阅读说明：本技术 应用径向技术色谱的系统和方法 (System and method for chromatography using radial techniques ) 是由 D·W·K·列文森 A·J·胡斯特 于 2018-12-07 设计创作，主要内容包括：本发明公开了一种使用多个珠粒的应用径向技术色谱的系统和方法,其中每个珠粒中包括一个或多个直径为约<Image he="57" wi="140" file="DDA0002587734000000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>至约<Image he="59" wi="167" file="DDA0002587734000000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>的孔,和每个珠粒的平均半径在约100pm至约250pm之间。还公开了选择用于径向流色谱柱中的珠粒的方法,以及使用径向流色谱柱纯化未澄清的进料流的方法。(A system and method for applying radial technique chromatography using a plurality of beads, each comprising one or more beads having a diameter of about To about And each beadThe pellets have an average radius of between about 100pm and about 250 pm. Also disclosed are methods of selecting beads for use in a radial flow chromatography column, and methods of purifying an unclarified feed stream using a radial flow chromatography column.)

1. A radial flow chromatography column, comprising:

a plurality of beads, each bead comprising one or more pores therein, and

the interstitial channels formed between the beads are,

wherein each hole has a diameter of aboutTo about

Wherein at least about 80% of the plurality of beads have a diameter of about 200pm to about 500pm,

wherein the beads have an average radius R of from about 100pm to about 250 pm.

2. The radial flow chromatography column of claim 1, wherein the beads are made of a polymer, glass, alumina, silica, Controlled Pore Glass (CPG), cellulose, encapsulated iron particles, encapsulated CPG, or encapsulated silica.

3. The radial flow chromatography column of claim 2, wherein the beads are polymer beads.

4. The radial flow chromatography column of any preceding claim, wherein any beads having R <0.414R have been removed.

5. The radial flow chromatography column of any preceding claim, wherein any beads having R <0.225R have been removed.

6. The radial flow chromatography column of any preceding claim, wherein the interstitial channel has: radius of tetrahedral position r_tet0.225R, octahedral site radius R_oct0.414R, or both.

7. The radial flow chromatography column of any preceding claim, wherein the interstitial channel has a narrowest channel radius R_cha＝0.155R。

8. The radial flow chromatography column of any one of claims 1-3, wherein the beads are monodisperse.

9. A method of selecting beads for use in a radial flow chromatography column, comprising: a) determining a narrow desired bead radius R range based on the components present in the feed stream; b) removing beads of a defined radius r outside the desired bead range; and c) defining the percentage of bead radius R within the desired bead range.

10. The method of claim 9, wherein the beads are made of polymer, glass, alumina, metal or other crystalline, semi-crystalline or amorphous material, silica, Controlled Pore Glass (CPG), cellulose, encapsulated iron particles, encapsulated CPG or encapsulated silica.

11. The method of claim 9, wherein the beads are polymeric beads.

12. The method according to any one of claims 9 to 11, wherein the beads of radius r are removed by wet or dry sieving and/or elutriation.

13. The method according to any one of claims 9 to 11, wherein beads of radius R <0.414R are removed.

14. The method of claim 13, wherein beads of radius R <0.225R are removed.

15. A method of purifying an unclarified feed stream using the radial flow chromatography column of claim 1, comprising the steps of:

a. packing the radial flow column with the beads;

b. treating the clarified feed stream containing the particles of interest to calibrate purification conditions;

c. determining from the results of step b the binding of the particles of interest;

d. an unclarified feed stream containing particles of interest is treated.

16. The method of claim 15, wherein the particle of interest is a protein, virus, VLP, DNA, RNA, antigen, liposome, oligosaccharide or polysaccharide, or any combination thereof.

Technical Field

The present invention relates to radial flow column chromatography, and more particularly, to a radial flow column containing beads of a particular size and parameters to enhance filtration of an unclarified feed stream, and a method of selecting beads.

