Patent ID: 12188911

DETAILED DESCRIPTION

A chromatography system is designed for purification of target products (such as proteins, biomolecules from cell culture/fermentation, natural extracts) using at least one packed column of resin, to create a purification step. Each column is switched between loading and non-loading steps, such as wash and elution.

InFIG.1, an overview of a bioprocess purification system10, configured to purify a target product using a separation process is shown. The bioprocess purification system comprises a number of steps related to Cell culture11, Hold12, Capture13, Viral inactivation14, Polish15and Delivery16.

The cell culture step11may be a perfusion type culture which comprises continuous addition of nutrients for cell growth in perfusion culture and continuous removal of product and waste through drain and filtration, e.g. using an Alternate Tangential Filtration (ATF) filter setup. The step may comprise process control for viable cell density (VCD), and the next step in the process starts when VCD reaches a pre-determined value. The VCD may be controlled by adapting the components of the cell culture media fed to the culture or by addition of certain components directly to the culture. Alternatively, the cell culture is of batch type.

The sample containing the target product is exploited in a cell free extraction process, e.g. by filtration, centrifugation or another technique.

The hold step12is an optional step depending on process needs, e.g. if a filter is in-line before capture step13. The step may comprise process control on weight, and the next step in the process starts when a pre-determined volume value is reached, or alternatively after a certain time period or when a pre-determined mass is reached. The hold step may be used both for collecting a volume of filtered feed from a perfusion cell culture or from a batch culture.

The capture step13comprises at least one chromatography column that may have a filter in-line before the capture step. The capture step13may comprise a continuous chromatography setup, as illustrated inFIG.2, which may be run e.g. as periodic counter current chromatography with a continuous feed of sample from the cell culture step11, directly or via the hold step12, containing the target product. The capture step comprises one or more batch elutions, and process control using in-line UV-sensors handles variation in feed concentration and resin capacity. The next step starts when a pre-determined amount value (e.g. volume, mass or time) is reached.

In the viral inactivation step14, different options for virus inactivation is available depending on process needs. One option is to use batch mode with low pH for 30-60 minutes in a hold up tank. The step may comprise process control on volume, time, temperature and pH. The next step starts when a pre-determined time is reached.

The polish step15may be straight through processing (STP) with a connected batch step or continuous chromatography with a continuous load step, or a combination thereof. The flow rate is adjusted to a perfusion rate required by producer cells, which means that the flow rate is determined by the preceding step. The step may comprise process control for UV, flow and volume, and the next step starts when a pre-determined volume and amount is reached, alternatively when a timeout is reached.

The delivery step16may comprise a virus removal step, e.g. a viral filter, before an ultra-filtration step. The delivery step may be used as concentration step for batch addition of sample from polish step. The delivery step may comprise continuous or batch delivery of product and may comprise continuous or batch removal of waste. The step may comprise process control for pH, conductivity, absorbance, volume and pressure, and delivery is achieved when a pre-determined product concentration in a pre-defined environment is reached.

An automation layer17is used for handling decision points for the next step in the process. Different types of sensors (not shown), both in-line sensors and off-line sensors, are integrated into the process flow to monitor different parameters that may be used for providing the automation layer17with data that could be used to handle the decision points. Sensors include but are not limited to only measure flow, VCD, weight, pressure, UV, volume, pH, conductivity, absorbance, etc.

It should be noted that UV absorption is an example of a parameter that could be monitored to detect the composition of the sample being purified. However, other parameters may be used operating in other frequency ranges, such as IR, fluorescence, x-rays, etc.

The capture step13may comprise a continuous chromatography setup20, as illustrated inFIG.2, or a single column240, as illustrated inFIG.4. Continuous chromatography supports process intensification by reducing footprint and improving productivity. In addition, continuous chromatography is especially suited for purification of unstable molecules, as the short process time helps to ensure stability of the target product.

InFIG.2, sample containing the target product is fed into the continuous chromatography20via inlet21and the eluted target product is available at outlet22. The continuous chromatography20comprises multiple columns A, B, N, and each column is provided with a column inlet23and column outlet24. The column inlet23and column outlet24of each column is connected to a valve system25configured to connect the columns cyclically to the inlet21and the outlet22to achieve continuous purification of the target product. Example of a system configuration having three columns is described in connection withFIG.3a-3c.

