Protein capture from primitive cell culture using protein attached in open tubular and annular helically coiled tubes

Abstract

Provided herein is a tubular protein capture device comprising a continuous tubular body having an inner surface and an outer surface, and methods of use thereof for cell clarification purposes. A protein selected from the group consisting of Protein A, Protein G, Protein L, or a combination thereof is attached to the inner surface of the tubular protein capture device described herein. The devices and methods provided herein can be used to improve the efficiency and / or ability of cell clarification to isolate proteins. In some embodiments, the continuous tubular body of the present technology has a spiral shape. In other embodiments, the continuous tubular body of the present technology includes a plurality of tubes aligned within a hollow housing.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 418,112, filed on October 21, 2022, the entire content of which is incorporated herein by reference. Technical field

[0003] The present technology generally relates to cell clarification devices and techniques for separating proteins from sample matrices (e.g., raw cell cultures or lysed cell cultures). Specifically, the present technology relates to a tubular protein capture device including a continuous tubular body having an inner surface and an outer surface, and methods of using the same. The tubular protein capture device of the present technology includes a protein selected from Protein A, Protein G, Protein L, or a combination thereof, attached to the inner surface of the tubular device. Background art

[0004] Biological macromolecules such as proteins constitute an important class of products in the food, biotechnology, pharmaceutical, and cosmetic industries. Generally, proteins are produced by cell culture using mammalian or bacterial cell lines that are engineered to produce the protein of interest by inserting a recombinant plasmid containing the gene for the protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium containing sugars, vitamins, amino acids, and growth factors, which is typically provided by animal serum preparations. Therefore, separating the desired protein from the mixture of compounds supplied to the cells and from the by - products of the cells themselves to a purity sufficient for analytical characterization poses a significant challenge. In addition, recent advances in bioreactor efficiency have significantly increased cell density, cell debris, and process - and product - related impurities. This improved upstream efficiency has led to new purification challenges caused by high product and contaminant concentrations and complex components.

[0005] Mechanical filtration is one of the most common strategies for removing cells after cell lysis to obtain a clarified sample containing the protein of interest. However, fouling of the filter membrane that results in the formation of a filter cake at the surface of traditional depth filters can limit the number of repeated sample draws that can be clarified. Membrane fouling is the process by which particulate, colloidal, or solute macromolecules deposit or adsorb onto membrane pores or surfaces through physical and chemical interactions or mechanical actions, which results in a decrease in membrane pore size or blockage. In addition, mechanical filtration poses a risk of loss of the target protein due to protein adsorption on the filter, and an increased risk of cell shear due to increased backpressure and reduced pore size.

[0006] The growing demand for developing efficient, rapid, and cost-effective protein purification methods is driving research and growth in this field. New strategies and pretreatment methods for protein clarification can improve clarification efficiency. Such methods result in better protein purity, thereby improving the overall efficiency of subsequent downstream purification steps.

[0007] Tangential flow filtration is advantageous for bioreactor clarification because the permeate flow can be directly introduced into subsequent product capture steps. However, its use is limited to large-scale production and it is difficult to implement effectively at the analytical scale due to the small volumes required for sample extraction. Summary of the Invention

[0008] One object of the present invention is to improve the efficiency and / or ability to clarify cells to isolate proteins.

[0009] This technology allows for the selective capture of proteins from both raw cell cultures and lysed cell cultures. Capturing proteins from raw cell cultures can result in a reduction in the number of steps in the protein purification process, thereby providing an increase in efficiency and a reduction in overall cost.

[0010] In one aspect, the present disclosure relates to a tubular protein capture device comprising: (i) a continuous annular tubular body having an inner surface and an outer surface, wherein the inner surface defines a fluid flow path and comprises a protein attached to at least a portion of the inner surface, the protein selected from Protein A, Protein G, Protein L, or a combination thereof; and (ii) an inlet and an outlet for flowing a sample matrix, the inlet and the outlet being in fluid communication with the fluid flow path, wherein the continuous tubular body has a helical shape that extends continuously from the inlet of the continuous annular tubular body to the outlet.

[0011] In another aspect, the present technology relates to a tubular protein capture device comprising: (i) a continuous tubular body having an inner surface and an outer surface, wherein the inner surface defines a fluid flow path and comprises a protein attached to at least a portion of the inner surface, the protein selected from Protein A, Protein G, Protein L, or a combination thereof; and (ii) an inlet and an outlet for flowing a sample matrix, the inlet and the outlet being in fluid communication with the fluid flow path.

[0012] In some embodiments, the continuous tubular body has a helical shape that extends continuously from the inlet to the outlet of the continuous tubular body. In some embodiments, the continuous tubular body has an annular geometry. In some embodiments, the continuous tubular body has an open tubular geometry.

[0013] In some embodiments, the protein attached to the inner surface of the tubular protein capture device is selected from Protein A, Protein G, Protein L, or a combination thereof. In some embodiments, the protein attached to the inner surface of the tubular protein capture device is Protein A. In some embodiments, the protein is a functional derivative, fragment, or variant of Protein A.

[0014] The above aspects may include one or more of the following features. In some embodiments, the sample matrix comprises at least one type of protein. In some embodiments, the sample matrix comprises a cell culture, cell material, cell extract, or a combination thereof.

[0015] In some embodiments, the sample matrix is a clarified sample. That is, the clarified sample has undergone a clarification step, for example, before centrifugation. In some embodiments, the sample matrix is lysed.

[0016] In some embodiments, the sample matrix is in the form of a solution. In some embodiments, the solution is heterogeneous. In some embodiments, the solution is homogenized.

