Method for depletion or removal of endotoxin from endotoxin-containing source or potentially endotoxin-containing source

Abstract

To provide a method for depletion or removal of endotoxins from a known or suspected endotoxin-containing source.SOLUTION: A method for depletion or removal of endotoxins from a known or suspected endotoxin-containing source comprises the steps of: in an essentially aqueous system, bringing the source into contact with a positively charged solid phase material having a surface on which ferric iron is immobilized by a (2-aminoethyl)amine (TREN) ligand; incubating the source for a period of time sufficient to bind the endotoxins to the porous solid phase material; separating the solid phase material from the essentially aqueous system; and optionally isolating the aqueous system freed or depleted from the endotoxins.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for depleting or removing endotoxins from an endotoxin-containing supply and to various fields in which the method of the invention may be used, as well as to fractions obtainable by the method of the invention. [Background technology]

[0002] Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria. When such bacteria die and degrade, LPS, also known as endotoxin, is released into the surrounding environment. Endotoxin is stable and persists for long periods of time even under extreme conditions. Endotoxin is a concern for the biopharmaceutical industry because it is highly toxic. Even amounts as low as a few ppm can cause a pyrogenic reaction. Larger doses can cause organ failure and death.

[0003] Potential sources of endotoxins in the field of bioprocessing include: Some products are grown in gram-negative bacteria and therefore are heavily contaminated with endotoxins. Cell cultures for producing biopharmaceuticals, protein solutions, buffers and other process solutions can become contaminated (infected) by gram-negative bacteria which release endotoxins into the solution. Endotoxins may be present even when viable gram-negative bacteria are absent or have been present recently. For example, dry chemical components used to formulate processing solutions can become contaminated with endotoxins. Process solutions can also become contaminated by accidental contact with contaminated supplies due to inadequate process hygiene. Again, actual infection with live bacteria is not necessary. Endotoxin supplies are much more diverse and widely distributed than supplies of live bacteria.

[0004] Regulatory authorities around the world recognize that endotoxin contamination can have serious adverse effects on people receiving treatment and therefore set strict limits for acceptable endotoxin concentrations. For example, recent publications have noted that the US Food and Drug Administration in particular is often not satisfied with the adequacy of manufacturing methods to adequately control endotoxin levels in final biopharmaceutical products [1,2].

[0005] This puts pressure on manufacturers in the biopharmaceutical industry to design processes that effectively remove endotoxins. More effective processes require more effective tools.

[0006] Currently, various solutions are available to reduce endotoxin levels in biopharmaceuticals. One of the oldest and most widely trusted methods is anion exchange chromatography. Anion exchangers have a positive charge, and they bind primarily to biomolecules through charge interactions. Specifically, biomolecules with sufficient negative charges bind to the positive charges on the anion exchanger. Endotoxins are negatively charged and bind strongly to the anion exchanger. In most cases, endotoxins bind more strongly to the anion exchanger than the biopharmaceutical product of interest. This allows for the selective elution of the product of interest, which is primarily endotoxin-free. Endotoxin removal performance by quaternary anion exchangers (Q, QA, QAE, TMAE) is the standard against which other endotoxin removal methods are compared [3-7].

[0007] There are many variations of anion exchange chromatography that function similarly, including so-called multimodal or mixed-mode chromatography media. The term multimodal means that they utilize a combination of two or more chemical mechanisms to bind biomolecules, such as, among others, positive and negative charges, aromatic and aliphatic hydrophobic groups, hydrogen donors and hydrogen acceptors. Virtually all multimodal media used to bind endotoxins are positively charged [3-5]. Examples include immobilized amino acids such as histidine or histamine. In other cases, synthetic chemical structures combine at least one positive charge with at least one hydrophobic residue. In some cases, the combination of positively charged and hydrophobic residues is further combined with residues that mediate hydrogen bonds. In other cases, complex biological proteins with high affinity for endotoxins are immobilized on chromatographic surfaces to selectively capture endotoxins. However, these ligands are still governed by their positive charge and their hydrophobic effects [8].

[0008] Metal affinity chromatography represents another class of tools for endotoxin removal. It is known to use iron or calcium immobilized on the chelating ligand iminodiacetic acid (IDA) for endotoxin removal [9,10]. However, this method has practical drawbacks. IDA has a net charge of 2-; calcium has a net charge of 2+, and iron has a net charge of 2+ or 3+. The resulting ligand:metal complex has a net charge of either 0 or a weak +1. As a practical consequence, many proteins and other biological agents cannot be bound; they are diluted by processing and require follow-on methods to be concentrated. Another drawback is that many non-endotoxin contaminants also do not bind and remain together with the product of interest in the unbound fraction. This is important since therapeutic applications require that endotoxins of host proteins be reduced to very small levels. As a practical consequence, further purification steps are required. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0009] It is an object of the present invention to provide an improved method for the depletion or removal of endotoxins in endotoxin-containing or potentially endotoxin-containing sources. Another object of the present invention is to provide a commercially applicable method for the depletion or removal of endotoxins, useful in the pharmaceutical or food industries. Yet another object of the present invention is to provide a product having a lower endotoxin content than previously manufactured products, which may be obtained by a process having processing steps that pose a risk of contaminating the product with endotoxins. [Means for solving the problem]

[0010] The object of the present invention is a method for the depletion or removal of endotoxins from an endotoxin-containing feed by solid phase material extraction in an essentially aqueous system, comprising: providing a known or suspected endotoxin-containing source; contacting the feed, known to contain endotoxins or suspected to contain endotoxins, with a positively charged solid phase material having a surface onto which ferric iron is immobilized, wherein the solid phase extraction material has the ferric iron immobilized via a (2-aminoethyl)amine (TREN) ligand; incubating the supply, known to contain endotoxin or suspected to contain endotoxin, for a time sufficient to allow endotoxin to bind to the porous solid phase material; separating the solid phase material from the aqueous system; This is achieved by a method which may optionally include the step of isolating said aqueous system which has been rendered free or depleted of endotoxins.

[0011] The method comprises: 3+ A solid phase is used that contains the immobilized ligand TREN carrying the ligand.

[0012] The term "endotoxin" includes compounds from gram-negative bacteria known under the term lipopolysaccharide (LPS). According to one embodiment of the invention, endotoxin is understood as an endotoxin or endotoxin fragment that is pathogenic. The terms "lipopolysaccharide", "endotoxin" or "endotoxin fragment" can be used interchangeably.

[0013] "Endotoxin-containing" source means any source that is or may be contaminated with endotoxins or endotoxin fragments. The source may be an essentially aqueous solution or dispersion suitable for solid phase extraction. Typically, an endotoxin-containing source or a source that may contain endotoxins may be, but is not limited to, a naturally occurring biological fluid that contains at least one component of interest that is or may be contaminated with endotoxins, such as, for example, a fermentation broth or a medium that has been used in a recombinant production process.

[0014] "Solid-phase extraction material having a surface on which ferric iron is immobilized" refers to a solid-phase extraction material in which the primary mechanism is a solid phase containing ferric iron bound by chelation, and "ferric ions immobilized by (2-aminoethyl)amine (TREN)" refers to a solid phase in which the primary mechanism is ferric iron bound by chelation, with the positive charge of the ferric ion-binding ligand serving as a secondary enhancing mechanism.

[0015] The term "solid phase extraction" refers specifically to a sample preparation method in which compounds dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, cell cultures, water, beverages, soil, and animal tissues. Solid phase extraction generally uses the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase) for a solid (known as the stationary phase) through which the sample is passed to separate the mixture into desired and undesired components. As a result, either the desired compounds of interest or the undesired impurities in the sample are retained on the stationary phase. The fraction that passes through the stationary phase is either collected or discarded depending on whether it contains the desired compounds or the undesired impurities. If the fractions retained on the stationary phase contain the compounds of interest, they can then be removed from the stationary phase for recovery in an additional step in which the stationary phase is rinsed with an appropriate eluent.