Background

As commonly used, chromatography is a technique used to separate the various components of a sample mixture. In a liquid chromatography system, a sample, followed by an eluent, is injected into a chromatographic separation column. The separation column contains packing, matrix media or substances that interact with the various components of the sample to be separated. The composition of the separation medium depends on the liquid that is introduced therethrough to achieve the desired separation. As the sample and eluent pass through the separation medium, different components of the sample will pass through the separation medium at different rates due to different interactions. The separation of these components from the separation medium occurs at the outlet or effluent.

Various types of vertical flow and horizontal flow separation columns are known in the art. Due to the need for high performance chromatography, horizontal flow type chromatography columns have been developed. Such horizontal flow or radial flow columns are described, for example, in U.S. patent nos. 4,627,918 and 4,676,898. In horizontal flow or radial flow type columns, the sample and eluent are introduced through a distributor to the outer or circumferential wall or surface of the separation medium or matrix, and the liquid passes horizontally or radially inward through the separation medium to a central or collection port, and then elutes from the column at different times and at different rates.

The chromatographic columns and methods were then developed as direct treatment of the crude feed for the separation of bioactive substances including cell/fermentation harvests, tissue extracts, algae, plant-derived cells and substances and plasma/blood. The large bead chromatography media was packed into a standard, low pressure chromatography column in which the end plate screens were replaced with large pore screens (60-180pm pores). Large pores prevent the column from clogging. Due to the large particle size, cellular material flows in the inter-particle cavities between the beads, and soluble products are captured by the functional groups on the beads.

Traditionally, downstream processing of cell culture/fermentation harvested biologicals requires two main operations: i) preparation of the feed stream and ii) recovery and purification. The sample should be prepared appropriately before application to the column. This is both time consuming and can be quite expensive. If a sample is to be prepared, the feed stream is typically diluted to reduce cell density, viscosity and salt concentration, all of which are beneficial for improved recovery and purification. Recovery involves the steps of removing cells and other particulate matter by centrifugation and/or microfiltration, and reducing the initial volume, typically ultrafiltration. Since conventional chromatographic media are rapidly contaminated with cell debris, the purification operation must be particle-free feed.

Not only are centrifugation and filtration operations lengthy and expensive, they can also be of a quality loss. Proteases released from the disrupted cells degrade the target protein, further complicating the task of developing purification methods and increasing purification costs. The longer the contact time with the concentrated cell debris, the more product is lost.

Capturing protein product directly from an unclarified feed minimizes product degradation, improves product quality, yield, and process economics. Furthermore, if the product capture and cell removal steps are combined into a single operation, capital intensive recovery operations would be greatly simplified.

There are two ways to capture product directly from an unclarified feed such as a cell culture/fermentation harvest or other biological sample (e.g., plasma). One method proposes fluidization (fluidization) of trapped resin particles. By fluidization, the individual particles are separated, allowing the fragments to leave the bed unobstructed.

There are several problems with this approach. Fluidized bed systems operate at a predetermined high flow rate and have no flexibility in the operation or method of changing the size of the column. The buffer consumption of this system is higher than that of packed bed systems, a major cost factor for high value pharmaceutical products, many of which require specialized buffers to purify them. The ratio of column volume to solid phase particle volume is very high. This will negatively affect the residence time and binding capacity within the column, since there is not enough time for the target molecule to diffuse completely into the solid phase. In addition, the particles collide within the bed and the solid phase fragments produce so-called "fines", reducing the performance and reuse of the column. The operation of a fluidized bed also requires specialized, expensive hardware and chromatographic media.

Another method is to use a packed bed column to remove the particles. This approach has not been explored to a large extent for the following reasons. The removal of cell debris from packed bed columns requires the use of large, preferably spherical, particles. These particles require sufficient space in the inter-particle lumen to allow cells or other particles of equivalent size to exit the column.

A disadvantage of using large particles (beads) is that the binding capacity of their proteins is a function of the available surface of the gel bed per unit volume. Thus, as the particle diameter increases, a loss of binding capacity can be observed. When the particle diameter is increased from 0.1mm to 1mm, such as for processing dense cell suspensions, about 90% of the protein binding capacity is lost. This makes packed bed columns impractical for handling crude process feed streams.

