Systems for purifying biologically active proteins and related methods

Abstract

A system for purifying a biologically active protein in a fluid, the system comprising at least one fractal subsystem and a filter press constructed and arranged to support the at least one fractal subsystem in a horizontal arrangement to provide a selectively adjustable bed depth, wherein each fractal subsystem is in fluid communication with each other fractal subsystem. The fractal subsystem may include a resin chamber and a pair of fractal packets, wherein the pair of fractal packets are disposed on opposite sides of the resin chamber. The resin chamber may include a frame, a pressurized space through the frame for containing resin, a pair of meshes for containing the resin within the pressurized space, and a pair of feed ports adapted to at least one of introduce fluid into or remove fluid from the pressurized space.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 329,606, filed on April 11, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Background Art

[0003] The present invention relates to systems and methods for purifying biologically active proteins, and more specifically, to modular, fast-flow ion exchange columns; related systems; and related methods for purifying biologically active proteins, such as trace amounts of whey proteins contained in, for example, high-fat raw whole milk or colostrum-based feeds. Although the present invention will be described in conjunction with purifying biologically active proteins, it will be understood by those of ordinary skill in the art that the systems and devices described herein can also be applied to processes for extracting metals (e.g., lithium, etc.) from liquids or brine.

[0004] Ion exchange is often required for the separation and purification of proteins (e.g., trace whey proteins, etc.) contained in liquids (e.g., high-fat milk, colostrum-based feeds, etc.). However, conventional ion exchange chromatography systems are limited by their design and functionality. For example, in addition to being unable to purify high-fat milk sources, conventional ion exchange chromatography systems are particularly expensive; have a fixed height (or length); have a relatively large non-modular footprint; have low flow rates; lack uniform feed flow through the resin contained in the system; lack bidirectional flow; lack the ability to adjust the bed depth height (or length); and lack a uniform elution gradient flow. Summary of the invention

[0005] In a first aspect, the present invention relates to a fractal subsystem for use with an ion exchange system. In some embodiments, the fractal subsystem includes a resin chamber and a pair of fractal bags, wherein the pair of fractal bags are disposed on opposite sides of the resin chamber. In some specific implementations, the resin chamber may include a frame, a pressurized space for accommodating resin through the frame, a pair of mesh screens for accommodating the resin in the pressurized space, and a pair of feed ports, the feed ports being suitable for introducing a fluid into the pressurized space or removing at least one of the fluid from the pressurized space.

[0006] In some variations, the mesh screen comprises a 200 mesh plain Dutch weave having a nominal opening in the mesh of 75 microns and / or having a geometric opening in the mesh of 85 microns.

[0007] In some applications, the fractal bag may include a frame, a plenum space passing through the frame, a fractal distributor disposed in the plenum space, a fractal flow plate disposed in the plenum space, and a feed input port adapted to introduce a fluid into the plenum space. A weir may be formed in the plenum space in each upper corner of the frame.

[0008] In a second aspect, the present invention relates to a system for the biologically active proteins in a purified fluid. In some embodiments, the system includes at least one fractal subsystem and a filter press, and the filter press is constructed and arranged to support at least one fractal subsystem in a horizontal arrangement mode to provide a bed depth that can be selectively adjusted, wherein each fractal subsystem is communicated with each other fractal subsystem each fractal subsystem fluid. In some embodiments, the fractal subsystem includes a resin chamber and a pair of fractal bags, wherein the pair of fractal bags are arranged on the opposite side of the resin chamber. In some specific implementations, the resin chamber may include a frame, a pressurized space for accommodating resin through the frame, a pair of mesh screens for accommodating resin in the pressurized space, and a pair of feed ports, and the feed port is suitable for introducing fluid into the pressurized space or removing at least one of the fluid from the pressurized space.

[0009] In some variations, the mesh screen comprises a 200 mesh plain Dutch weave having a nominal opening in the mesh of 75 microns and / or having a geometric opening in the mesh of 85 microns.

[0010] In some applications, the fractal bag may include a frame, a plenum space passing through the frame, a fractal distributor disposed in the plenum space, a fractal flow plate disposed in the plenum space, and a feed input port adapted to introduce a fluid into the plenum space. A weir may be formed in the plenum space in each upper corner of the frame.

[0011] In some implementations, a filter press may include a pair of vertical supports; a pair of horizontal supports fixedly attached to the vertical supports, wherein the horizontal supports are constructed and arranged to support a fractal bag; and a filter press subsystem constructed and arranged to apply a compressive force to the fractal bag. In some variations, the filter press subsystem may include a selectively movable rod, a follower disposed at a distal end of the movable rod, and a hydraulic press disposed at a proximal end of the movable rod.

[0012] Advantageously, the selectively adjustable bed depth can range between one (1) inch and eight (8) feet; the system is capable of bi-directional flow; the system produces laminar flow in the fluid; and / or the fluid can contain a supply feed having a fat content greater than 0.1% and less than five (5) percent.

[0013] While the invention will be described in conjunction with proteins containing trace amounts of whey protein and liquids containing high fat raw whole milk or colostrum-based feeds, etc., one of ordinary skill in the art will appreciate that the concepts described herein may also be applied to food, wastewater, water treatment, electronic devices, industrial catalysts, carbohydrate refining, extraction of metals from liquids or brines, and active pharmaceutical ingredient manufacturing environments where ion exchange may be desired. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated as a part of this specification, illustrate preferred embodiments of the invention, and together with the general description given above and the detailed description of the preferred embodiments given below, serve to explain and teach the principles described herein.

