector, and the outlets of column a, column C and column D are connected to the waste. As this second step continues, the flow of buffer into the inlet of column B causes the target to travel through column B and into column C. The detector monitors the fluid exiting column B and, when a signal is detected indicating that the target has passed the detector and entered column C, the second step of the first cycle is completed. This is shown in the right-hand end state of fig. 8b), in which column C is now substantially filled with cleaning fluid. The multi-position valve is then adjusted so that the third step of the first cycle can begin.

shortly after the initial state of the third step of the first cycle, as shown on the left in fig. 8C), column a and column C are filled with buffer, column B has started to be filled with wash fluid, and column D filled with wash fluid is flushed with the contents from column C. The multi-position valve is arranged such that the inlets of column a and column C are connected to the buffer stream, the inlet of column B is connected to the washing fluid stream, the outlet of column C is connected to the inlet of column D via a UV detector, and the outlets of column a, column B and column D are connected to the waste. The detector monitors the fluid exiting the column C and, when a signal is detected indicating that the target has passed the detector and entered the column D, the third step of the first cycle is completed. This is shown in the right-hand final state of fig. 8c), in which column B is now substantially filled with the cleaning fluid. The multi-position valve is then adjusted so that the fourth step of the first cycle can begin.

shortly after the initial state of the fourth step of the first cycle, as shown on the left in fig. 8D), column a and column D are filled with buffer, while column B, which is filled with wash fluid, is flushed with buffer, and column C is filled with wash fluid. The multi-position valve is arranged such that the inlets of column a and column D are connected to the buffer stream, the inlet of column C is connected to the washing fluid stream, the outlet of column a is connected to the inlet of column B via a UV detector, and the outlets of column B and column C are connected to the waste. However, the outlet of column D is connected via a second detector to an outlet valve (not shown) that can direct the fluid exiting column D to a waste or container for collecting the target. Initially, the outlet valve directs the fluid exiting the column D to a waste. Once this step begins, the flow of buffer into the inlet of column D causes the target to travel through column D. Once the second detector detects the presence of target molecules in the fluid exiting the column D, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected, the fourth step of the first cycle is complete. This is shown in the right-hand end state of fig. 8d), in which column C is now substantially filled with cleaning fluid.

If further separation is to be performed on the system, a second cycle may begin. The steps of this cycle are shown in fig. 9a) to 9 d).

Fig. 9a) shows the initial and final state of the four columns in the first step of the second cycle. The multi-position valve is arranged such that the inlets of column a and column B are connected to the buffer stream, column D is connected to the washing fluid stream, the outlet of column B is connected to the inlet of column C via a UV detector, and the outlets of column a, column C and column D are connected to the waste. After starting the first step of the second cycle, as shown on the left in fig. 9a), column a and column B are filled with buffer, column D filled with buffer is filled with wash fluid, and column C filled with wash fluid is filled with buffer. The first step is initiated by injecting the sample into the buffer stream entering the inlet of column B. This causes the sample to travel through column B. The detector monitors the fluid exiting column B and, when a signal is detected indicating that the target has passed the detector and entered column C, the first step of the first cycle is complete. This is shown in the right terminating state of fig. 9 a). Column a, column B and column C contained buffer, and column D contained wash fluid. The multi-position valve is then adjusted so that the second step of the second cycle can begin.

In a second step of the second cycle shown in fig. 9B), the multi-position valve is arranged such that the inlets of column B and column C are connected to the buffer stream, the inlet of column a is connected to the washing fluid stream, the outlet of column C is connected to the inlet of column D via a UV detector, and the outlets of column a, column B and column D are connected to the waste. This second step starts and the flow of buffer into the inlet of column C causes the target to travel through column C and into column D. After the second step of the second cycle has started, as shown on the left in fig. 9B), columns B and C are filled with buffer, column D is flushed with washing fluid, and column a is filled with washing fluid. The detector monitors the fluid exiting the column C and, when a signal is detected indicating that the target has passed the detector and entered the column D, the second step of the second cycle is completed. This is shown in the right terminating state of fig. 9 b). Column B, column C and column D contained buffer, and column a contained wash fluid. The multi-position valve is then adjusted so that the third step of the second cycle can begin.