[0016] The term "optionally isolating the concentrated product from an aqueous system from which endotoxins, host cell proteins, and host cell DNA have been removed or depleted" refers to a step in which the product concentrated on the surface of the solid phase is selectively released by increasing the concentration of salt to disrupt the interaction between the positive charges of the ligands and the product, while endotoxins and DNA remain bound by their affinity for the chelated ferric iron.

[0017] The term "suspected endotoxin-containing source" means a source that is not yet known to contain endotoxin, but that is at risk of containing endotoxin.

[0018] According to the present invention, endotoxins interact with ferric iron immobilized on the surface of a solid phase material (stationary phase) by chelation. As a result, the mobile phase flowing through the solid phase extraction material depletes or removes endotoxins. Also according to the present invention, negatively charged endotoxins interact with the surface of the solid phase, which is positively charged by being covalently coated with the electropositive chelating ligand tris(2-aminoethyl)amine, also known as TREN. It is understood that the cooperativity between these two interaction modes is the basis for the expanded utility of the present invention.

[0019] According to the present invention, TREN-Fe 3+ The surface of the solid phase material having the structure preferably is positively charged by at least +1 or +2 or +3 per ligand.

[0020] "Chelation" refers to the stable chemical entrapment of metal ions. It involves the formation or existence of two or more separate coordinate bonds between a multidentate chelating ligand and a single central metal atom. Usually, these ligands are organic compounds and are called chelants, chelators, chelating agents or sequestering agents. In most cases, the forces of chelation are many times stronger than simple ionic interactions, e.g., 15-60 times stronger, allowing metal ions to remain trapped by the chelating ligands over a wide range of pH values ​​and even in the presence of high concentrations of salt.

[0021] In accordance with the present invention, the solid phase material has immobilized ferric iron by means of the positively charged chelating ligand tris(2-aminoethyl)amine (TREN).

[0022] According to one embodiment of the present invention, the inherent electropositive charge of the chelating ligand TREN is a key contributor to the improved performance of the present invention compared to metals immobilized on negatively charged chelating ligands. It will be appreciated that the purely positive charge of TREN gives it an affinity for negatively charged endotoxins, even if it is independent of the affinity mediated by the chelated ferric ions. Positively charged anion exchange chromatography media, which are understood to work only by charge interactions, are known to exert such interactions with endotoxins. Similarly, it will be appreciated that the electropositivity of TREN gives it a strong affinity for contaminating DNA and RNA that may be present in the sample. In the absence of metal affinity, negatively charged ligands tend to repel negatively charged biomolecules such as endotoxins, DNA, and RNA. A further practical and important advantage of the positively charged TREN compared to negatively charged chelating ligands such as IDA and NTA is that the majority of biological products from which it may be desired to remove endotoxins can be bound by electrostatic interactions and thereby concentrated from a dilute feed stream. In the original description of ferric ions immobilized on negatively charged chelating ligands, it was shown that most of the products did not bind, which ultimately led to a dilution of the products compared to the samples to which they were applied [9, 10].

[0023] In one embodiment, the endotoxin-contaminated product is contacted with a solid surface comprising a negatively charged chelating ligand bearing iron (such as IDA-Fe or NTA-Fe). This reduces the endotoxin content of the liquid phase containing the product of interest. The liquid phase is then contacted with a positively charged chelating solid phase such as TREN-Fe, which has the effect of concentrating the product of interest from its dilute state, following the previous step. Selective elution of the product from TREN-Fe leaves behind endotoxins that are still bound. It will be appreciated that this approach may be useful when the product of interest is heavily contaminated with endotoxins to the extent that no single method can achieve the desired low endotoxin levels in a single run.

[0024] According to another embodiment, the solid phase material can have immobilized ferric iron by means of the more positively charged chelating ligands, diethylenetriamine, triethylenetetramine, tetraethylpentamine, N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl)propanoylaminopentyl]-N-hydroxy-butanediamide (deferoxamine), 3-hydroxy-1,2-dimethylpyridin-4(1H)-one (deferiprone), 4-[(3Z,5E)-3,5-bis(6-oxo-1-cyclohexa-2,4-dienylidene)-1,2,4-triazolidin-1-yl]benzoic acid (deferasirox), bound to the surface of the solid phase material. These ligands are positively charged.

[0025] According to further embodiments of the invention, the solid phase material may comprise a chromatographic material that depletes or removes endotoxins by ferric chelation and / or complementary electrostatic interactions from supplies known to contain endotoxins or suspected to contain endotoxins.

[0026] According to an embodiment of the invention, the solid phase extraction is in particular a method selected from the group consisting of chromatography, filtration on chromatographic materials, co-precipitation and combinations thereof. Co-precipitation can include the situation where particles of a positively charged solid phase extraction material containing ferric ions are added to an endotoxin-containing or potentially endotoxin-containing feed. The positively charged solid phase extraction material containing ferric ions binds to the endotoxin and the particles coprecipitated with the endotoxin can then be filtered or sedimented, including by sedimentation with the aid of centrifugation or ultrasonic precipitation.

[0027] According to the present invention, the term "chromatographic material" includes any solid phase whose surface has been chemically modified for the purpose of adsorbing biomolecules, including porous particles, including porous chromatographic particles, non-porous particles, including non-porous chromatographic particles, membranes, including porous and non-porous membranes, macroreticular (three-dimensional scaffold) materials, including but not limited to monoliths and hydrogels, columns, or individual fibers packed amorphously into a web that is a sheet, rolled sheet, pleated sheet, or other solid phase material with a physical configuration that can be contacted with aqueous solutions that contain or may contain endotoxins by passing a fluid through it.

[0028] In yet another embodiment of the present invention, the solid phase extraction material advantageously removes entrained viruses from supplies containing or potentially containing endotoxins.

[0029] In some embodiments, monolithic chromatography formats are particularly advantageous due to their low internal dispersion, lack of turbulent shear forces, and high capacity for large solutes such as endotoxins, DNA, and viruses. Its higher capacity for large solutes compared to other chromatography formats is particularly important since endotoxins are known to exist as very large aggregates. Higher capacity translates into higher confidence that the capacity will not be the limiting factor in endotoxin removal efficiency. The utility of monoliths also increases when the desired product itself is large, for example, a virus, including especially bacteriophages, since high capacity for both bacteriophages and endotoxins is required.

[0030] In another embodiment of the invention, the pH value may be adjusted by a physiologically acceptable acid or base and / or the ionic strength is adjusted by a physiologically acceptable buffer salt, which may be selected from the group consisting of acetate, citrate, phosphate, TRIS (tris-hydroxyaminomethane), imidazole, histidine, histamine, triethylamine, and salts of zwitterionic (positively and negatively charged at the same time) buffer components such as HEPES (hydroxyethylpiperazineethanesulfonic acid) and MES (morpholinoethanesulfonic acid), among others.

[0031] In most embodiments, the recommended operating pH for use in the methods of the invention may be within the range of pH 7.0±1.5, or pH 7.0±1.0, or pH 7.0±0.5. The pH range may be extended if necessary, for example, to pH 7.0±2.0, or broader if necessary if the product of interest is known to tolerate these ranges. The ability of the method to remove endotoxins and nucleic acids remains within these ranges.

[0032] In most embodiments, the conditions for equilibrating the TREN-Fe3+ solid phase are near physiological, including a pH in the range of 7.0±0.5 and a sodium or potassium chloride concentration of about 150 mM±100 mM. The salt concentration may be lower, if desired, such as in the range of 0 mM to 50 mM, or 0 mM to 25 mM, or 0 mM to 10 mM, or 0 mM to 5 mM. The ability of the method to remove endotoxins and nucleic acids remains within these ranges.

[0033] In some embodiments, it may be desirable to use higher concentrations of salt during equilibration, such as 250 mM to 1000 mM, or 250 mM to 750 mM, or 250 mM to 500 mM, and the ability of the method to remove endotoxins and nucleic acids remains within these ranges.