[0015] Figure 1 shows an exemplary fractal subsystem according to some embodiments of the present invention;

[0016] Figure 2A Some embodiments according to the present invention include Figure 1 A first side perspective view of an ion exchange system of a fractal subsystem; and

[0017] Figure 2B Some embodiments according to the present invention include Figure 1 A second side perspective view of the fractal subsystem of the ion exchange system. DETAILED DESCRIPTION

[0018] method:

[0019] Although one of ordinary skill in the art may utilize the functions and principles taught in the present disclosure for many applications, including but not limited to the extraction of metals (e.g., lithium, etc.) from liquids or brine, these functions and principles will be illustratively described in the context of purifying fluids (such as raw milk for human consumption), pharmaceutical API manufacturing, etc. Whereas conventional systems are limited to purifying feeds having a fat content of no more than 0.1%, advantageously, the systems and methods of the present invention are capable of processing high viscosity feeds having a fat content of up to about five (5) percent. Such high fat content fluids would clog conventional systems.

[0020] In the first step, high resolution of separation of undesirable proteins having positive or negative charges from raw milk is desirable. Therefore, before the raw milk becomes skim milk, the fluid (e.g., raw milk) can be directed to an ion exchange system, as described in more detail below, to remove one or more (e.g., whey trace) charged proteins. Advantageously, the ion exchange system comprises a (e.g., oppositely charged chromatographic) resin suitable for separating proteins having different charges from a liquid.

[0021] It is clear to those skilled in the art that the net surface charge of a liquid is a function of pH and that proteins have no net charge at their isoelectric point (pi). Thus, if the pi is greater than the pH, the protein will bind to positively charged ions in an anion exchanger, while if the pi is less than the pH, the protein will bind to negatively charged ions in a cation exchanger. Once these charged proteins are removed, the liquid can be returned to a conventional (e.g., dairy) purification process where it is initially turned into skim milk.

[0022] In more detail, the method includes four stages. First, the system column is prepared under the desired pH or the system column is prepared to the desired pH. Next, the liquid can be introduced into the column and subjected to an ion exchanger so that the target molecule in the liquid is combined with the ion exchanger. Unbound substances can be washed out from the column, and the bound biomolecules are eluted so that they are gradually released from the ion exchanger due to, for example, changes in the buffer composition. Finally, in the regeneration step, the target molecule that is kept combined with the ion exchanger can be removed. More details about the exemplary method are provided in the following paragraphs and U.S. Provisional Patent Application No. 63 / 486,437, which is incorporated herein by reference in its entirety.

[0023] The purification method can reduce, minimize or eliminate chemical treatment, enzyme treatment, acid treatment and / or heat treatment. The purification method can reduce chemical treatment, enzyme treatment, acid treatment and / or heat treatment. The purification method can minimize chemical treatment, enzyme treatment, acid treatment and / or heat treatment. The purification method can eliminate chemical treatment, enzyme treatment, acid treatment and / or heat treatment. The purification method can reduce, minimize or eliminate chemical treatment. The purification method can reduce, minimize or eliminate enzyme treatment. The purification method can reduce, minimize or eliminate acid treatment. The purification method can reduce, minimize or eliminate heat treatment. The purification method can reduce each of chemical treatment, enzyme treatment, acid treatment and heat treatment. The purification method can minimize each of chemical treatment, enzyme treatment, acid treatment and heat treatment. The purification method can eliminate each of chemical treatment, enzyme treatment, acid treatment and heat treatment. The purification method can eliminate each of chemical treatment, enzyme treatment and acid treatment. The purification method can eliminate each of chemical treatment, enzyme treatment and heat treatment.

[0024] In general, the methods provided herein for producing purified lactoferrin do not include typical heat treatments (e.g., pasteurization) in industrial purification processes, which may destroy the native conformational state (e.g., denaturation) with reference to the conformational state found in the natural milk source used for lactoferrin purification. Typical heat treatments used in industrial purification processes may be about 63°C or higher. The heat treatment may be 50°C or higher, 51°C or higher, 52°C or higher, 53°C or higher, 54°C or higher, or 55°C or higher. The heat treatment may be 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, or 100°C or higher. The purification method may include performing the process at a temperature that reduces, minimizes, or eliminates the destruction of the native conformational state relative to typical heat treatments in industrial purification processes, with reference to the conformational state present in the natural milk source used for lactoferrin purification. The purification method may include performing the process at a temperature that reduces, minimizes or eliminates the biological activity of lactoferrin relative to typical heat treatments in industrial purification processes, with reference to the biological activity present in the natural milk source used for lactoferrin purification. The purification method may include receiving the natural milk source at a refrigerated temperature (e.g., at a temperature below 15°C, such as within a temperature between 2-15°C). The purification method described herein may include a heat treatment below 50°C. For example, the milk source may be heated during one or more purification steps, for example, to a temperature above 37°C but not exceeding 55°C. The heating may include a temperature above 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C. The heating may include a temperature above 37°C and a temperature below 55°C. The heating may include a temperature above 40°C and a temperature below 55°C. The heating may include a temperature above 45°C and a temperature below 55°C. Heating may include temperatures above 50°C and temperatures below 55°C. Heating may include temperatures above 37°C and temperatures of 50°C or less, 51°C or less, 52°C or less, 53°C or less, 54°C or less, or 55°C or less. Heating may include temperatures above 40°C and temperatures of 50°C or less, 51°C or less, 52°C or less, 53°C or less, 54°C or less, or 55°C or less. Heating may include temperatures above 45°C and temperatures of 50°C or less, 51°C or less, 52°C or less, 53°C or less, 54°C or less, or 55°C or less. The purification methods described herein may include heat treatments of 50°C or less, 51°C or less, 52°C or less, 53°C or less, 54°C or less, or 55°C or less. The purification methods described herein may comprise maintaining the temperature at less than 50°C or less, 51°C or less, 52°C or less, 53°C or less, 54°C or less, or 55°C or less during purification.Maintaining the temperature during purification may include maintaining the temperature throughout the purification process. Maintaining the temperature during purification may include maintaining the temperature in one or more individual steps (e.g., chromatography, filtration, and / or drying steps) of the purification process. The maintained temperature may include varying temperatures (e.g., different temperature ranges) specific to one or more individual steps of the purification process.