In a third step of the second cycle, as shown in fig. 9C), the multi-position valve is arranged such that the inlets of column B and column D are connected to the buffer stream, the inlet of column C is connected to the washing fluid stream, the outlet of column D is connected to the inlet of column a via a UV detector, and the outlets of column a, column B and column C are connected to the waste. At the beginning of this step, column B, column C and column D were filled with buffer, while column C was filled with wash fluid. As shown on the left in fig. 9c), once this third step begins, the flow of buffer into the inlet of column D causes the target to travel through column D. The detector monitors the fluid exiting the column D and, when a signal is detected indicating that the target has passed the detector and entered the column a, the third step of the second cycle is completed. This is shown in the right terminating state of fig. 9 c). Column a, column B and column D contained buffer, column C contained wash fluid, and the target was located in column a. The multi-position valve is then adjusted so that the fourth step of the second cycle may begin.

in a fourth step of the second cycle, as shown in fig. 9D), the multi-position valve is arranged such that the inlets of column a and column B are connected to the buffer stream, the inlet of column D is connected to the washing fluid stream, the outlet of column B is connected to the inlet of column C via a UV detector, the outlets of column B and column D are connected to a waste section, and the outlet of column B is connected via a second detector to an outlet valve (not shown) that can direct the fluid leaving column B to the waste section or to a container for collecting the target. Initially, the outlet valve directs fluid exiting column B to a waste. Once this step begins, the flow of buffer into the inlet of column B causes the target to travel through column B. Once the second detector detects the presence of target molecules in the fluid exiting the column B, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected, the fourth step of the first cycle is complete. At the beginning of this step, column a, column B and column D are filled with buffer, while column C is filled with wash fluid. As shown on the left in fig. 9d), once this fourth step has started, the flow of buffer into the inlet of column a causes the target to travel through column a. Once the second detector detects the presence of target molecules in the fluid exiting column a, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected, the fourth step of the second cycle is complete. This is shown in the right-hand end state of fig. 9D, where column a, column B and column C contain buffer and column D contains wash fluid.

If further separation is to be performed on the system, a third cycle may begin. The steps of this cycle are shown in fig. 10a) to 10 c). Fig. 10a) shows the intermediate and final states of the four columns in the first step of the third cycle. The multi-position valve is arranged such that the inlets of column B and column C are connected to the buffer stream, column a is connected to the washing fluid stream, the outlet of column C is connected to the inlet of column D via a UV detector, and the outlets of column a, column B and column D are connected to the waste. After starting the first step of the third cycle, as shown on the left in fig. 10a), columns B and C are filled with buffer, column a filled with buffer is filled with wash fluid, and column D filled with wash fluid is filled with buffer. The first step is initiated by injecting the sample into the buffer stream entering the inlet of column C. This causes the sample to travel through column C. The detector monitors the fluid exiting the column C and, when a signal is detected indicating that the target has passed the detector and entered the column D, the first step of the first cycle is complete. This is shown in the terminating state on the right in fig. 10 a). Column B, column C and column D contained buffer, and column a contained wash fluid. The multi-position valve is then adjusted so that the second step of the third cycle can begin.

In a second step of the third cycle, the multi-position valve is arranged such that the inlets of column C and column D are connected to the buffer stream, the inlet of column B is connected to the washing fluid stream, the outlet of column D is connected to the inlet of column a via a UV detector, and the outlets of column a, column B and column C are connected to the waste. This second step starts and the flow of buffer into the inlet of column D causes the target to travel through column D and into column a. After the second step of the third cycle has started, as shown on the left in fig. 10B), columns C and D are filled with buffer and column B is filled with wash fluid. The detector monitors the fluid exiting the column D and, when a signal is detected indicating that the target has passed the detector and entered the column a, the second step of the third cycle is completed. This is shown in the right-hand end state of FIG. 10B), in which column B is filled with washing fluid, the other columns contain buffer, and the target is located in column A. The multi-position valve is then adjusted so that the third step of the third cycle can begin.