[0017] In some embodiments, the sample matrix is substantially cell-free. In other embodiments, the sample matrix is a raw cell culture.

[0018] In other embodiments, the continuous tubular body comprises a plurality of tubes aligned within a hollow housing, wherein an inlet and an outlet for flowing a sample (e.g., a solution) are in fluid communication with the plurality of tubes.

[0019] In some embodiments, the inner surface of the continuous tubular body (e.g., at least a portion of the inner surface in fluid communication with the sample) is coated with a polymer, and a protein selected from Protein A, Protein G, Protein L, or a combination thereof (i.e., a capture protein) is attached to the polymer. In some embodiments, the protein is selected from functional derivatives, fragments, or variants of Protein A. In some embodiments, the polymer comprises polyethylene glycol, fluorinated ethylene propylene, or ethylene tetrafluoroethylene. In some embodiments, the polymer comprises functional hydroxyl groups. In some embodiments, the protein is attached to the polymer by a covalent bond. In some embodiments, the protein is attached to the polymer by non-covalent interactions.

[0020] In some embodiments, a protein selected from Protein A, Protein G, Protein L, or a combination thereof is capable of binding to at least one type of protein within the sample matrix.

[0021] In some embodiments, the continuous tubular body of the present disclosure is configured to maintain a laminar flow with an apparent Reynolds number between 3 and 60.

[0022] In some embodiments, the helical continuous tubular body includes more than one turn, wherein the distance (e.g., pitch) between successive coiled tubes is less than 10 mm.

[0023] In some embodiments, the outer surface of the continuous tubular body disclosed herein is made of a material selected from metals, glass, and polymers. In some embodiments, the polymer is selected from materials including polyethylene glycol, fluorinated ethylene propylene, or ethylene tetrafluoroethylene. In one aspect, the present disclosure relates to a method for isolating and / or purifying proteins from a sample matrix (e.g., a lysed cell culture or a raw cell culture).

[0024] In another aspect, the present technology relates to a method for capturing at least one type of protein from a sample matrix. The method includes: a) providing a tubular device according to one or more embodiments disclosed herein; b) flowing the sample matrix through the continuous tubular body, wherein a protein selected from Protein A, Protein G, Protein L, or a combination thereof can bind to at least one type of protein within the sample matrix; c) binding at least one type of protein within the sample matrix to a protein selected from Protein A, Protein G, Protein L, or a combination thereof, thereby capturing at least one type of protein from the sample matrix. In some embodiments, the sample matrix includes a cell culture, cell material, cell extract (e.g., a lysed cell culture), or a combination thereof.

[0025] In another aspect, the present technology relates to a method for extracting at least one type of protein from a sample matrix including a cell culture, cell material, cell extract, or a combination thereof, the method including: (i) performing the method for capturing at least one type of protein from a sample matrix according to multiple embodiments of the present disclosure; and (ii) eluting the tubular device with a mobile phase having a pH less than 6.

[0026] The above aspects may include one or more of the following features. In some embodiments, the sample matrix includes less than 100 μg of total protein.

[0027] In some embodiments, the sample matrix flows through the continuous tubular body at an apparent Reynolds number between 3 and 60.

[0028] The devices and methods of the present technology offer many advantages. For example, the devices and methods provide improved interaction between the sample matrix and the clarification medium / material. In particular, many embodiments utilize the geometry of the flow path to improve the capture of specific proteins. In some embodiments, the present technology allows for the customization of the Dean number of the fluid flow to improve the radial mixing of the sample matrix with the protein coated on the inner surface of the tubular device. The Dean number can be customized by changing the flow rate.

[0029] The design of the device of the present technology (e.g., spiral design) utilizes Dean vortices to improve radial mixing. In some embodiments, the improved radial mixing increases protein capture efficiency. Compared with conventional devices (e.g., devices using dead-end filtration technology or devices with an open tubular design), the design of the device of the present technology (e.g., annular design) further provides a higher surface area to length ratio, resulting in higher protein capture efficiency.

[0030] The devices and methods provided herein using open tubular geometries and annular geometries help to minimize shear forces on cell membranes, resulting in a reduction in cell breakage. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0032] Figure 1A and Figure 1B show a cross-sectional view of an annular geometry ( Figure 1A ) and an open tubular geometry ( Figure 1B ).

[0033] Figure 1C show a cross-section of an open tubular body of the present technology, the open tubular body including a protein capture attached to the inner surface of the open tubular body.

[0034] Figure 1D , Figure 1E and Figure 1F show a cross-section of an annular tubular body of the present technology, the annular tubular body including a protein capture (110) attached to at least a portion of the inner surface of the annular tubular body. Collectively, Figures 1D to 1F present different embodiments of the present technology, wherein the protein capture (110) is attached to different portions of the inner surface within the annulus.

[0035] Figure 2 show the change in wall shear rate (s -1 ) relative to the change in volumetric flow rate (μL / min) in a device having an open tubular geometry with a diameter of 145 μm (diamond), an open tubular geometry with a diameter of 203 μm (square), an annulus with a diameter of 51 μm (cross), and an open tube with a diameter of 254 μm (triangle).

[0036] Figure 3 show examples of substantially coiled continuous tubular bodies according to various embodiments of the present disclosure.

[0037] Figure 4A , Figure 4B , Figure 4C ,Figure 4D , Figure 4E , Figure 4F , Figure 4G and Figure 4H illustrate various multi-lumen tube configurations in accordance with multiple embodiments of the present disclosure.

[0038] Figure 5A and Figure 5B illustrate the effects of flow rates of 200 μL / min ( Figure 5A ) and 100 μL / min ( Figure 5B ) on the Dean number, as well as the effects of coiled tube diameters (10 mm and 5 mm) and internal tube diameters (150 μm, 200 μm, 250 μm) on the Dean number.