[0025] The purification method may include an acid treatment. Without wishing to be bound by theory, the acid treatment may be used to remove casein, such as treating a natural milk source or derivative at a pH that causes casein to become insoluble in solution prior to lactoferrin purification. Generally speaking, the acid treatment provided herein for producing purified lactoferrin does not include an acid treatment that destroys the native conformational state (e.g., denatures lactoferrin) and / or reduces the biological activity of lactoferrin (respectively with reference to the conformational state or biological activity present in the natural milk source for lactoferrin purification). The purification method may include an acid treatment at a pH of 4.0 or higher. The purification method may include an acid treatment at a pH of 3.0 or higher.

[0026] Purification methods may include chromatography. Purification methods may include ion exchange chromatography. Chromatographic methods, such as ion exchange chromatography are known to those skilled in the art. Purification methods described herein will generally include cation exchange chromatography. Purification methods may include ion exchange chromatography steps other than cation exchange chromatography, such as both cation and anion exchange chromatography, including one or more additional purification processes in any order and / or separate. Resins and matrices for ion exchange chromatography are known in the art. For example, cation exchange resins include, but are not limited to, polymethacrylates and agarose matrices. Purification methods may include high pressure liquid chromatography (HPLC).

[0027] Chromatographic methods generally include one or more equilibration and / or regeneration steps. Illustrative non-limiting examples of equilibration and regeneration steps include flushing with (1) reverse osmosis water; (2) flushing with chemically pure 1M NaCl; and (3) flushing again with reverse osmosis water.

[0028] Chromatographic methods typically include a loading step. Generally, the volume loaded is based on the predetermined binding capacity of the resin and an estimate of the native lactoferrin content of the raw milk feed material.

[0029] Chromatographic methods typically include one or more elution steps, such as eluting the purified lactoferrin from the resin / column in ion exchange chromatography. Elution methods are known to those skilled in the art. Elution methods may include two or more elution steps. Without wishing to be bound by theory, multiple elution steps may be used to first elute contaminants (e.g., any other product other than lactoferrin) and then elute the desired product (e.g., lactoferrin).

[0030] The elution method may include two or more elution steps at different salt concentrations. Without wishing to be bound by theory, one or more initial elution steps (eg, an elution step prior to an elution step containing the desired purified lactoferrin) may be performed to remove unwanted proteins and other contaminants.

[0031] The elution method may include a first elution step using a chemically pure NaCl solution between 0.2-0.7M. Typically, a first elution step of a chemically pure NaCl solution between 0.2-0.7M is performed to remove unwanted proteins and other contaminants. Without wishing to be bound by theory, the completion of the first elution may be monitored by UV-Vis spectroscopy and / or colorimetry for the presence of contaminants such as lactoperoxidase and other enzymes native to the raw milk feed material. For example, a first elution step of a chemically pure NaCl solution between 0.2-0.7M may be performed until a lack of protein eluted from the resin is detected by UV-Vis spectroscopy.

[0032] The elution method may include a first elution step of a 0.2M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25M chemically pure NaCl solution. The elution method may include a first elution step of a 0.30M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45M chemically pure NaCl solution. The elution method may include a first elution step of a 0.50M chemically pure NaCl solution. The elution method may include a first elution step of a 0.55M chemically pure NaCl solution. The elution method may include a first elution step of a 0.6M chemically pure NaCl solution. The elution method may include a first elution step of a 0.65M chemically pure NaCl solution. The elution method may include a first elution step of a 0.7M chemically pure NaCl solution. The elution method may include a first elution step of a chemically pure NaCl solution less than 1M.

[0033] The elution method may include a second elution step with a 1 M chemically pure NaCl solution. Typically, elution with a 1 M chemically pure NaCl solution will elute lactoferrin from the resin. The elution method may include a second elution step with about a 1 M chemically pure NaCl solution.

[0034] The elution method may include a first elution step of a chemically pure NaCl solution between 0.2-0.7M and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.2M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.25M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.3M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.35M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.40M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of a 0.45M chemically pure NaCl solution and a second elution step of a 1M chemically pure NaCl solution. The elution method may include a first elution step of 0.50M chemically pure NaCl solution and a second elution step of 1M chemically pure NaCl solution. The elution method may include a first elution step of 0.55M chemically pure NaCl solution and a second elution step of 1M chemically pure NaCl solution. The elution method may include a first elution step of less than 1M chemically pure NaCl solution and a second elution step of 1M chemically pure NaCl solution.