[0034] Elution of the biological product of interest should be attempted first with salts that have little or no known tendency to strongly interact with metals, such as halide salts or acetate salts. Such salts will minimize disruption of the interaction between immobilized Fe3+ ions and phosphatidic acid residues of endotoxins and nucleic acids. For example, elution may be initially assessed with a gradient up to 2.0 M NaCl, or up to 3.0 M NaCl, or up to 4.0 M NaCl.

[0035] Alternatively, elution of the biological product of interest may be accomplished by application of phosphate salts, including sodium and potassium phosphate salts, in a single step, or in multiple steps with increasing phosphate concentration, or in a linear gradient over a range. For example, elution may be performed in a single step of exposure to 500 mM phosphate; by increasing the phosphate concentration in increments of 100 mM, or 50 mM, or less, or more, to a final concentration of 500 mM phosphate; or by a linear gradient from 0 mM to 500 mM phosphate. As a general matter, the lowest concentration that elutes the biological product of interest will most effectively reduce endotoxins and nucleic acids. It is rarely necessary to exceed 500 mM phosphate, but higher increments may be evaluated if necessary. If the phosphate concentration is intended to exceed 500 mM, potassium phosphate is preferred over sodium phosphate, since the solubility of sodium phosphate is limited at high concentrations. Citrate may be substituted for phosphate, but endotoxin removal may be compromised. Elution can be achieved using chelating agents such as EDTA (ethylenediaminetetraacetic acid), but there is a high risk of compromising the ability of the method to remove endotoxins.

[0036] Further embodiments of the invention use the methods of the invention to remove viruses in virus-containing supplies; to prepare bacteriophage-containing compositions with low endotoxin content; to purify recombinantly produced proteins, particularly for therapeutic use; to remove endotoxins and / or viruses from cell culture components used in the preparation of recombinantly produced proteins; and to remove endotoxins from in vitro diagnostic assay reagents where endotoxins may interact with sample components in a manner that interferes with the ability of the assay to produce accurate and precise results.

[0037] The subject of the present invention is also the essentially aqueous endotoxin-free or endotoxin-depleted fraction obtainable by the method according to the invention, in particular 9This is a fraction that has an endotoxin concentration of less than 1 EU per infectious bacteriophage particle (1 EU per billion phage particles).

[0038] In some embodiments, the concentration of endotoxin units in a sample can be determined by some form of assay based on the Limulus Amoebocyte Lysate assay. Briefly, Limulus is the genus name for horseshoe crabs. Their blood contains cells called amoebocytes. When the amoebocytes are lysed and exposed to endotoxin, a blood clot is generated. Such assays are made in a wide variety of physical formats that allow for simple, accurate, and reproducible endotoxin quantification. Such assays are well known in the art, and many automated analytical systems are available to perform such tests on a routine, reproducible basis. One example includes the EndoSafe system manufactured by Charles River Laboratories. The complete analytical method can be found at www.criver.com / sites / default / files / resources / Endosafe(R)nexgen-PTS TMAssayGuide.pdf. To allow comparisons between different preparations, it is useful, and sometimes necessary, to express them in terms of a fixed concentration of product or number of product units. In one such case, endotoxin contamination can be expressed, for example, as the number of endotoxin units (EU) per million infectious phage, the number of endotoxin units per billion infectious phage, or the number of endotoxin units per some other fixed standard number of infectious phage. It will be understood that this approach to expressing endotoxin contamination precludes unbalanced comparisons based on endotoxin units per unit volume (e.g., per mL) and not taking into account the relative concentration of infectious phage. In another such case, endotoxin contamination can be expressed as endotoxin units per milligram (mg) or gram (g) of product. In another such case, endotoxin contamination can be expressed as endotoxin units per dose.

[0039] In embodiments where it is desirable to express the amount of endotoxin relative to a certain number of infectious viral particles, such as bacteriophages, it will be necessary to estimate the number of such viral particles. Estimates of the number of infectious viral particles are generally developed by a set of methods called plaque assays. Plaque assays for bacteriophages are typically performed in petri dishes or multi-well plates. They involve first creating a gel layer impregnated with a bacterial species that a particular bacteriophage species is known to infect. A dilute solution of bacteriophage is applied to a surface, soaked in a first gel layer, and then a second gel layer is added to prevent uncontrolled spreading of the liquid on the surface. Wherever a bacteriophage is present in contact with a bacterial cell, it will infect and eventually kill that cell, releasing thousands to millions of bacteriophages. These will eventually infect many more bacterial cells over a large area that is sufficient to create a visible plaque, an area that is visually distinct from the area where still-living bacterial cells are clustered. Several such experiments are performed with different concentrations of bacteriophage, up to the point where it is possible to show with reasonable confidence that one plaque corresponds to one initial infecting bacteriophage. This initial infecting bacteriophage is called a plaque forming unit (pfu). The number of pfu per unit of volume of the original sample, e.g. per milliliter (mL), is understood to represent the concentration of infectious bacteriophage in that original sample. Knowing the number of pfu / mL makes it possible to express the relative concentration of endotoxin, e.g. 10 endotoxin units per million pfu, 10 endotoxin units per million infectious phage, or 10EU / 106 pfu.

[0040] The subject of the present invention is also a kit comprising at least one component for carrying out the method of the invention. In one embodiment of the present invention, the kit comprises at least one component of the solid phase extraction material of the invention, in particular in the form of a particulate material or a monolith or a combination thereof.

[0041] The kits of the invention may further comprise instructions for carrying out the methods of the invention.

[0042] In closely related embodiments, the kit may optionally include other monoliths that have some ability to remove endotoxins, such as one or more of a monolith coated with a negatively charged chelating agent, a monolith coated with a non-metal binding anion exchanger, and / or a monolith coated with hydroxyl groups for high salt applications.

[0043] The kit of the present invention can be used as an endotoxin removal kit, which includes the immobilized ferric ion solid phase TREN-Fe in combination with a hydrogen bond solid phase (e.g., H-Bond ADC monolith). The kit can be used for the production of bacteriophages, but also in many other contexts, especially involving antibodies. Endotoxins are a problem for many product sectors in the pharmaceutical industry, including biotechnology.

[0044] Another such kit, also directed towards phages, may comprise one or more immobilized ferric ion solid phases TREN-Fe in combination with a hydroxylated solid phase (such as an OH monolith), where the hydroxylated solid phase is used to concentrate the bacteriophages and reduce the contaminant load (e.g., proteins, nucleic acids, and endotoxins).

[0045] In another embodiment, a kit comprising at least one component for carrying out the method of the invention comprises a TREN monolith and may further comprise a hydroxyl-coated monolith, where the two monoliths are particularly intended for the purification of viruses, including bacteriophages, with low endotoxin, DNA and host protein contamination.

[0046] In another embodiment, a kit containing at least one component for carrying out the method of the invention may contain only the TREN monolith. In one such embodiment, the kit may be directed to the specific application of removing endotoxins from antibodies. Alternatively, it may be used to remove endotoxins from bacteriophage preparations that have been partially purified by other means.

[0047] Another embodiment of the present invention is a method for depleting or removing pyrogens from a pyrogen-containing supply by solid phase material extraction in an essentially aqueous system, comprising: providing a known or suspected pyrogen-containing source; contacting said feed, known to contain pyrogens or suspected to contain pyrogens, with a solid phase material having a surface onto which ferric iron is immobilized; incubating said supply known to contain pyrogens or suspected to contain pyrogens for a time sufficient to allow the pyrogens to bind to the porous solid phase material; - separating said solid phase material from said essentially aqueous system, - optionally comprising the step of isolating said essentially aqueous system that has been rendered pyrogen-free or depleted. [Brief description of the drawings]

[0048] [Figure 1] FIG. 1 shows the chromatogram according to Example 2. [Diagram 2] FIG. 2 shows the chromatogram according to Example 3. [Diagram 3] FIG. 3 shows the chromatogram from Example 6. [Figure 4] FIG. 4 shows the chromatogram from Example 7. [Diagram 5] FIG. 5 shows the chromatogram from Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention utilizes the combined ability of ferric iron and the ability of electrostatic interactions to interact preferentially with endotoxin or endotoxin fragments. In the following, the present invention is described in detail by using the method of the present invention for endotoxin removal from bacteriophage produced in E. coli culture. In view of the teachings of the present invention, a person skilled in the art can easily develop a similar method for removing endotoxin or endotoxin fragments from any sample that contains or is suspected of containing endotoxin.

