Patent Description:
Column chromatography is a separation and/or purification technique in which a stationary "bed" of a packing medium is contained within a rigid tube. The packing medium can be in the form of particles of a solid ("stationary phase") or a solid support material coated with a liquid stationary phase. Either way, the packing medium typically fills the inside volume of the column tube.

In separation chromatography, as a liquid sample ("mobile phase") passes through the column, different compounds in the sample can associate differentially with the stationary phase (e.g., packing medium) such that they are slowed relative to the mobile phase and move through the column at different speeds. Thus, those compounds that associate more with the stationary phase move more slowly through the column than those that associate less, and this speed differential results in the compounds being separated from one another as they pass through the column. Features of the stationary phase that promote differential association can be, e.g., ionic charge (ion exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), and porosity (size exclusion chromatography).

In another type of column chromatography, affinity chromatography, the packing medium includes binding agents, such as antigens, antibodies, or ligands, that specifically bind to one or more desired compounds or molecules in the liquid sample. Thus, as the liquid sample flows through the packing medium, only the desired compounds or molecules remain in the column. A subsequent flow through the packing medium of an eluting liquid separates the desired compounds or molecules from the binding agents attached to the packing medium, or separates the binding agents from the packing medium. Either way, the desired compounds or molecules are rinsed out of the column and collected in the eluting fluid. Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and purification from blood.

The main components of a chromatography column are the tube, which is often made of a metal, glass, or highly rigid plastic material, and a pair of flow distributors, which are typically inserted into the two ends of the tube to form a space or chamber in the tube between the flow distributors into which the packing medium is loaded. The flow distributors are designed with integrated porous surfaces such as screens, mesh, or frits that retain the chromatography media.

Chromatography columns can be pre-packed and used upon demand for development and commercial bioprocess manufacturing. These columns are produced and fixed in bed height such that flow distributors are irreversibly aligned within the chromatography tube. New developments in bioprocessing, such as continuous processing, or multi-product facilities, require increased stringency in microbial control. Aseptic connection strategies and flow paths from cell culture into downstream purification are essential to reduced risk of contamination. Sterilization by steam and radiation has been applied to disposable bioreactors, flow paths, and recently chromatography columns for biologics purification. However, in some instances off-gassing of the packing medium solution can occur during the gamma irradiation process, creating gas pockets and pressurization within the column's packed bed. In this case, the packed bed can negatively affect the fluid flow dynamics. Thus, there is a need to design a pre-packed chromatography column that is aseptic or sterile and free of gasses and that does not impair the separation performance of the column or increase the risk of increasing unwanted bioburden during normal use of the column.

In accordance with its abstract, <CIT> relates to relates to axial flow chromatography columns, methods for separating one or more analytes in a liquid by the use of such columns, and systems employing such columns. The column comprises a first port and a second port, the first port and said second port being at essentially the same level or elevation above the level of the bed space on the chromatography column.

In accordance with its abstract, <CIT> relates to methods for sterilizing chromatography columns, stating 'If one sterilizes pre-packed, plastic chromatography columns with an appropriate level of gamma irradiation, the resulting sterile chromatography columns maintain sufficient packing media function and maintain column mechanical properties and pressure ratings.

In accordance with its abstract, <CIT> relates to an automated installation procedure for assembling a disposable flow path: providing a disposable flow path comprising tubing and a plurality of sensors onto a re-usable instrument; qualifying said tubing and said plurality of sensors to be on the flow path based on a standard; and determining if the tubing and the plurality of sensors comply with characteristics and performance according to limits for specifications or acceptance criteria.

The invention is based, at least in part, on the discovery that if you connect a tubing and valve system as specified herein to a pre-packed chromatography column to create a closed system, you can then sterilize the closed system, e.g., with gamma irradiation, and thereafter use the tubing and valve system to simply and easily purge any gas bubbles and/or pressure that may have formed inside the closed system during sterilization and yet maintain sterility of the closed system. As a result, the new systems and methods can maintain the performance of the packed column and avoid disruption of the chromatography flow path during use due to bubbles and/or pressure formed from off-gassing within the column during irradiation and prior to use.

The columns can be packed with chromatography media of various types and can be used to manufacture biologics. Pre-packed columns prepared according to this invention can be sterilized by a gamma radiation dose greater than or equal to <NUM> kGy. Functional fluid distribution (e.g., measured by Height Equivalent to the Theoretical Plate (HETP) or HETP and asymmetry testing) following gamma irradiation is similar to original values following gas removal.

The present disclosure describes the functionality of efficient priming of the connected lines and then providing backflow, also known as upflow (e.g., flow in reverse to the typical direction of forward flow during column use without having to have a chromatography system capable of reverse flow) to the column to remove entrapped air and restore the packed bed. The effluent flow can exit via an outlet, it can exit via a fluid path connected to a sterile or aseptic filter, or it can be collected in an expansion bag attached for that purpose. A sterilizing grade hydrophobic vent filter could also be incorporated to relive pressure build up or to evacuate trapped gasses. This vent filter in combination with a check valve would ensure venting in one direction without backflow into the system.