[0035] The elution method may include a first elution step of a chemically pure NaCl solution between 0.25-0.7M and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of a 0.20M chemically pure NaCl solution and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of a 0.25M chemically pure NaCl solution and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of a 0.30M chemically pure NaCl solution and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of a 0.35M chemically pure NaCl solution and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of a 0.40M chemically pure NaCl solution and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of a 0.45M chemically pure NaCl solution and a second elution step of a chemically pure NaCl solution of about 1M. The elution method may include a first elution step of 0.50M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of 0.55M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of 0.60M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of 0.65M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution. The elution method may include a first elution step of 0.70M chemically pure NaCl solution and a second elution step of about 1M chemically pure NaCl solution.

[0036] The elution method may include two or more elution steps at different pH levels, elution gradients. The elution method may include two or more elution steps at different salt concentrations, different pH levels, and combinations thereof. The elution method may include an elution gradient. The elution method may include a salt elution gradient. The elution method may include a pH elution gradient. The elution method may include both salt and pH elution gradients.

[0037] For each of the above-described ion exchange chromatography steps (e.g., equilibrium regeneration, loading and / or elution), one skilled in the art will recognize appropriate fluid rates, e.g., depending on the choice of resin, equipment, raw milk feed material, etc.

[0038] The purification method may include filtration. The filtration method is known to those skilled in the art. The purification method may include microfiltration (generally referring to filtration using a membrane pore size of 0.1 to 10 μm). The microfiltration may include a membrane pore size of 1-10 μm. The microfiltration may include a membrane pore size of 10 μm. The microfiltration may include a membrane pore size of 1-10 μm. The microfiltration may include a membrane pore size of 0.1-1 μm. The microfiltration may include a membrane pore size of 0.1 μm. The microfiltration may include a membrane pore size of 1 μm. The microfiltration may include a membrane pore size of 0.1, 0.2, 0.3, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9 and / or 1 μm. The microfiltration may include a ceramic filter.

[0039] The purification method may include ultrafiltration (generally refers to filtration using a membrane pore size of 0.01 to 0.1 μm). Ultrafiltration systems may also be referred to by the molecular weight cutoff size designed for size-based separation between permeate and retentate. Ultrafiltration systems may include 5-30 kDa systems. Ultrafiltration systems may include 5 kDa systems. Ultrafiltration systems may include 10 kDa systems. Ultrafiltration systems may include 15 kDa systems. Ultrafiltration systems may include 20 kDa systems. Ultrafiltration systems may include 25 kDa systems. Ultrafiltration systems may include 30 kDa systems.

[0040] The purification method may include both microfiltration and ultrafiltration, including one or more additional purification processes in any order and / or separately. The purification method may include multiple microfiltration and ultrafiltration steps, including one or more additional purification processes in any order and / or separately. As an illustrative non-limiting example, a first pre-filtration microfiltration step (e.g., using a 10 μm filter) may be used before ion exchange chromatography, a second microfiltration step (e.g., using a 0.1-1.4 μm filter) may be used after ion exchange chromatography, followed by an ultrafiltration step (e.g., using a 5-30 kDa).

[0041] Purification process may include a combination of chromatography and filtration, including one or more additional purification processes in any order and / or separately. Purification process may include a combination of multiple chromatography and / or filtration steps, including one or more additional purification processes in any order and / or separately. Purification process may include a combination of microfiltration, ultrafiltration and ion exchange chromatography, including one or more additional purification processes in any order and / or separately. In an illustrative non-limiting example, purification process may include ion exchange chromatography (including wash-out), followed by microfiltration, followed by ultrafiltration. In another illustrative non-limiting example, purification process may include microfiltration, followed by ion exchange chromatography (including wash-out), followed by other microfiltration, followed by ultrafiltration.

[0042] The purification method may include separating the raw dairy product into dairy derivatives, such as separating a natural milk source into skim milk and cream. For example, the raw dairy product may be separated into dairy derivatives prior to chromatography and / or filtration. Methods for separating the raw dairy product into dairy derivatives are known to those skilled in the art and include, but are not limited to, cold bowl separation.

[0043] After the lactoferrin is purified, the purified product can be dried. In general, the drying methods provided do not include treatments that destroy the native conformational state (e.g., denature the lactoferrin) and / or reduce the biological activity of the lactoferrin (with reference to the conformational state or biological activity present in the natural milk source used for the purification of the lactoferrin, respectively). Methods for drying lactoferrin are known to those skilled in the art and include, but are not limited to, freeze drying / lyophilization, fluidized bed drying, and / or low temperature spray drying.

[0044] In some embodiments, purity can be assessed. Natural milk sources (including cow's milk) generally contain a variety of protein components other than lactoferrin, including but not limited to lactoperoxidase, lysozyme, casein, immunoglobulin, whey protein and lactoglobulin. Natural milk sources may also contain other components, such as fat and endotoxins. In general, the purification methods provided herein reduce, minimize or eliminate components other than lactoferrin. Without wishing to be bound by theory, removing one or more additional components may improve lactoferrin biological activity and / or improve safety.

[0045] Provided herein are lactoferrin compositions having an increased percentage of lactoferrin mass relative to the ratio present in an unprocessed lactoferrin-containing dairy product.

