System and method of applied radial technology chromatography

Abstract

To provide improved chromatographic materials and methods to achieve direct processing of crude feeds, such as cell culture / fermentation harvests, tissue extracts, cell fragments, viruses, blood 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.SOLUTION: The foregoing problem is solved by a radial flow chromatography column of the present invention.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to radial flow column chromatography, and more particularly to radial flow columns containing beads of specific sizes and parameters that enhance filtration of unclarified feed streams, and methods for selecting the beads. [Background technology]

[0002] Chromatography, as it is commonly used, is a technique for separating the various components of a sample mixture. In a liquid chromatography system, a chromatographic separation column is injected with a sample followed by an eluent. The separation column contains a packing or matrix medium or material that interacts with the various components of the sample to be separated. The composition of the separation medium is determined by the fluids introduced therein to achieve the desired separation. As the sample and eluent pass through the separation medium, the various components of the sample undergo different interactions and move through the separation medium at different rates. These components emerge separated at an outlet or effluent from the separation medium.

[0003] Various types of vertical and horizontal flow separation columns are known in the art. The need for high performance chromatography has led to the development of horizontal flow chromatography columns. Such horizontal or radial flow columns are described, for example, in U.S. Pat. Nos. 4,627,918 and 4,676,898. In horizontal or radial flow columns, the sample and eluent are introduced to the outer periphery or peripheral wall or surface of the separation medium or matrix through a distributor, and the fluid 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.

[0004] Chromatography columns and methods were then developed for the direct processing of crude feeds to isolate biologically active materials, including cells / fermentation harvests, tissue extracts, algae, plant-derived cells and materials, and plasma / blood. Large bead chromatography media is packed into a standard low-pressure chromatography column in which the end plate screens are replaced with large pore screens (60-180 pm pores). The large pores prevent clogging of the column. Due to the large particle size, cellular material flows between the beads in the interparticle spaces, while soluble products are captured by functional groups on the beads.

[0005] Traditionally, downstream processing of biology from cell cultures / fermentation harvests has required two major operations: i) feed stream preparation, and ii) recovery and purification. Samples must be properly prepared before being applied to the column, which can be a significant burden in terms of time and cost. If sample preparation is required, the feed stream is typically diluted to reduce cell density, viscosity, and salt concentration, all of which aid in improved recovery and purification. Recovery includes removal of cells and other particulate material by centrifugation and / or microfiltration, as well as an initial volume reduction step, typically ultrafiltration. Because conventional chromatographic media is rapidly contaminated by cellular debris, a particle-free feed must be prepared for the purification operation.

[0006] Centrifugation and filtration are not only time-consuming and costly, but also reduce quality. Proteases released from broken cells can degrade the target protein, further complicating the purification method development challenge and increasing purification costs. More product may be lost due to longer contact time with concentrated cell debris.

[0007] Direct capture of protein products from unclarified feeds would minimize product degradation and improve product quality, yield, and process economics, and combining the product capture and cell removal steps into a single operation would greatly simplify capital-intensive recovery operations.

[0008] There are two approaches to directly capture product from non-clarified feeds such as cell culture / fermentation harvests or other biological samples (e.g. plasma). One approach proposes fluidization of the capture resin particles, which separates the individual particles and allows the debris to exit the column bed unhindered.

[0009] There are several problems with this approach. Fluidized bed systems operate at a fixed high flow rate and have no flexibility of operation or the means to change the column size. The buffer consumption of this system is higher than that of packed bed systems, which is a major cost factor for expensive pharmaceuticals, many of which require special buffers for purification. The ratio of column volume to solid phase particle volume is very high. This will adversely affect the residence time and binding capacity in the column, as the target molecules will not have enough time to completely diffuse into the solid phase. Furthermore, particles will collide in the column bed and the solid phase fragments into so-called "fines", reducing both the performance and reusability of the column. Fluidized bed operation also requires specialized and expensive hardware and chromatographic media.