In one aspect, the present disclosure provides methods of removing entrapped gas, pressure, or both gas and pressure from a pre-packed chromatography column that comprises a column tube having a column tube inlet and a column tube outlet and first and second flow distributors arranged within the column tube to form a chamber filled with a packing medium. The methods include (a) obtaining a tubing and valve set comprising tubing and at least one valve configured to define at least two different fluid paths through the tubing; (b) either: (i) attaching tubing of the tubing and valve set to the column tube inlet and the column tube outlet and then sterilizing the connected pre-packed chromatography column and the tubing and valve set, or (ii) sterilizing the pre-packed chromatography column and the tubing and valve set individually and then attaching the sterile tubing of the tubing and valve set to the sterile column tube inlet and the sterile column tube outlet in a manner that maintains sterility of both the column and the tubing and valve set; (c) attaching the sterile tubing and valve set to a sterile or aseptic fluid source and to a fluid outlet; and (d) pumping sterile or aseptic liquid from the sterile or aseptic fluid source along the tubing and valve set, wherein the at least one valve is in a first position, into the column tube inlet and out from the column tube outlet along a first flow path, thereby removing any entrapped gas, pressure, or both gas and pressure from the chamber, the tubing, and the at least one valve along the first flow path without breaching the sterility of the pre-packed chromatography column and the tubing and valve set.

In these methods, attaching the sterile tubing of the tubing and valve set to the sterile column tube inlet and the sterile column tube outlet in a sterile manner can include using aseptic or sterile connectors or weldable tubing. In these methods, the sterilizing can be achieved with irradiation, e.g., with gamma radiation. For example, by applying a gamma radiation dose of at least <NUM> kGy.

In some embodiments, attaching tubing to the column tube inlet and the column tube outlet includes connecting ends of the tubing to a connector fixed to the column tube inlet and to a connector fixed to the column tube outlet. For example, the connectors can be screwed into, clamped onto, or welded to the column tube inlet and to the column tube outlet.

In certain implementations, the methods can further include moving the at least one valve from the first position to a second position to direct the sterile or aseptic liquid along a second flow path. In other implementations, the methods further include moving the at least one valve from the first position to a second position and pumping sterile or aseptic liquid from the sterile or aseptic liquid source into the column tube outlet, through the pre-packed column tube, and out of the column inlet. For example, these methods can include pumping a volume of sterile or aseptic liquid equivalent to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more volumes of the chamber.

In some embodiments, the methods further include attaching the tubing to a sterile or aseptic filter and pumping sterile or aseptic liquid through the sterile or aseptic filter. In some implementations, the methods can further include collecting the sterile or aseptic liquid pumped through the sterile or aseptic filter and testing one or more properties of the sterile or aseptic liquid.

In some implementations, the methods can include venting pressure from the tubing and valve set with a vent filter. In some implementation, the methods further include attaching tubing from the tubing and valve set to an expansion bag.

In some embodiment, the methods are used with column tubes that are pre-packed for a single use and that are disposable. In general, the methods can be used when the pre-packed chromatography column and tubing and valve set are connected to a chromatography system.

In another aspect, the disclosure features systems for aseptic purification of biomolecules. The systems include a sterile pre-packed chromatography column comprising a column tube having a column tube inlet and a column tube outlet and first and second flow distributors arranged within the column tube to form a chamber filled with a packing medium; a sterile tubing and valve set attached to the column tube inlet and the column tube outlet, wherein the tubing and valve set comprises tubing and at least one valve configured to define at least two different fluid paths fluidly connected to the column tube inlet and the column tube outlet; and a pump configured to pump sterile or aseptic liquid from a sterile or aseptic fluid source along the sterile tubing and valve set, wherein the at least one valve comprises a first position that enables the sterile or aseptic liquid to flow into the column tube inlet and out of the column tube outlet along a first flow path to remove any entrapped gas, pressure, or both gas and pressure from the chamber, the tubing, and the at least one valve along the first flow path.

In these systems, the sterile column can be pre-packed with resin and the column, resin, and tubing have a sterility assurance level (SAL) of <NUM>-<NUM> or better.

In some implementations, the systems further include a controller configured and arranged to control the pump and the position of the at least one valve. For example, in some embodiments, the controller includes a processor and a memory storing instructions for execution by the processor for opening and closing the at least one valve.

In some embodiments, the systems further include connectors at the column inlet and outlet configured to form sterile or aseptic connections and to permit fluid to flow through the chamber formed between the first and second flow distributors. For example, in some embodiments the connectors are pre-installed to form sterile or aseptic connections with the tubing and valve set.

In certain implementations, the systems further include a second pump fluidly attached to the tubing and valve set. In some implementations, the systems further include a vent filter arranged in the tubing and valve set to enable venting gas, pressure, or gas and pressure from the pre-packed chromatography column and the tubing and valve set. For example, such vent filters can be or include a hydrophobic vent filter and a check valve.

In certain embodiments or these systems, the chromatography column tube is formed of one or more of stainless steel, glass, polypropylene (PP), polyethylene (PE), polyamides, acetals, or glass-filled or carbon-filled plastics or elastomeric components. In different embodiments, the chromatography packing medium is one of glass, plastic, cellulose, agarose, ceramic, or polymer, in the form of rough particles, fibers, membranes, or beads. In certain implementations, the chromatography column tubes are pre-packed for a single use and are disposable.