However, packed bed column operation offers simplicity, efficiency and economy. It is flexible and relatively easy to scale up. No special granules, equipment or training of the operator is required. Standard chromatography production takes up relatively little space and does not require adjustment of the height of the production equipment to accommodate the fluidized bed equipment.

Product utilization is another important issue in terms of operational throughput. For fluidized bed systems this is predetermined, but for packed column systems only the reaction binding kinetics is the rate limiting factor. This allows higher fluxes, 3-10 times higher than fluidized bed systems.

After product capture, residual cellular material is removed by a brief high-speed wash pulse. The product is then eluted using classical elution methods. Thus, known macrobead chromatography resins can directly process cell cultures or fermentation broths and other unclarified feeds in packed bed columns by combining cell removal and simultaneous product capture.

Us patent No. 5,466,377 proposes a method and large bead chromatography particles for capturing the desired product directly from an unclarified process liquid on a standard, low pressure packed bed chromatography column.

There remains a need for improved chromatographic materials and methods for the direct treatment of crude feed materials, such as cell culture/fermentation harvests, tissue extracts, cell debris, viruses, plasma, waste feed streams derived from vegetable or fruit extracts, or waste feed streams derived from milk processing or other natural material sources, on packed bed columns.

Disclosure of Invention

The invention discloses a radial flow chromatographic column, comprising: a plurality of beads, each bead comprising one or more pores therein, and interstitial channels formed between the beads. Each hole having a diameter of aboutTo about

At least about 80% of the beads have a diameter of about 200pm to about 500pm and the average radius R of the beads is between about 100pm to about 250 pm. The beads may be monodisperse (i.e., all beads have a radius of ± about 10% of the target or marker radius) or may have r<0.414R or R<0.225R has been removed.

A method of selecting beads for use in a radial flow chromatography column is also disclosed. The method comprises the following steps: a) determining a narrow desired bead radius R range based on the components (or particles of interest) present in the feed stream; b) removing beads of a defined radius r outside the desired bead range; and c) defining the percentage of bead radius R within the desired bead range. Beads of radius r can be removed by wet or dry sieving and/or elutriation. The beads removed may be beads with a radius R <0.414R or R < 0.225R.

Also disclosed herein is a method for purifying an unclarified feed stream using a radial flow chromatography column comprising: a plurality of beads, each bead comprising one or more pores therein, and interstitial channels formed between the beads, wherein eachThe diameter of the hole is about

To about

At least about 80% of the plurality of beads have a diameter of about 200pm to about 500pm and the average radius R of the beads is between about 100pm to about 250 pm. The method comprises the following steps: a) packing the radial flow column with beads; b) treating the clarified feed stream containing the particles of interest to calibrate the purification conditions; c) determining from the results of step b the binding of the particles of interest; d) an unclarified feed stream containing particles of interest is treated.

Detailed Description

The present invention provides radial flow columns and methods of making and using the same for direct filtration (i.e., treatment) of a crude biological feed stream, such as an unclarified (i.e., unfiltered) cell culture. The presently disclosed subject matter enables purification at substantially lower cost and faster processing, particularly compared to methods such as packed bed chromatography and expanded bed chromatography. Exemplary applications include the separation of plasma; biological treatment of cell cultures to isolate and purify proteins (particularly drugs such as herceptin, insulin, avastin, etc.); capture and purification of viruses and virus-like (vims-like) particles; the purification of waste feed streams derived from vegetable or fruit extracts and waste feed streams derived from milk processing or other natural sources.

The disclosed chromatography surprisingly and effectively allows whole cells and other particles to pass around and between the disclosed beads without damage, without clogging the gel bed, and allows the direct passage of an unclarified (unfiltered) feed stream of cell cultures including whole cells, cell debris, homogenates of natural or recombinant plant matter, nanoparticle solutions and/or other particles (typically clogging columns with small beads). It is also capable of selectively binding molecular targets of interest in the feed stream, which can subsequently be recovered. For example, for IgG purification, the beads can be functionalized by covalently binding protein a or protein G to the surface of the beads, allowing reversible IgG binding, followed by washing and subsequent elution. For virus and VLP capture, the beads can be modified to have a high outer surface area and a high (positive) charge density.