[0010] Another approach is the use of packed bed columns to remove particles. This avenue remains largely unexplored for the following reasons: Removal of cellular debris in packed bed columns requires the use of large, preferably spherical, particles. These particles require sufficient interparticle space to allow cells or other particles of comparable size to exit the column.

[0011] The disadvantage of using large particles (beads) is that the protein binding capacity is a function of the available surface per unit volume of the gel bed. Thus, as particle size increases, a loss in binding capacity is observed. When particle size increases to 0.1 mm to 1 mm, as is necessary to handle dense cell suspensions, approximately 90% of the protein binding capacity is lost. For this reason, packed bed columns have been impractical for processing crude feed streams.

[0012] However, packed bed column operation offers simplicity, efficiency, and economy. It is flexible and relatively easy to scale. No special particles, equipment, or operator training is required. For standard chromatography, the work area is relatively small and no changes to the work facility height are required to accommodate fluidized bed equipment.

[0013] The rate of product application is another important issue in terms of the throughput of the operation. This is pre-determined in a fluidized bed system, whereas in a packed column system, only the binding rate of the reaction is the rate-limiting factor. This allows for throughputs up to 3-10 times higher than in a fluidized bed system.

[0014] After product capture, residual cellular material is removed by a short, fast wash pulse. The product is then eluted by a typical elution method. Thus, known large bead chromatography resins allow for the direct processing of cell cultures or fermentation broths, as well as other non-clarified feeds, in packed bed columns by combining simultaneous product capture with cell removal.

[0015] US Pat. No. 5,466,377 proposed a method and large bead chromatography particles for capturing the desired product directly from the non-clarified process liquid in a standard low pressure packed bed chromatography column.

[0016] There remains a need for improved chromatographic materials and methods to achieve direct processing in packed bed columns of crude feeds such as cell culture / fermentation harvests, tissue extracts, cell fragments, viruses, plasma, waste streams from vegetable or fruit extracts, or waste streams from milk processing or other natural sources. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Patent No. 4,627,918 [Patent Document 2] U.S. Patent No. 4,676,898 [Patent Document 3] U.S. Patent No. 5,466,377 Summary of the Invention [Problem to be solved by the invention]

[0018] Disclosed herein is a radial flow chromatography column comprising a plurality of beads, each bead containing one or more pores, and interstitial channels formed between the beads. [Means for solving the problem]

[0019] Disclosed is a radial flow chromatography column comprising a plurality of beads, each bead comprising one or more holes, and interstitial channels formed between the beads. Each hole has a diameter of about 250 Å to about 5000 Å, at least about 80% of the plurality of beads have a diameter of about 200 pm to about 500 pm, and the beads have an average radius R of about 100 pm to about 250 pm. The beads may be monodisperse (i.e., the radius of all beads is within ± about 10% of the target or tagged radius), or may be removed with r<0.414R or r<0.225R.

[0020] Also disclosed is a method for selecting beads for use in a radial flow chromatography column. The method includes the steps of: a) identifying a narrow range of desired bead radii R based on components (or particles of interest) present in a feed stream; b) removing beads of a given radius r that are outside the desired bead range; and c) defining a percentage of bead radii R within the desired bead range. Beads of radius r can be removed by wet or dry sieving and / or elutriation. The removed beads can be beads with a radius r<0.414R or r<0.225R.

