Patent Application
20210170303
The present invention relates to packed bed columns useful for liquid chromatography separation of biomolecules, to a method of packing such columns and to methods of separating biomolecules with the columns.
Columns used in liquid chromatography typically comprise a tubular body enclosing a packed bed of porous chromatography medium through which a carrier liquid flows, with separation taking place by partitioning between the carrier liquid and solid phase of the porous medium.
Prior to any separation process, the bed has to be prepared by starting from the particulate medium that is to be introduced into the column. The process of bed formation is called ‘the packing procedure’ and a correctly packed bed is a critical factor influencing the performance of a column containing a packed bed. Typically, the packed bed is prepared by slurry packing, i.e. consolidating a suspension of discrete particles in liquid, known as slurry that is pumped, poured, or sucked into the column. Once the predetermined volume of slurry has been delivered into the column it needs to be further consolidated and compressed by moving a movable adapter down the longitudinal axis of the column towards the bottom of the column, normally at a constant speed. The excess liquid during this procedure is expelled at the column outlet, while the media particles are retained by means of a filter material, a so-called ‘bed support’, with pores too small to allow the media particles to pass though. The packing process is complete once the packed bed has been compressed by the optimum degree of compression. Another approach for column slurry packing is the flow packing method, where compression of the porous structure is primarily achieved by applying a high flow rate over the column, hereby forming a porous structure starting at the outlet bed support. The resulting drag force on the particles in the porous structure causes eventually a pressure drop and a compression of the bed. The compressed bed is finally confined by bringing the adapter into position.
The efficiency of subsequent chromatographic separation relies strongly on 1) the liquid distribution and collection system at the fluid inlet and outlet of the packed bed, 2) the special orientation (also know as the packing geometry) of the media particles in the packed bed, and 3) the compression of the packed bed. If the compression of the packed bed is too low then chromatographic separations performed on that bed suffer from “tailing” and, generally, such insufficiently compressed beds are unstable. If the compression of the packed bed is too high then chromatographic separations performed by the bed suffer from “leading” and such over-compressed beds can affect throughput and binding capacity, and, in general, give much higher operating pressures. If the compression is optimum, then the separation peaks formed during use exhibit much less leading or tailing and are substantially symmetrical. The optimum degree of compression is also crucial for achieving good long-term stability of the porous structure, hereby securing optimal performance throughout a number of process cycles. The optimum degree of compression required for a column is determined experimentally for each column size (width or diameter), bed height, and media type.
An alternative packing method is called “dry packing”, where the column is filled with dry particles of the porous medium and liquid is introduced in the column afterwards. This has advantages in prepacked columns that can be delivered dry to the customer without having to add any preservatives to the packing liquid and minimizing weight during transport. Dry packing is typically used for silica media aimed at separation of small molecules, as described e.g. by G Guiochon J Chromatogr A 704 (1995) 247-268, although fairly poor column efficiencies are obtained. For swellable chromatography media, such as dextran or agarose-based media commonly used in separation of biomolecules, dry packing has however been avoided due to a perception that the swelling of the particles will cause poor performance of the packed bed. U.S. Pat. No. 4,353,801 mentions dry packing of solvent-swellable styrene-divinylbenzene particles as being inferior to wet packing.
One aspect of the present invention is to provide a packing method for a high efficiency chromatography column starting from dry swellable particles. This is achieved with a method comprising the steps of a) transferring to a column chamber 2 in a chromatography column 1 an amount of said dry swellable particles 5 sufficient to give a swollen volume Vs in a liquid of about 105-120% of the column chamber volume, b) closing the column and c) providing said liquid to the column. In other words, the method comprises metering out (e.g. by weighing) an aliquot of dry swellable particles 5, providing said aliquot to a column chamber 2 in a chromatography column 1, closing the column chamber and supplying a liquid to the column chamber, wherein the swollen volume Vs of said aliquot is about 105-120% of the column chamber volume.
A specific aspect of the invention is a storage stable prepacked column that is easily shipped to the user and equilibrated by the user. This is achieved by a column comprising dry swellable particles, wherein the amount of particles is sufficient to give a swollen volume Vs in an aqueous liquid of about 105-120% of the column chamber volume.