The continuous chromatography20is further provided with buffer inlet26and waste outlet27in order to be able to perform the required operations. An in-line sensor28may be provided after the column outlet24of each column or be assigned to the process flow and integrated into the valve system25. Important parameters, such as UV, are measured to control the process, as described below. Another in-line sensor28′ may be provided before the column inlet23of each column in order to be able to directly evaluate performance of each column. An in-line inlet sensor26may also be provided to monitor the composition of the sample fed into the continuous chromatography20.

The continuous chromatography may also comprise off-line sensors29, which are designed to extract material from the process and thereafter evaluate selected parameters before the material is disposed of as waste.

The continuous chromatography comprises at least two, such as at least three, columns and the principle of operations in a three columns (3C) setup is described in connection withFIGS.3a-3c. The 3C setup features two parallel flows: one for loading of the two columns in the loading zone, and one for the non-loading steps, e.g. elution and regeneration of the third column.

InFIG.3a, illustrating step1, columns A and B are in the loading zone. Column A can be overloaded without sample loss, as column B catches the breakthrough from column A. In this way, the utilization of the resin binding capacity is maximized.

InFIG.3b, illustrating step2, the overloaded column A is switched and column B becomes the first column and column C becomes the second column in the loading zone. The overloaded column A will now be subjected to the non-loading steps, such as elution and regeneration in a parallel workflow.

InFIG.3c, illustrating step3, the overloaded column B in the loading zone is switched. Now column C becomes the first column and column A the second column in the loading zone, while column B is subjected to elution and regeneration in the parallel workflow. These three steps are repeated in a cyclic manner until required target product volume, mass or amount is reached (or until resin lifetime is reached and columns needs to be repacked or exchanged).

The continuous chromatography setup illustrated inFIG.2may utilize more than three columns, and in a four column (4C) setup, the same principle applies. However, the non-loading steps can become limiting in a 3C setup, and the non-loading steps can be split on two columns and run in parallel utilizing a third flow path in the 4C setup. The 4C setup allows for balancing the loading and non-loading steps. More columns will lead to a more flexible system, while the complexity of the valve system25becomes increasingly complicated. However, some continuous chromatography have sixteen or more columns.

FIG.4schematically shows one embodiment of a chromatography system190comprising two 3-way input-valves160and161, arranged to select the input fluid from fluid sources A1, A2, B1, B2for two system pumps150and151. The chromatography system190may further comprise:a pressure sensor200for registering the system pressure in the flow path after the system pumps, anda mixer210to ensure appropriate mixing of the fluids supplied by the pumps.

These correspond to the cell culture block11illustrated inFIG.1, as indicated by the dashed lines110.

The system further comprises:an injection valve220for injecting a sample into the fluid path,a column connection valve230for selectively connecting/disconnecting a column240in the fluid path.a pre-column pressure sensor235and a post-column pressure sensor236an ultraviolet (UV) monitor250for detecting the output from the column.a conductivity monitor260, anda pH monitor265.

These correspond to the capture block13illustrated inFIG.1, as indicated by the dashed lines130.

The system further comprises:an output selection valve270with two or more output positions, e.g. connected to a fraction collector280, a waste receptacle or the like, which correspond to delivery block16inFIG.1, as indicated by dashed lines160, anda system controller300connected to pumps and valves for controlling the liquid flow through the system, and to sensors and monitors for monitoring the flow, connections being illustrated by dotted lines310, which correspond to Automation block17inFIG.1, as indicated by dashed lines170.

The chromatography system ofFIG.4represents a general example of how a single column chromatography system may be designed, and other embodiments may be of different design comprising two or more of some components and potentially lack some of the components. E.g. components corresponding to Hold12, Viral inactivation14and polish15as illustrated inFIG.1.