[0021] 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, and interstitial channels formed between the beads, wherein each pore has a diameter of about 250 Å to about 5000 Å, at least about 80% of the plurality of beads have a diameter of about 200 pm to about 500 pm, and the beads have an average radius R of about 100 pm to about 250 pm. The method includes the steps of a) packing the beads into the radial flow column, b) treating a clarified feed stream containing a particle of interest to adjust purification conditions, c) determining binding of the particle of interest from the results of step b, and d) treating the unclarified feed stream containing the particle of interest. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Radial flow columns and methods for making and using same are provided for the direct filtration, i.e., processing, of crude biological feedstreams, such as unclarified (i.e., unfiltered) cell cultures. The subject matter of the present disclosure provides for substantially lower cost and faster purification, particularly compared to methods such as packed bed chromatography and expanded bed chromatography. Exemplary applications include fractionation of plasma; bioprocessing of cell cultures to isolate and purify proteins (e.g., pharmaceuticals such as Herceptin, insulin, Avastin, etc.); capture and purification of viruses and virus-like particles; purification of waste streams from vegetable or fruit extracts, and waste streams from milk processing or other natural sources.

[0023] The chromatography disclosed herein surprisingly and advantageously allows whole cells and other particles to pass undamaged around and between the disclosed beads without clogging the gel bed, allowing the direct passage of unclarified (unfiltered) feed streams of whole cells, cell fragments, homogenates of natural or recombinant plant materials, nanoparticle solutions, and / or cell cultures containing other particles (which would normally clog columns with small beads). Also, the molecular targets of interest in the feed stream can be selectively bound and subsequently recovered. For example, in IgG purification, the beads can be functionalized by covalently binding A or G proteins to the surface of the beads to allow reversible IgG binding, followed by washing and subsequent elution. For virus and VLP capture, the beads can be modified to have a high external surface area and a high (positive) charge density.

[0024] Disclosed herein is a radial flow chromatography column comprising a plurality of beads, each bead containing one or more pores, and interstitial channels formed between the beads.

[0025] The radial flow column can be made according to any known radial flow column, except for the differences specifically described herein with respect to the beads, gel bed, holes, and channels. The radial flow column can have a bed length of about 3 cm to about 50 cm, and the bed volume (V) can range from about 5 ml to about 1000 liters. The radial flow column can be shaped as a "doughnut" (a full size radial flow column with a perfectly circular hollow cylindrical shape), a "cake" slice (having the shape of a trapezoidal prism, i.e., a small piece of a doughnut, with the same bed length / radius and curvature, but with a smaller volume), or a truncated "cone" (originating from a small cylindrical core piece taken from the cake or doughnut, and having the shape of a cone or truncated cone, with the same bed length / radius and curvature, but with an even smaller volume than the "cake").

[0026] The column may include one or more porous filter frits. Often there is an external frit and an internal frit. Each frit may be designed to have a pore size of about 40 pm to about 300 pm, about 80 pm to about 250 pm, or about 100 pm to about 200 pm. The frit may be made of any material known in the art. Optionally, 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).

[0027] Radial flow columns of "doughnut", "cake", or "cone" geometry all have a constant ratio of external frit area to internal frit area. This ratio can range from 1.5:1 to 10:1. The preferred range is 2:1 to 4:1. The flow dynamics for the three different geometries with the same bed length and external frit to internal frit area ratio are virtually identical.

[0028] The beads may be spherical or nearly spherical. They may be made of polymers, glass, alumina, metals, or other crystalline, semi-crystalline, or amorphous materials, 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, such as polyacrylates, such as methacrylates, polystyrene, or polysaccharides, such as dextran, pullulan, agarose, etc., or natural or bound polysilicates. The beads may be composed of two or more homogeneously or heterogeneously blended polymers. The polymer beads may be spherical (or nearly spherical) polysaccharide beads.

[0029] The beads can have an average diameter of about 200 pm to about 1000 pm, or about 200 pm to about 500 pm. In one embodiment, at least about 80% of the polymeric beads have a diameter of about 200 pm to about 500 pm, or at least about 85%, or at least about 90% of the polymeric beads have a diameter of about 200 pm to about 500 pm.

[0030] The beads can have an average radius (R) of from about 100 pm to about 500 pm, or from about 100 pm to about 250 pm.