In some instances, the chromatography column with attached tubing and valve set as described herein can be attached to a chromatography system (e.g., AKTA™ Ready, GE Healthcare) through an aseptic or sterile connection or by welding. Any overpressure and/or gasses or bubbles in the chromatography column and tubing and valve set cause by the sterilization process, e.g., by gamma irradiation, can be vented through the chromatography system by opening the valve set or using a priming sequence described herein.

In some instances, the tubing is weldable to form closed process flow paths. The tubing and valve set can form an aseptic or sterile connection pre- or post-gamma irradiation.

As used herein, the terms "resin," "separation media," "chromatography media," and "medium" all refer to particles suspended in a liquid that are used to fill chromatography columns. Examples of these resins include materials made of glass, plastics, polymers, cellulose, agarose, and other substances. The resins may be rough particles or beads. The beads may be monodispersed or have a population of sizes and may vary, for example, from <NUM> to <NUM> in size. The resin may be modified to contain various substituents that affect binding to the particles to achieve separation of proteins and other substances in a feed stream. The substituents may be positively or negatively charged, or hydrophobic, or a specific affinity moiety (such as protein A) or a combination of these properties. In some instances, the chromatography columns are filled with fibers, membranes, or monoliths that are retained with a fluid distribution system.

As used herein, the term "connector" refers to a joining device that enables an aseptic or sterile connection, for example, either a mechanical mating or one or more locations suitable for joining with a sterile or aseptic tubing weld.

As used herein, the term "aseptic" refers to a condition or process designed to reduce the likelihood of contamination from pathogens (e.g., pathogenic bacteria, viruses, fungi, and/or parasites or harmful spores).

The term "sterile" generally refers to a condition or process designed to achieve an environment that is free or essentially free from all living microorganisms (harmful or otherwise) and their spores. As used herein, "sterile" means a sterility assurance level (SAL) of <NUM>-<NUM> or better.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The present disclosure describes new methods and systems for gamma irradiation of pre-packed chromatography columns arranged in closed systems including a series of valves and tubing, where the performance through the packed column bed remains suitable for its intended use and wherein the system of valves and tubing can be used to purge gas from the closed systems that may arise during sterilization, e.g., by gamma irradiation.

Pre-packed or disposable chromatography columns can be sterilized by gamma radiation and are shown to maintain packed bed integrity, e.g., as measured by pulse injection of a non-interacting small molecule. However, in some instances off-gassing of the packing medium solution can occur during the gamma irradiation process, creating undesirable gas of air pockets or bubbles and/or pressure within the column's packed bed and connected sterile or aseptic tubing that can negatively affect the fluid flow dynamics. In some instances, pressurization of the columns occurs. The current disclosure solves the problem of the trapped pressure, gas, or air by the use of tubing and valve systems and vent filters installed to the inlet and outlet of the pre-packed column. These tubing, valve systems, and vents permit a closed aseptic connection to the chromatography system, allowing for the removal of entrapped gas from the column while maintaining a closed system and sterility. These tubing valve systems and venting methods result in pre-packed columns that have packed bed performance comparable to original pre-irradiation values. The tubing can be any type of tubing, such as silicone, braided / silicone, C-FLEX®, etc..

Disruption of the chromatography flow path due to bubbles and/or pressure from off-gassing within the column can thus be avoided. The columns can be packed, for example, with chromatography media with a silica, agarose, ceramic, or polymeric backbone, which can be functionalized with an affinity ligand (e.g., protein A-recombinant native structure, or engineered functional domains), ionic interaction ligands, mixed mode ligands, or hydrophobic ligands. Columns are packed and stored in aqueous buffers that may contain organic components (e.g., <NUM>% benzyl alcohol, <NUM>% ethanol). These columns can be used to manufacture biologics such as proteins, viruses, virus-like particles, exosomes, and others. Pre-packed columns prepared as described herein are sterilized by a gamma radiation dose typically greater than or equal to <NUM> kGy. Functional fluid distribution measured by Height Equivalent to the Theoretical Plate (HETP) and asymmetry tests following gamma irradiation show values similar to original values following gas removal.

The column tubes are hollow, cylindrical members, and are typically round cylinders that permit a fluid (e.g., a liquid) to flow from a first end (e.g., an upper end) to a second end (e.g., a lower end). The inner diameter of the tubes are sized and configured to receive flow distributors for delivering fluid to and removing fluid from the tube. Based on various chromatography column performance specifications, the tubes can be made in a variety of different sizes and configurations and include the OPUS® line of chromatography columns and other columns used in biopharmaceutical applications (Repligen Corp. , Waltham, MA).

The chromatography components described herein can be made from any of various structurally and chemically suitable materials. For example, the components can be made from various plastics, such as thermoplastics (e.g., acrylonitrile butadiene styrene (ABS), acrylic (PMMA), polypropylene (PP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), other thermoplastics, or composites) and thermosetting plastics (e.g., epoxy resins, and fiber reinforced plastics. The columns can be also made entirely from metals, e.g., stainless steel, or from other glass or rigid plastics such as polyamides (such as various nylons), acetals, or glass-filled or carbon-filled plastics, e.g., glass-fiber and carbon-fiber plastics) or elastomeric components. Material selection considerations can include the specific mechanical properties of the materials and whether the materials will withstand the induced internal operating pressures of the system. The columns' design is such that they can be packed with various types of chromatography packing media, e.g., resins, other porous or non-porous materials to a "bed height" with infinite variability between <NUM> and <NUM> and longer. The internal diameter can be, for example, but not limited to, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> or larger, up to about <NUM>, <NUM>, or <NUM>, or larger.