[0046] The lactoferrin percentage can be assessed by mass spectrometry, including but not limited to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry and / or linear trap quadrupole Orbitrap Velos mass spectrometry. Typically, the assessment by mass spectrometry includes quantifying the area under the peak corresponding to lactoferrin and the area under the peak not corresponding to lactoferrin. In some cases, lactoferrin may be associated with multiple peaks, such as an ionization peak corresponding to lactoferrin. The peak corresponding to lactoferrin may include a peak corresponding to full-length post-translationally modified (e.g., glycosylated) lactoferrin. The peak corresponding to full-length post-translationally modified lactoferrin is generally in the range of about 80,000-86,000 m / z. The exact peak of the full-length post-translationally modified may vary, such as reflecting different glycosylation states. In some cases, for inclusion, the peak between 79000-90000 of m / z may be considered to correspond to lactoferrin. The area under the peak not corresponding to lactoferrin includes all other peaks, with the possible exception of the ionization peak of about 41,500 m / z associated with lactoferrin (e.g., having an m / z between 41000-42000). The area under the peak not corresponding to lactoferrin may include peaks with m / z between 18000-80000 (in addition to the peak with m / z between 41000-42000). A specific comparison may also be made between lactoferrin and the peak corresponding to lactoperoxidase (e.g., having an m / z between 77000 and 78000). The area under the peak not corresponding to lactoferrin may include peaks with m / z between 18000-45000 (in addition to the peak with m / z between 41000-42000). Linear trap quadrupole Orbitrap Velos mass spectrometry can also quantify the percentage of lactoferrin relative to other components in the sample. For example, quadrupole Orbitrap Velos mass spectrometry can quantify the percentage of lactoferrin by determining the percentage of peptide spectrum matches (PSMs).

[0047] The percentage of lactoferrin may be assessed by liquid chromatography, such as high performance liquid chromatography (HPLC). Assessment by liquid chromatography may include quantifying the area under the peak corresponding to lactoferrin and the area under the peak not corresponding to lactoferrin.

[0048] For assessments involving peak quantification, one or more areas under the peaks that do not correspond to lactoferrin present in the unprocessed lactoferrin-containing dairy product may be below the detection limit of the purified lactoferrin composition. In some cases, each area under the peaks that do not correspond to lactoferrin may be below the detection limit.

[0049] The percentage of lactoferrin can be assessed by enzyme-linked immunosorbent assay (ELISA). In some cases, ELISA can distinguish the percentage of lactoferrin in the native protein conformation by using antibodies that specifically bind to the native protein conformation of lactoferrin.

[0050] When purifying lactoferrin, a specific component that is typically present in unprocessed milk sources to be reduced, minimized or eliminated is lactoperoxidase. In addition, lactoperoxidase is often present in detectable amounts in purified lactoferrin compositions produced by typical industrial processes.

[0051] Provided herein are lactoferrin compositions wherein at least 70% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 75% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 80% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 85% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 90% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 95% of the composition is purified lactoferrin. Compositions include those compositions wherein at least about 100% of the composition is purified lactoferrin. Compositions include those compositions wherein at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the composition is purified lactoferrin. Compositions include those wherein at least 91% of the composition is purified lactoferrin. Compositions include those wherein at least 92% of the composition is purified lactoferrin. Compositions include those wherein at least 93% of the composition is purified lactoferrin. Compositions include those wherein at least 94% of the composition is purified lactoferrin. Compositions include those wherein at least 95% of the composition is purified lactoferrin. Compositions include those wherein at least 96% of the composition is purified lactoferrin. Compositions include those wherein at least 97% of the composition is purified lactoferrin. Compositions include those wherein at least 98% of the composition is purified lactoferrin. Compositions include those wherein at least 99% of the composition is purified lactoferrin.

[0052] Provided herein are lactoferrin compositions having an increased lactoferrin:lactoperoxidase ratio relative to the ratio in an unprocessed lactoferrin-containing dairy product. The ratio of lactoferrin:lactoperoxidase can be assessed according to methods known to those skilled in the art, such as the purity assessment methods described herein (e.g., mass spectrometry, HPLC, and / or ELISA). For example, the assessment can include quantifying one or more areas under a peak corresponding to lactoferrin and an area under a peak corresponding to lactoperoxidase.

[0053] Endotoxins present or potentially present in natural milk sources may be reduced, minimized or eliminated. Without wishing to be bound by theory, removal of endotoxins may improve the safety of the purified lactoferrin composition. Methods for assessing endotoxin levels are known to those skilled in the art.

[0054] Also provided herein are methods of assessing the purity of a purified lactoferrin composition, such as using any of the purity assessment methods described herein.

[0055] Fractal Subsystems:

[0056] See also Figure 1 , Figure 2A and Figure 2B , an exemplary embodiment of a fractal subsystem 100 for use in conjunction with an ion exchange chromatography system 200 is shown. Unlike conventional mixing systems that include a columnar shape of approximately fixed height and have a substantially vertical orientation and further use turbulence to mix fluids, the customizable fractal subsystem 100 uses a scaling structure whose shape can be arranged in any practical desired size (e.g., length) in a substantially horizontal orientation. More specifically, the fractal subsystem 100 can be constructed and arranged to achieve fluid scaling and mixing using multiple engineered channels that ensure that scaling is completed before the components to be mixed contact each other. Advantageously, fractal scaling eliminates turbulence, large-scale process non-uniformities, and / or side reactions.