Disclosed is a radial flow chromatography column comprising a plurality of beads, each bead comprising one or more pores therein, and interstitial channels formed between the beads.

The radial flow column may be made according to any known radial flow column, except for the differences specifically discussed herein with respect to beads, gel beds, wells and channels. The radial flow column crystalline state has a bed length of about 3cm to about 50cm and a bed volume (V) in the range of about 5ml to about 1000 liters. The shape of the radial flow column may be "donut" (full-size radial flow column in the form of a right circular hollow cylinder), "cake" (sheet in the form of a trapezoidal prism, i.e. a small part of a circular ring, with the same bed length/radius and curvature but smaller volume), or truncated "cone" (in the form of a frustum or truncated cone, resulting from the removal of a small cylindrical core segment from the cake or ring, with the same bed length/radius and curvature but with a smaller volume than the "cake").

The column may include one or more porous filter frits. Typically having an outer core frit and an inner core frit. The pore size of each filter frit can be between about 40pm to about 300pm, between about 80pm to about 250pm, or between about 100pm to about 200 pm. The core frit may be made of any conventionally known material. Alternatively, it may be made of stainless steel or stainless steel and one or more polymers such as, but not limited to, Polyethylene (PE) or polypropylene (PP).

The "donut", "block" or "cone" shaped radial flow columns all have a constant ratio of the outer core frit area to the inner core frit area. This ratio may be between 1.5: 1 to 10: 1, in the above range. The preferred range is 2: 1 to 4: 1. the hydrodynamics of the three different shapes with the same bed length and external to internal external core frit area ratio are virtually identical.

The beads may be spherical or nearly spherical. The beads may be made of polymer, glass, alumina, metal or other, semi-crystalline or amorphous material, silica, Controlled Pore Glass (CPG), cellulose, encapsulated iron particles, encapsulated CPG, encapsulated silica, or any combination thereof. The polymer beads may be made of any polymer known for use in the art, for example polyacrylates (such as methacrylates), polystyrenes or polysaccharides (such as dextran, pullulan, agarose) or natural or bound polysilicates. The beads may be composed of two or more polymers, either homogeneously or heterogeneously mixed. The polymer beads may be spherical (or nearly spherical) polysaccharide beads.

The beads may have an average diameter of between about 200pm and about 1000pm, or between about 200pm and about 500 pm. In one embodiment, at least 80% of the polymer beads have a diameter of about 200pm to about 500pm, or at least about 85%, or at least about 90% of the polymer beads have a diameter of about 200pm to about 500 pm.

The beads have an average radius (R) of between about 100pm and about 500pm, or between about 100pm and about 250 pm.

Most beads are generally not monodisperse (all of the same size, the same diameter), but have a range of diameters outside the given range. Thus, for example, such a measurement means that 80% of the total mass (or volume) of the beads falls within 200-500 pm. The remaining 20% (regardless of how the percentages are defined) are outside the range, smaller or larger. In order to keep the interstitial channels from clogging, it is important to remove or at least deplete 20% of the smaller beads outside the given range for a certain time before column filling. If smaller beads of about the same size as the channel diameter are not depleted or removed, a gel bed of between 200 and 500pm beads may contain enough small beads to partially or completely block the channel.

Some chromatography beads are commercially available and are typically sold by bead diameter. However, the given diameter is the average of the bead diameters; this does not mean that all beads have a given diameter. Other companies may sell beads by listing a range of bead diameters. However, this is a range in which a specific, sometimes undefined, percentage of beads will fall. Then, the total percentage of beads with a diameter outside the given range is unknown; there are few beads falling above or below this range in a given percentage.