Another aspect is to provide a method for preparation of a plurality of columns connected in parallel from dry particles of a chromatography medium. This is achieved by a method as defined in the claims, which allows the preparation of columns with remarkably low differences in permeability. This is important in order to avoid overall band broadening due to flow rate differences between the columns.
A further aspect is to provide a method for manufacturing a chromatography subsystem comprising a plurality of chromatography columns connected in parallel, using dry particles of a chromatography medium to pack the columns. This is achieved by a method as defined in the claims and enables a chromatography system which is scalable by the addition of further chromatography columns connected in parallel.
Other aspects are a plurality of columns connected in parallel as described above and their use for separation of biomolecules, where their unique scalability is advantageous.
One or more of the aspects above may be achieved by the present invention as defined by the appended claims. Additional aspects, details and advantages of the invention will appear from the detailed description and claims that follow.
The term “swellable particles” means herein particles that increase in size upon immersion in a liquid. This can be observed as a sediment volume in the liquid which is significantly larger than the bulk volume of the same amount of dry particles.
The term “swollen volume” (Vs) means herein the sediment volume of an aliquot of particles suspended and equilibrated in a liquid. The sediment volume can be measured by suspending the aliquot of particles, equilibrating for up to 24 h, resuspending the particles if needed, letting the particles sediment and measuring the sediment volume in a graded vessel (e.g. a measuring cylinder).
The term “liquid uptake” (Vs/md) means herein the ratio between the swollen volume Vs of an aliquot of particles as defined above and the dry weight and of the particle aliquot before immersion in the liquid.
In one embodiment of the invention, illustrated in
In a further embodiment, depicted in
In an alternative embodiment, the liquid uptake is determined on basis of dry particle volume instead of using the weight of the dry particles as reference. Equally, preparation of aliquots to be filled into at least one column of a given specific column may be conducted on basis of dry particle volume instead of dry particle mass.
In certain embodiments the dry swellable particles in the column chamber can be manipulated to allow for a more even spatial distribution before the providing of liquid to the column. In one embodiment, illustrated in
In one embodiment the liquid is provided to the column in the upflow direction. The liquid will then rise evenly in a vertical direction from the bottom inlet through the dry particle bed, resulting in a more even particle distribution than if the liquid is pouring down from the top of the column. Once the particles have been swollen by the liquid, the column may then be operated in any direction (upflow, downflow, tilted etc.) In a specific embodiment the rate of liquid addition to the dry swellable particles does not exceed the rate of capillary suction by the particles. The rate of capillary suction can be determined e.g. by placing a column with dry swellable particles in a trough containing liquid up to the lower end of the dry bed and measuring the rate of liquid rising through the bed by optical, gravimetric or other methods. Keeping the rate of liquid addition lower or equal to the rate of capillary suction has the advantage of giving a more homogeneous particle distribution after swelling and hence better column performance. The rate of liquid addition can be expressed as the liquid velocity in the column chamber (the liquid flow rate divided by the internal cross section area of the column chamber). In a specific embodiment the liquid velocity in the column chamber is less than 100 cm/h, such as between 5 and 70 cm/h, during the addition of liquid to the dry particles. In one embodiment the liquid is aqueous and it can then comprise a wetting agent (an additive that decreases the surface tension), such as a lower alcohol, e.g. ethanol, or a surfactant. An advantage of this is that a more uniform distribution of the particles after swelling can be obtained.
In one embodiment, depicted in
In one embodiment the method comprises, after the transfer of dry swellable particles to the column chamber 8 and before the closure of the column, a step of transferring the column chamber 8 to a rigid housing 11 providing dimensional stability for the column chamber during swelling of the gel and/or operation of the column. In this context, the free-standing column chamber 8 may deflect somewhat during swelling of the gel or due to pressurization during operation of the column. A rigid housing 11 will prevent any such deflection and any associated deterioration of the column performance.
In one embodiment the method comprises before the addition of liquid to the column a step of radiation sterilising the column or the flexible container with the dry swellable particles. This step may be carried out at any stage of the method. The radiation sterilisation can be carried out through gamma or electron beam irradiation.