The flow distributors used in these columns are in the form of a cylindrical disc with one or more inlet/outlet openings that enable liquids to flow into and through the disc. In addition, the flow distributors can include a bed support, screen, and/or filter that is attached to the packing medium side of the flow distributor disc. The column also may or may not incorporate O-rings between the flow distributors and the inner wall of the column tube. The flow path of the flow distributors can be designed according to standard practices and known designs, and the flow distributors themselves can be made, for example, of the same or a similar plastic material as the tubes, but can also be made of metal, ceramics, and other rigid materials that are inert to the liquids and reagents that are to be flowed through the columns.

The tubes of the chromatography columns described herein can be packed with any solid phase medium material that is used in column chromatography as specified by the end-user. This diversity of potential packing medium materials extends to both the composition of base particles as well as their functional chemistries (e.g., affinity, ion exchange, and hydrophobic interaction). Packing medium materials can include a slurry of stationary phase particles added to a mobile phase liquid or solvent. Stationary phase particles can include silica gel (SiO<NUM>), ceramic, alumina (Al<NUM>O<NUM>), cellulose, agarose, polymeric and other suitable materials in various particle sizes. The mobile phase can include one or more of various solvents, such as deionized water, buffered salt solutions, ethanol, or other common solutions used for chromatographic separations.

The tubing and valve sets described herein for removal of gases and priming of a chromatography column can be attached to an OPUS® column or any chromatography column in a pre-packed format that can be gamma irradiated for sterilization purposes. Generally, a radiation dose of <NUM> kGy or greater is effective. In some implementations, a dose range of <NUM>-<NUM> kGy can be used. As described above, the columns are constructed of materials that are able to withstand such irradiation. Additionally, the tubing and valve sets connected to the columns are also constructed of gamma-stable materials.

<FIG> shows a sterile or aseptic chromatography column system <NUM> that can be used for the sterile or aseptic purifying of biomolecules (e.g., proteins such as antibodies, viruses, virus-like particles, exosomes, etc.). A chromatography column <NUM>, such as an OPUS® column, is attached to a tubing and valve system or set <NUM>. An upstream tubing portion <NUM> of the tubing set <NUM> is attached to a fluid source or fluid inlet <NUM> at one end and to a column inlet <NUM> at another end. An additional part of the tubing set <NUM>, the downstream tubing portion <NUM>, is attached to a fluid sink or fluid outlet <NUM> at a first end and to a column outlet <NUM> at its second end. The tubing and valve set <NUM> includes additional tubing branches <NUM>. These tubing branches <NUM> can connect the upstream tubing <NUM> to the downstream tubing <NUM> to create multiple flow paths for liquid flowing through the tubing and valve set <NUM>.

The column inlet <NUM> and column outlet <NUM> are connectors that attach to the column <NUM> and to the tubing and valve system <NUM>. In some instances, the connectors of column inlet <NUM> and column outlet <NUM> are integral with the column <NUM>. In other instances, the connectors of column inlet <NUM> and column outlet <NUM> are integral with the tubing and valve system <NUM>. The connectors of column inlet <NUM> and column outlet <NUM> can attach the tubing and valve set <NUM> to the column <NUM> in a sterile or aseptic manner. For example, the connectors of column inlet <NUM> and column outlet <NUM> can be welded to the column <NUM>, and then can be gamma irradiated post-welding. Alternatively, the connectors of column inlet <NUM> and column outlet <NUM> can be welded to the tubing and valve set <NUM>. In all instances, the tubing and valve set <NUM>, the connectors of column inlet <NUM> and column outlet <NUM>, and the column <NUM> can be gamma irradiated after they are attached to each other. In all instances, the tubing can be attached to the column before or after sterilization of the column. In some instances, the column <NUM> includes a weldable sterile or aseptic connection on inlet <NUM> and outlet <NUM>, and the tubing and valve set <NUM> can include weldable tubing or a connector on the upstream tubing portion <NUM> and/or on the downstream tubing portion <NUM>. In different scenarios, the weldable sterile or aseptic connectors are attached to column <NUM> and tubing set <NUM> before, during, or after the gamma irradiation process, and they enable an aseptic or sterile connection to be made and maintained.

The tubing and valve set <NUM> includes multiple valves, e.g., in <FIG> there are valves <NUM>-<NUM>. The valves are positioned at various locations along the upstream <NUM> and downstream <NUM> portions of the tubing and valve set <NUM> as well as along the tubing branches <NUM>. Opening and closing the valves <NUM>-<NUM> can permit or prevent fluid entering the tubing and valve set from the fluid inlet <NUM> from flowing along different portions of the tubing and valve set <NUM>, e.g., opening and closing valves <NUM>-<NUM> directs fluid along different fluid paths that may include the upstream tubing and may or may not include the column <NUM>, the downstream tubing <NUM> or one or more of the tubing branches <NUM>. A controller <NUM> (shown schematically) can be connected to the valves <NUM>-<NUM> and pump <NUM> or (pumps) that propel the liquid through the tubing and valve set <NUM> from the fluid inlet <NUM>. The controller <NUM> can synchronize the opening and closing of the valves <NUM>-<NUM> (or more valves, depending on the overall the system) and the speed of the pumps to perform the steps of priming and using the sterile or aseptic chromatography column system <NUM> as described below.