[0031] Most beads are generally not monodisperse (all the same size and have the same diameter), but have a range of diameters beyond the given range. So, for example, this measurement means that 80% of the total mass (or volume) of the beads is within the range of 200-500 pm. The remaining 20% ​​(regardless of how the percentage is defined) is outside the range, either smaller or larger than this. In order to avoid clogging of the interstitial channels, it is important that the 20% of smaller beads outside the given range are removed or at least depleted at some point before packing the column. If the smaller beads of approximately the same size as the channel diameter are not depleted or removed, a gel bed of 200-500 pm beads may contain enough small beads to partially or completely block the channel.

[0032] Some chromatography beads are commercially available, and are often sold by bead diameter. However, the specified diameter is an average of the bead diameters, and does not mean that all beads have that specified diameter. Other companies may sell beads with a range of bead diameters listed. However, this is a range that includes a certain, sometimes unclear, percentage of beads. In that case, the total percentage of beads that have a diameter outside the given range is unknown, but it is rare that there is a given percentage of beads that are above or below the range.

[0033] The beads may have functionalized groups (e.g., ion exchange groups, hydrophobic interaction groups, etc.) that allow for selective binding of molecular targets of interest (for possible subsequent recovery). Potential targets include viruses, virus-like particles, proteins (specifically, but not limited to, IgG, IgM, IgY, and blood proteins), DNA, RNA, oligonucleotides, polypeptides, and cells.

[0034] The beads have one or more pores, each having a diameter of about 250 Å to about 5000 Å, and each pore may extend partially through the bead or may extend through the bead to a separate exit point.

[0035] Interstitial channels are formed between the packed beads, partially constituting the void volume, and if these channels are wide enough to allow cells and cell fragments to pass through the gel bed without clogging, and if the channels do not contain smaller beads that may restrict or impede the passage of the cells and cell fragments due to their size, users can utilize the packed beads to avoid harmful filtration or centrifugation, precipitation, or other costly, time-consuming, and potentially product-losing steps prior to the chromatographic purification step.

[0036] Monodisperse spherical beads are ideally packed in either a hexagonal close-packed (HCP) or cubic close-packed (CCP) configuration. Both packing configurations have the same maximum bead (sphere) density and both have similar packing energies, so neither is clearly energetically favored over the other. Although the preferred packing configuration of polymer, e.g., polysaccharide, beads cannot be predicted, both packing configurations will form channels (interstitial spaces or channels) between the beads, the minimum size of which is critical to the success or failure of the devices and systems disclosed herein.

[0037] The interstitial channels should be: 1) large enough to allow cells, cell fragments, and other interfering particles to pass through without clogging the gel bed; 2) similar in size to the interstitial channel itself, and therefore free of smaller beads that could clog the channel; and 3) formed from a population of beads with as narrow a range of bead diameters as possible, so that monodisperse beads are as close to the ideal size as possible.

[0038] Furthermore, the gap channels should not be compressed too narrowly, which may result in i) too high flow rates, ii) too high pressure, or iii) too high bead packing density, clogging the gap channels. The gap channels should be open (i.e., unclogging) to provide a continuous path to process the feed stream. Also, too high a flow rate will narrow the gap channels of a non-rigid gel bed. Thus, the gel bed must be sufficiently rigid to allow a flow rate of 0.1 to 10 column volumes per minute without narrowing the gap channels (by bead compression) to allow cells and cell fragments to pass through the gel bed.

[0039] The stiffness of the gel bed also depends on the stiffness of the beads themselves. In order to minimize changes in the diameter / size of the interstitial channels between the beads (assuming all beads are spherical or nearly spherical and with a given size distribution), it is important that the morphology of the beads does not change under the flow conditions.

[0040] One option is to use very rigid beads that are inert to the buffers used in the chromatographic separation and therefore do not show any change in bead size or shape. However, for example, beads made from silica or CPG (controlled pore glass) have excellent mechanical rigidity and stability, but little or no resistance to NaOH.