These beads may have functional groups (e.g., ion exchange groups, hydrophobic interaction groups, etc.) that enable them to selectively bind the molecular targets of interest (for potential later recovery). Potential targets include viruses, virus-like particles, proteins (particularly, but not limited to, IgG, IgM, IgY, and blood proteins), DNA, RNA, oligonucleotides, polypeptides, and cells.

The beads have one or more pores therein. Each hole having a diameter of about

To aboutEach hole may extend partially through the bead to form a terminus (dead-end) or may extend through the bead to another exit point.

Interstitial channels are formed between the filler beads, partially comprising void volumes. When these channels are wide enough to allow cells and cell debris to pass through the gel bed without clogging, and when there are no smaller beads (which, due to their size, can limit or prevent the passage of cells and cell debris), the user can utilize these packed beads to avoid adverse filtration or centrifugation, sedimentation, or other expensive, time consuming, and potential product loss steps prior to the chromatographic purification step.

Monodisperse spherical beads will ideally be packed in a hexagonal close-packed (HCP) or cubic close-packed (CCP) configuration. Both packing configurations have the same maximum bead (sphere) density and similar packing energies, so there is no apparent energy advantage of one over the other. While the preferred packing configurations of the polymers (e.g., polysaccharides, beads) are unpredictable, both packing configurations form channels (interstitial spaces or channels) between the beads, the minimum size of which is important to the success or failure of the devices and systems disclosed herein.

The clearance channel should be: 1) large enough to allow cells, cell debris and other interfering particles to pass through without clogging the gel bed; 2) without the smaller beads, these beads are similar in size to the interstitial channels themselves, and thus may clog the channels; and 3) formed from populations of beads having a bead diameter range as narrow as possible, the size of the monodisperse beads being as close as possible to the ideal size.

Furthermore, the clearance channel cannot be compressed too narrowly, which results in: i) too high a flow rate, ii) too high a pressure, or iii) too high a packing density of the beads, thereby clogging the interstitial channels. The interstitial channels should be open (i.e., free of blockages) to provide a continuous path for the treatment feed stream. In addition, too high a flow rate narrows the interstitial channels of the non-rigid gel bed. Therefore, the gel bed must be sufficiently rigid to allow a flow rate of 0.1 to 10 column volumes per minute without narrowing the interstitial channels (by bead compression) to allow cells and cell debris to pass through the gel bed.

The rigidity of the gel bed also depends on the rigidity of the beads themselves. In order to minimize the variation in diameter/size of the interstitial channels between beads (assuming that all beads are spherical or nearly spherical and have a given size distribution), it is important that the morphology of the particles does not change under flow conditions.

One option is to use very rigid beads which are inert to the buffer used for chromatographic separations and therefore do not change their size and shape; however, beads made of, for example, silicon or CPG (controlled pore glass) have excellent mechanical rigidity and stability but little or no resistance to sodium hydroxide.

Polymer beads, such as but not limited to methacrylate beads or polystyrene beads, can be stable to more reagents and remain rigid. Polysaccharide beads, such as but not limited to dextran beads or agarose beads, are less rigid but may be more stable to some reagents (e.g., NaOH). However, polysaccharide beads tend to be "softer" than other polymer beads and therefore more likely to become compressed. The extent to which compression occurs depends on the pressure exerted by the liquid flowing through the packed gel bed. Too much compression results in a non-porous bed through which no fluid can pass (the internal pore network and interstitial spaces are closed). The key factors that typically lead to an increase in pressure and compression are the flow rate of the applied liquid (typically expressed in mL/min, CV/min or cm/hr) and the viscosity of the liquid.

The mechanical stability and rigidity of the soft polysaccharide beads can be increased by applying certain means. For example, the following methods will improve the mechanical stability and rigidity of the beads:

1. cross-linking within the polysaccharide structure. This will make the beads more pressure resistant, thereby preserving the morphology of the beads. However, depending on the crosslinking chemistry applied, the size of the internal pores within the bead structure may be affected.