In a third step of the third cycle, as shown in fig. 10C), the multi-position valve is arranged such that the inlets of column a and column C are connected to the buffer stream, the inlet of column D is connected to the washing fluid stream, the outlet of column a is connected to the inlet of column B via a UV detector, and the outlets of column B, column C and column D are connected to the waste. At the beginning of this step, column a, column C and column D were filled with buffer, while column B was filled with wash fluid. As shown on the left in fig. 10c), once this third step begins, the flow of buffer into the inlet of column a causes the target to travel through column a. The detector monitors the fluid exiting column a and, when a signal is detected indicating that the target has passed the detector and entered column B, the third step of the third cycle is completed. The starting state of the column for the fourth step is shown in the right-hand end state of fig. 10c), in which the target is located in column B. The multi-position valve is then adjusted so that the fourth step of the third cycle may begin.

At the beginning of the fourth step, column a, column B and column C are filled with buffer, while column D is filled with wash fluid. In a fourth step of the third cycle, as shown in fig. 10D), the multi-position valve is arranged such that the inlets of column B and column C are connected to the buffer stream, the inlet of column a is connected to the washing fluid stream, the outlet of column C is connected to the inlet of column D via a UV detector, the outlets of columns a and D are connected to a waste section, and the outlet of column B is connected via a second detector to an outlet valve (not shown) that can direct the fluid leaving column B to the waste section or to a container for collecting the target. Initially, the outlet valve directs fluid exiting column B to a waste. Once this step begins, the flow of buffer into the inlet of column B causes the target to travel through column B. Once the second detector detects the presence of target molecules in the fluid exiting the column B, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected, the fourth step of the first cycle is complete. This is shown in the terminating state on the right in fig. 10 d).

if further separation is to be performed on the system, a fourth cycle may begin. The steps of this cycle are shown in fig. 11a) to 11 d). Fig. 11a) shows the intermediate and final states of the four columns in the first step of the fourth cycle. Initially, column B, column C and column D were packed with buffer, and column a was packed with wash fluid. The multi-position valve is arranged such that the inlets of column C and column D are connected to the buffer stream, column B is connected to the washing fluid stream, the outlet of column D is connected to the inlet of column a via a UV detector, and the outlets of column B, column C and column D are connected to the waste. This first step is initiated by injecting the sample into the buffer stream entering the inlet of column D. This causes the sample to travel through column D. After starting the first step of the third cycle, as shown on the left in fig. 11a), columns C and D are filled with buffer, column a filled with washing fluid is filled with buffer, and column B filled with buffer is filled with washing fluid. The detector monitors the fluid exiting the column D and, when a signal is detected indicating that the target has passed the detector and entered the column a, the first step of the first cycle is complete. This is shown in the terminating state on the right in fig. 11 a). Column a, column C and column D contained buffer, and column B contained wash fluid. The target is located in column a. The multi-position valve is then adjusted so that the second step of the fourth cycle can begin.

in a second step, the multi-position valve is arranged such that the inlets of column a and column D are connected to a buffer stream, the inlet of column C is connected to a washing fluid stream, the outlet of column a is connected to the inlet of column B via a UV detector, and the outlets of column B, column C and column D are connected to a waste. This second step starts and the flow of buffer into the inlet of column a causes the target to travel through column a and into column B. After the second step of the third cycle has started, as shown on the left in fig. 11b), column a and column D are filled with buffer, and column C is filled with wash fluid. The detector monitors the fluid exiting column a and, when a signal is detected indicating that the target has passed the detector and entered column B, the second step of the fourth cycle is completed. This is shown in the right-hand terminal state of FIG. 11B), where the target is located in column B, the buffer is located in columns A and D, and the wash fluid is located in column C. The multi-position valve is then adjusted so that the third step of the fourth cycle can begin.