[0039] Figure 6 depict the effect of loading flow rates (1 μL / sec to 9 μL / sec) on the binding ability of the open tubular capture devices of the present disclosure.

[0040] Figure 7 depict the effect of loading flow rates (1 μL / sec to 50 μL / sec) on the binding ability of the annular cross-section protein capture devices of the present disclosure. DETAILED DESCRIPTION

[0041] Various concepts related to devices and methods for separating proteins from a sample matrix, particularly using a continuous tubular body that captures proteins, and embodiments of such devices and methods are described in more detail below. The captured protein can selectively bind to or interact with one or more proteins present in the sample matrix. It should be understood that the various concepts introduced above and discussed in more detail below can be implemented in any of a variety of ways, as the disclosed concepts are not limited to any particular implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

[0042] The present disclosure provides new and improved methods for purifying one or more proteins of interest from a sample matrix containing one or more proteins of interest. In certain cases, the disclosed devices and methods reduce the number of steps available in the protein purification process, thereby reducing the overall operating cost and saving time.

[0043] As used herein, the terms "purify", "separate", or "isolate", which are used interchangeably herein, refer to increasing the purity of a target molecule (e.g., a protein) from a composition or sample matrix (e.g., a solution containing the target molecule and one or more impurities). Generally, the purity of the target molecule is increased by removing at least one impurity from the composition (either completely or partially).

[0044] The methods and devices provided herein can be used for cell clarification. As used herein, the term "clarification" refers to the process of reducing the turbidity (measured, e.g., in nephelometric turbidity units (NTU)) of a proteinaceous matrix (e.g., a solution) by removing suspended particles. Clarification can be achieved by a variety of means, including batch and continuous centrifugation, depth filtration, vertical and tangential flow filtration, and precipitation (including flocculation with small molecule and polymeric species), or any combination of these methods. The disclosed methods can be performed before or after any clarification technique used in the art for cell clarification.

[0045] The process for purifying proteins from cell debris initially depends on the site of protein expression. Some proteins are secreted directly from the cell into the surrounding growth medium; other proteins are produced intracellularly and remain within the cell. For the latter proteins, the first step in the purification process involves cell lysis, which can be carried out by a variety of methods, including mechanical shearing, osmotic shock, or enzymatic treatment. Such disruption releases all of the contents of the cell into the homogenate and additionally produces subcellular fragments that are difficult to remove due to their small size. These are typically removed by centrifugation or filtration. The same problem also occurs, albeit to a lesser extent, for proteins that are directly secreted due to the natural death of the cells and the release of intracellular host cell proteins during the protein production run. Thus, typical purification methods currently in use include the steps of: (i) cell lysis to recover intracellular proteins or, in the case of secreted proteins, to recover the proteins from the medium; (ii) using, e.g., differential centrifugation or filtration to remove cell debris to obtain a clarified sample containing one or more proteins of interest; and (iii) using multiple chromatography media in a multi-step method to separate the protein of interest from other proteins and / or various other impurities in the sample matrix. The methods and devices of the present disclosure can be performed / used during step (iii). Since the methods and devices disclosed herein allow for the clarification of the original cell culture, in some cases, steps (i) and (ii) can be omitted. Thus, the present technology can result in a reduction in the number of steps that can be used in the protein purification process, thereby increasing protein recovery from the sample matrix.

[0046] In various methods using the tubular protein capture device provided herein, the yield loss of the protein of interest is less than about 20%, less than about 10%, less than about 5% of the total protein mass. In other words, the methods provided herein using the protein capture device of the present technology produce a yield (e.g., 97%, 98%, 99%) of the protein of interest that is 80% or greater, 90% or greater, 95% or greater, where 100% is the total protein mass.

[0047] In some embodiments, the one or more proteins of interest are antibodies or Fc region-containing proteins. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody. In some embodiments, the sample matrix comprising the one or more proteins of interest is a clarified cell culture feed. In some examples, the clarified cell culture feed is obtained via depth filtration and / or centrifugation. In other examples, the clarified cell culture is obtained via precipitation with salt, acid, polymer, or stimulus-responsive polymer. In some embodiments, the sample matrix comprising the one or more proteins of interest is a raw cell culture feed. The sample matrix can comprise cell culture, cell material, cell extract, or a combination thereof.

[0048] In one aspect, the present technology relates to a tubular protein capture device comprising a continuous tubular body having an inner surface and an outer surface. The inner surface defines a fluid flow path through which the sample matrix can flow. A capture protein selected from Protein A, Protein G, Protein L, or a combination thereof can bind to or interact with (e.g., reversibly) one or more proteins present in the sample matrix. The tubular protein capture device provided herein also includes an inlet and an outlet for flowing the sample matrix, the inlet and the outlet being in fluid communication with the fluid flow path.

[0049] As used herein, unless otherwise indicated, the term "sample" refers to any composition or mixture containing one or more proteins of interest. The sample can be derived from biological sources or other sources. Biological sources include eukaryotic and prokaryotic sources such as plant and animal cells, tissues, and organs. The sample can also include diluents, buffers, detergents, and contaminants, debris, etc. found mixed with the one or more proteins of interest. The sample can be "partially purified" (i.e., having undergone one or more purification steps such as filtration steps) or can be obtained directly from a host cell or organism that produces the one or more proteins of interest (e.g., the sample can comprise harvested cell culture broth).

[0050] As used herein, the term "continuous body" refers to a physical structure having an uninterrupted protrusion in space.

[0051] As used herein, the term "comprising" means including but not limited to, and the term "including" means including but not limited to.