FIG.5is a simplified flow chart for a liquid chromatography system190according toFIG.4. InFIG.5the flow path has been straightened out and some components have been removed to achieve a more simplistic view. InFIG.5the system controller is shown connected only to the pump150, the pressure sensor200, the pre-column pressure sensor235and the post-column pressure sensor236, but it may be connected to other components as discussed above. InFIG.5, the system comprises both the pre-column pressure sensor235and the post-column pressure sensor236, whereby the column pressure is directly measured by the pre-column sensor235, and the delta-column pressure by subtracting the pressure registered by the post-column sensor236from the column-pressure.

As briefly mentioned above, some systems do not have other pressure sensors than the system pressure sensor200.FIG.6is a simplified flow chart of such a liquid chromatography system190with one single pressure sensor200for registering the system pressure. As mentioned above, the pressure control in such a system only relies on the registered system pressure, by sensor200.FIG.7is a simplified flow chart of a liquid chromatography system according to one embodiment of the present invention, wherein the controller300is arranged to estimate the pre-column pressure based on the registered system pressure, the characteristics of the flow path, and the viscosity and flow-rate of the liquid in the system. The estimated pre-column pressure may be referred to as a “virtual pressure sensor” schematically shown inFIG.7by faint dotted lines.

According to one embodiment, the calculation of the virtual pressure signal may be based on Bernoulli's formula for pressure drop in a flow channel.

Flow channel ΔP [MPa]=0.000000000679*L*Q*V/D <4>where

L=length [mm]

D=diameter [mm]

Q=flow rate [ ml/min]

V=viscosity [cP]

By providing length and diameter of the flow path, and the viscosity of the liquid in the system, to the system controller, it may be arranged to calculate the pressure drop caused by the flow path up to the column at the current flow rate. In some systems, the length and size of the flow path between the system pressure sensor200and the column240, may be standardized, so that the predefined parameters may be used for the calculations. In other systems, (which is the most common situation), the flow path between components in the chromatography system is user defined, whereby a user of the system has to enter the parameters using by a user interface.

According to one embodiment, the major part of the flow path between the system pressure sensor200and the column240may be comprised of capillary tubing of the same diameter, then the flow path characteristics may be estimated as the total length of the tubing, thus excluding contributions from other components, like valves or the like from the calculations. In other embodiments, the contribution from valves or the like in the flow path are taken into consideration and may be system defined, whereas, tubing or the like is user defined. It should be noted that, in case the flow path comprises sections of different size (e.g. tubing of different inner diameter), the pressure drop over each section has to be calculated individually and eventually added together to provide the total pressure drop.

When the pressure drop in the flow path up to the column240is estimated by the above calculations, the virtual Pre-column pressure is calculated by subtracting the pressure drop from the system pressure registered by system pressure sensor200.

Example

If System pressure is 5 bar and the calculated pressure drop over the flow path is 2 bar then the calculated virtual pre-column pressure is estimated to 3 bar.

All pressure contributions after the virtual pressure sensor will automatically be compensated for, since these will directly affect the measured system pressure. So, e.g. if a flow restrictor is added or removed, the measured System pressure will change as well as the calculated Pre-column pressure. Changes in the flow path between the System pressure sensor and the column must be taken care of in the estimation.

According to one embodiment, in case the viscosity is not known, the controller may assume that water is used whereby the viscosity can be estimated for different temperatures using a known expression like:
V[cP]=A×10B/(T-C),
where T=temperature [K]; A=0.02414; B=247.8 K; C=140 K.

In the real situation there may be some factors that may affect the accuracy of the virtual pressure estimation. If the viscosity of the liquid is unknown and it is assumed to be water, but it has a higher viscosity, then the estimated value for flow path ΔP becomes too low. Then the calculated value for the virtual pressure signal becomes higher than the actual value whereby a pressure alarm will trigger before the actual pressure becomes too high for the column. This is also the case if other components in the flow path (mixer, valves etc.) generate some back pressure. Consequently, for liquids with viscosity lower than water, the estimation will give a virtual pre-column pressure that is lower than the actual pressure. However, such liquids are mostly used for high pressure columns where the high accuracy of the pressure signal is not required since most such column withstand higher pressures than they are normally used with. According to one embodiment, the system is arranged to estimate the delta-column pressure by using the same principles for the flow path after the column a virtual post-column pressure may be estimated and used to calculate a virtual delta-column pressure.