[0041] Polymer beads, such as but not limited to methacrylate or polystyrene beads, can provide better reagent stability and retain rigidity. Polysaccharide beads, such as but not limited to dextran or agarose beads, are less rigid but more stable to reagents such as NaOH. However, polysaccharide beads are often "softer" than other polymer beads and therefore more susceptible to compression. 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 that cannot flow (both the internal pore network and the interstitial spaces are closed). Typically, the primary factors that cause this increased pressure and compression are the flow rate of the applied liquid (usually expressed in mL / min, CV / min, or cm / hr) and the viscosity of the liquid.

[0042] The mechanical stability and stiffness of the softer polysaccharide beads can be increased by applying certain procedures. For example, the following techniques improve the mechanical stability and stiffness of the beads: 1. Cross-linking within the polysaccharide structure, which makes the beads more resistant to pressure and therefore maintains their morphology, however, depending on the cross-linking chemistry used, the size of the internal pores within the bead structure may be affected. 2. Increase the density of the amount of polysaccharide used in the bead formulation. This is often done with agarose-based particles. Standard commercially available agarose beads have an agarose percentage of about 2-10% (20-100 grams of agarose per liter of formulated beads). Beads with increased density may have ≥ about 6% agarose. However, this density increase may reduce the internal pore size of the beads: 4% of the beads have an average molecular weight cutoff of approximately 20 million daltons · 6% of the beads have an average molecular weight cutoff of approximately 4 million daltons.

[0043] The optimal interstitial channel size depends on the bead size but ultimately depends on the components present in the feed stream. Specifically, the optimal size of the interstitial channel depends on the particles that desirably pass through the gel bed. A method for determining the optimal size of the interstitial channel can be as follows: 1. Determination of bead size: From the radius of the cells or fragments passing through the gel bed, estimate the required radius of the channel formed by three beads (the "narrowest channel radius") and calculate the required minimum bead size. 2. Determination of the size fraction to be removed from the gel: Calculate the lengths of the radii of the tetrahedral and octahedral sites from the radius of the beads (the radius of monodisperse beads or the average of the bead radii of polydisperse beads). The radii of these sites are the radii of the largest beads that must be removed from the gel.

[0044] Next, the theoretical maximum diameter of the small beads that can somehow pass through the smallest channel formed between three identical spheres was calculated. The diameter of the channel may be about 3 to about 10 times larger, or about 4 to about 6 times larger, than the largest particles present in the non-clarified feed stream (i.e., cells, cell fragments, or other particles).

[0045] For cubic close-packed, hexagonal close-packed, or Barlow packing of completely stabilized and vibrated monodisperse beads with no measurable change in packing density, the following applies: · The radius r of the tetrahedral site tet = 0.225R (R is the radius of the bead, and r tet is the radius of the tetrahedral site) · The radius r of the octahedral site oct = 0.414R (R is the radius of the bead, and r oct is the radius of the octahedral site) · The radius r of the narrowest channel cha = 0.155R (R is the radius of the bead, and r cha is the radius of the channel) · Beads with a radius x where 0.155R < x < 0.414R fit into the octahedral site and are of a size that can permanently occupy it. · Beads with a radius x where 0.155R < x < 0.225R fit into the tetrahedral sites and are of a size that can be permanently occupied.

[0046] Both "holes" of tetrahedra and octahedra are always present in the bed of filled beads. The holes are called tetrahedra or octahedra based on the number of beads forming around the holes.

[0047] In the random close packing of polydisperse beads, the above values are the minimum values, and the actual values may be larger. The radius r cha = 0.155R channels should be at least 1.1 times (preferably about 2 to about 4 times) the radius of cells, fragments, or particles to easily pass through the gel bed.