[0052] Select the selected material, as well as the size and shape, of the continuous body of the annular tube described herein to achieve the capture of proteins from a flowing sample matrix (including but not limited to raw cell cultures, lysed cell cultures, cell extracts, clarified cell cultures). For example, the continuous body of the annular tube can be configured in any shape and / or size capable of capturing or separating a certain amount of protein from the sample matrix. The total amount of protein in the sample matrix can be less than 900 μg, 800 μg, 700 μg, 600 μg, 500 μg, 400 μg, 300 μg, 200 μg, 100 μg, 50 μg, 25 μg, or 10 μg.

[0053] In one example, the continuous tubular body of the protein capture device can be in a substantially coiled shape (e.g., formed in a concentric ring configuration). In other examples, the continuous tubular body can be substantially non-uniform in shape (i.e., include coiled portions and / or straight portions extending in multiple different directions defined by radially different curvatures from the inlet to the outlet of the continuous body).

[0054] As another example, the continuous tubular body can be substantially straight in shape and include multiple tubes aligned within a hollow housing. In any example according to the principles herein, the continuous tubular body can be formed in a tangled configuration. As used herein, the term "tangled" refers to a tubing body having at least a portion that includes multiple coils, creases, and / or loops. In one example, the multiple coils, creases, and / or loops can be overlapping. Such a tangled tubing body can be obtained at least in part by positioning and fabricating the tubing in multiple directions. The exemplary tangled tubing herein can exhibit a greatly increased surface area of the resulting tubing body, which can lead to an increase in capture efficiency.

[0055] As a non-limiting example, the tangled tubing body can consist of continuous small-radius bends. In one embodiment, the tangled tubing body can have an overall compact structure.

[0056] Curved and helical microchannels have a wide range of applications in microfluidic devices, such as the isolation of circulating tumor cells, cell enrichment, bacterial separation, fluid mixing, microfiltration, and the inertial aggregation and separation of particles. A fluid flowing in the axial direction of a curved channel experiences a pressure gradient in the radial direction. Without being bound by theory, it is believed that the curved fluid channel causes the fluid to continuously circulate in the radial direction and subsequently form two or more reverse vortices in the channel. This lateral recirculation flow is referred to as secondary (or Dean) flow. The formation of Dean flow in curved and helical channels is inevitable, but their presence can be dominant and useful in some applications, while in other applications it is undesirable.

[0057] The disclosed tubular protein capture device requires maximizing Dean flow while promoting radial mixing to increase the capture efficacy of the captured proteins attached to the inner surface of the tubular protein capture device.

[0058] The disclosed tubular protein capture device utilizes Dean vortices stimulated by a coiled helical geometry to improve radial mixing. Attachment of the captured proteins on the inner surface of the coiled tube allows the cell material to flow freely to waste while optimizing radial mixing and protein capture along the inner surface of the coiled tube.

[0059] Captured protein

[0060] The continuous tubular body of the present disclosure includes a capture protein, such as a protein capable of binding (e.g., reversibly) to one or more proteins of interest present in a sample matrix or interacting with the one or more proteins of interest. The capture protein can selectively bind to the protein of interest and / or other impurities among other proteins. The capture protein of the present disclosure is attached to the inner surface of the tubular protein capture device.

[0061] In some cases, the capture protein can be attached to the inner surface of the tubular body via a polymer coating the inner surface of the continuous tubular body. That is, in some cases, the inner surface of the tubular body is coated with a polymer, and the capture protein is attached to the polymer by covalent bonds, non-covalent bonds, or a combination thereof.

[0062] In some embodiments, the capture protein is attached to the polymer coating the inner surface of the continuous tubular body via a single-point attachment. Single-point attachment generally means that the protein moiety is attached to the polymer via a single covalent bond. Such single-point attachment can also be carried out by using suitable reactive residues that are placed at exposed amino acid positions, i.e., in loops, near the N-terminus or C-terminus, or elsewhere on the periphery of the protein fold. Suitable reactive groups are, for example, thiol or amino functional groups.

[0063] The polymer coated or attached to the inner surface of the continuous tubular body can be any polymer having free functional groups (e.g., -OH, -NH 2 , -COOH) that can interact or react with the capture protein. In some embodiments, the polymer comprises polyethylene glycol.

[0064] In some embodiments, the capture protein is selected from Protein A, Protein G, Protein L, their functional variants, or combinations thereof. In some embodiments, the protein attached to the inner surface of the tubular protein capture device is Protein A. In some embodiments, the protein is a functional derivative, fragment, or variant of Protein A.

[0065] Protein A is a bacterial cell wall protein from Staphylococcus aureas that binds to mammalian IgG primarily through their Fc regions. In its native state, Protein A has five IgG-binding domains and other domains of unknown function.

[0066] As used herein, the terms "immunoglobulin," "Ig," "IgG," or "antibody" (used interchangeably herein) refer to a protein having a basic four-polypeptide chain structure consisting of two heavy chains and two light chains, the chains being stabilized, for example, by interchain disulfide bonds, which can specifically bind an antigen. The terms "Fc region" and "Fc region-containing protein" mean a protein containing the constant region or domain of the heavy and / or light chains of an immunoglobulin (such as the CH and CL regions as previously defined). A protein containing an "Fc region" can have effector functions of an immunoglobulin constant region. The "Fc region" (such as the CH2 / CH3 region) can selectively bind to an affinity ligand (such as Protein A or a functional variant thereof). In some embodiments, an Fc region-containing protein specifically binds Protein A or a functional derivative, variant, or fragment thereof. In other embodiments, an Fc region-containing protein specifically binds Protein G or Protein L or a functional derivative, variant, or fragment thereof.