2. Increasing the density of the amount of polysaccharide used in the bead formulation. This is usually done with agarose based particles. The agarose percentage of standard commercially available agarose beads is between about 2% and 10% (20 g to 100g agarose per liter of bead prepared). The beads with increased density may contain ≧ about 6% agarose. However, this increase in density may reduce the internal pore size of the beads:

the average molecular weight cut-off of 4% of the beads was about 2000 kilodaltons;

the average molecular weight cut-off for 6% of the beads was about 400 kilodaltons.

The optimal interstitial channel size depends on the size of the beads and ultimately on the components present in the feed stream. In particular, the optimum size of the interstitial channels depends on the particles that are intended to pass through the gel bed. The method of determining the optimal size of the clearance channel is as follows:

1. determination of bead size: from the radius of the cells or debris that will pass through the gel bed, the radius required for the channel formed by the three beads is estimated ("narrowest channel radius"), and the minimum bead size required is calculated.

2. Determination of the size fraction removed from the gel: from the radius of the beads (monodisperse bead radius or average of the polydisperse bead radii), the radius lengths of the tetrahedral and octahedral positions are calculated. The radius of these locations is the radius of the largest bead that must be removed from the gel.

The maximum diameter of a globule that can theoretically pass through the smallest channel formed between three identical spheres is then calculated. The diameter of the channel may be about 3 to about 10 times, or about 4 to about 6 times, larger than the diameter of the largest particle (i.e., cell debris, or other particle) present in the unclarified feed stream.

For cubic close-packed, hexagonal close-packed or barlow-packed, fully fixed, shaking to no measurable change in packing density of monodisperse beads (kepler's hypothesis), the following holds:

radius of tetrahedron position r_tet0.225R (R is the radius of the beads, R)_tetIs the radius of the tetrahedral position);

octahedral site radius r_oct0.414R (R is the radius of the beads, R)_octRadius at the octahedral position);

narrowest passage radius r_cha0.155R (R is the radius of the beads, R)_chaIs the radius of the channel);

beads with radius x, where 0.155R < x <0.414R, which can fit and permanently occupy one octahedral position.

Beads with radius x, where 0.155R < x <0.225R, which can be sized to fit and permanently occupy one tetrahedral site.

Tetrahedral and octahedral "holes" are always present in the bed of packed beads. These holes are based on the number of beads surrounding and forming the hole, and are named tetrahedral or octahedral.

For random close packing of polydisperse beads, the above values are the minimum, and the actual values may be larger. Radius of passage r_cha0.155R should be a minimum of 1.1 times (preferably about 2 to about 4 times) the radius of the cells, debris or particles so that the cells, debris or particles can easily pass through the gel bed.

Monodisperse beads can be prepared by:

a. producing monodisperse beads having an optimum radius and being completely free of small beads;

b. in the emulsification process, narrow-range polydisperse beads are prepared by tightly controlling the conditions. These conditions include: adding an optimal type and amount of emulsifier, maintaining an optimal stirring speed, maintaining an optimal temperature, all of which contribute to a narrowing of the size distribution of the beads formed;

c. wet or dry screening to remove particles smaller (or larger) than a selected size; and/or

d. Elutriation to remove particles smaller than a certain size.

To improve filtration, smaller beads that may clog interstitial channels can be removed prior to packing the gel bed.

The form and function of the clearance channel may be improved by taking some or all of the following steps:

a. for example, using the information shown in tables 1-3 below, the desired average bead radius R is calculated/determined to allow cells, cell debris, or other particles to pass through.

b. A gel bed is formed in which beads of radius R <0.414R have been removed.

i. Removal of all beads with radius R <0.414R will form a gel bed with maximum flow and porosity, shorter path. Its advantage is high speed of purifying.

c. A gel bed is formed in which beads of radius R <0.225R have been removed.

i. Removal of all beads with radius R <0.225R will form a gel bed with somewhat reduced porosity, increased path length and increased residence time. Its advantage is high effect.

d. Removing small beads with radius R <0.225R or R <0.414R prevents the interstitial channels of the gel bed from becoming congested/clogged. This will also reduce the amount of gel bed washing required during the purification cycle.

e. The radius r of the beads to be removed is increased by at most 25% (i.e. the radius r of the beads to be removed is 25% larger than the radius r of the other beads).

f. The size distribution of the beads is narrowed as much as possible to achieve a reduction in random close-packed density, and close to cubic/hexagonal close-packed density.