[0048] Monodisperse beads can be prepared as follows: a. Producing monodisperse beads with an optimal radius and no smaller beads present; b. Producing a narrow range of polydisperse beads by carefully controlling the conditions during the emulsification process. These conditions include adding the optimal type and amount of emulsifier, maintaining the optimal stirring speed, and maintaining the optimal temperature, all of which contribute to narrowing the size distribution of the beads formed; c. Removing fractions of particles smaller (or larger) than the selected size by wet or dry sieving; and / or d. Decanting to remove fractions of particles smaller than a determined size.

[0049] To improve filtration, smaller beads that may clog the interstitial channels can be removed before filling the gel bed.

[0050] The interstitial channels can have their shape and function improved by performing some or all of the following steps: a. Calculate / determine the desired average bead radius R to allow cells, cell fragments, or other particles to pass through, for example, using the information provided in Tables 1-3 below. b. Form a gel bed from which beads having a radius r<0.414R have been removed. i. Removing all beads with a radius r<0.414R creates a gel bed with maximum fluidity and porosity, and shorter paths, the advantage of which is a faster purification process. c. Form a gel bed from which beads having a radius r<0.225R have been removed. i. Removing all beads with a radius r<0.225R will result in a gel bed with slightly less porosity, longer path length, and longer residence time. The advantage is that purification will be more efficient. d. Removing smaller beads with radii r<0.225R or r<0.414R prevents blockage / clogging of interstitial channels in the gel bed. This also reduces the amount of gel bed cleaning required between purification cycles. e. Increase the radius r of the beads to be removed by up to 25% (i.e., the radius r of the beads to be removed is 25% more than otherwise shown). f. The bead size distribution should be as narrow as possible to achieve a reduction in random close-packed density and approach cubic / hexagonal close-packed density.

[0051] [Table 1]

[0052] [Table 2]

[0053] [Table 3]

[0054] Another embodiment is a method of selecting beads for use in a radial chromatography gel bed, comprising the steps of: a) identifying a narrow desired bead diameter (or radius) range based on components (or particles of interest) present in a feed stream; b) removing beads of a specified diameter (or radius) that are outside the desired bead diameter range; and c) specifying a percentage of bead diameters (radii) within the desired bead diameter range.

[0055] Yet another embodiment is a method for filtering an unclarified feed stream using the radial flow column described above, comprising the steps of: a. packing the beads into a radial flow column; b. treating the clarified feed stream containing the particles of interest to adjust purification conditions; c. determining the binding of the particles of interest from the results of step b; d. treating the unclarified feed stream containing the selected protein; The method includes:

[0056] The particle of interest can be a whole cell, a cell fragment, a virus, or a protein. It can be a VLP, DNA, RNA, an antigen, a liposome, an oligosaccharide, or a polysaccharide.

[0057] After the beads are packed into a radial flow column, the clarified feed stream is used to tune the optimal purification conditions, followed by actual routine purification of the non-clarified feed stream. This allows understanding of how the particle of interest, such as a protein, binds to the column (RFC, Zetacell™). In the purification of the non-clarified stream, both forward and backward washes can be used to remove traces of cells / cell fragments. A cake or cone geometry can be used for small scale optimization, followed by a donut geometry for scale-up. This method can be used in combination with "simulated moving bed" ("SMB") / continuous methods.

[0058] Depending on the reagent system used during chromatography, the polymer beads shrink and swell, thereby increasing and decreasing both the internal pore diameter and the interstitial space between the (spherical) beads. This does not happen with silica and CPG particles, but does happen with all polymer and polysaccharide particle based gel beds. The effects of swelling and shrinking can be controlled totally, nearly totally, or at least partially by the skilled artisan's careful selection of the buffer system used to pack the column and during routine operation.