[0067] Protein A can be recovered from its natural source or can be produced synthetically (e.g., by peptide synthesis or by recombinant techniques). Functional derivatives, fragments, or variants of Protein A according to the present disclosure can be characterized by a binding constant to the Fc region of murine IgG2a or human IgG1 of at least K = 10 -8 M, and preferably K = 10 -9 M. In this context, an interaction that conforms to such values for the binding constant is referred to as "high-affinity binding." Preferably, such functional derivatives or variants of Protein A contain at least a portion of the functional IgG-binding domain of wild-type Protein A, which is selected from the native domains E, D, A, B, C, or engineered mutants thereof that have retained IgG-binding functionality.

[0068] "Binding" refers to the affinity between two molecules (e.g., a capture protein and a target protein, i.e., the protein of interest). When binding occurs between the target protein and the capture protein of the present disclosure, the target protein is reversibly immobilized on the capture ligand by means of ligand-protein interactions. Non-limiting examples of ligand-protein interactions include antibody-antigen binding, enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation, DNA-binding protein-DNA binding, regulatory protein-protein interactions, etc. Binding may be due to, for example, the combined effects of spatial complementarity between the target protein and the capture protein at the binding site and electrostatic, hydrogen bonding, hydrophobic, and / or van der Waals forces at the binding site. Generally, the greater the spatial complementarity and the stronger the other forces at the binding site, the greater the binding specificity of the protein for its corresponding ligand.

[0069] In some cases, the binding constant between the capture protein and one or more target proteins is between K = 10 -4 M and K = 10 -9 M.

[0070] Tubular protein capture device

[0071] In one aspect, provided herein is a tubular protein capture device that includes a continuous tubular body having an inner surface and an outer surface, wherein the inner surface defines a fluid flow path and includes a capture protein attached to the inner surface; and (ii) an inlet and an outlet for flowing a sample matrix (e.g., a solution), the inlet and the outlet being in fluid communication with the fluid flow path.

[0072] In some embodiments, the outer surface and / or the inner surface of the continuous tubular body is made of a material selected from metals, glass, polymers, and combinations thereof. In some embodiments, the metal is selected from titanium, nickel, copper, brass, and stainless steel. In some embodiments, the polymer is selected from materials comprising polyethylene glycol, fluorinated ethylene propylene, ethylene tetrafluoroethylene, or combinations thereof.

[0073] In some embodiments, the outer surface and / or the inner surface of the continuous tubular body is made of polyether ether ketone.

[0074] In some embodiments, the outer surface and / or the inner surface of the continuous tubular body is made of a fluorinated compound selected from ethylene tetrafluoroethylene (ETFE) and fluorinated ethylene propylene (FEP).

[0075] In some embodiments, the outer surface and / or the inner surface of the continuous tubular body is made of a polymer made by using thermoforming processing techniques.

[0076] The device of the present technology allows for optimizing radial mixing by varying the geometric parameters of the device disclosed herein. For example, promoting radial mixing (e.g., laminar flow) increases the capture efficiency of the captured protein because the opportunity for the protein of interest to bind to or interact with the captured protein increases when the sample matrix flows in a laminar flow with a customized Dean number.

[0077] Compared to conventional filters (e.g., dead-end filters), the open tubular design of the tubular protein capture device according to one or more embodiments of the present disclosure helps to minimize the shear force on the cells within the sample.

[0078] In other embodiments, the annular design of the tubular protein capture device provides a high surface area to length ratio, which is beneficial for increasing the capture efficiency. Due to the increased area of each cross-section above which the affinity capture protein (e.g., Protein A, Protein G, Protein L, or a combination thereof) can be attached, the high surface area to length ratio is expected to result in a higher capture efficiency.

[0079] To this end, it is desirable that the sample matrix flow through the annular geometry of the tubular design consisting of cross-sectional rings to produce improved results. Such a design will also benefit from a coiled helical configuration as Dean vortices are generated across the annulus. Figure 1A and Figure 1B shows the cross-sections of the annular geometry ( Figure 1A ) and the open tubular geometry ( Figure 1B ). The area to length ratio of the annular design with a 51 μm annulus corresponding to Figure 1A (cross-sectional area of the annular capillary: 0.155 mm2; surface area of the annular capillary: 6063 μm / L) is 0.0392, while the area to length ratio of the open tubular design with a 145 μm inner diameter corresponding to Figure 1B (cross-sectional area of the annular capillary: 0.0177 mm2; surface area of the annular capillary: 471 μm / L) is 0.0267. That is, compared to the open tubular design, the continuous annular tubular body of the present disclosure provides a larger surface area to attach more captured proteins to the inner surface of the tubular body. A cross-sectional view of the continuous tubular body of the present technology is provided in Figures 1C to 1F to show how the captured protein (110) (e.g., Protein A, Protein G, Protein L, or a combination thereof) attaches to the inner surface of the tubular body.

[0080] Figure 1C shows how the captured protein (110) attaches to the inner surface of the open tubular body.

[0081] Figures 1D to 1FIllustrates different variants of the present technology, where the capture protein is attached to different portions of the inner surface within the annular geometry of the tubular body.

[0082] Using a wall-centered approach, where one or more proteins of interest are directly captured from the sample matrix, eliminates the need for mechanical filtration and the associated risks of protein absorption and cell wall shearing.

[0083] As used herein, "annular" or "annulus" refers to the space between two substantially concentric objects (or between two substantially concentric regions, such as between a sleeve and a pipe) through which fluid can flow. Annular shapes suitable for the present invention include annular, oval, elliptical, toroidal, etc.