One embodiment of the invention is a column comprising dry swellable particles, wherein the amount of particles is sufficient to give a swollen volume Vs in an aqueous liquid of about 105-120% of the column chamber volume. In one embodiment this liquid is a 0.1 mol/l solution of sodium chloride (NaCl) in water at 20° C. Such a solution has a conductivity of approx. 10 mS/cm which is in the range of many buffers used in biomolecule/biopharmaceutical separations.
In one embodiment the liquid uptake Vs/md of the dry swellable particles (in 0.1 M aqueous NaCl at 20 C) is between 5 and 25 ml/g, such as between 5 and 15 ml/g. An advantage of this embodiment is a good control of the compression factor and minimised variability in swelling (and column performance) with composition of the liquids applied during operation of the column. In one embodiment the liquid uptake of the dry swellable particles in distilled water is less than 1.5 times the liquid uptake in 0.1M aqueous NaCl. An advantage of this is that the variability in swelling with liquid composition is kept low.
In one embodiment the column volume is fixed. A fixed volume column can be constructed without any moving parts (adaptors etc), which simplifies construction and is advantageous from a cost point of view. In one embodiment the column comprises molded or extruded parts. Molding and extrusion methods such as injection molding, blow molding, rotation molding, compression molding etc are convenient and cost-efficient methods to manufacture column parts from e.g. thermoplastic materials.
In one embodiment the column chamber volume is at least one litre. Columns with more than one litre bed volume are frequently used in industrial biopharmaceutical purification. They are often complicated and expensive steel constructions with moving parts, so solutions allowing use of simple plastic constructions are desirable.
In one embodiment illustrated by
In one embodiment illustrated by
In one embodiment the dry swellable particles comprise a polysaccharide, such as crosslinked agarose. Crosslinked agarose particles are highly useful for biomolecule and biopharmaceutical purification due to their high throughput and low non-specific adsorption. They are normally supplied in aqueous ethanol solution and an advantage of using dry agarose particles is that no flammable ethanol has to be handled. Drying of agarose particles can be done using e.g. freeze drying, spray drying or vacuum drying.
In embodiment the dry swellable particles comprise charged ligands. Such ligands are predominantly used in ion exchange and multimodal separations, both of which are highly useful in biomolecule and biopharmaceutical separations. Both cation exchangers, anion exchangers and multimodal ion exchangers are contemplated.
In one embodiment the dry swellable particles comprise ligands comprising hydrophobic functionality. Such ligands are used in hydrophobic interaction chromatography and many multimodal separations, both of which are highly useful in biomolecule and biopharmaceutical separations. Examples of ligands and ligand parts with hydrophobic functionality are alkyl and aryl groups.
In one embodiment the dry swellable particles comprise affinity ligands. Such ligands are highly useful for high selectivity separation of biomolecules and biopharmaceuticals. Examples of affinity ligands are protein A, protein G, protein L, lectins, enzyme substrates, lectins, biotin, avidin, antibodies, antibody fragments, antigens etc.
In one embodiment the liquid provided to the column with the dry swellable particles comprises at least one biomolecule, such as a biopharmaceutical. Examples of such liquids include protein solutions and virus suspensions, e.g. feeds to be purified in bioprocessing operations. An advantage of swelling the dry particles directly with a feed is that a separate swelling step can be eliminated and that the swelling in itself can provide a separation and concentration effect for species not penetrating into the swollen structure.
One embodiment of the invention is a column comprising swollen particles, packed according to the previously described embodiments. In a specific embodiment the column has a reduced plate height h less than 15, such as less than 10 for a test species. The test species can be a non-retained small molecule, such as an inorganic salt or acetone. In a further embodiment the asymmetry factor As of the column is less than 3, such as less than 2.5 for a test species selected as above.