[0059] After optimizing the process using the clarified feed stream directly in a miniature radial flow chromatography column containing polymeric beads as a gel bed, linear scale-up to larger process scales can be achieved by maintaining the bed length, maintaining the area ratio of the outer frit to the inner frit, and maintaining all operational parameters (flow rate, number of column volumes per unit time, buffer composition, residence time, solid phase, pressure, and temperature) for each step of the method (loading of clarified or unclarified feed stream, forward and reverse washes to remove cells, cell debris, and unbound materials, elution to release captured targets directly from the solid phase, regeneration to clean and prepare the column and solid phase for subsequent reuse). If at any time the large-scale purification system needs to be retuned, reoptimized, or otherwise modified, linear scale-down to smaller, more manageable RFC columns is also achieved by maintaining the bed length, maintaining the external to internal frit area ratio, and maintaining all the operating parameters (flow rate, number of column volumes per unit time, buffer composition, residence time, solid phase, pressure, and temperature) of each step of the method (loading of clarified or unclarified feed stream, forward and reverse washes to remove cells, cell debris, and unbound materials, elution to release captured target directly from the solid phase, regeneration to clean and prepare the column and solid phase for subsequent reuse).

[0060] The above illustrates some possibilities for implementing the present invention. Thus, while certain exemplary embodiments have been described, it is apparent that various modifications and changes can be made to these embodiments without departing from the broad scope of the present invention, and many other embodiments are possible within the scope and spirit of the present invention. For the purpose of simplifying the disclosure, various features have been grouped together in a single embodiment. This method of disclosure should not be interpreted as reflecting that the claimed embodiments have more features than are expressly recited in each claim. Rather, as the following claims reflect, the subject matter of the present invention 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.

[0061] It is noted that it is also contemplated that any feature or element expressly identified in this document may be specifically excluded as a feature or element of an embodiment of the invention defined in the claims. It is also noted that it is contemplated that any feature or element that is expressly identified (or excluded, whether specifically or implicitly) may be used in combination with any other feature or element that is expressly identified (or excluded, whether specifically or implicitly).

Claims

1. 1. A radial flow chromatography column for the purification of a particle of interest, which is a virus or a VLP, comprising: The radial flow chromatography column includes a plurality of beads packed in the column and non-clogged interstitial channels formed between the beads, The radial flow chromatography column has a bed length of 3 cm to 50 cm; each bead contains one or more pores, each pore having a diameter between 250 Å and 5000 Å; at least 80% of the plurality of beads have a diameter between 200 μm and 500 μm; the beads have an average radius R between 100 μm and 250 μm; The beads maintain their size at flow rates up to 10 column volumes per minute. The bead radius is ±10% of the target radius, and beads with a radius r<0.225R are removed prior to column packing; The system provides for purification of viruses or VLPs by direct filtration from a non-clarified or crude biological feedstream; Radial flow chromatography columns.

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

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

4. 4. A radial flow chromatography column according to any one of claims 1 to 3, wherein any beads having r<0.414R have been removed.

5. The gap channel is a tetrahedral section with radius r tet =0.225R, octahedral part radius r oct 5. The radial flow chromatography column of claim 1, having a R = 0.414R, or both.

6. The gap channel has the narrowest channel radius r cha 6. The radial flow chromatography column of claim 1, having R = 0.

155.

7. 7. A radial flow chromatography column according to any one of claims 1 to 6, wherein the beads have a high positive charge density.

8. 8. A radial flow chromatography column according to any one of claims 1 to 7, wherein the beads have functionalized groups for selectively binding to the target virus.

9. 9. The radial flow chromatography column of claim 8, wherein the functionalized groups are ion exchange groups or hydrophobic interaction groups.

10. 10. The radial flow chromatography column of claim 1, wherein the target virus is recovered after binding.

11. 10. A method for selecting beads for use in a radial flow chromatography column according to claim 1 or 8, comprising the steps of: a) identifying a narrow range of desired bead radii R based on components present in a feed stream; b) removing beads of a given radius r that are outside the desired bead range; and c) defining a percentage of bead radii R within the desired bead range.

12. 12. The method of claim 11, 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.

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

14. 14. The method according to any one of claims 11 to 13, wherein the beads of radius r are removed by wet or dry sieving and / or elutriation.