In some implementations, the new systems include valves, e.g., valves <NUM>-<NUM>, with manual opening and closing abilities without a controller <NUM>. In some implementations, the systems include a controller <NUM> that includes one or more processors and volatile or nonvolatile memory containing software instructions for execution by the one or more processors that include software instructions for operating the valves and pumps. The software instructions are stored on any type of non-transitory computer-readable medium and are executable by the one or more processors to perform the instructions, including instructions to synchronize the opening and closing of the valves <NUM>-<NUM> (or more valves, depending on the overall the system) and the speed of the pumps to perform the steps of priming and using the sterile or aseptic chromatography column system <NUM> as described herein and as shown in the figures (e.g., <FIG>, <FIG>, and <FIG>), which show specific sequences of opening and closing difference valves according to different protocols for different sets of valves and different priming, upflow, and downflow sequences.

The sterile or aseptic chromatography column system <NUM> of <FIG> can be used to prime the chromatography column <NUM> while removing any gas bubbles and/or pressure that might be present in the column <NUM>, the tubing <NUM>, and any connectors such as the column inlet <NUM> and column outlet <NUM>. For example, a first priming sequence is shown in <FIG>. This first priming sequence provides priming initialization and upflow (e.g., backflow) conditioning of the column, as described below for forward flow pumps.

In <FIG>, valves <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> are open, while valves <NUM> and <NUM> are closed as indicated by the "X. " This configuration permits flow through, e.g., down through, the upstream tubing <NUM>, e.g., fluid flowing from the fluid inlet <NUM> downwards to prime upstream tubing <NUM>. This configuration also permits upflow (e.g., backflow) conditioning of the column <NUM>, e.g., flow from the column outlet <NUM> through the column <NUM>, and out through inlet <NUM> and with the buffer or storage solution being used to prime the column <NUM> then exiting out of the fluid outlet <NUM>. Typically, one or more column volumes (CVs) of fluid are pumped through the column (e.g., 1CV, <NUM> CVs, <NUM> CVs, or <NUM> CVs). Less than 1CV could also be effective. A column volume is defined as the volume of the chamber formed within the column tube between the first and second flow distributors and in this application is filled with a packing medium while the priming operation is taking place. As shown by the arrows in <FIG>, fluid proceeds from fluid inlet <NUM> along the upstream tubing <NUM> through the top tubing branch <NUM> to the downstream tubing <NUM>, and then from column outlet <NUM> through the column <NUM>, out from column inlet <NUM>, along the bottom tubing branch <NUM> and exits the system at fluid outlet <NUM>.

A second priming sequence is shown in <FIG>. This sequence primes the top portion of the tubing and valve set <NUM>, e.g., the portions of the tubing and valve set <NUM> closest to the fluid inlet <NUM> and the fluid outlet <NUM>. In this instance, valves <NUM>, <NUM>, <NUM>, and <NUM> are open while valves <NUM>, <NUM>, <NUM>, and <NUM> are closed. Fluid downflow proceeds from the inlet and then exits out of the fluid outlet <NUM> via the upper branch <NUM>. Fluid flow is controlled to continue at least until the portion of the tubing nearest valve <NUM> is filled with fluid.

A third priming sequence is shown in <FIG>. This sequence primes the remaining portion of the tubing and valve set <NUM>, e.g., the portion of the tubing and valve set <NUM> closest to valve <NUM>. In this instance valves <NUM>, <NUM>, <NUM>, and <NUM> are open while valves <NUM>, <NUM>, <NUM>, and <NUM> are closed. Fluid downflow proceeds from the inlet <NUM> and along upstream tubing <NUM> including valve <NUM> to tubing branch <NUM> with valve <NUM>, then to the latter portion of downstream tubing <NUM> to fluid outlet. Fluid flow is controlled to continue at least until the portion of the tubing nearest valve <NUM> is filled with fluid.

In some instances, and because it is likely that post-gamma irradiation there are air bubbles and/or pressure in the upper portions of the tubing set, the priming steps of <FIG> and <FIG> are performed first and second in a post-gamma irradiated column, before any flow through the column. Doing so ensures no further air will enter the column. In various scenarios, the priming step of <FIG> can come before the priming step of <FIG>, and vice versa. In general, the priming steps of <FIG> and <FIG> can be carried out in any order before <FIG>, as long as no air enters the column in the downflow.

A fourth priming sequence is shown in <FIG>. This sequence primes the downstream portion of the tube <NUM>. This configuration is also used for downflow operation using the tubing and valve set <NUM>. Here, valves <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> are open while valves <NUM> and <NUM> are closed. With this valve set up, downflow operation is achieved by downflow from inlet <NUM> and the solution will exit from the fluid outlet <NUM> as shown by the arrows.