As mentioned, the virtual pre-column pressure and the delta-column pressure may be used to control the operation of the chromatography system, e.g. by monitoring the pressures with respect to predefined or user-defined pressure limits, or by running the chromatographic system at a predefined column pressure or the like.

The disclosure illustrates a chromatography process configured to operate with at least one column and configured for purification of a sample comprising a target product using a predefined process. The predefined process may be a generic process, a validated process or a special process, and can be predefined from the manufacturer or generated by the end-user.

FIG.9illustrates data flow in a system adapted for manufacturing columns and a liquid chromatography system. As described in more detail below, column data is generated during manufacturing of columns and is stored in a database dB accessible to the chromatography system90. Data flow is indicated by dotted lines, and control signals by dashed lines.

The liquid chromatography system comprises a controller91configured to control the operation of the chromatography system to run the predefined process, retrieve column data accessible from a data storage dB, the column data being specific to each column, and adapt at least one process parameter of the predefined process for each column based on column data, whereby the predefined process is adapted to each column to obtain the target product and maintain the performance of the liquid chromatography system.

The data storage may be a database (integrated in the chromatography system or accessible from a source outside the chromatography system, such as a cloud based implementation). Another alternative is to store column data on individual column, e.g. as a memory chip and communication with the chromatography system via RFID.

The process parameter comprises: pressure over each column, flow of sample into the column, flow of residue out from the column and/or processed volume of sample/time period (column volume/hour).

According to some embodiments, the liquid chromatography system is configured to operate with a single column for purification of the sample, and according to some embodiments the liquid chromatography system is configured to operate with at least three columns for continuous purification of the sample.

FIG.8illustrates the concept of assigning the columns to a type400(also referred to as family). Each individual column401-1,401-2,401-nbelongs in this example to a specific type400having predetermined production parameters related to the column. In this example, the production parameters defines a range of different physical properties of the used column components and different process properties used to manufacture the column.

The column comprises a vessel for holding a resin and filters, and column components may include hardware specific properties, physical dimensions of the vessel (such as height of the vessel), material properties of the resin, such as grain size and distribution, physical properties of the filter, etc.

Process properties relates to the manufacturing process of the column, such as height of resin bed, pressure boundaries, flow specification, etc. The height of the resin bed may be the actual height or the height of the vessel.

The production parameters may further comprise actual volume of resin in each column.

In some example embodiments, the liquid chromatography system further comprises sensors92a,92badapted to read sensor parameters, wherein the adaptation of the at least one process parameters further is based on sensor readings. In some embodiments, the sensor readings comprise any of: UV, Flow and Pressure.

In some embodiments. the production parameters further comprise actual volume of resin in each column.

The database, which is accessible by the system may comprise historic data for each individual column and/or columns belonging to the same specific type (i.e. the same family) previously used in the liquid chromatography system for purification of the sample. Historic data information from the controller is stored in the database for this purpose.

FIG.10is a flow chart illustrating a method for manufacturing a column for a liquid chromatography system. The disclosure also comprises a method for manufacturing a column for a liquid chromatography system, the column having an inlet and an outlet and comprises a vessel for holding a resin. The method comprises:1) Selecting, step S2, a type of column configured to be used in a process for purification of a sample comprising a target product.2) Selecting, step S3, the resin (which is the media) based on the type of column, the resin having media properties. Media properties comprise measured and calculated properties of the media that is intended to be used in the column. The media parameters are affected by the media manufacturing process and tolerances of different media components used when manufacturing the media.3) Selecting, step S4, the vessel (i.e. the hardware) for holding the resin, the vessel having hardware properties. Hardware properties comprise measured and calculated properties of the different components used to manufacture the column, that affects the function of the column, such as physical dimensions (with tolerances) and filter properties (if included).4) Packing, step S5, the resin to form a resin bed in the vessel using a pressure based on the type of column to establish a height of the resin bed.5) Determining production parameters, step S6, based on the media properties and the hardware properties to define column data for the column, and storing the column data in a data storage, which is accessible for the liquid chromatography system. The data storage may be integrated in the column or be a data base accessible to the chromatography system, as mentioned above.