15. 14. The method of claim 11, wherein beads having r<0.414R are removed.

16. 16. The method of any one of claims 11 to 15, wherein the beads have functionalized groups for selectively binding to the target virus.

17. The method of claim 16, wherein the functionalized groups are ion exchange groups or hydrophobic interaction groups.

18. 13. A method for purifying an unclarified feed stream containing a particle of interest using the radial flow chromatography column of claim 1, wherein the particle is a virus or a VLP; a. packing the beads into a radial flow column; b. treating the clarified feed stream containing the viruses or VLPs to adjust purification conditions; c. determining the binding of the virus or VLP from the results of step b; d. Treating the non-clarified feed stream containing the virus or VLPs; A method comprising:

19. 20. The method of claim 18, wherein the beads have functionalized groups for selectively binding to the target virus.

20. 20. The method of claim 19, wherein the functionalized groups are ion exchange groups or hydrophobic interaction groups.

21. 21. The method of any one of claims 18 to 20, wherein the target virus is recovered after binding.

22. 1. A radial flow chromatography column for the purification of a particle of interest that is IgG or IgM, comprising: The radial flow chromatography column includes a plurality of beads packed in the column and non-clogged interstitial channels formed between the beads, The radial flow chromatography column has a bed length of 3 cm to 50 cm; each bead contains one or more pores, each pore having a diameter between 250 Å and 5000 Å; at least 80% of the plurality of beads have a diameter between 200 μm and 500 μm; the beads have an average radius R between 100 μm and 250 μm; The beads maintain their size at flow rates up to 10 column volumes per minute. The bead radius is ±10% of the target radius, and beads with a radius r<0.225R are removed prior to column packing; The system provides for purification of IgG or IgM by direct filtration from a non-clarified or crude biological feedstream. Radial flow chromatography columns.

23. 23. The radial flow chromatography column of claim 22, wherein the beads are made of polymer, glass, alumina, silica, controlled pore glass (CPG), cellulose, encapsulated iron particles, encapsulated CPG, or encapsulated silica.

24. 24. The radial flow chromatography column of claim 23, wherein the beads are polymeric beads.

25. 25. A radial flow chromatography column according to any one of claims 22 to 24, wherein any beads having r<0.414R have been removed.

26. The gap channel is a tetrahedral section with radius r tet =0.225R, octahedral part radius r oct 26. The radial flow chromatography column of claim 22, having a R = 0.414R, or both.

27. The gap channel has the narrowest channel radius r cha 27. The radial flow chromatography column of any one of claims 22 to 26, having R = 0.

155.

28. 28. A radial flow chromatography column according to any one of claims 22 to 27, wherein the beads are functionalised by covalently binding Protein A or Protein G to the surface of the beads.

29. 23. A method for selecting beads for use in a radial flow chromatography column according to claim 22, comprising the steps of: a) identifying a narrow range of desired bead radii R based on components present in a feed stream; b) removing beads of a given radius r that are outside the desired bead range; and c) defining a percentage of bead radii R within the desired bead range.

30. 30. The method of claim 29, 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.

31. The method of claim 30, wherein the beads are polymeric beads.

32. 31. The method according to any one of claims 28 to 30, wherein beads of radius r are removed by wet or dry sieving and / or elutriation.

33. 33. The method of any one of claims 28 to 32, wherein beads having r<0.414R are removed.

34. 23. A method for purifying an unclarified feed stream containing a particle of interest using the radial flow chromatography column of claim 22, wherein the particle is IgG or IgM; a. packing the beads into a radial flow column; b. treating the IgG or IgM containing clarified feed stream to adjust purification conditions; c. determining IgG or IgM binding from the results of step b; d. treating the non-clarified feed stream containing IgG or IgM; A method comprising:

35. 35. The method of claim 34, wherein the beads are functionalized by covalently attaching Protein A or Protein G to the surface of the beads.

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