One embodiment is at least two columns which are prepared from the same batch of dry swellable particles. In a specific embodiment the columns are connected in parallel. Connection in parallel of columns with the same bed volume, particle type and bed height is a convenient way of increasing the total cross-sectional area and throughput. When columns connected in parallel are used, it is however of great importance that the fluid velocities are identical in the columns to ensure that the same separation is achieved in each column. This puts high demands on the reproducibility of the columns with respect to hydraulic resistance and pressure-flow behavior. With dry swellable particles from the same batch in the columns, it is possible to control the hydraulic resistance and pressure-flow behavior well enough to allow parallel use.
In one embodiment the column comprising swollen particles is used for separation of at least one biomolecule such as a biopharmaceutical. Suitable biomolecules can be proteins, peptides, nucleic acids, carbohydrates, virus particles etc. Suitable biopharmaceuticals can be immunoglobulins (e.g. monoclonal antibodies), immunoglobulin fragments and other constructs, insulin and other therapeutic peptides, erythropoietin, plasma proteins, oligonucleotides, plasmids, vaccines etc. In a specific embodiment the biomolecule or biopharmaceutical is a protein.
In one embodiment the biomolecule or biopharmaceutical binds to the particles and at least one impurity is removed by washing with a washing liquid. The biomolecule or biopharmaceutical may then be eluted from the particles with an elution liquid. This mode is often called bind-elute separation and is particularly useful when the amount of impurities is significant and/or when a very high separation selectivity is required.
In another embodiment at least one impurity binds to the particles and the biomolecule or biopharmaceutical is recovered in the flow-through of the column. This mode is often called flow-through separation and provides a very high throughput, particularly when the amount of impurities is relatively low.
In a further aspect the invention discloses a method for preparing a connected plurality of packed chromatography columns, comprising the steps of:
The chromatography medium particles can e.g. be the dry swellable particles discussed above, but they can also be other types of chromatography media. The aliquots can suitably be prepared by weighing, which easily allows the provision of aliquots which differ from each other by less than 2.0 percent by weight, such as by less than 1.0 percent or even less than 0.5 percent by weight. In contrast, the preparation of aliquots from chromatography media suspended in liquids is much less accurate due to sedimentation and segregation effects. The weighing and any determination of the weight differences can suitably be done when the particles are in equilibrium with the surrounding atmosphere, to avoid any effects of differences in moisture uptake after drying processes. The aliquots can e.g. be prepared from a single homogeneous batch of dry chromatography medium particles. This batch can either be a single manufacturing batch or a homogeneous blend of several manufacturing batches. The homogeneity can for example be ensured by tumbling or other type of mixing. If any segregation effects should be observed, for example in cases of very wide particle size distributions or density distributions, the homogeneity of the aliquots might be improved by using for example a riffler or rotary sample divider to divide the particles into rough aliquots, weighing the aliquots and making fine adjustments of their weights.
Alternatively, the aliquots can be prepared from different batches. In this case, the batches have suitably been tested and found to be sufficiently similar to allow for use in parallel columns. Suitable testing parameters include volume average particle size (diameter), width of the particle size distribution and liquid uptake as discussed above. The width of the particle size distribution can for example be measured as a standard deviation or as a ratio between the volume and number average sizes (diameters). The acceptance values for the testing parameters can be selected such that the permeability or flow velocity differences between columns packed from the different batches are less than 20, 10, 5, 2 or 1 percent.
In one embodiment the method further comprises, after step b), c) or d), a step b′) of supplying a reswelling liquid to said plurality of packed chromatography columns. This step allows the swelling of the particles and can provide compression of the column as described in the embodiments above.
In one embodiment the method comprises, after step a) and before step b), a step a′) of separately suspending the aliquots in a packing liquid. This embodiment allows a wet packing of the columns from resuspended particles. As the aliquots are prepared with a low weight difference from dry particles, this embodiment still allows the preparation of columns with low differences in permeability and other properties.
In one embodiment step c) of the method further comprises fluidically connecting the plurality of chromatography columns to a single sample inlet. The connection may be as disclosed in
In a further aspect the invention discloses a method for manufacturing a chromatography subsystem comprising a plurality of chromatography columns connected in parallel, said method comprising the steps of:
The embodiments discussed above apply equally to this method. The subsystem support structure may e.g. be a rack or skid arranged to receive a plurality of chromatography columns. The chromatography columns can be individual self-supporting columns as shown e.g. in
In one embodiment all the columns are connected in parallel. In other embodiments, the subsystem comprises a plurality of serial column trains connected in parallel.