[0057] Due to the large number of applications in which it can be used, the various components of the fractal subsystem 100 can be made from a variety of materials (e.g., metals, plastics, carbon fiber, etc.). Advantageously, the ability to use a large number of materials ensures that for discrete applications, custom materials can be used so that the material meets the requirements of the raw materials, in-situ cleaning chemicals, solvents, or elution buffers. However, for use with food and drugs, federal law requires that the components of the fractal subsystem 100 be made from FDA 3A approved materials. In an exemplary embodiment, the components of the fractal subsystem 100 can be made from polypropylene, such as manufactured by Boedeker Plastics, Inc, Shiner, Texas P natural homopolymer.

[0058] In some embodiments, the fractal subsystem 100 includes a (e.g., modular) resin chamber 20 disposed between a pair of fractal packages 10a, 10b. This arrangement in a single system 200 enables multiple fractal subsystems 100 and correspondingly multiple resin chambers 20. Advantageously, the flow volume can be selectively (e.g., evenly) divided between multiple resin chambers 20, and the flow rate in each resin chamber 20 can be kept equal on all resin beds. Having fractal packages 10a, 10b on opposite sides of the resin chamber 20 enables the flow direction to be reversed during processing, which provides greater resin utilization and reduces single-point inlet clogging. In some specific implementations, a single fractal package combined with a resin chamber 20 can be provided for the purpose of easier cleaning. (e.g., modular) The resin chamber 20 allows more efficient resin filling and removal. In fact, enhanced resin filling and removal extend the service life of the resin because the backwashing (e.g., in a cleaning-in-place operation) and removal process of the resin improve the success rate of cleaning.

[0059] Advantageously, each of the fractal packages 10a, 10b may include a frame 12 surrounding an internal pressurized space 14. In some specific implementations, the fractal distributor 11 and the fractal flow plate 13 may be disposed within the internal pressurized space 14 so that the fractal distributor 11 and the fractal flow plate 13 can be releasably or removably attached to the frame 12. In some applications, the fractal distributor 11 may be constructed and arranged to provide a channel array that evenly divides the incoming fluid with a laminar flow, further ensuring that the flow is evenly provided to the resin chamber 20. In some variations, the fractal flow plate 13 receives the fluid from the fractal distributor 11 and then supplies the fluid to the resin chamber 20. In some variations, the fractal distributor 11 may be a low volume distributor, which advantageously can produce better peak resolution and less gradient mixing. More specifically, the low system / component retention volume in the fractal distributor 11 allows for more precise solvent control and minimal mixing with the previous step fluid. Furthermore, the low volume distributor provides a more uniform elution gradient contact with the resin and supply feed.

[0060] To prevent fluid from bypassing the resin, a (e.g., 3 / 8 inch) weir may be incorporated or integrated into the upper or top corners of the internal plenum space 14. Advantageously, providing a weir on the edge of the resin chamber 20 ensures 100% contact of the fluid with the raw resin, a degree of fluid contact that cannot be achieved using conventional columnar arrangements. For purposes of illustration only, the frame 12 and internal plenum space 14 have a generally rectangular or rounded rectangular shape.

[0061] Around the outer periphery of the frame 12, one or more (e.g., two) support arms 16 and one or more feed ports 18 are fixedly attached to the frame 12. The support arms 16 are constructed and arranged to enable a user to manipulate the frame 12 and support the frame 12 on the filter press 200, while the feed ports 18 are constructed and arranged to introduce fluid into the fractal distributor 11 and / or the fractal flow plate 13 and / or remove fluid from the fractal distributor and / or the fractal flow plate.

[0062] In some implementations, the resin chamber 20 includes a frame 22 around an internal pressurized space 24. For illustrative purposes only, the frame 22 and the internal pressurized space 24 have a generally rectangular or rounded rectangular shape. In some implementations, one or more (e.g., two) mesh screens 23 may be arranged in the internal pressurized space 24 so that the mesh screen 23 can be releasably or removably attached to the frame 22. The size of the opening in the mesh screen 23 depends to a large extent on the type of resin used. In an exemplary implementation, the mesh screen 23 may include a 200 mesh plain Dutch weave (316SS) having a geometric opening of 85 microns in the mesh and a nominal opening of 75 microns in the mesh. Table 1 summarizes the maximum, minimum, and nominal mesh sizes.

[0063] Advantageously, the mesh size facilitates the passage of larger materials and more viscous products through the resin and mesh 23. In operation, in one implementation, a fluid may be introduced into the system 100 via a first feed port 18 in a first fractal bag 10a, and caused to flow through the resin chamber 20, and removed from the system 100 via a second feed port 18 in a second fractal bag 10b. Advantageously, the system 100 is bidirectional; thus, a fluid may be introduced into the system 100 via a second feed port 18 in a second fractal bag 10b, and caused to flow through the resin chamber 20, and removed from the system 100 via a first feed port 18 in a first fractal bag 10a.

[0064] Around the outer periphery of the frame 22, one or more support arms 26 and one or more resin ports 28a, 28b are fixedly attached to the frame 22. The support arms 26 may be constructed and arranged to enable a user to manipulate the frame 22 and support the resin chamber 20 on the filter press 200, while the resin ports 28a, 28b may be constructed and arranged to introduce resin into the mesh 23 and remove resin from between the meshes.

[0065] Table I

[0066]

[0067] system:

[0068] See also Figure 2A and Figure 2B, an exemplary filter press 200 for use with a fractal subsystem 100 is shown. Unlike conventional systems, the exemplary filter press 200 has an adjustable bed depth, the depth of which depends on the number of fractal subsystems 100 introduced or incorporated into the filter press 200. The adjustable bed depth allows high flow rates and low back pressure. The exemplary filter press 200 using multiple fractal subsystems 100 can also achieve directional flow, which minimizes channeling and therefore provides greater resin utilization. Table I provides exemplary nominal flow rates through the system 100. It will be appreciated by those skilled in the art that achievable and desired flow rates can be based on the design of the fractal packs 10a, 10b and the resin chamber 20. Therefore, due to the inherent modular capabilities of the system 200, the resin chamber 20 can be designed to handle even faster speeds.