In some examples, the column further comprises hardware in the form of a top filter arranged between the inlet and the resin bed, and the method further comprises selecting the top filter based on the type of column, the top filter having top filter properties, and determining production parameters based further on the top filter properties to define the column data.

In some examples the column further comprises hardware in the form of a bottom filter arranged between the outlet and the resin bed, and the method further comprises selecting the bottom filter based on the type of column, the bottom filter having bottom filter properties, and determining production parameters based further on the bottom filter properties to define the column data.

The disclosure also comprises a column for a liquid chromatography system, the column having an inlet and an outlet and comprises a vessel for holding a resin, wherein the column is manufactured according to the method mentioned above. A system for manufacturing the columns is illustrated inFIG.9, where the output from the system is columns and column data, which is stored in a database dB accessible to the chromatography system.

In some examples, the column further comprises hardware in the form of a data storage, e.g. a data chip, configured to store column data, and a communication device configured to communicate the column data to the liquid chromatography system.

In some embodiments, the communication device is configured to communicate with the liquid chromatography system using RFID.

FIG.11is a flow chart illustrating a method for controlling a liquid chromatography system configured to operate with at least one column. The disclosure also comprises a method for controlling a liquid chromatography system configured to operate with at least one column and configured for purification of a sample comprising a target product using a predefined process, wherein the method comprises:A) controlling, step S12, the operation of the chromatography system to run the predefined process.B) retrieving column data, step S13, accessible from a data storage, the column data being specific to each column, and adapting, step S15, at least one process parameter of the predefined process for each column based on column data.

Whereby the predefined process is adapted to each column to obtain the target product and maintain the performance of the liquid chromatography system from a sample.

In some examples, each column is of a specific type and the data storage comprises historic data for each column and/or columns belonging to the same specific type previously used in the liquid chromatography system for purification of the sample, wherein the method comprises further adapting the at least one process parameter of the predefined process based on the historic data, step S13a.

In some examples, the data storage comprises column data related to production parameters when producing each column, the production parameters comprises, height of resin bed, pressure boundaries, flow specification, material properties, hardware specific properties, filter properties, and physical dimensions of the vessel, wherein the method comprises further adapting the at least one process parameter of the predefined process based on the production parameters, step S13b.

In some examples, the liquid chromatography system further comprises sensors adapted to read sensor parameters, wherein the method further comprises further adapting the at least one process parameters based on sensor readings, step S14a. The sensor readings may comprise any of: UV, Flow and Pressure.

The data storage may be selected to be a database, which may be integrated in the liquid chromatography system. An alternative is to integrate the data storage in each column.

In addition to the above, the preparation and qualification of packed columns are important steps to ensure robustness and safety for both the purification process and the final product. Column efficiency

testing plays a central role in the qualification and monitoring of packed bed performance. Even though it cannot be used as a single parameter to predict purity and recovery, it is a

quick way to test the column and equipment performance before starting the purification process. This test can also be used inbetween runs to check for changes of the bed integrity.

This note provides a brief overview of the theory behind and experimental test practices used in column efficiency testing. Test conditions are recommended and critical parameters

that influence the measured efficiency are discussed in order to facilitate the development of robust test protocols.

Efficiency testing is the analysis of the residence time distribution for a tracer substance passing through the column. Typical test signals applied to the column are pulse or step signals. In order to characterize the chromatography column without interference, tracer substance and eluent conditions are selected such that chemical interactions with the medium and disturbances of the fluid flow are avoided. The most common type of test signal applied is a pulse function. A small volume of a tracer substance is added to the liquid flow close to the column inlet and the broadening of this pulse is analyzed when measured as a chromatographic peak at the column outlet.

Column efficiency is typically defined in terms of two parameters:Peak broadening over the column is described by an equivalent number of theoretical plates (equilibrium stages)Peak symmetry is described by a peak asymmetry factor As

Peak broadening is typically described as plate number N or as height equivalent to a theoretical plate (HETP). This concept is equivalent to a tanks-in-series model reflecting the number of equilibrium stages represented by the column.