[0084] Depending on the inner diameter, both the annular geometry and the open tubular geometry proposed herein contribute to reducing the wall shear rate compared to conventional filtration techniques, which results in reduced cell rupture and subsequent reduction of host cell protein contaminants ( Figure 2 ).

[0085] In some embodiments, the continuous tubular body has a helical shape that extends continuously from the inlet of the continuous tubular body to the outlet.

[0086] Figure 3 Shows an example of a substantially coiled continuous tubular body 300. As Figure 3 shown, the diameter of the outer dimension (320 or OD) is greater than the diameter of the inner dimension (315 or ID). Referring to Figure 3 , the outer peripheral wall is located at, for example, OD or 320, while the inner peripheral wall is located at ID or 310.

[0087] As Figure 3 shown, the continuous tubular body may be characterized by one or more of the following: the length or distance L or 310, the distance between consecutive coils (pitch) P or 305.

[0088] In various non-limiting exemplary embodiments, the continuous tubular body may be formed with a length (L) that varies between about 40 mm and about 5 mm. In particular, the length (L) may vary between about 20 mm and about 1 mm, between about 20 mm and about 5 mm. Generally speaking, the length may be any length that adapts to the system preference.

[0089] In various non-limiting exemplary embodiments, the continuous tubular body can be formed with an average coiled diameter that varies between about 1 mm and about 20 mm. In particular, the average coiled diameter can vary between about 5 mm and about 15 mm. More specifically, the average coiled diameter can vary between about 5 mm and about 10 mm. Generally speaking, the coiled diameter of the tubing is selected according to the flow rate and resistance requirements of a particular system. Thus, a wide variety of inner diameters can be used.

[0090] In various non-limiting exemplary embodiments, the continuous tubular body can be formed with a pitch (P) that varies between about 1 mm and about 10 mm, according to system preferences.

[0091] In some exemplary embodiments, Figure 3 the continuous tubular body shown can be formed with a dimensional parameter (e.g., OD, ID, P) that remains substantially constant over a length L. In other embodiments, one or more of these dimensions can vary over the length L. For example, the pitch P can vary over the length L of the continuous tubular body such that the pitch P exhibits a constant decrease or increase over L, or an intermittent decrease or increase over L, or both. Also, for example, the diameter ID of the internal dimension can vary over the length L of the continuous tubular body such that the change in ID can decrease or increase constantly over L, or decrease or increase intermittently over L, or both. Also, for example, the diameter OD of the external dimension can vary over the length L of the continuous tubular body such that the change in OD can decrease or increase constantly over L, decrease or increase intermittently over L, or both. Also, for example, the continuous body of the breathable tubing can be formed such that both the ID and OD increase over the length L, i.e., the coil can change size over L. The percentage increase of any one of these dimensions (ID, OD, and P) over L can be up to 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In one embodiment, the continuous body of the breathable tubing can be constructed with a tightly coiled flow path such that over L, all three dimensional parameters (ID, OD, and P) increase over the length L.

[0092] In some embodiments, the continuous tubular body is configured to maintain a laminar flow with an apparent Reynolds number between 2 and 60, between 3 and 50, between 5 and 40, between 5 and 30, between 5 and 20, between 3 and 10, between 3 and 7, between 3 and 60, or between 3 and 60.

[0093] In some embodiments, the mobile fluid includes a flow rate between 100 μL / min and 1250 μL / min. The flow rate of the sample matrix disclosed herein can be adjusted to obtain an optimal Dean number to promote capture efficiency.

[0094] In some embodiments, the continuous tubular body is configured to maintain a laminar flow with a Dean number between 1.5 and 6, between 1.5 and 5, or between 2 and 4.

[0095] Provided herein is a tubular protein capture device according to multiple embodiments disclosed herein, wherein the continuous tubular body includes a plurality of tubes aligned within a hollow housing, and wherein an inlet and an outlet for fluid flow are in fluid communication with the plurality of tubes ( Figures 4A to 4H ). The inner diameter of each tube within the hollow housing can be designed into various geometric structures, such as star-shaped internal fibers, screws, slotted channels ( Figures 4A to 4H ).

[0096] In some embodiments, the continuous tubular body can include from about 3 to about 500 tubes within the hollow housing of the continuous tubular body. The number of tubes included in the hollow housing can vary according to the inner diameter and / or outer diameter of the tubes within the hollow housing of the continuous tubular body and the diameter of the hollow housing.

[0097] In some embodiments, each tube is a single-channel tube. That is, there is only one channel in each tube for the sample to flow through ( Figure 4A ). In other embodiments, each tube within the hollow housing of the continuous tubular body has a plurality of internal cavities. Various cross-sectional configurations can be used to form the internal cavities. For example, Figures 4A to 4H illustrates cross-sectional configurations that result in the incorporation of a single channel ( Figure 4A )(i.e., a single internal cavity) or multiple internal cavities (e.g., 1Web that provides two different internal cavities ( Figure 4B ), 2T that provides 4 internal cavities ( Figure 4C ), 2W that provides at least 110 internal cavities ( Figure 4D ), 3T that provides 17 internal cavities ( Figure 4E ), 3W that provides 11 internal cavities ( Figure 4F ), 3Y that provides 22 internal cavities ( Figure 4G ), 4+ that provides 21 internal cavities ( Figure 4H )). As used herein, an "internal cavity" refers to a channel within a tube.

[0098] Multi-lumen tubes can provide side-by-side flow of the sample. Multi-lumen tubes provide a high surface area-to-length ratio, which is beneficial for increasing the capture efficiency.