[0050] Bacteriophages for therapeutic and other commercial uses are produced by first culturing the bacteria they target. An appropriate cell culture medium is inoculated with the desired species of bacteria. When the bacterial cell density approaches its highest level, the bacteria are then inoculated with the particular species of bacteriophage that is desired to be purified. One or more such individual phage particles infect the bacterial cell. The bacterial cell is injected with DNA, producing hundreds to thousands or more individual bacteriophages inside the bacterial cell. The bacterial cell breaks open, releasing the phages, which then infect more bacterial cells. At some point, virtually all of the bacterial cells have been infected and died, and there are now large amounts of bacteriophages present in the cell culture along with dead bacterial debris. The cell culture process is terminated, and the bacteriophages are harvested. More specifically, bacterial debris is removed, for example by filtration or centrifugation, thus rendering the bacteriophage preparation in a state in which the bacteriophage can be purified from soluble contaminants such as proteins, DNA, endotoxins, and cell culture components.

[0051] The bacteriophage species of greatest interest are those that generally infect and kill the most dangerous species of bacteria. These include the so-called Gram-negative species, including, inter alia, Escherichia, Salmonella, Pseudomonas, Neisseria, Legionella, Shigella, and Klebsiella. Such species impose another burden in addition to their pathogenic potential. Their cell walls contain lipopolysaccharides (LPS), also known as endotoxins. LPS is pathogenic even independently of the parent bacteria. In some cases, even fragments of LPS, such as the lipid A region of the molecule, are pathogenic. High concentrations of LPS are released into the cell culture medium from dying bacteria. For bacteriophages to be used safely in humans, endotoxins must be removed to extremely low levels. Endotoxin levels in bacteriophage harvests can routinely exceed 50,000 endotoxin units per milliliter (EU / mL) and often must be reduced to below 10 EU / mL to make them safe for human use.

[0052] Bacteriophages produced by non-gram-negative species, particularly Staphylococcus spp. and Streptococcus spp., may also be contaminated with endotoxins from various sources, but generally have much lower levels of contamination and may benefit substantially from treatment according to the present invention.

[0053] The core method of the present invention involves the use of chelating agents, particularly ferric ions (Fe 3+ The present invention includes a solid phase whose surface has been chemically modified to include a chelating agent capable of chelating ferric iron. The chelating agent is a positively charged iron chelator, such as tris(2-aminoethyl)amine (TREN), among others. The positively charged chelating solid phase is exposed to a solution of ferric ions, such as 100 mM ferric chloride in water, to charge the chelating groups and leave the surface of the solid phase coated with chelated ferric iron.

[0054] Without being bound by any theory, it is believed that the chelated ferric ions have a strong preferential affinity for the phosphate-associated oxygen atoms of the lipid A and core polysaccharide regions of the LPS (endotoxin) molecule. It is understood that amino and amide nitrogen atoms also contribute to iron affinity when present in those regions. It is understood that the positively charged chelating ligands impart a positive charge to the surface of the solid phase, which is known to have a strong electrostatic affinity for endotoxins and acts in concert with the metal affinity of iron for endotoxins. When a solution containing endotoxin is exposed to the chelated ferric ions on the solid phase surface, the endotoxin binds. The binding is strong enough to withstand a wide range of salt concentrations and pH values. However, bacteriophages bind weakly or not at all to the chelated ferric ions. In the absence of a positively charged ligand, this allows the bacteriophage to pass through the surface of the solid phase. The positive charge of the solid phase imparted by the electropositive chelating ligand binds the bacteriophage, which has the beneficial effect of concentrating the bacteriophage from a dilute feed stream. This method does not require the solid phase to be in a column format. Any physical format of the solid that allows the endotoxin to come into direct contact with the chelating ferric iron solid phase will achieve the desired results.

[0055] TREN-Fe 3+ The surface area of ​​the solid phase material having the formula (I) is positively charged, preferably at least +1 or +2 or +3 per ligand.

[0056] It is also understood that the positive charges of the chelating agent increase the affinity of the solid phase for endotoxin, since the negatively charged phosphate-associated oxygen atoms of lipid A and the core polysaccharide region experience strong electrostatic attraction to the positive charges on the chelating ligand.

[0057] In many embodiments of the methods of the invention, contaminating nucleic acids, including DNA and RNA, are reduced or removed concomitantly with the reduction or removal of endotoxins. It will be appreciated that their strong binding to the solid phase is mediated by both the affinity of their phosphate groups for immobilized iron and their electrostatic affinity for the positively charged solid phase.

[0058] Experimental data show for all embodiments that immobilized ferric ions are a factor that allow very strong binding of endotoxins and furthermore separation of endotoxins from bacteriophages, which bind weaker. Endotoxin binding appears to be uniformly strong for all endotoxins. The relative weak binding of bacteriophages to chelated ferric ions on the solid phase may be explained by the observation that the phages lack phosphate residues that could facilitate strong binding of endotoxins. However, apart from the lack of phosphate residues, there is diversity among bacteriophages with respect to their structure and chemistry. Many have a positive charge that mediates interaction with negatively charged solid phases. Many have a negative charge that mediates interaction with positively charged solid phases.

[0059] The chelator TREN has up to four positive charges. When it binds to the three positively charged ferric ion, its net charge is hypothetically +7.

[0060] This method is particularly noteworthy for its exceptional ability to reduce endotoxin contamination, but also for its extremely tolerant nature with respect to operating conditions. This allows for greater flexibility than alternative methods. For example, anion exchange chromatography requires that salt concentrations be kept low during sample application. High salt concentrations interfere with endotoxin binding, thus limiting the usefulness of the anion exchange chromatography process. Salts for ion exchange must generally be monovalent, such as sodium chloride, whose individual ions have only one charge for Cl and 1+ for Na. The loading concentration of NaCl must generally be kept below about 50 mM. The use of multivalent ions, such as phosphate, which has two or three negative charges depending on the pH, prevents effective endotoxin removal by ion exchangers even at very low concentrations, such as about 25 mM or less. Utilizing a positively charged solid phase coated with chelated ferric ions in TREN allows for endotoxin removal even at NaCl concentrations of about 100 mM to about 1000 mM and phosphate concentrations of at least 500 mM. The operating pH range of the ferric ion method is much broader than that of anion exchange, for example, about pH 3-8, whereas the operating range of anion exchange chromatography is required to be about pH 7-8.

[0061] The ability of chelated ferric ions on positively charged surfaces to separate bacteriophage from endotoxin over a wide range of conditions is one of the surprising aspects of the present invention, expanding its usefulness by allowing it to be utilized in a variety of formats. In some embodiments, the buffer conditions allow bacteriophage to bind weakly to the solid phase. Thus, bacteriophage accumulate on the surface of the solid phase. When the bacteriophage are later separated from the solid phase, they elute at high concentrations, which can be advantageous, especially when the original feed has a large volume containing phage at low concentrations.

[0062] For bacteriophages, there is no known affinity for iron moieties on the solid surface. However, bacteriophages have a strong electrostatic attraction to the positive charges on the immobilized chelator. This causes the bacteriophage to bind to the surface of the solid phase along with the endotoxin. This has practical importance as it allows the bacteriophage to be rapidly concentrated from a large volume of a dilute sample. Because the bacteriophages have no significant affinity for immobilized iron, they can be selectively eluted by a buffer that leaves behind the strongly bound endotoxin, thereby separating the bacteriophage from the endotoxin.