[0069] In some embodiments, the filter press 200 includes a pair of (e.g., vertical) supports 110, 120 that are, among other things, constructed and arranged to support a pair of horizontal or substantially horizontal supports or side bars 130 that are constructed and arranged to support one or more fluid-coupled fractal subsystems 100. For example, in some variations, the side bars 130 support support arms 16, 26 disposed on opposite sides of the frames 12, 22 of the fractal packs 10a, 10b and the resin chamber 20.

[0070] In some implementations, the filter press subsystem 150 can be disposed between and supported by the side bars 130. In some variations, the filter press subsystem 150 can include a selectively movable rod 140 operably coupled to a hydraulic press 160 at a proximal (e.g., fixed) end of the rod 140 and operably coupled to a follower 170 at a distal (e.g., movable) end of the rod 140. A hydraulic cylinder 180 can also be disposed at the proximal end of the rod 140 such that operation of the hydraulic cylinder 180 advances the rod 140 toward the distal end or retracts it to the proximal end.

[0071] In operation, one or more fractal subsystems 100 may be disposed between the follower 170 and the distal vertical support 120. Advantageously, unlike conventional systems, the bed depth height (or length) is selectively adjustable and is not fixed. In fact, any practical number of fractal subsystems 100 may be disposed between the follower 170 and the distal vertical support 120 on the filter press 200. Thus, the column produced by the fractal subsystem 100 is mobile, modular, and horizontally rather than vertically oriented. In addition, the bed depth of the column is selectively adjustable (e.g., between about 1 inch and eight feet). Advantageously, the fractal subsystem 100 and the filter press 200 also allow bidirectional flow through the column.

[0072] Once the desired number of fractal subsystems 100 are disposed on the filter press 200, the feed ports 18 are fluidly connected to a fluid source or fluid discharge port, and the resin ports 28a, 28b are fluidly connected to a resin source or resin discharge port, operation of the hydraulic press 160 may cause the follower 170 to compress the fractal subsystem 100 toward the distal end of the rod 140. Advantageously, when the follower 170 compresses the fractal subsystem 100 and when an incompressible liquid is introduced into the fractal subsystem 100 (e.g., via one or more of the feed ports 18), the liquid is forced through the fractal distributor 11, the fractal flow plate 13, and the resin contained in the resin chamber 20. Advantageously, the combination of the filter press 200 and the fractal subsystem 100 provides a laminar flow, a uniform elution gradient flow, and a uniform feed flow across and through the resin disposed between the mesh screens 23 of the resin chamber 20. Since in some applications, the fluid flow may be bidirectional, single outlet fouling of the feed at the inlet to the resin may be reduced.

[0073] Example:

[0074] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the values ​​used (e.g., quantities, temperatures, etc.), but of course some experimental errors and deviations should be taken into account.

[0075] Unless otherwise indicated, the implementation of the present invention will adopt conventional protein chemistry, biochemistry, recombinant DNA technology and pharmacological methods belonging to the skills of the art. Such technology is fully described in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A Lehninger, Biochemistry (Worth Publishers, Inc., latest edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan ed., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd edition (Plenum Press) Volumes A and B (1992).

[0076] Example 1. Purification of lactoferrin

[0077] like Figure 1 In brief, raw milk (untreated milk, e.g., milk that has not been chemically, enzymatically, acidically, or thermally treated prior to lactoferrin purification) is (1) transferred prior to entering an industry standard milk processing workflow; (2) passed through an ion exchange resin / column, wherein the effluent is typically returned to the standard milk processing workflow and the lactoferrin-containing eluate is collected; (3) the collected eluate is filtered; and (4) the purified lactoferrin is processed. Notably, all other known lactoferrin purification processes begin with a milk source obtained after an industry standard milk processing workflow that typically includes heat pasteurization. Furthermore, the use of natural, unprocessed dairy products (e.g., raw milk) as source material results in lactoferrin containing relevant post-translational modifications that may be involved in biological activity, in contrast to recombinantly produced lactoferrin.

[0078] Chromatographic columns were used to separate and purify bovine lactoferrin to preserve its native post-translational modifications, glycosylation, and bound iron. The columns were loaded with selected cation exchange resins (either polymethacrylate or agarose matrix) and equilibrated and regenerated by (1) washing with reverse osmosis water; (2) washing with chemically pure 1 M NaCl; and (3) washing again with reverse osmosis water.

[0079] Raw, untreated, unprocessed bovine whole milk (less than 24 hours after milking) is obtained directly from dairy transport or silo containers and then separated, skimmed, heated and / or pasteurized. Lactoferrin is purified from raw colostrum (RC) and raw whole milk (RM).

[0080] The milk is filtered of large particles with a 10 μm filter and heated to above 37°C and maintained at a temperature below 63°C (generally considered the starting temperature for pasteurization) before loading into the column. The volume loaded is based on a predetermined binding capacity and an estimate of the native lactoferrin content of the raw milk feed material. During the loading process, all flow-through is returned to the pasteurizer balance tank to return the feed to the plant to a point prior to separation and pasteurization.