[0099] Based on system preferences, the internal cavities within the continuous tube can have different sizes and shapes (e.g., 1Web, 2T, 2W, 3T, 3W, 3Y, 4+ as Figures 4A to 4H shown). Multi-lumen tubes provide multiple channels within a single tube structure.

[0100] In various non-limiting exemplary embodiments, the tube diameter varies between about 0.1 mm and about 20 mm. Specifically, the tube diameter varies between about 0.15 mm and about 10 mm.

[0101] Method for clarification

[0102] In one aspect, provided herein is a method of capturing at least one type of protein from a sample matrix using a tubular protein capture device according to various embodiments of the present disclosure.

[0103] As used herein, the chromatographic "capture" step comprises binding a protein of interest to a capture protein. The primary function of this step is to bind one or more proteins of interest from the sample matrix while allowing impurities (e.g., contaminating proteins, lipids, carbohydrates, etc.) to flow through. The protein of interest is then eluted with a buffer for further downstream processing.

[0104] As used herein, the term "impurity" or "contaminant" refers to any foreign or unwanted molecule, including biomacromolecules (such as DNA, RNA, one or more host cell proteins, endotoxins, lipids, and one or more additives), which may be present in a sample containing a target molecule, e.g., a protein isolated from one or more foreign or unwanted molecules using the methods of the present invention. Additionally, such impurities may include any reagents used in steps that may have occurred prior to the methods disclosed herein.

[0105] The method provided herein for capturing at least one type of protein from a sample matrix comprises: (i) providing a tubular device according to various embodiments disclosed herein; (ii) flowing the sample matrix through a continuous annular tubular body, wherein the capture protein is capable of binding to at least one type of protein within the sample matrix; (iii) binding at least one type of protein within the sample matrix to the capture protein, thereby capturing at least one type of protein from the sample matrix.

[0106] Also provided herein is a method of extracting at least one type of protein from a sample matrix. After performing the method of capturing at least one type of protein from a sample matrix according to various embodiments of the present technology, the tubular device is eluted with a mobile phase having a pH less than 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.0, or 4.5. "Eluting" a molecule (e.g., a polypeptide of interest or an impurity) from a chromatographic resin means removing the molecule therefrom by changing the solution conditions such that the buffer competes with the molecule of interest for binding to the chromatographic resin. A non-limiting example is eluting a molecule from an ion exchange resin by changing the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for charged sites on the ion exchange material.

[0107] In some embodiments, the sample matrix comprises less than 100 μg of total protein.

[0108] In some embodiments, the sample matrix flows through the continuous tubular body at an apparent Reynolds number between 3 and 60.

[0109] In some embodiments, the sample matrix flows onto the open tubular body at a loading flow rate between 3 μL / sec and 7 μL / sec. In some embodiments, the loading flow rate is 3 μL / sec, 4 μL / sec, 5 μL / sec, 6 μL / sec, or 7 μL / sec.

[0110] In some embodiments, the sample matrix flows onto the annular cross-section tubular body at a loading flow rate between 20 μL / sec and 50 μL / sec. In some embodiments, the loading flow rate is between 20 μL / sec and 25 μL / sec, 25 μL / sec and 30 μL / sec, 30 μL / sec and 35 μL / sec, 35 μL / sec and 40 μL / sec, 40 μL / sec and 45 μL / sec, or 45 μL / sec and 50 μL / sec.

[0111] In some embodiments, the sample matrix flows through the continuous tubular body at a Dean number between 2.5 and 6.0, 3.0 and 5.0, or 3.5 and 4.5.

[0112] The Dean number can be optimized by varying the flow rate of the matrix solution.

[0113] Example

[0114] Example 1: Influence of flow rate, coil diameter and inner tube diameter on Dean number

[0115] The flow rate, coil diameter, and inner tube diameter are obtained mathematically and are derived from the table

[0116] (Table 1). The value of the Dean number is obtained by the following formula:

[0117]

[0118] where Re is the Reynolds number, d is the tube diameter, D is the coil diameter, and ρ is the pitch in mm.

[0119] Figure 5A and Figure 5B are graphical representations of these calculated values. Figure 5A and Figure 5BThe 10 and 5 given in [reference] refer to the coil diameters in mm; 150, 200, 250 each correspond to the inner tube diameters in μm; and 200 μL / min and 100 μL / min refer to the flow rates. Figure 5A and Figure 5B show that smaller tube diameters and coil diameters result in higher Dean numbers, which are expected to yield higher recovery rates due to more contact between the protein and the modified surface. Additionally, Figure 5A and Figure 5B show that the Dean number increases with the volumetric flow rate. This information is useful for considerations regarding cell shear associated with the flow rate.

[0120] Table 1: Variation of Dean number based on tube ID, coil D, pitch and flow rate

[0121]

[0122]

[0123] Example 2: Open tubular and annular cross-section loading flow rates

[0124] The ability of the tubular protein capture device disclosed herein to bind and elute protein samples was determined for a range of loading flow rates. The tightly coiled open tubular capture device was connected to a liquid chromatography system with a UV detector. A 15 μL or 20 μL sample (IgG) was loaded onto the coil at 1 μL / sec, 2 μL / sec, 3 μL / sec, 4 μL / sec, 5 μL / sec, 6 μL / sec, 7 μL / sec, 8 μL / sec, or 9 μL / sec (loading flow rate). As a comparator, a loosely coiled capture device was used. As Figure 6 shown, the tightly coiled open tubular capture device (squares and diamonds) provided verifiable evidence of the Dean vortex effect, where binding was reduced at low flow rates (1 μL / sec to 3 μL / sec) below the critical linear velocity required to form secondary flow. After the critical point, binding increased due to the emergence of secondary flow (4 μL / sec to 7 μL / sec). Due to backpressure, the system began to leak at a loading rate of approximately 8 μL / sec, resulting in a decrease in binding capacity. In contrast, the loosely coiled (triangles) did not allow secondary flow and did not show an increase in binding capacity or recovery rate with increasing flow rate.