In a third step of the fourth cycle, as shown in fig. 11C), the multi-position valve is arranged such that the inlets of column B and column D are connected to the buffer stream, the inlet of column a is connected to the washing fluid stream, the outlet of column B is connected to the inlet of column C via a UV detector, and the outlets of column B, column C and column D are connected to the waste. At the beginning of this step, column a, column B and column D are filled with buffer, while column C is filled with wash fluid. As shown on the left in fig. 11c), once this third step begins, the flow of buffer into the inlet of column B causes the target to travel through column B. The detector monitors the fluid exiting column B and, when a signal is detected indicating that the target has passed the detector and entered column C, the third step of the fourth cycle is completed. The starting state of the column for the fourth step is shown in the right terminating state of fig. 11C), in which the target is located in column C. The multi-position valve is then adjusted so that the fourth step of the fourth cycle may begin.

At the beginning of the fourth step, column B, column C and column D were filled with buffer, while column a was filled with the wash fluid. In a fourth step of the fourth cycle, as shown in fig. 11D), the multi-position valve is arranged such that the inlets of column C and column D are connected to the buffer stream, the inlet of column B is connected to the washing fluid stream, the outlet of column D is connected to the inlet of column a via a UV detector, the outlets of column a and column B are connected to the waste section, and the outlet of column C is connected via a second detector to an outlet valve (not shown) that can direct the fluid exiting column C to the waste section or to a container for collecting the target. Initially, the outlet valve directs fluid exiting column C to a waste. Once this step begins, the flow of buffer into the inlet of column C causes the target to travel through column C. Once the second detector detects the presence of target molecules in the fluid exiting the column C, the outlet valve is operated to direct the flow to a receptacle for collecting the target, and the target is collected. Once the target has been collected, the fourth step of the fourth cycle is complete. This is shown in the terminating state on the right in fig. 11 d).

If further separation is to be performed on the system, a new series of cycles may begin. Since the contents of the column in the final state of the fourth cycle (i.e. the buffer in columns a, B and C and the wash fluid in column D) are the same as the contents of the column in the initial state of the first cycle, the new series of cycles preferably follows the same sequence as the previous series of cycles.

If four SEC or desalination cycles are performed in the order described above, each column has been used once for the initial injection of the sample and each column has been used once for the final fractionation of the target. Thus, the load on each column has been substantially equal over four cycles. Thus, in each set of four cycles using four columns, at the start of each new cycle, the labels of the individual columns may be considered to have been reduced by one in base four, such that when the next cycle is started, the second column (i.e., column B) is reassigned to the first column (i.e., column a), the third column (column C) is reassigned to the second column (column B), the fourth column (column D) is reassigned to the third column (column C), and the first column (column a) is reassigned to the fourth column (column D).

thus, when N columns are used, then in each subsequent cycle of the set of N cycles, the column designations are reduced by one in the base number N. For example, in a system using three columns, N =3, and at the end of the first cycle, column 1 is reassigned to column 3, column 2 is reassigned to column 1, and column 3 is reassigned to column 2.

In a system with three columns (N =3), it is possible to use only two of the columns (N-1) for SEC or desalination, in which case each cycle has only two steps, namely a first step of passing the sample through the first column to the second column while washing the third column, and a second step of collecting the target from the second column while flushing the third column with a buffer and filling the first column with washing fluid from the third column.

In a system with three columns (N =3), it is possible to use all three (N) of the columns for SEC or desalination, in which case each cycle has three steps, namely a first step of passing the sample through the first column to the second column while washing the third column, a second step of transferring the target from the second column to the third column while flushing the first column with a washing fluid, and a third step of collecting the target from the third column while filling the second column with the washing fluid from the first column. A new cycle may then start from the first column. However, if the load on the column is to be made equal, the next cycle should start with loading onto the second column. Preferably, in order to reduce the risk of the target coming into contact with the wash fluid in the second column, the loading of the sample should be delayed until after a suitable portion (e.g. 20% or 25% or 50% or even more) of the column volume of buffer has entered the second column.