[0063] It will be appreciated that negatively charged phosphate-associated oxygen atoms on nucleic acids such as DNA and RNA are also strongly bound by immobilized ferric ions. Similarly, their negative charge enhances their affinity for positively charged chelators. This extends the usefulness of positively charged iron chelators such as TREN, as they can enhance the removal of DNA and RNA along with endotoxins.

[0064] The unique utility of the solid phase with a positively charged chelator with immobilized ferric ions is further extended by its ability to be eluted with a salt gradient, allowing for more efficient removal of host cell proteins. For contaminants lacking affinity for iron, the surface of the solid phase is functionally equivalent to an anion exchanger and can be used as is.

[0065] Experimental data show that immobilized ferric ions are a necessary factor for very strong endotoxin binding for all embodiments.

[0066] In some embodiments, conditions can be developed such that the bacteriophage bind to the chelated ferric ions on the solid phase. This can be achieved by avoiding the presence of excess salt, including in particular avoiding an excess of phosphate. Many phages bind to TREN, which would serve the purpose of concentration. By increasing the conductivity of the salt, the phages can then be recovered. The endotoxins remain bound to the solid phase under conditions that allow for the recovery of the phages. This mode of chromatography is known in the art as a bind-elute mode, in which the product of interest is bound from the crude oil feed stream while some contaminants flow through, and the product of interest is then later eluted in a concentrated form while another set of contaminants may still remain bound. In this case, the contaminants that remain bound include endotoxins, DNA, and RNA, among others.

[0067] In some such embodiments, the buffer conditions used to equilibrate the sample and solid phase may be defined by the absence of salt beyond the presence of the buffer compound, e.g., containing about 20 mM to about 50 mM acetate, MES, phosphate, Tris or other buffer, but lacking additional salts such as sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, ammonium sulfate, sodium sulfate or potassium sulfate. In other such embodiments, the concentration of buffer salts at concentrations greater than about 20 mM to about 50 mM may be limited to concentrations less than about 50 mM, less than about 100 mM, or less than about 100 mM for any of the above or other salts. In all of the above cases, the intent is to allow binding of the bacteriophage.

[0068] In some embodiments, the bacteriophage may be eluted in a single step of increasing salt concentration. Similar embodiments may include a pre-elution step to remove weakly bound contaminants before the bacteriophage is eluted with a higher concentration of salt. In some such embodiments, a third step may be applied to remove contaminants that are more strongly bound than the phage from the solid phase with a higher concentration of salt. In some embodiments, instead of separate steps of salt concentration, the salt concentration may be changed gradually and continuously to perform a so-called linear gradient elution. In some embodiments, the pH may be changed to adjust the binding conditions and the amount of salt required to achieve elution of the bacteriophage. Generally, the binding between bacteriophage and endotoxin becomes stronger as the pH decreases, which means that at low pH values ​​the binding is more resistant to salt and more salt may be required to elute the bacteriophage. Such relationships and how to manage them are well known in the art.

[0069] It is important to recognize that despite the wide range of conditions and modes of operation that can be embodied by the present invention, the present invention meets the objective of removing or reducing the amount of endotoxin in a bacteriophage preparation, regardless of their variations. Variation of conditions within the specified range is not determinative of whether the present invention works. The ability of the present invention to achieve the claimed endotoxin reduction or removal is defined by the presence of chelated ferric ions on the surface of the solid phase. This is important because it allows the use of conditions that result in high recovery of infectious bacteriophage. This characteristic is even more important to understand, as it contrasts with the requirements and capabilities of other endotoxin removal methods, such as anion exchange chromatography, which requires a very narrow range of pH and salt conditions. The ability of the present invention to achieve good results over a wide range of conditions highlights an important aspect of its usefulness.

[0070] In some embodiments, it may be desirable to use the method of the present invention to remove endotoxins from biological preparations that do not bind well to the positively charged chelator-ferric ion complex. One example is IgG antibodies. Most bind poorly to positively charged media because electrostatic repulsion caused by their electrically positive charge prevents effective binding. In such cases, the biological product flows through the column during sample application, while endotoxins and nucleic acids are removed.

[0071] In some embodiments, the contact time required for endotoxin binding varies depending on the type of solid phase to which the chelated ferric ions are bound. When the solid phase is in the form of a membrane, fiber, sheet, hydrogel, or monolith used in a flow-through format such as a chromatography device, the required contact time may be less than about 10 seconds, less than about 5 seconds, or less than about 1 second. For practical purposes, such low contact times are generally not achievable due to instrumental limitations, including pump flow rates and / or pressure tolerances. When the solid phase consists of non-porous particles packed in a column, the required contact time may be less than about 10 seconds, less than about 30 seconds, less than about 1 minute, or less than about 2 minutes. When the solid phase consists of porous particles packed in a column, the required contact time may be about 2 minutes to about 5 minutes or more, with longer contact times, such as about 10 minutes to about 20 minutes, ensuring more effective endotoxin removal. If the solid phase consists of loosely distributed porous particles in an open vessel, the required contact time may be greater than about 30 minutes, greater than about 60 minutes, or greater than about 120 minutes, and may continue to benefit from further exposure, such as continuing contact for about 4 hours, about 8 hours, or about 16 hours. Contact time parameters are well known in the art, and different contact times that may be required for different materials or operating formats are also well known and well within the ability of one of ordinary skill in the art to optimize without undue experimentation.

[0072] In certain embodiments, endotoxins may be removed from the solid phase after use by treatment with about 1 M NaOH for at least about 60 minutes. Optionally, sodium chloride may be included, resulting in a compound formulation of 1 M NaOH plus 2 M NaCl in one example. The ferric coating may be regenerated, if necessary, by passing about one column volume of about 100 mM ferric chloride over it prior to equilibration for the next processing cycle.

[0073] In all embodiments, optimization of endotoxin binding capacity and removal efficiency should be balanced with the need to preserve the ability of the bacteriophage to infect its bacterial target. In some such embodiments, one or more stabilizing compounds may be included in the buffer to aid in preserving viral infectivity. In one such embodiment, the buffer may contain about 10% glycerin, or about 5% to about 15% glycerin, or about 2% to about 20% glycerin, or about 1% to about 25% glycerin, or higher concentrations of glycerin up to about 50% as a stabilizer to preserve viral infectivity. It will be appreciated that additives such as glycerin will increase viscosity. This may disqualify certain solid phases, such as particles packed in a column, since shear stress increases directly proportional to viscosity. In a monolith, this constraint may be suspended since the flow through the chromatographic bed is laminar. This contributes to the conclusion that monoliths are the preferred physical format of the solid phase, especially where the inclusion of a viscosity-increasing additive such as glycerin may be required to stabilize the phage.

[0074] In some embodiments, the buffer may contain an amino acid as a stabilizer that is positively charged under physiological conditions. The buffer may specifically contain about 100 mM to about 700 mM arginine, or about 200 mM to about 600 mM arginine, or about 300 mM to about 500 mM arginine, or about 400 mM to about 500 mM arginine. In some such embodiments, the buffer may contain about 100 mM to about 200 mM histidine as a stabilizer, or about 50 mM to about 250 mM, or about 75 mM to about 150 mM, or about 25 mM to about 225 mM.

[0075] In other embodiments, the buffer may contain about 50 mM to about 250 mM sorbitol, xylose, or mannitol, or about 100 mM to about 200 mM, or about 150 mM to about 200 mM sorbitol, xylose, or mannitol as a stabilizer. In some embodiments, the buffer may contain about 1% to about 15% sucrose, or about 5% to about 10% sucrose. In some embodiments, the buffer may contain about 50 mM to about 2.0 M, or about 100 mM to about 1.0 M, or about 200 mM to about 800 mM, or about 400 mM to about 700 mM, or about 500 mM to about 600 mM of glycine, taurine, or betaine as a stabilizer.