[0125] Similar experiments were conducted on the annular cross-section capture device. The tightly coiled annular capture device (4 mm inner coil diameter) was connected to a liquid chromatography system with a UV detector. A 20 μL sample was loaded onto the coil at a flow rate between 1 μL / sec and 50 μL / sec (loading flow rate). As a comparator, a 73 mm loosely coiled capture device was used. As Figure 7As shown, the tightly coiled annular capture device (triangle) results in a reduced recovery rate at loading flow rates up to 20 μL / sec, which is consistent with diffusion-limited binding. Above 20 μL / sec loading flow rate, the recovery rate increases, which is consistent with the secondary current improving the binding efficiency. The annular capture device can withstand higher flow rates at equivalent back pressures compared to the open tubular device (about less than 150 psi). The 73 mm loosely coiled capture device (diamond) results in significantly lower recoveries, with no increase at flow rates above 20 μL / sec.

Claims

1. A tubular protein capture device, the tubular protein capture device comprises: (i) A continuous tubular body having an inner surface and an outer surface, wherein the inner surface defines a fluid flow path and comprises a protein attached to at least a portion of the inner surface, the protein being selected from Protein A, Protein G, Protein L, or a combination thereof; and (ii) An inlet and an outlet for flowing a sample matrix, the inlet and the outlet being in fluid communication with the fluid flow path, wherein the continuous tubular body has a helical shape extending continuously from the inlet to the outlet of the continuous tubular body.

2. The tubular protein capture device according to claim 1, wherein the continuous tubular body has an annular geometry.

3. The tubular protein capture device according to claim 1, wherein the continuous tubular body has an open tubular geometry.

4. The tubular protein capture device according to claim 1, wherein the sample matrix comprises at least one type of protein.

5. The tubular protein capture device according to claim 1, wherein the sample matrix comprises a cell culture, cell material, cell extract, or a combination thereof.

6. The tubular protein capture device according to claim 1, wherein the continuous tubular body comprises a plurality of tubes aligned within a hollow housing, and the inlet and the outlet for flowing the sample matrix are in fluid communication with the plurality of tubes.

7. The tubular protein capture device according to claim 1, wherein at least a portion of the inner surface of the tubular body is coated with a polymer, and the protein selected from Protein A, Protein G, Protein L, or a combination thereof is attached to the polymer.

8. The tubular protein capture device according to claim 7, wherein the polymer comprises polyethylene glycol, fluorinated ethylene propylene, or ethylene tetrafluoroethylene.

9. The tubular protein capture device according to claim 7, wherein the protein is attached to the polymer by a covalent bond.

10. The tubular protein capture device according to claim 7, wherein the protein is attached to the polymer by non-covalent interactions.

11. The tubular protein capture device according to claim 4, wherein the protein selected from Protein A, Protein G, Protein L, or a combination thereof is capable of binding to at least one type of protein present within the sample matrix.

12. The tubular protein capture device according to any one of claims 1 to 3 or 6, wherein the continuous tubular body is configured to maintain a laminar flow with an apparent Reynolds number between 3 and 60.

13. The tubular protein capture device according to claim 1, wherein the continuous tubular body comprises more than one turn, and the distance between consecutive coiled tubes is less than 10 mm.

14. The tubular protein capture device according to claim 1, wherein the outer surface of the continuous tubular body is made of a material selected from metal, glass, polymer, or a combination thereof.

15. The tubular protein capture device according to claim 14, wherein the polymer is selected from materials comprising polyethylene glycol, fluorinated ethylene propylene, or ethylene tetrafluoroethylene.

16. A method for capturing at least one type of protein from a sample matrix, wherein the sample matrix comprises a cell culture, cell material, cell extract, or a combination thereof, the method comprising: a) providing a tubular device according to any one of claims 1 to 3; b) flowing the sample matrix through the continuous tubular body, wherein the protein selected from Protein A, Protein G, Protein L, or a combination thereof is capable of binding to at least one type of protein within the sample matrix; c) binding the at least one type of protein within the sample matrix to the protein selected from Protein A, Protein G, Protein L, or a combination thereof, thereby capturing at least one type of protein from the sample matrix.

17. A method for extracting at least one type of protein from a sample matrix comprising a cell culture, cell material, cell extract, or a combination thereof, the method comprising: (i) performing the method according to claim 16; and (ii) eluting the tubular device with a mobile phase having a pH less than 6.

18. The method according to claim 16 or 17, wherein the sample matrix comprises less than 100 μg of total protein.

19. The method according to claim 16 or 17, wherein the sample matrix flows through the continuous tubular body at an apparent Reynolds number between 3 and 60.

20. A tubular protein capture device, the tubular protein capture device comprising: (i) a continuous annular tubular body having an inner surface and an outer surface, wherein the inner surface defines a fluid flow path and comprises a protein attached to at least a portion of the inner surface, the protein selected from Protein A, Protein G, Protein L, or a combination thereof; and (ii) an inlet and an outlet for flowing a sample matrix, the inlet and the outlet being in fluid communication with the fluid flow path, wherein the continuous tubular body has a helical shape that continuously extends from the inlet of the continuous annular tubular body to the outlet.

21. The tubular protein capture device according to claim 20, wherein the sample matrix comprises at least one type of protein that is capable of binding to at least one type of protein selected from Protein A, Protein G, and Protein L.

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