After the priming sequences shown in <FIG> are completed (<FIG> shows an upflow sequence and <FIG> shows a downflow sequence), the tubing and valve set <NUM> and the column <NUM> are completely filled with liquid and ready for chromatography operations, with any bubbles and/or pressure that may have been present being removed. Any air remaining in the outlet portion <NUM> will be removed by the downflow.

The system <NUM> can also be used for upflow operation while using the tubing set. For upflow operation valves <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> are open while valves <NUM> and <NUM> are closed. This configuration allows the same fluid flow path as shown in <FIG> for upflow conditioning of the column. In both the system of <FIG> and <FIG> upflow occurs without additional changes such as flipping the column upside down, or making other changes to the system such as reversing the direction of the pump <NUM> that propels fluid from fluid inlet <NUM> to fluid outlet <NUM>. In addition, this upflow sequence does not need the column to be physically re-plumbed, thereby maintaining the aseptic/sterile nature of the column and tubing and valve set, e.g., if the column was gamma irradiated.

Referring to <FIG>, a second embodiment of a sterile or aseptic chromatography column system <NUM> is shown. The components of the sterile or aseptic chromatography column system <NUM> are similar to those of <FIG> and the same reference numbers refer to the same system elements. The embodiment in <FIG> has a similar flow path as shown as in <FIG>, but includes only valves <NUM>, <NUM>, <NUM>, and <NUM>. When in downflow operative use, valves <NUM> and <NUM> are closed (as indicated by the X on each valve in the figure) and the fluid path is as shown by the arrows. Referring to <FIG>, when in upflow operative use, valves <NUM> and <NUM> are closed (as indicated by the X on each valve in the figure) and the fluid path is as shown by the arrows.

In some embodiments, fluid exiting the column <NUM> does not flow out of the fluid outlet <NUM>. <FIG> shows a sterile or aseptic chromatography column system <NUM> similar to the previous embodiments and with the same elements numbered with the same reference numbers. However, the tubing and valve set includes only valves <NUM>-<NUM> and the tubing branch <NUM> that includes valve <NUM> does not connect the upstream tubing <NUM> to the downstream tubing <NUM>. Instead, that tubing branch <NUM> is fluidly connected to the upstream tubing <NUM> on one end and to an inline sterile or aseptic filter <NUM> at the second end. The inline sterile or aseptic filter <NUM> can be used as an outlet to prime the tubing and valve set <NUM> rather than fluid outlet <NUM>. In some embodiments, inline sterile or aseptic filter <NUM> can be connected to a fluid collection container <NUM>. Fluid collected in the fluid collection container <NUM> can be used for testing purposes, e.g., to determine the condition or properties of the fluid and/or resin within the column <NUM>.

The sterile or aseptic chromatography column system <NUM> of <FIG> can be used to prime the chromatography column <NUM> while, e.g., removing any gas bubbles and/or pressure that might be present in the column <NUM>, the tubing <NUM>, and any connectors such as the column inlet <NUM> and column outlet <NUM>. A first priming sequence for system <NUM> is shown in <FIG>. Valves <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> are open, as indicated, while valves <NUM> and <NUM> are closed. This configuration permits liquid downflow of the upstream tubing <NUM> and upflow conditioning of the column <NUM>. Typically, three or more column volumes (CVs) of fluid are pumped through the column. As shown by the arrows in <FIG>, fluid proceeds from fluid inlet <NUM> along the upstream tubing <NUM> through connective tubing branch <NUM> to the downstream tubing <NUM>, and then from column outlet <NUM> through the column <NUM> and out from column inlet <NUM>. Due to valve <NUM> being closed the fluid then is diverted along the tubing branch <NUM> to the inline sterile or aseptic filter <NUM> and optionally to a collection container <NUM>.

A second priming sequence is shown in <FIG>. This sequence primes the top portion of the tubing and valve set <NUM>, e.g., the portions of the tubing and valve set <NUM> closest to the fluid inlet <NUM> and the fluid outlet <NUM>. In this instance, valves <NUM>, <NUM>, and <NUM> are open while valves <NUM>, <NUM>, <NUM>, and <NUM> are closed (or alternatively only valves <NUM> and <NUM> are closed). Fluid downflow proceeds from the fluid inlet <NUM>, across the tubing branch <NUM> with valve <NUM> and then exits out of the fluid outlet <NUM>. Fluid is controlled to continue to flow at least until the portion of the tubing including valve <NUM> is filled with fluid.

A third priming sequence is shown in <FIG>. This sequence primes the remaining portion of the tubing set <NUM>, e.g., the portion of the tubing set <NUM> closest to valve <NUM>. In this instance valves <NUM>, <NUM>, and <NUM> are open while valves <NUM>, <NUM>, <NUM>, and <NUM> are closed. Fluid downflow proceeds from the inlet <NUM> and along upstream tubing <NUM> including valve <NUM> to tubing branch <NUM> with valve <NUM>, and out to the inline sterile or aseptic filter <NUM>. Fluid flows at least until the portion of the tubing nearest valve <NUM> is filled with fluid. In some instances, the priming steps of <FIG> and <FIG> are performed first and second in a post-gamma irradiated column, before any flow through the column. Doing so ensures no further air will enter the column. These steps can also be reversed.