[0076] In particular, in the method of the present invention, a monolithic solid phase is used. The monolith ensures faster binding kinetics and higher capacity for endotoxins than other solid phase configurations. Without being bound by this theory, an explanation for this can be found in the molecular design of endotoxins, especially their large size. Endotoxins in bacterial cell cultures are not pure, as even pure endotoxins form large aggregates in the presence of metal ions and in the absence of detergents. They can be coaggregated with a range of other components, including cell debris, such as cell wall and cell membrane fragments, proteins, nucleic acids, lipids, and carbohydrates, among others. These heteroaggregates can sometimes exceed hundreds of nm in size. Some of these aggregates are too large to enter the pores where most of the bound substrate is located, thus affecting the removal performance in porous particle chromatography media. Even those small enough to enter the pores still have slow diffusion constants that slow their entry into the pores.

[0077] Any solid phase used to practice the present invention may optionally be used once and then discarded, or may be used multiple times.

[0078] A limitation of all solid phase methods for endotoxin removal is related to their capacity. Even highly effective removal methods can be overwhelmed by the excessive amount of sample. For this reason, endotoxin removal, especially from high endotoxin feeds such as bacteriophage preparations, often includes a preliminary step to bring the endotoxin level into the range of the final polishing method. Loading 10 million endotoxin units onto a solid phase with a nominal capacity of 10 million endotoxin units will exceed its capacity and leave the processed preparation contaminated with endotoxin.

[0079] In some embodiments, the capacity limitations of the chelating ferric ion solid phase, if significant, can be circumvented by pre-treating the sample with an alternative method that reduces the endotoxin content sufficiently to the extent that the endotoxin content can be further reduced by the methods of the invention to meet the requirements for suitable purification of bacteriophage for human use.

[0080] Solid-phase extraction materials for binding ferric iron to their surfaces are chemically modified with a positively charged chelating ligand, namely tris(2-aminoethyl)amine (TREN).

[0081] Alternatively, the positively charged iron chelating ligand may be diethylenetriamine, triethylenetetramine, tetraethylpentamine, N'-[5-(acetyl-hydroxy-amino)pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl)]propanoylamino-pentyl]-N-hydroxy-butanediamide (deferoxamine), 3-hydroxy-1,2-dimethylpyridin-4(1H)-one (deferiprone), or 4-[(3Z,5E)-3,5-bis(6-oxo-1-cyclohexa-2,4-dienylidene)-1,2,4-triazolidin-1-yl]benzoic acid (deferasirox). Solid phase materials with immobilized TREN ligands are available from Bioworks Workbeads. TM40 TREN. It is not commercially available in the iron-loaded form required to carry out the method of the invention. A variety of other solid phase materials can be synthesized on which TREN itself is immobilized and ferric ions are subsequently captured by a TREN ligand or another positively charged iron chelating ligand. Such solid phase materials can be provided in the form of monoliths, hydrogels, filters, fibers, free particles, but are not limited to any of these, which can be provided in the form of a ready-to-use chromatography device. In further embodiments, the method of the invention using ferric ions immobilized on a solid phase by positively charged TREN can be combined with additional purification methods to achieve a higher degree of purification (e.g., a higher degree of contaminating protein removal, a higher degree of contaminating DNA removal, and / or a higher degree of endotoxin removal). In some such embodiments, the method of the invention using ferric ions immobilized on a solid phase by a positively charged TREN may be combined with a precipitation step in which the product of interest is precipitated by known techniques, such as salt precipitation with ammonium sulfate or potassium phosphate, or PEG precipitation with polyethylene glycol, for example, with a molecular weight of 4-10 kDa. In other such embodiments, the method of the invention using ferric ions immobilized on a solid phase by a positively charged TREN may be combined with a chromatographic method, such as ion exchange chromatography, or hydrophobic interaction chromatography, or hydrogen bond chromatography, or affinity chromatography, or steric exclusion chromatography, or any other chromatographic method. In some such embodiments, the method of the invention using ferric ions immobilized on a solid phase by a positively charged TREN may be operated in a column format. In other such embodiments, the method of the invention using ferric ions immobilized on a solid phase by a positively charged TREN may be operated in a batch format. In some such embodiments, both the method of the invention using ferric ions immobilized on a solid phase by a positively charged TREN and the additional purification method may be carried out in a batch format.

[0082] In one embodiment in which the method of the present invention using ferric ions immobilized on a solid phase by positively charged TREN is combined with other chromatographic methods, the other chromatographic methods include the use of a solid phase extraction material with hydroxyl groups (-OH) on its surface. In one such embodiment, this approach is used for the purpose of purifying viruses (e.g., lipid enveloped viruses, or non-lipid enveloped viruses, or bacteriophages). In one such embodiment, the hydroxylated solid phase extraction material may be combined with viruses in the presence of a precipitating salt, such as one or a combination of ammonium sulfate, potassium phosphate, sodium or potassium sulfate, or sodium or potassium citrate, among others, where the precipitating salt is present in a concentration sufficient to push the virus out of solution and accumulate on the surface of the solid phase, while smaller contaminants do not accumulate and are therefore excluded. The virus is then recovered by reducing the concentration of the precipitating salt, and the virus is eluted. The eluted fraction containing the virus can then be processed by the method of the present invention using ferric ions immobilized on a solid phase by positively charged TREN.

[0083] The kit of the present invention includes at least one solid phase extraction material in a suitable form that can be used to carry out the method of the present invention. The solid phase extraction material is a granular material, a monolith, or a combination thereof. The solid phase extraction material may be arranged in a cartridge, a column, or may be packed in loose form. The solid phase extraction material may also be provided in the form of fibers and / or fibrous materials, or one or more membranes.

[0084] In one embodiment, the component for carrying out the method of the invention is a TREN monolith. In such an embodiment, the kit may optionally include other monoliths with some ability to remove endotoxins, such as one or more of a monolith coated with a negatively charged chelator, a monolith coated with a non-metal binding anion exchanger, and / or a monolith coated with hydroxyl groups for high salt applications.

[0085] In another embodiment, a kit comprising at least one component for carrying out the method of the invention comprises a TREN monolith and optionally a hydroxyl-coated monolith, where the two monoliths are particularly intended for the purification of viruses, including bacteriophages, with low endotoxin, DNA and host protein contamination.

[0086] In another embodiment, the kit of the present invention may further comprise at least one component for the preparation of a buffer solution used in the method of the present invention. Such components may be substances for adjusting the ionic strength or pH value of the solutions used during the implementation of the method of the present invention. The kit of the present invention may also comprise materials for carrying out an assay for measuring the endotoxin content or an assay for determining the quality of the product to be isolated, such as measuring the infectivity of the bacteriophage to be purified. The kit of the present invention may further comprise instructions for carrying out the method of the present invention.

[0087] The kit of the present invention can be used as an endotoxin removal kit, which includes one or more immobilized ferric ion solid phases TREN-Fe in combination with a hydrogen bond solid phase (H-Bond ADC monolith). The kit can be used for the production of bacteriophages, but also in many other contexts, especially involving antibodies.

[0088] Another such kit, also directed to phages, may include one or more immobilized ferric ion solid phases TREN-Fe in combination with a hydroxylated solid phase (such as an OH monolith), where the hydroxylated solid phase is used to concentrate the bacteriophages and reduce the contaminant load (e.g., proteins, nucleic acids, and endotoxins). The kit may also include one or more immobilized ferric ion solid phases.

[0089] All references cited herein are incorporated by reference to the extent they do not contradict an explicit teaching herein.