A widely used method for evaluating a pulse test (to determine the plate number) involves the measurement of peak width at half of the maximum peak height. This approach is an alternative to numerical curve integration when applying the method of moments, where the first moment is the average and the second moment is the variance of the retention volume/time. As outlined inFIG.1, the pulse response is plotted against time or eluted volume and the peak width at half peak height is measured and related to the elution time or, preferably, the eluted volume at maximum peak height. The retention time or retention volume measured at the maximum peak height corresponds to the average residence time or volume found at a symmetric (Gaussian) peak shape. A dimensionless and thus convenient parameter for efficiency characterization is the reduced plate height h. This parameter facilitates the comparison of column efficiency irrespective of column length and particle diameter of the medium.

Optimal column efficiency typically corresponds to an experimentally determined reduced plate height of h≤3 for the porous media employed in bioprocess chromatography. This efficiency is achieved when testing a well-packed bed with an optimized set-up of column and system under optimal test conditions.

A detailed explanation of column efficiency testing is provided in the Application note 28-9372-07 AA published by GE Healthcare, which is incorporated herein in its entirety.

In one embodiment of the present invention, the chromatography system is arranged to perform automated column efficiency testing to determine efficiency parameters such as h and Asand to further provided guidance to the user with respect to column efficiency. The automated test here presented gives all the possible data to calculate the reduced plate height since the software automatically uses the specific parameters for the given column and resin to calculate the efficiency of the column. This give the possibility to directly know if the given column is good enough for application use or not. If not, the user will be informed so that a more appropriate column can be used. A suitable cleaning procedure can be applied, the column can be repacked, a the column can be replaced by a new column or any other necessary action can be taken. This gives a more secure and robust procedure for the liquid chromatography step since the efficiency of the column can be guaranteed.

In accordance with one embodiment, the chromatography system is arranged to automatically determine one or more efficiency parameters, such as reduced plate height h and Asand based on the result guide a user of the system to a relevant action or alternatively, the system may be configured to initiate some or all relevant actions automatically based on the efficiency parameters.

In one example, the system may be configured to automatically or semi-automatically initiate one or more of the following actions in order to improve column efficiency:A standard column clean procedure (e.g. NaOH 0.5M) when 3<h<3.5 (level 1)An intense column cleaning procedure (e.g. NaOH 1M, acid, and/or isopropanole) when 3.5<h<5 (level 2)A column repacking procedure when 5<h<7 (level 3)Cancel process and instruct user to replace the resin when h<7 (level 4)

Once an action is completed, the system is preferably configured to automatically determine one or more efficiency parameters and to verify the improved efficiency.

In alternative embodiments, the system may be configured to repeat one or more of the above actions two or more times in case the desired result is not reached. The system may further be configured to escalate to a higher level after a predetermined attempt at one level.

It should be noted that the above ranges for the different levels may be different depending on the resin type and column type. The ranges may also be different depending on the chromatographic process step that the column will be used in. for example a column that is prepared for a capture step may have a higher h compared to a column prepared for an intermediate or a polishing step.

The chromatography system may be arranged to retrieve the particle size data for the determination of the efficiency parameters either from a predefined standard data file in a database or from measurement data of the particle size of the specific resin lot used. The lot specific particle size data may be determined by a separate or interconnected measurement unit or alternatively retrieved from a database comprising lot specific particle size data—e.g. a cloud-based database.

According to one embodiment the automated column verification further comprises determination of the column delta pressure to verify the column packing performance. In case the determined delta pressure is determined to be too low there is an increased risk for gap building whereas in case the delta pressure is determined to be too high, it may fall outside of the system pressure range. The relevant ranges for delta pressure may be retrieved from the column database and or the system settings.

The disclosure further comprises a computer program for controlling a liquid chromatography system, comprising instructions which, when executed on at least one processor, cause the at least one processor to carry out the method according to method mentioned above.

The disclosure further comprises a computer-readable storage medium carrying a computer program for controlling a liquid chromatography system as defined above.