After the priming sequences shown in <FIG>, the tubing and valve set <NUM> and the column <NUM> are completely filled with liquid and ready for chromatography operations. A configuration for a downflow operation using the tubing set <NUM> is shown in <FIG>. Here, valves <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> are open while valves <NUM> and <NUM> are closed. With this valve set up, downflow operation is achieved by downflow from inlet <NUM> and solution will exit out of the fluid outlet <NUM> as shown by the arrows while traversing the column <NUM>.

<FIG> shows an additional sterile or aseptic chromatography column system <NUM>. In addition to the elements that are the same as discussed in the embodiments above, system <NUM> includes a second fluid inlet <NUM> and a second fluid outlet <NUM>, with associated additional valves. Various fluid sources and sinks are therefore possible, in addition to the optional fluid container connected to inline sterile or aseptic filter <NUM>. A second pump <NUM> is shown connected to second fluid inlet <NUM>, however in system <NUM> as in all sterile or aseptic systems described herein, more than one or two flow pumps are possible. In some instances, one or more pumps are used, such that an operator can use one set of the inlet and outlet connectors and pre-prime the tubing set and column to remove all the air and then clamp off those tubing. Afterwards, the column can be used on a later date without priming the column again. In addition, because there is still one set of unused sterile or aseptic connectors, the column can be hooked up under sterile or aseptic or aseptic conditions again. Alternatively, an operator can use the two sets of sterile or aseptic connections to use the column twice.

<FIG> shows an additional sterile or aseptic chromatography column system <NUM>. In addition to the elements that are the same as discussed in the embodiments above, system <NUM> includes an expansion bag <NUM>. The expansion bag <NUM> can be partially filled with buffer. The expansion bag <NUM> can optionally include a vent external to system <NUM> that includes a sterile or aseptic filter. Sterile or aseptic chromatography column system <NUM> also can include a secondary pump <NUM>. The secondary pump <NUM> is fluidly connected to the tubing and valve system <NUM> and can push or pull liquid through the tubing <NUM>, in addition to the in-place pumping system <NUM>. The secondary pump can be manual, e.g., a syringe, or bellows pump. Rather than redirecting fluid through use of valves, the secondary pump <NUM> can direct fluid upflow if desired.

<FIG> shows an additional sterile or aseptic chromatography column system <NUM>. In addition to the elements that are the same as discussed above with respect to <FIG>, the system <NUM> includes the features of the embodiments of <FIG> and <FIG>. These features include a second fluid inlet <NUM> and a second fluid outlet <NUM>, an expansion bag <NUM>, and a sterile or aseptic filter <NUM>. Additional valves connect the various tubing elements and controller <NUM> can direct fluid to all these elements.

<FIG> shows an additional sterile or aseptic chromatography column system <NUM>. In addition to the elements that are the same as discussed above with respect to <FIG>, the system <NUM> includes a sterilizing grade hydrophobic vent filter <NUM> incorporated to relieve pressure build up or to evacuate trapped gasses. The hydrophobic vent filter <NUM> in combination with a check valve <NUM> ensures venting in one direction without backflow into the system <NUM>.

<FIG> shows an additional sterile or aseptic chromatography column system <NUM>. In addition to the elements that are the same as discussed above with respect to <FIG>, the system <NUM> includes a tubing set <NUM> and column <NUM> that include weldable tubing or sterile or aseptic connections A, B, C, D that allow the tubing set and column to be connected independently, and in a sterile or aseptic manner, after the gamma irradiation. The column <NUM> and tubing set <NUM> can be gamma irradiated separately as long as a method of sterile or aseptically connecting the two parts post-gamma irradiation is available. <FIG> illustrates weld-able tubing <NUM> present on the column <NUM> and tubing set <NUM>. A sterile or aseptic connection can be welded (A to C and B to D) between the weld-able tubing <NUM> or can be made through sterile or aseptic connectors (not shown). The post-gamma sterile or aseptic attachment of a tubing set <NUM> to a chromatography column <NUM> can be applied to any tubing set <NUM>, provided a method of sterilely or aseptically connecting the two parts post-gamma irradiation is available.

The following examples illustrate, but do not limit, the systems and methods described herein.

During the process of gamma irradiation gasses and pressurization occurs within the closed column, which can negatively affect column usability. Through a tubing and valve set, these gasses and pressure can be removed from the column as described herein. The goal of this experiment was to demonstrate air removal efficiency for tubing and valve set <NUM>. Two pre-packed OPUS® columns (Repligen Corporation) were used. Each pre-packed column had dimensions of <NUM> inner diameter and a <NUM> bed height packed with GE Healthcare Capto™ S, an agarose based cation exchange resin. For this experiment, the tubing set was not primed and (full of air) to demonstrate a worst-case scenario to model effects of gamma processing.

The columns with tubing sets were connected to an ÄKTApilot® (<FIG>). The tubing set was primed with the following sequence:.

Several observations were made during the process. The first two sequences primed the majority of the tubing set. The upflow sequence primed the inlet line while not introducing air into the bottom of the column due to the outlet line that is positioned at the same height as the inlet line, which is located at the top of the column. The downflow sequence primed the outlet and the last leg of the tubing set.

In conclusion, after the priming sequence was completed, the tubing set did not contain any entrapped air as determined by visually inspection. The valve combination set-up was demonstrated to be functional in removing air from the columns and tubing system via described methods.