[0090] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0091] Example 1 Preparation of an experimental control group to provide a baseline for demonstrating the relative efficacy of the present invention. 10 mL of clarified harvest of E. coli cell culture containing bacteriophage T4 was partially purified by hydrophobic interaction chromatography (HIC). The bacteriophage elution peak from the HIC step was diluted with 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM NaCl, 10% glycerol, pH 7.7 to a final conductivity of 7.42 mS / cm. 100 μL of CIMmac QA (strong anion exchange) monolith (BIA Separations) was equilibrated with 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM NaCl, 10% glycerol, pH 7.7. The diluted bacteriophage was loaded onto the QA monolith. The monolith was washed with 60 bed volumes of equilibration buffer, followed by a linear gradient elution over 50 bed volumes (5 ml) to 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 2 M NaCl, 10% glycerol (pH 7.7). The column was washed and sanitized with 1 M sodium hydroxide, 2 M sodium chloride. Fractions were collected throughout the experiment for subsequent analysis. The bacteriophage eluted in a single peak with a conductivity value of approximately 25 mS / cm (measured at the peak center). 55% of the infectious bacteriophage eluted in the main peak. Testing of the peak by Endosafe showed a concentration of 141 endotoxin units per milliliter (EU / mL), which corresponds to 11 EU per billion phages.

[0092] Example 2 (Comparative Example) Experimental endotoxin reduction with IDA-Fe (a negatively charged chelating ligand) (CIM IDA, BIA Separations) following an initial hydrophobic interaction chromatography (HIC) step. HIC was performed on the T4 bacteriophage harvest as described in Example 1, but on a larger scale with 160 mL of lysate loaded onto an 8 mL HIC monolith. The 31 mL elution fraction contained 138,167 EU / mL of endotoxin. This fraction was diluted with 20 mM KH2PO4, 10% glycerol, pH 7.0 to a final conductivity of 6.7 mS / cm. 340 μL of IDA monolith was prepared by washing with 20 CV of deionized water; then 20 CV of 100 mM acetic acid, pH 3.0; 20 CV of 200 mM ferric chloride, then 20 CV of 50 mM acetic acid, 2.0 M sodium chloride, pH 4.5; 20 CV of deionized water. The column was then equilibrated to 20 mM potassium phosphate, 10% glycerol, pH 7.0. A 2 mL fraction from the HIC was diluted with 40 mL of mobile phase A to a conductivity of 6.7 mS / cm. The sample was loaded onto the IDA monolith and then the column was washed with 20 CV of 20 mM potassium phosphate, 10% glycerol, pH 7.0 to displace any remaining unbound material. The column was subjected to a linear gradient elution with 20 CV ending with 500 mM potassium phosphate, pH 7.0. Fractions were collected throughout the experiment for subsequent analysis. The chromatogram is shown in Figure 1. 100% of the infectious bacteriophage was found in the flow-through fraction from the 0.0 mL mark to approximately 45 mL, indicating about a 50% dilution from volume eluted from the HIC column. Endotoxin concentration was measured to be 315 EU / mL, which corresponds to 5.75 EU per billion phage.

[0093] Example 3 Experimental demonstration that ferric ions immobilized on the surface of an experimental monolithic tris(2-aminoethyl)amine (TREN) solid phase surface manufactured by BIA Separations achieves superior endotoxin reduction compared to a strong anion exchanger (CIMmultus QA, BIA Separations). HIC was performed on the T4 bacteriophage harvest as described in Example 2. Endotoxin testing showed that the bacteriophage fraction contained 138167 EU / mL endotoxin. A 2 mL fraction from the HIC was diluted with 40 mL of mobile phase A to a final conductivity of 9.3 mS / cm. A 1 mL TREN monolith was prepared as described in Example 2 by running the reagents over it at a flow rate of 5 CV / min. The column was equilibrated with 20 mM potassium phosphate, pH 7.0. 40 mL of the diluted sample was loaded and then the column was washed with 15 CV of 20 mM potassium phosphate, pH 7.0. The column was subjected to a linear gradient elution with 20 CV ending with 500 mM potassium phosphate, pH 7.0. Fractions were collected throughout the experiment for subsequent analysis. The chromatogram is shown in Figure 2. No infectious bacteriophage was found in the column effluent corresponding to the column loading phase. 125% of the bacteriophages were found in the peak eluting at a conductivity of 16 mS / cm. Testing with the LAL assay (Endosafe) showed a concentration of less than 10 endotoxin units per milliliter (EU / mL) in the elution peak, which is understood to represent a value close to but not exceeding 10 EU / mL, equivalent to less than 0.4 EU per billion phages. This is approximately 25 times better than achieved with the industry standard method using a strong anion exchanger.

[0094] Example 4 (Comparative Example) Preparation of an experimental control to provide a comparative example for the method of the present invention. Clarified harvest of E. coli culture containing bacteriophage T4 was partially purified by ammonium sulfate precipitation. 1 mL of harvest was combined with 1.5 mL of 3 M ammonium sulfate, buffer 50 mM MES, pH 6.5. The mixture was centrifuged at 9000×g for 10 min and the supernatant was discarded. The precipitate was resuspended in 0.8 mL of 500 mM potassium phosphate, pH 7.0. Free (unloaded) porous particles with iminodiacetic acid on the surface (iminodiacetic acid Sepharose, Sigma-Aldrich) were prepared by washing twice with 20 mL of deionized water, centrifuging at 100×g for 5 min, then discarding the supernatant, then washing in the same way with 20 mL of 100 mM sodium acetate, pH 3.0, then treating with 20 mL of 200 mM ferric chloride, then washing with 50 mM sodium acetate, 2 M sodium chloride, pH 4.5, then washing with 20 mL of deionized water. The particles were then equilibrated with 50 mM MES, pH 6.5. Experiments were performed with a 50% slurry of these particles. 0.2 mL of the 50% slurry was added to 0.8 mL of the resuspended ammonium sulfate precipitate and incubated at 2-8 °C with mixing three times a day to allow endotoxin binding. The particles were sedimented by centrifugation and removed. The remaining liquid containing the bacteriophage contained 15.8 EU / mL of endotoxin.

[0095] Example 5 Experimental demonstration that ferric ions immobilized on the surface of a tris(2-aminoethyl)amine (TREN) solid phase achieves superior endotoxin reduction, independent of the purification method and physical form of the solid phase combined. Clarified harvest of E. coli culture containing bacteriophage T4 was partially purified by hydrophobic interaction chromatography (HIC). 1 ml of the eluted fraction containing 5876 EU was used for the next step after dilution 100-fold with 50 mM MES, pH 6.0. Free (unloaded) porous particles with TREN on their surface (Workbeads TREN 40, BioWorks) were charged with ferric ions. 1 mL of 50% slurried TREN particles was added to 0.25 mL of the diluted HIC fraction and incubated for 120 min with occasional mixing to allow endotoxin binding. The particles were sedimented by centrifugation and removed. The remaining liquid containing the bacteriophage contained 9.73 EU / mL of endotoxin.

[0096] Example 6 Preparation of experimental controls to provide a baseline for demonstrating the relative effectiveness of the present invention. First purification of bacteriophage T4 by preferential exclusion chromatography and then purification by anion exchange chromatography. Anion exchange chromatography using strong anion exchangers is considered the industry standard method for endotoxin removal. 10 mL of clarified harvest of E. coli cell culture containing bacteriophage T4 was partially purified by preferential exclusion chromatography on 1 mL of CIMmultus OH monolith. The bacteriophage elution peak was diluted with 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM NaCl, 10% glycerol, pH 7.7 to a final conductivity of 7.42 mS / cm. 100 μL of CIMmac QA (strong anion exchange) monolith was equilibrated with 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM NaCl, 10% glycerol, pH 7.7. The diluted bacteriophage was loaded onto the QA monolith. The monolith was washed with 60 bed volumes of equilibration buffer, followed by a linear gradient elution with 50 bed volumes (5 mL) to 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 2 M NaCl, 10% glycerol, pH 7.7. The column was washed and sanitized with 1 M sodium hydroxide, 2 M sodium chloride. Fractions were collected throughout the experiment for subsequent analysis. Endotoxin was reduced from 177,524 EU per phage 1e+10 in the harvest to 12054 EU per phage 1e+10 after the preferential exclusion step and 78 EU per phage 1e+10 after anion exchange. Host protein was reduced from 6059 ng per phage 1e+10 in the harvest to 153 ng per phage 1e+10 after the preferential exclusion step and 115 ng per phage 1e+10 after anion exchange. The results are shown in Figure 3.