In another aspect the invention discloses a chromatography subsystem comprising a plurality of chromatography columns connected in parallel, manufactured by the above method. This subsystem can be used for separation of at least one biomolecule, such as a biopharmaceutical.
In one embodiment the biomolecule or biopharmaceutical binds to the particles, at least one impurity is removed by washing with a washing liquid and the biomolecule or biopharmaceutical is recovered from the column by elution with an elution liquid (bind-elute mode), while in another embodiment at least one impurity binds to the particles and the biomolecule or biopharmaceutical is recovered in the flow-through of the column or in a wash fraction (flowthrough mode). In both these embodiments it is advantageous with no or minimal flow velocity difference between the columns. In bind-elute mode it is advantageous to avoid premature breakthrough of the biomolecule/biopharmaceutical, which can occur if the flow velocity is higher in one particular column. It is also advantageous to have the same flow velocity in all columns during elution, to get a narrow elution peak and a correspondingly high concentration of the biomolecule/biopharmaceutical in the elution pool. In flowthrough mode it is advantageous to avoid premature breakthrough of the impurity.
In some embodiments the (relative) flow velocity difference between any two of said plurality of packed columns is less than 20 or less than 10 percent, such as less than 5, less than 2 or less than 1 percent. The flow velocity difference may be measured directly by flow meters in line with the different columns, but it can also be assessed indirectly e.g. by measuring the difference in retention time of a substance, such as a substance which does not bind to the chromatography medium. The flow velocity difference can be a direct function of the difference in permeability between the different columns and hence, the (relative) permeability difference between any two of said plurality of packed columns can be less than 20 or less than 10 percent, such as less than 5, less than 2 or less than 1 percent. The permeability may be calculated as the flow velocity through a column multiplied by the viscosity of the liquid and divided by the back pressure over the column.
Other features and advantages of the invention will be apparent from the following examples and from the claims.
A tracer (acetone 2% v/v in DI water) is applied as tracer with a volume of 1-1.5% of the column volume to analyze the residence time distribution at the column outlet (UV signal @ 280 nm). The residence time distribution should be represented as a single peak, compare
Column efficiency is typically defined in terms of two parameters:
The relative peak width is defined as number of (theoretical) plates N, height equivalent of a theoretical plate HETP or preferably as reduced plate height h:
The asymmetry factor As describes the deviation from an ideal Gaussian peak shape and calculates from the peak width at 10% of peak height:
A
S
=b/a
Plate number N
Mean residence time1 μ1
Variance1 σ2
Retention time2 tR
Retention volume2 VR
Peak width at half peak height wh
Bed height L
Particle diameter dp
Height equivalent of a theoretical plate HETP
Reduced plate height h=HETP/dp
Asymmetry factor As 1 first and second moments in residence time distribution (RTD) curve derived from integration of tracer signal2 retention time (retention volume) corresponding to time (or eluted volume) at maximum peak height
The dry swellable particles used were Q SEPHAROSE HP (GE Healthcare, Sweden), an anion exchanger with tetramethylammonium group ligands coupled to crosslinked agarose beads of 34 micron average diameter when swollen in water. The Q SEPHAROSE HP particles had been transferred to acetone and then vacuum-dried at room temperature.
129 mg dry particles per ml column chamber volume were transferred to two vertically standing columns of different types: an XK26 column (GE Healthcare, Sweden) having a column chamber of diameter 26 mm, height 30 mm and volume 15.93 ml and an XK50 column (GE Healthcare, Sweden) having a column chamber of diameter 50 mm, height 31 mm and volume 60.87 ml.
A water solution of 20% ethanol was pumped into the bottom inlet of each column with a fluid velocity of 60 cm/h. The pumping was continued until three column chamber volumes had passed the column and three column volumes of distilled water were then pumped through at a fluid velocity of 200 cm/h. The columns were conditioned by 4×3 column chamber volumes of distilled water at 300 cm/h, pumped in alternating upflow and downflow mode.