[0081] Once loading is complete, the resin is rinsed with reverse osmosis water to remove any remaining milk compounds that are not bound to the resin. The first elution involves washing the resin with a chemically pure NaCl solution (0.2-0.7 M) and is monitored and assessed for completion (i.e., lack of protein eluted from the resin) by UV-Vis spectroscopy and / or colorimetric assays that monitor the presence of contaminants such as lactoperoxidase and other enzymes native to the raw milk feed material. In particular, the first NaCl is performed until lactoperoxidase (usually the major contaminant) is no longer present in the eluted fractions, as assessed by a peroxidase colorimetric assay using a colorimetric peroxidase substrate. The fractions from the first elution are retained in a separation vessel. The resin is then rinsed with reverse osmosis water. A second elution is performed with 1 M NaCl to remove the separated lactoferrin. The fractions from the second elution are retained in a separation vessel and then (1) filtered through a ceramic microfiltration filter (0.1-1.4 μm); and (2) concentrated using an ultrafiltration system (5-30 kDa).

[0082] The desalted eluate is then freeze dried, but can also be directly passed into a fluid bed dryer to dry into a pharmaceutical grade excipient. Purified lactoferrin produced according to the methods described herein is referred to as: Hyacinth lactoferrin ("Hyacinth"); Lactoferrin (only without the words "laboratory grade" or "commercial supplement grade" attached; ODT-SC210; and API-E2. Various names may refer to purified lactoferrin produced according to variations of the methods described herein.

[0083] Having thus described several aspects of at least one embodiment of the present invention, it will be appreciated that various changes, modifications and improvements will readily occur to those skilled in the art. Such changes, modifications and improvements are intended to be part of this disclosure and are intended to fall within the spirit and scope of the present invention. Therefore, the foregoing description and accompanying drawings are intended only as examples.

Claims

1. A fractal subsystem for use with an ion exchange system, the fractal subsystem comprising: Resin room; as well as A pair of fractal packs, wherein the pair of fractal packs are disposed on opposite sides of the resin chamber.

2. The fractal subsystem of claim 1, wherein the resin chamber comprises: frame; a plenum space extending through the frame, the plenum space being adapted to contain resin; a pair of mesh screens for containing the resin within the pressurized space; as well as A pair of resin ports adapted to at least one of introduce fluid into the plenum or remove fluid from the plenum.

3. The fractal subsystem of claim 2, wherein the mesh screen comprises a 200 mesh plain Dutch weave.

4. The fractal subsystem of claim 2, wherein said mesh screen has a nominal opening in said mesh of 75 microns.

5. The fractal subsystem of claim 2, wherein said mesh screen has a geometric opening of 85 microns in said mesh.

6. The fractal subsystem of claim 1, wherein each of the fractal packages comprises: frame; a plenum space extending through said frame; A fractal distributor, the fractal distributor being arranged in the pressurized space; A fractal flow plate, the fractal flow plate being arranged in the pressurized space; as well as A feed input port is adapted to introduce fluid into the pressurized space.

7. The fractal subsystem of claim 6, wherein a weir is formed within the plenum space in each upper corner of the frame.

8. A system for purifying a biologically active protein in a fluid, the system comprising: At least one fractal subsystem, each fractal subsystem comprising: Resin chamber; and a pair of fractal packs, wherein the pair of fractal packs are disposed on opposite sides of the resin chamber; and A filter press constructed and arranged to support at least one fractal subsystem in a horizontal arrangement to provide a selectively adjustable bed depth, wherein each fractal subsystem is in fluid communication with each other fractal subsystem.

9. The system of claim 8, wherein the resin chamber comprises: frame; a plenum space extending through the frame, the plenum space being adapted to contain resin; a pair of mesh screens for containing the resin within the pressurized space; as well as A pair of resin ports adapted to at least one of introduce fluid into the plenum or remove fluid from the plenum.

10. The system of claim 9, wherein the mesh screen comprises a 200 mesh plain Dutch weave.

11. The system of claim 9, wherein the mesh screen has a nominal opening in the mesh of 75 microns.

12. The system of claim 9, wherein the mesh screen has a geometric opening of 85 microns in the mesh.

13. The system of claim 8, wherein each of the fractal packages comprises: frame; a plenum space extending through said frame; A fractal distributor, the fractal distributor being arranged in the pressurized space; A fractal flow plate, the fractal flow plate being arranged in the pressurized space; as well as A feed input port is adapted to introduce fluid into the pressurized space.

14. The system of claim 13, wherein a weir is formed within the plenum space in each upper corner of the frame.

15. The system of claim 8, wherein the filter press comprises: a pair of vertical supports; a pair of horizontal supports fixedly attached to the vertical supports, wherein the horizontal supports are constructed and arranged to support the fractal bag; as well as A filter press subsystem is constructed and arranged to apply a compressive force to the fractal pack.

16. The system of claim 15, wherein the filter press subsystem comprises: a rod capable of selective movement; a follower disposed at a distal end of the movable rod; as well as A hydraulic press is disposed at the proximal end of the movable rod.

17. The system of claim 8, wherein the selectively adjustable bed depth ranges between one (1) inch and eight (8) feet.

18. The system of claim 8, wherein the system is capable of bi-directional flow.

19. The system of claim 8, wherein the system generates laminar flow in the fluid.

20. The system of claim 8, wherein the fluid comprises a supply feed having a fat content greater than 0.1% and less than five (5) percent.

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