The following experiment was carried out to evaluate the sterile or aseptic chromatography column system shown in <FIG> to confirm that this embodiment can fully prime and remove trapped air in the tubing set <NUM> post gamma irradiation. Additionally, the test was carried out to confirm the valve combination set-up for priming upflow and downflow operations, and to determine that the addition of the tubing and valve set <NUM> does not affect column performance.

The two pre-packed columns used in Example <NUM> were also used for this experiment. These two columns were tested on an ÄKTApilot® chromatography system (GE Healthcare) for HETP (N/m) and asymmetry with and without tubing set prior to gamma sterilization. The columns were tested at <NUM>/hr in <NUM> NaCl with a <NUM>% CV <NUM> NaCl spike injection. The addition of the tubing set did not affect the column performances of these columns (Table # <NUM>).

<FIG> shown the tubing and valve set <NUM> attached to an OPUS® <NUM> inner diameter column. <FIG> shows the tubing and valve set <NUM> attached to an ÄKTApilot®.

In preparation for gamma irradiation, each tubing set and pre-packed column assembly was primed and stored with phosphate buffered saline (PBS) containing <NUM>% Benzyl Alcohol. No air was observed in the tubing assembly after storage and prior to gamma irradiation.

The columns were gamma irradiated to a target range of <NUM>-<NUM> Gy.

Post-gamma sterilization, entrapped air was observed within each tubing set assembly in the following locations:.

Both columns were tested post-gamma irradiation for column performance.

The tubing set was primed with <NUM> NaCl at a flow rate of <NUM>/hour. Priming Sequence:.

Air was observed to be removed from column outlet <NUM> during the downflow sequence. Both columns were tested and decreased HETP (N/m) were observed for both columns when compared to before gamma irradiation. Asymmetry was tailing more for column # <NUM> post-gamma irradiation (Table <NUM>).

An upflow sequence was then performed (valves <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> open and valves <NUM> and <NUM> closed) on both columns with tubing sets and air was observed to exit the inlet of the column. The columns were tested, which resulted in improved HETP (N/m) for both columns and improved asymmetry for column # <NUM> when compared to the initial post-gamma irradiation test.

In conclusion, the tubing and valve set successfully removed air bubbles found in the tubing set post-gamma irradiation. Furthermore, the tubing and valve set also helped recover lost HETP (N/m) through upflow operation. The following figures show the following results:.

During the process of gamma irradiation, gasses and pressurization occur within the closed column and tubing set assembly, which can negatively affect column usability. As describe in Example <NUM> the use of a tubing and valve set as described herein can remove these gasses and excess pressure from the column.

The goal of the present experiment was to demonstrate that gamma irradiation results in pressurization of the column, tubing and valve set, and/or column and tubing and valve set assembly. Two pre-packed OPUS® columns (Repligen Corporation) were used. Each pre-packed column had dimensions of <NUM> inner diameter and a <NUM> bed height packed with GE Healthcare Capto™ S, an agarose based cation exchange resin. For this experiment, both columns were attached a length of tubing to the inlet and outlet of the column with a pressure sensor attached to the inlet tubing.

Post-gamma irradiation, both column pressure sensors were read and pressurization of the column with tubing set were observed (Table <NUM>).

In conclusion, gamma irradiation results in the pressurization of the column tubing set assembly. With the attachment of the tubing set <NUM> described thus far, successful removal of gas bubbles would also aides in the de-pressurization of the column, tubing set, or both.

Claim 1:
A method of removing entrapped gas, pressure, or both gas and pressure from a pre-packed chromatography column (<NUM>) that comprises a column tube having a column tube inlet (<NUM>) and a column tube outlet (<NUM>) and first and second flow distributors arranged within the column tube to form a chamber filled with a packing medium, the method comprising:
(a) obtaining a tubing and valve set (<NUM>) comprising tubing and multiple valves (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) configured to define at least two different fluid paths through the tubing;
(b) either:
(i) attaching tubing of the tubing and valve set (<NUM>) to the column tube inlet (<NUM>) and the column tube outlet (<NUM>) and then sterilizing the connected pre-packed chromatography column (<NUM>) and the tubing and valve set (<NUM>), or
(ii) sterilizing the pre-packed chromatography column (<NUM>) and the tubing and valve set (<NUM>) individually and then attaching the sterile tubing of the tubing and valve set (<NUM>) to the sterile column tube inlet (<NUM>) and the sterile column tube outlet (<NUM>) in a manner that maintains sterility of both the column and the tubing and valve set (<NUM>);
(c) attaching the sterile tubing and valve set (<NUM>) to a sterile or aseptic fluid source (<NUM>) and to a fluid outlet (<NUM>); and
(d) pumping sterile or aseptic liquid from the sterile or aseptic fluid source (<NUM>) along the tubing and valve set (<NUM>), wherein the multiple valves (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) is in a first position, into the column tube inlet (<NUM>) and out from the column tube outlet (<NUM>) along a first flow path, thereby removing any entrapped gas, pressure, or both gas and pressure from the chamber, the tubing, and the multiple valves (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) along the first flow path without breaching the sterility of the pre-packed chromatography column (<NUM>) and the tubing and valve set (<NUM>).