The performance of the columns was evaluated by the column efficiency test described above. The performance results are given in Table 1.
The dry swellable particles used were CAPTO S (GE Healthcare, Sweden), a cation exchanger with sulfonate group ligands coupled to dextran-extended crosslinked agarose beads of 90 micron average diameter when swollen in water. The CAPTO S particles had been transferred to acetone and then vacuum-dried at room temperature.
3.34 g dry particles were transferred to two a vertically standing XK26 column (GE Healthcare, Sweden) having a column chamber of diameter 26 mm, height 30 mm and volume 15.93 ml
A water solution of 20% ethanol was pumped into the bottom inlet of the column with a fluid velocity of 30 cm/h. The pumping was continued until three column chamber volumes had passed the column and 1.5 column volumes of distilled water were then pumped through at a fluid velocity of 100 cm/h.
The performance of the columns was evaluated by the column efficiency test described above. A stability test was also run with 340 cm/h distilled water in upflow mode for 20 h. The performance and stability results are given in Table 2.
As dry swellable particles, a single batch of the chromatography medium CAPTO Q (GE Healthcare Bio-Sciences AB, Sweden) was used. This is an anion exchanger with tetramethylammonium group ligands coupled to crosslinked agarose beads of 90 micron average diameter when swollen in water. The medium was obtained as a slurry in 20% aqueous ethanol solution and was dried by sequential washing on a glass filter with distilled water, 99.5% ethanol and acetone, followed by vacuum drying at 20° C. for 3 days.
Two aliquots of 12.70 g (calculated to give a free swollen volume of 66 ml) each were weighed out from the air-dry particles. Each of these was poured into an empty HISCALE 50 column (GE Healthcare Bio-Sciences AB, Sweden), empty column with high pressure stability vertically arranged with the adaptor positioned in the middle of the column. The adaptor in each column was then moved downwards to a position corresponding to a bed height of 30 mm. The columns were wetted with an upwards flow of 30 cm/h distilled water. After 6 column volumes had passed each column, the flow was raised to 300 cm/h for an additional 20 column volumes. A downward flow of 1000 cm/h distilled water was then applied for 60 column volumes.
The two columns were arranged in parallel, as shown in
The relative difference in retention time of the acetone peak is a measure of the relative flow rate difference between the two columns and is within the 2.2-2.3% range for all three global flow rates.
The columns were then conditioned with three column volumes of 50 mM Tris buffer, pH 8.00 and the feed was switched to 5 mg/ml bovine serum albumin (BSA) dissolved in 50 mM Tris buffer, pH 8.00. About 80 column volumes of BSA solution was pumped in, before the BSA was eluted with 10 column volumes of 50 mM Tris+1 M NaCl, pH 8.00. This test was repeated at three different flow rates and the dynamic binding capacity at 10% breakthrough (Qb10) was calculated individually for each column according to standard methods. The results are presented in Table 4.
The relative capacity differences between the two columns are within the 4-6% interval.
The packing and efficiency tests of Example 3 were repeated with a single batch of CAPTO ImpRes Q (GE Healthcare Bio-Sciences AB, Sweden) as the chromatography medium, dried under the same conditions as in Example 3. Aliquots of 11.60 g (calculated to give a free swollen volume of 66 ml) air-dry particles were weighed out and otherwise the conditions were the same. The efficiency results are presented in Table 5.
In this case the relative retention time difference between the two columns is less than 0.7%.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0951003-3 | Dec 2009 | SE | national |
The present application is a continuation of U.S. application Ser. No. 14/201,175, filed Mar. 7, 2014, which is a continuation-in-part of U.S. application Ser. No. 13/518,029, now U.S. Pat. No. 9,958,421, issued May 1, 2018, which is a filing under 35 U.S.C. § 371 and claims priority to international application number PCT/SE2010/051424 filed Dec. 20, 2010, which claims priority to application No. 0951003-3 filed in Sweden on Dec. 22, 2009, the entire contents of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14201175 | Mar 2014 | US |
| Child | 17169699 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13518029 | Jun 2012 | US |
| Child | 14201175 | US |