In order to make the load on the columns equal, it is of course possible to change the order of the columns by increasing or decreasing the number of individual columns by any number smaller than N, provided that the steps of the cycle are modified accordingly to ensure that the cleaning of the columns is achieved between targets from two different samples entering the columns.

a method according to the invention for performing size exclusion chromatographic Separation (SEC) or desalting of a target from a sample in a system comprises the steps of:

i) A step of providing the system with at least three columns (A, B, C, …) that can be connected, for example, wherein only one pair of columns are connected in series at the same time;

ii) a step of connecting the column containing the target in series with another column,

ii) a step of washing at least one further column with a washing fluid while transferring the target from the column containing the target to the further column by a flow of the buffer fluid.

Additional steps of the method include: the outlet of the other column is connected to the inlet of the other column being cleaned, and then the target is collected from the outlet of the cleaned column.

An alternative additional step of the method comprises the steps of: the target is transported from the outlet of the further column successively through the washed further column and one or more additional columns in any order, and finally the target is collected from the last additional column or the washed further column.

An example of a method according to the invention for a Size Exclusion Chromatography (SEC) or desalination system comprising four columns for separating a target from a sample, wherein the target passes through the four columns before being collected,

The method comprises the following steps:

a) i) positioning a valve such that the inlet of the third column is connected to a source of wash fluid, the outlet of the first column is connected to the inlet of the second column, the inlet of the first column and optionally the inlet of the fourth column are connected to a source of the first buffer, and the outlets of the second, third and fourth columns are directed to a waste assembly,

ii) arranging a detector between the outlet of the first column and the inlet of the second column,

iii) injecting a sample S comprising the target substance into an inlet of the first column;

iv) subsequently, pumping a wash fluid into the third column and pumping a first buffer into the inlets of at least the first and optionally the fourth column until the target molecule has passed the detector and has entered the second column;

b) i) positioning a valve such that the outlet of the second column is connected to the inlet of the third column, the inlet of the second column and optionally the inlet of the first column is connected to a source of the first buffer, the inlet of the fourth column is connected to a source of a wash fluid,

ii) arranging the detector between the outlet of the second column and the inlet of the third column,

iii) subsequently pumping a wash fluid into the fourth column and a first buffer into the inlet of at least the second column until the target molecule has passed the detector and has entered the third column;

c) i) connecting the outlet of the third column to the inlet of the fourth column, the inlet of the third column and optionally the inlet of the second column to a source of the first buffer, the inlet of the first column to a source of a washing fluid,

ii) arranging the detector between the outlet of the third column and the inlet of the fourth column,

iii) subsequently pumping a washing fluid into the first column and a first buffer into at least the inlet of the third column until the target molecule has passed the detector and has entered the fourth column,

d) i) connecting the outlet of the fourth column to a valve member for diverting fluid to a waste or target collection assembly, the inlet of the third column being connected to a source of cleaning fluid, the outlet of the first column being connected to the inlet of the second column, and the outlets of the second and third columns being connected to a waste assembly;

ii) arranging the first detector between an outlet of the first column and an inlet of the second column, and arranging the second detector between an outlet of a fourth column and an inlet of a collection assembly,

iii) pumping a wash fluid into the third column and pumping a first buffer into the inlet of at least a fourth column until the target molecule has passed the detector and has entered the collection assembly.

in all examples of the method according to the invention, if it is determined that washing between each injection of the sample is not necessary (e.g. it is possible to run multiple samples before washing the column), the method may be provided with a step in which the washing fluid is replaced by a buffer fluid or indeed there is no fluid at all.

The foregoing description of the present disclosure has been provided for the purpose of illustration, and it will be understood by those skilled in the art that various changes and modifications may be made without changing the concept and essential features of the present disclosure. It is therefore clear that the embodiments described hereinabove are illustrative in all respects and not limitative of the disclosure.

the scope of the present disclosure is defined by the claims below, not by the detailed description of the embodiments. It should be understood that all modifications and embodiments that come within the meaning and range of equivalency of the claims are to be embraced within their scope.