TABLE 1

TABLE 2

TABLE 3

Another embodiment is a method of selecting beads for a radial chromatography gel bed, the method comprising: a) determining a narrow desired bead diameter (or radius) range based on the components (or particles of interest) present in the feed stream; b) removing beads of a defined diameter (or radius) outside the desired bead diameter range; and c) defining the percentage of bead diameter (or radius) within the desired bead diameter range.

Another embodiment is a method of filtering an unclarified feed stream using the radial flow column disclosed above, comprising the steps of:

a. packing the radial flow column with beads;

b. treating the clarified feed stream containing the particles of interest to calibrate purification conditions;

c. determining from the results of step b the binding of the particles of interest; and

d. the treatment includes selecting an unclarified feed stream of the protein.

The particle of interest may be a whole cell, a cell fragment, a virus, or a protein. It may also be a VLP, DNA, RNA, antigen, liposome, oligosaccharide or polysaccharide.

After filling the radial flow column with beads, the unclarified feed stream is subjected to conventional purificationThe clarified feed stream is used to calculate optimal purification conditions. This helps to understand how particles of interest (e.g., proteins) interact with the column (RFC, Zetacell)^TM) And (4) combining. For purification of the clarified stream, forward and reverse washing may be used to remove traces of cells/cell debris. A block or cone shape can be used for small scale optimization before scaling up/using the torus shape. The process may be carried out in conjunction with a "simulated moving bed" ("SMB")/continuous process.

Depending on the reagent system used in chromatography, the polymer beads will shrink and expand, increasing and decreasing the internal pore diameter and the interstitial space between the beads (spherical). This does not occur with particles having silica and CPG, but does occur with all gel beds based on polymer and polysaccharide particles. By careful selection of the buffer system used for packing the column and in normal operation by the skilled person, the effect of expansion and contraction can be controlled (wholly, almost wholly, or at least partially).

After optimizing the process using a clarified feed stream directly on a small radial flow chromatography column containing polymer beads as a gel bed, linear scaling up to larger process scale can be achieved by: maintaining bed length, maintaining ratio of external to internal frit area, and maintaining all operating parameters (flow rate, amount of column volume per unit time, buffer composition, retention time, solid phase, pressure and temperature) for each step of the process (loading of clarified or unclarified feed stream, forward and reverse washing to remove cells, cell debris and unbound material, elution to release directly captured targets from solid phase, regeneration to clean and prepare column and solid phase for subsequent reuse). If the large scale purification system needs to be recalibrated, re-optimized, or otherwise changed at any time, the linearity is scaled down to be controllable, and small RFC columns can also be achieved by: maintaining bed length, maintaining ratio of external to internal frit area, and maintaining all operating parameters (flow rate, amount of column volume per unit time, buffer composition, retention time, solid phase, pressure and temperature) for each step of the process (loading of clarified or unclarified feed stream, forward and reverse washing to remove cells, cell debris and unbound material, elution to release directly captured targets from the solid phase, regeneration to clean and prepare the column and solid phase for subsequent reuse).

The foregoing illustrates some of the possibilities for practicing the invention. Thus, while particular example embodiments have been described, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader scope of the invention; many other embodiments are possible within the scope and spirit of the invention. Various features are grouped together in a single embodiment for the purpose of streamlining the invention. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments have more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the above description of the invention, with each claim standing on its own as a separate exemplary embodiment.

It should be noted that it is contemplated that any feature or element explicitly identified herein may also be specifically excluded from a feature or element of an embodiment of the present invention as defined in the claims. It should also be noted that it is contemplated that any feature or element that is explicitly identified (or explicitly or implicitly excluded) may be used in combination with any other feature or element that is explicitly identified (or explicitly or implicitly excluded).