[0097] Example 7 Initial purification of bacteriophage T4 by preferential exclusion chromatography and purification by hydrogen bonding chromatography. The hypothesis was tested that hydrogen bond chromatography would ensure more effective endotoxin removal than anion exchange, and thus provide a more competitive reference for the method of the present invention. An aliquot of bacteriophage eluted from the preferential exclusion chromatography step in Example 6 was purified by hydrogen bond chromatography instead of anion exchange chromatography. 100 μL of CIMmac H-Bond monolith was equilibrated with 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 20 mM NaCl, 10% glycerol, pH 7.7. Diluted bacteriophage was loaded onto the QA monolith. The monolith was washed with 60 bed volumes of equilibration buffer, and then a linear gradient elution with 50 bed volumes (5 ml) was performed to 50 mM Tris, 1 mM CaCl2, 0.5 mM MgCl2, 2 M NaCl, 10% glycerol, pH 7.7. The column was washed and sanitized with 1M sodium hydroxide, 2M sodium chloride. Fractions were collected throughout the experiment for subsequent analysis. Endotoxin was reduced from 177,524 EU per 1e+10 phage in the harvest to 12054 EU per 1e+10 phage after the preferential exclusion step and 19 EU per 1e+10 phage after hydrogen bond chromatography. Host protein was reduced from 6059 ng per 1e+10 phage in the harvest to 153 ng per 1e+10 phage after the preferential exclusion step and 26 ng per 1e+10 phage after hydrogen bond chromatography. The results are shown graphically in Figure 4. Overall, endotoxin removal by hydrogen bond chromatography was approximately 4 times better than anion exchange, and host cell protein removal was approximately 5 times better.

[0098] Example 8 Experimental comparison of purification performance by the method of the present invention with anion exchange chromatography and hydrogen bonding chromatography. An aliquot of bacteriophage eluted from the preferential exclusion chromatography step in Example 6 was purified on a 100 μL TREN monolith. Equilibrated with 20 mM potassium phosphate, pH 7.0, 40 mL of diluted sample was loaded, and the column was then washed with 15 CV of 20 mM potassium phosphate, pH 7.0. The column was subjected to a linear gradient elution with 20 CV ending with 500 mM potassium phosphate, pH 7.0. Fractions were collected throughout the experiment for subsequent analysis. Endotoxin was reduced from 177,524 EU per 1e+10 phage in the harvest to 12054 EU per 1e+10 phage after the preferential exclusion step, and less than EU per 1e+10 phage (below the sensitivity of the assay) after the method of the present invention. Host protein was reduced from 6059 ng per 1e+10 phage in the harvest to 153 ng per 1e+10 phage after the preferential exclusion step, to less than 1 ng per 1e+10 phage (below the sensitivity of the assay) after the method of the invention. The results are shown graphically in Figure 5. Overall, endotoxin removal by the method of the invention was approximately 80 times better than anion exchange and 20 times better than hydrogen bond chromatography, and host cell protein removal was approximately 100 times better than anion exchange and 25 times better than hydrogen bond chromatography.

[0099] References 1: Lolas A. Microbial Control Strategies in Bioprocessing Falling Short of Assur-ing Product Quality and Satisfying Regulatory Expectations. American Pharm. Rev. (2013); www.americanpharmaceuticalreview.com / Featured-Articles / 134040-Microbial-Control-Strategies-in-Bioprocessing-Falling-Short-of-Assuring-Product-Quality-and-Satisfying-Regulatory-Expectations. 2: Suvarna K, et al. Case Studies of Microbiological Contamination in Biological Product Manufacturing. American. Pharm. Rev. 14(1) 2011: 50-56. 3: Issekutz A. Removal of Gram-Negative Endotoxin from Solutions By Affinity Chromatography. J. Immunol. Met. 61(3) 1983: 275-281. 4: Anspach F. Endotoxin Removal By Affinity Sorbents. J. Biochem. Biophys. 449(1-3)2001: 665-681. 5: Guo W, et al. Removal of Endotoxin from Aqueous By Affinity Membrane. Biomed. Chromatogr. 11(3) 1997: 164-166. 6: Chen H, et al. Factors Affecting Endotoxin Removal from Recombinant Therapeutic Proteins By Anion Exchange Chromatography. Prot. Expr. Purif. 64(1) 2009:76-81. 7: Hirayama C, et al. Cross-linked N,N Dimethylaminopropylacrylamide Spherical Particles for Selective Removal of Endotoxin. J. Chromatogr. A 676(2) 1994: 267-275. 8: Hoess A, Lidington R. Lipopolysaccharide-Binding and Neutralizing Peptides. World Patent WO 1995005393 A2 1993l; www.google.com / patents / WO1995005393A2?cl=en:Method. 9: Kang Y. Endotoxin removal from protein solutions by immobilized metal affinity chromatography, 2000, archives.njit.edu / vol01 / etd / 2000s / 2000 / njit-etd2000-023 / njit-etd2000-023.pdf. 10: Luo R, Kang Y. Methods for endotoxin removal from biological solutions using immobilized meta affinity, United States Patent US6365147B1, 1999.

Claims

1. 1. A method for depleting or removing endotoxins from a known or suspected endotoxin-containing source by solid phase extraction material in an essentially aqueous system, comprising: Providing a supply known to contain endotoxins or suspected to contain endotoxins; contacting the supply, known to contain endotoxins or suspected to contain endotoxins, with a positively charged solid phase material having a surface onto which ferric iron is immobilized, wherein the solid phase extraction material immobilizes the ferric iron via a (2-aminoethyl)amine (TREN) ligand; - incubating the supply known to contain endotoxin or suspected to contain endotoxin for a time sufficient to allow endotoxin to bind to the porous solid phase material; - separating the solid phase material from the essentially aqueous system, - A method which may optionally include the step of isolating said essentially aqueous system which has been rendered free or depleted of endotoxins.

2. 2. The method of claim 1, wherein the solid phase extraction is a method selected from the group consisting of chromatography, filtration, co-precipitation, and combinations thereof.

3. 3. The method of claim 1 or claim 2, wherein the solid phase material comprises a chromatographic material that depletes or removes endotoxins from the supply known to contain endotoxins or suspected to contain endotoxins by ferric chelation.

4. The method according to any one of claims 1 to 3 for the production of low endotoxin bacteriophage containing compositions, in particular for the purification of recombinantly produced proteins, for therapeutic applications, for the removal of endotoxins and / or viruses from cell culture components used in the preparation of recombinantly produced proteins, and for the removal of endotoxins from in vitro diagnostic assay reagents.

5. The method according to any one of claims 1 to 4, carried out in combination with other purification methods.

6. 6. The method of claim 5, wherein the other purification method is selected from hydrophobic interaction chromatography, preferential exclusion chromatography, and anion exchange chromatography.

7. A bacteriophage-containing, endotoxin-removed or endotoxin-depleted fraction obtained by the process of the present invention, comprising bacteriophages in an essentially aqueous system, the endotoxin concentration being 10 times that of infectious bacteriophage particles. ９ A fraction that is less than 1 EU per individual.

8. A kit comprising at least one component for carrying out the method according to any one of claims 1 to 6.

9. 9. The kit of claim 8, wherein the at least one component is at least one of the solid phase extraction materials, the solid phase extraction material having a positive charge and immobilizing ferric iron through a (2-aminoethyl)amine (TREN) ligand.

10. 10. The kit of claim 9, wherein the solid phase extraction material is in the form of a particulate material or monolith, a fiber, a fibrous material, one or more membranes, or a combination thereof.

11. A kit according to any one of claims 8 to 10, comprising instructions for carrying out the method according to any one of claims 1 to 6.

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